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THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 


Editorial  Board 


JOHN  B.  BUCK,  National  Institutes  of  Health 
PHILIP  B.  DUNHAM,  Syracuse  University 
SALLY  HUGHES-SCHRADER,  Duke  University 

LIBBIE  H.  HYMAN,  American  Museum  of 

Natural  History 

SHINYA  INOUE,  University  of  Pennsylvania 
J.  LOGAN  IRVIN,  University  of  North  Carolina 


JOHN  H.  LOCHHEAD,  University  of  Vermont 
ROBERTS  RUGH,  Columbia  University 
MELVIN  SPIEGEL,  Dartmouth  College 

WM.  RANDOLPH  TAYLOR,  University  of 

Michigan 

ANNA  R.  WHITING,  Oak  Ridge  National 

Laboratory 


CARROLL  M.  WILLIAMS,  Harvard  University 

DONALD  P.  COSTELLO,  University  of  North  Carolina 
Managing  Editor 


VOLUME  131 

JULY  TO  DECEMBER,  1966 


Printed  and  Issued  by 

LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 
LANCASTER,  PA. 


11 

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

Subscriptions  and  similar  matter  should  be  addressed  to  The 
Biological  Bulletin,  Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts.  Agent  for  Great  Britain :  Wheldon  and  Wesley, 
Limited,  2,  3  and  4  Arthur  Street,  New  Oxford  Street,  London, 
W.  C.  2.  Single  numbers,  $3.75.  Subscription  per  volume  (three 
issues),  $9.00. 

Communications  relative  to  manuscripts  should  be  sent  to  Dr. 
Donald  P.  Costello,  Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts,  between  June  1  and  September  1,  and  to  Dr. 
Donald  P.  Costello,  P.O.  Box  429,  Chapel  Hill,  North  Carolina 
27514,  during  the  remainder  of  the  year. 


Second-class  postage  paid  at  Lancaster,  Pa. 


LANCASTER  PRESS,  INC.,  LANCASTER,  PA. 


CONTENTS 


No.  1.     AUGUST,  1966 

PAGE 

Annual  Report  of  the  Marine  Biological  Laboratory 1 

BEVELANDER,  GERRIT,  AND  HIROSHI  NAKAHARA 

Correlation  of  lysosomal  activity  and  ingestion  by  the  mantle  epithelium  .     76 

COOK,  J.  R. 

Adaptations  to  temperature  in  two  closely  related  strains  of  Euglena  gra- 
cilis 83 

HAYWARD,  JOHN  S.,  AND  ERIC  G.  BALL 

Quantitative  aspects  of  brown  adipose  tissue  thermogenesis  during  arousal 
from  hibernation 94 

KANATANI,  HARUO,  AND  MIWAKO  OHGURI 

Mechanism  of  starfish  spawning.     I.  Distribution  of  active  substance  re- 
sponsible for  maturation  of  oocytes  and  shedding  of  gametes 104 

KELLY,  MAHLON  G.,  AND  STEVEN  KATONA 

An  endogenous  diurnal  rhythm  of  bioluminescence  in  a  natural  popula- 
tion of  dinoflagellates 115 

MAUZEY,  KARL  PERRY 

Feeding  behavior  and  reproductive  cycles  in  Pisaster  ochraceus 127 

RUGH,    R.,    L.    DUHAMEL,    C.    SoMOGYI,    A.    CHANDLER,    W.    R.    COOPER,    R. 

SMITH  AND  G.  STANFORD 

Sequelae  of  the  LD/50  x-ray  exposure  of  the  pre-implantation  mouse 

embryo :  Days  0.0  to  5.0 145 

STANLEY,  JON  G.,  AND  W.  R.  FLEMING 

The  effect  of  hypophysectomy  on  sodium  metabolism  of  the  gill  and  kid- 
ney of  Fundulus  kansae 155 

STEINBACH,  H.  BURR 

The  effects  of  glycerol  and  other  organic  solutes  on  motility  and  respira- 
tion of  some  invertebrate  spermatozoa 166 

STEPHENS,  GROVER  C.,  AND  RAGHUNATH  A.  VIRKAR 

Uptake  of  organic  material  by  aquatic  invertebrates.     IV.  The  influence 

of  salinity  on  the  uptake  of  amino  acids  by  the  brittle  star,  Ophiactis  arenosa  172 

ZEIN-ELDIN,  ZOULA  P.,  AND  GEORGE  W.  GRIFFITH 

The   effect   of   temperature    upon    the    growth    of    laboratory-held    post- 
larval  Penaeus  aztecus 186 

REITE,  OLA  BODVAR 

Mechanical  forces  as  a  cause  of  cellular  damage  by  freezing  and  thawing  197 

TYLER,  ALBERT,  JORAM  PIATIGORSKY  AND  HIRONOBU  OZAKI 

Influence  of  individual  amino  acids  on  uptake  and  incorporation  of  valine, 
glutamic  acid  and  arginine  by  unfertilized  and  fertilized  sea  urchin  eggs. . .  204 


iv  CONTENTS 

No.  2.     OCTOBER,  1966 

BEERS,  C.  DALE 

Distribution  of  Urceolaria  spinicola  (Ciliata,  Peritrichida)   on  the  spines 

of  the  sea  urchin  Strongylocentrotus  droebachiensis 219 

BOWEN,  SARANE  T.,  JEAN  HANSON,  PHILIP  DOWLING  AND  MAN-CHIU  POON 

The  genetics  of  Artemia  salina.     VI.  Summary  of  mutations 230 

BRANHAM,  JOSEPH  M. 

Motility  and  aging  of  Arbacia  sperm 251 

GROSCH,  DANIEL  S. 

The  reproductive  capacity  of  Artemia  subjected  to  successive  contamina- 
tions with  radiophosphorus 261 

GROSS,  WARREN  J.,  AND  RONALD  L.  CAPEN 

Some  functions  of  the  urinary  bladder  in  a  crab 272 

HUNTER,  W.  RUSSELL,  AND  DAVID  C.  GRANT 

Estimates  of  population  density  and  dispersal  in  the  naticid  gastropod, 
Polinices  duplicatus,  with  a  discussion  of  computational  methods 292 

JOHNSON,  LELAND  G. 

Diurnal  patterns  of  metabolic  variations  in  chick  embryos 308 

KOHL,  D.  M.,  AND  R.  A.  FLICKINGER 

The  role  of  DNA   synthesis  in  the  determination   of  axial  polarity   of 
regenerating  planarians 323 

MILKMAN,  ROGER,  AND  BERTIL  HILLE 

Analysis  of  some  temperature  effects  on  Drosophila  pupae 331 

HILLE,  BERTIL,  AND  ROGER  MILKMAN 

A  quantitative  description  of  some  temperature  effects  in  Drosophila 346 

POTTS,  W.  T.  W.,  AND  D.  H.  EVANS 

The  effects  of  hypophysectomy  and  bovine  prolactin  on   salt   fluxes   in 
fresh-water-adapted  Fundulus  heteroclitus 362 

TRUEMAN,  E.  R. 

The  mechanism  of  burrowing  in  the  polychaete  worm,  Arenicola  marina 
(L.) ' ' 369 

Abstracts  of  papers  presented  at  the  Marine  Biological  Laboratory 378 

No.  3.     DECEMBER,  1966 

EARTH,  LESTER  G. 

The  role  of  sodium  chloride  in  sequential  induction  of  the  presumptive 
epidermis  of  Rana  pipiens  gastrulae 415 

CALABRESE,  ANTHONY,  AND  HARRY  C.  DAVIS 

The  pH  tolerance  of  embryos  and  larvae  of  Mercenaria  mercenaria  and 
Crassostrea    virginica 427 

DILLER,  WILLIAM  F.,  AND  DEMETRIUS  KOUNARIS 

Description  of  a  zoochlorella-bearing  form  of  Euplotes,  E.  daidaleos  n.  sp. 
(Ciliophora,  Hypotrichida) 437 

KONIJN,  THEO  M.,  AND  KENNETH  B.  RAPER 

The    influence   of    light    on    the    size    of    aggregations    in    Dictyostelium 
discoideum.  .  446 


CONTENTS  V 

MCLAREN,  IAN  A. 

Predicting  development  rate  of  copepod  eggs 457 

NOVALES,  RONALD  R.,  AND  BARBARA  J.  NOVALES 

Factors  influencing  the  response  of  isolated  dogfish  skin  melanophores 

to  melanocyte-stimulating  hormone 4/0 

ROCKSTEIN,  MORRIS,  AND  PREM  LATA  BHATNAGAR 

Duration  and  frequency  of  wing  beat  in  the  aging  house  fly,  Musca 
domestica  L 479 

STRAND,  JOHN  A.,  JOSEPH  T.  CUMMINS  AND  BURTON  E.  VAUGHAN 

Artificial  culture  of  marine  sea  weeds  in  recirculation  aquarium  systems  .   487 

STUNKARD,  HORACE  W. 

The  morphology  and  life-history  of  Notocotylus  atlanticus  n.  sp.,  a 
digenetic  trematode  of  eider  ducks,  Somateria  mollissima,  and  the  desig- 
nation, Notocotylus  duboisi  nom.  nov.,  for  Xotocotylus  imbricatus 
(Looss,  1893)  Szidat,  1935 501 

TWEEDELL,  KENYON  S. 

Oocyte  development  and  incorporation  of  H3-thymidine  and  H3-uridine 

in  Pectinaria  (Cistenides)  gouldii 516 


Vol.  131,  No.  1  August,  1966 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED   BY   THE    MARINE   BIOLOGICAL  LABORATORY 


THE  MARINE  BIOLOGICAL  LABORATORY 

SIXTY-EIGHTH  REPORT,  FOR  THE  YEAR  1965 — SEVENTY-EIGHTH  YEAR 

I.     TRUSTEES  AND  EXECUTIVE  COMMITTEE  (AS  OF  AUGUST  14,  1965) 1 

II.     ACT  OF  INCORPORATION    4 

III.  BYLAWS  OF  THE  CORPORATION  5 

IV.  REPORT  OF  THE  DIRECTOR 7 

Addenda : 

1.  Memorials  9 

2.  The  Staff 12 

3.  Investigators,  Lalor  and  Grass  Fellows,  and  Students 22 

4.  Fellowships  and  Scholarships   36 

5.  Training  Programs   36 

6.  Tabular  View  of  Attendance,  1961-1965  39 

7.  Institutions  Represented 39 

8.  Evening  Lectures 42 

9.  Evening  Seminars   42 

10.  Members  of  the  Corporation  44 

V.     REPORT  OF  THE  LIBRARIAN 68 

VI.     REPORT  OF  THE  TREASURER  69 


I.     TRUSTEES 

GERARD  SWOPE,  JR.,  Chairman  of  the  Board  of  Trustees,  570  Lexington  Avenue,  New 

York  22,  New  York 

*ARTHUR  K.  PARPART,  President  of  the  Corporation,  Princeton  University 
*JAMES  H.  WICKERSHAM,  Treasurer,  791  Park  Avenue,  New  York  21,  New  York 
PHILIP  B.  ARMSTRONG,  Director,  State  University  of  New  York,  College  of  Medicine 

at  Syracuse 
ALEXANDER  T.  DAIGNAULT,  Assistant  Treasurer,  7  Hanover  Street,  New  York  5,  New 

York 
GEORGE  W.  DE  VILLAFRANCA,  Clerk  of  the  Corporation,  Smith  College 


*  Deceased. 


Copyright  ©  1966,  by  the  Marine  Biological  Laboratory 
Library  of  Congress  Card  No.  A38-518 


MARINE  BIOLOGICAL  LABORATORY 

EMERITI 

WILLIAM  R.  AMBERSON,  Marine  Biological  Laboratory 

C.  LALOR  BURDICK,  The  Lalor  Foundation 

C.  LLOYD  CLAFF,  Randolph,  Massachusetts 
*W.  C.  CURTIS,  504  West  Mount  Avenue,  Columbia,  Missouri 

PAUL  S.  GALTSOFF,  Woods  Hole,  Massachusetts 
*E.  B.  HARVEY,  Woods  Hole,  Massachusetts 

M.  H.  JACOBS,  University  of  Pennsylvania 

CHARLES  W.  METZ,  Woods  Hole,  Massachusetts 

CHARLES  PACKARD,  Woods  Hole,  Massachusetts 

A.  C.  REDFIELD,  Woods  Hole,  Massachusetts 

CARL  C.  SPEIDEL,  University  of  Virginia 

A.  H.  STURTEVANT,  California  Institute  of  Technology 

ALBERT  SZENT-GYORGYI,  Marine  Biological  Laboratory 


TO    SERVE    UNTIL    1969 

MAC  V.  EDDS,  JR.,  Brown  University 
STEPHEN  W.  KUFFLER,  Harvard  Medical  School 
ARNOLD  LAZAROW,  University  of  Minnesota 
CHARLES  B.  METZ,  University  of  Miami 
KEITH  R.  PORTER,  Harvard  University 
GEORGE  T.  SCOTT,  Oberlin  College 
GEORGE  WALD,  Harvard  University 
EDGAR  ZWILLING,  Brandeis  University 


TO    SERVE    UNTIL    1968 

E.  G.  BUTLER,  Princeton  University 
A.  C.  CLEMENT,  Emory  University 
ARTHUR  L.  COLWIN,  Queens  College 
DONALD  P.  COSTELLO,  University  of  North  Carolina 
JAMES  D.  EBERT,  Carnegie  Institution  of  Washington 
DOUGLAS  A.  MARSLAND,  Marine  Biological  Laboratory 
ROBERTS  RUGH,  College  of  Physicians  and  Surgeons 
H.  BURR  STEINBACH,  University  of  Chicago 


TO    SERVE    UNTIL    1967 

LESTER  G.  BARTH,  Columbia  University 

JOHN  B.  BUCK,  National  Institutes  of  Health 

AURIN  M.  CHASE,  Princeton  University 

SEYMOUR  S.  COHEN,  University  of  Pennsylvania  School  of  Medicine 

TERU  HAYASHI,  Columbia  University 

LEWIS  KLEINHOLZ,  Reed  College 

ALBERT  I.  LANSING,  University  of  Pittsburgh 

S.  MERYL  ROSE,  Tulane  University 

*  Deceased. 


TRUSTEES 

TO    SERVE    UNTIL    1966 

FRANK  A.  BROWN,  JR.,  Northwestern  University 

F.  D.  CARLSON,  The  Johns  Hopkins  University 

SEARS  CROWELL,  Indiana  University 

W.  D.  McELROY,  The  Johns  Hopkins  University 

C.  LADD  PROSSER,  University  of  Illinois 
*E.  A.  SCHARRER,  Albert  Einstein  College  of  Medicine 
*SISTER  FLORENCE  MARIE  SCOTT,  Seton  Hill  College 

WM.  RANDOLPH  TAYLOR,  University  of  Michigan 

STANDING  COMMITTEES 

EXECUTIVE  COMMITTEE  OF  THE  BOARD  OF  TRUSTEES 

*  ARTHUR  K.  PARPART,  ex  officio,  Chairman  KEITH  R.  PORTER,  1968 
GERARD  SWOPE,  JR.,  ex  officio  E.  G.  BUTLER,  1967 

*  JAMES  H.  WICKERSHAM,  ex  officio  H.  BURR  STEINBACH,  1967 
ALEXANDER  T.  DAIGNAULT,  ex  officio  TERU  HAYASHI,  1966 
PHILIP  B.  ARMSTRONG,  ex  officio  WILLIAM  D.  MCELROY,  1966 
MAC  V.  EDDS,  JR.,  1968 

THE  LIBRARY  COMMITTEE 

KEITH  R.  PORTER,  Chairman  ERIC  G.  BALL 

ALEX  B.  NOVIKOFF  JAMES  W.  LASH 

STANLEY  WATSON  MORDECAI  GABRIEL 
C.  LADD  PROSSER 

THE  APPARATUS  COMMITTEE 

ALBERT  I.  LANSING,  Chairman  ELOISE  E.  CLARK 

PHILIP  B.  DUNHAM  EUGENE  BELL 

EUGENE  COPELAND  L.  I.  REBHUN 

WILLIAM  D.  MCELROY  DAVID  POTTER 

THE  SUPPLY  DEPARTMENT  COMMITTEE 

RUDOLF  KEMPTON,  Chairman  HARRY  GRUNDFEST 

WILLIAM  J.  ADELMAN  GEORGE  T.  SCOTT 

FRANK  M.  FISHER  MAC  V.  EDDS,  JR. 

HOWARD  A.  SCHNEIDERMAN  ARNOLD  LAZAROW 
WALTER  HERNDON 

THE  INSTRUCTION  COMMITTEE 

TERU  HAYASHI,  Chairman  BOSTWICK  KETCHUM 

ANTHONY  C.  CLEMENT  LEWIS  H.  KLEINHOLZ 

DEWITT  STETTEN  ROGER  O.  ECKERT 

LESTER  G.  BARTH  MAIMON  NASATIR 

*  Deceased. 


4  MARINE  BIOLOGICAL  LABORATORY 

THE  BUILDINGS  AND  GROUNDS  COMMITTEE 

EDGAR  ZWILLING,  Chairman  MELVIN  SPIEGEL 

E.  G.  BUTLER  J.  WOODLAND  HASTINGS 

DANIEL  GROSCII  FRANCIS  D.  CARLSON 
JONATHAN  P.  GREEN 

THE  RADIATION  COMMITTEE 

PAUL  R.  GROSS,  Chairman  S.  J.  COOPERSTEIN 

H.  BURR  STEINBACH  GEORGE  SZABO 

ROBERTS  RUGH  DAVID  SHEMIN 

THE  RESEARCH  SPACE  COMMITTEE 

EDGAR  ZWILLING,  Chairman  SEARS  CROWELL 

TERU  HAYASHI  ANTHONY  C.  CLEMENT 

THE  COMMITTEE  FOR  NOMINATION  OF  OFFICERS 

E.  G.  BUTLER  TERU  HAYASHI 

MAC  V.  EDDS,  JR.  H.  BURR  STEINBACH 

WILLIAM  D.  MCELROY  KEITH  R.  PORTER 

II.     ACT  OF  INCORPORATION 

No.  3170 

COMMONWEALTH  OF  MASSACHUSETTS 

Be  It  Known,  That  whereas  Alpheus  Hyatt,  William  Sanford  Stevens,  William  T. 
Sedgwick,  Edward  G.  Gardiner,  Susan  Minns,  Charles  Sedgwick  Minot,  Samuel  Wells, 
William  G.  Farlow,  Anna  D.  Phillips,  and  B.  H.  Van  Vleck  have  associated  themselves 
with  the  intention  of  forming  a  Corporation  under  the  name  of  the  Marine  Biological 
Laboratory,  for  the  purpose  of  establishing  and  maintaining  a  laboratory  or  station  for 
scientific  study  and  investigation,  and  a  school  for  instruction  in  biology  and  natural  his- 
tory, and  have  complied  with  the  provisions  of  the  statutes  of  this  Commonwealth  in  such 
case  made  and  provided,  as  appears  from  the  certificate  of  the  President,  Treasurer,  and 
Trustees  of  said  Corporation,  duly  approved  by  the  Commissioner  of  Corporations,  and 
recorded  in  this  office ; 

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

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

Secretary  of  the  Commonwealth. 


BYLAWS  OF  THE  CORPORATION  5 

III.     BYLAWS  OF  THE  CORPORATION  OF  THE  MARINE 
BIOLOGICAL  LABORATORY 

(Revised  August  15,  1963) 

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  Chairman  of  the  Board  of 
Trustees,  President,  Director,  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 
members  may  be  called  by  the  Trustees  to  be  held  at  such  time  and  place  as  may  be 
designated. 

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

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

VI.  Inasmuch  as  the  time  and  place  of  the  Annual  Meeting  of  members  are  fixed  by 
these  bylaws,  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,  Massachusetts.     Special 
meetings  of  the  Trustees  shall  be  called  by  the  Chairman,  the  President,  or  by  any  seven 
Trustees,  to  be  held  at  such  time  and  place  as  may  be  designated,  and  the  Secretary 
shall  give  notice  thereof  by  written  or  printed  notice,  mailed  to  each  Trustee  at  his 
address  as  shown  on  the  records  of  the  Corporation,  at  least  one   (1)   week  before  the 
meeting.     At  such  special  meeting  only  matters  stated  in  the  notice  shall  be  considered. 
Seven  Trustees  of  those  eligible  to  vote  shall  constitute  a  quorum  for  the  transaction 
of  business  at  any  meeting. 

VIII.  There  shall  be  three  groups  of  Trustees : 

(A)  Thirty-two  Trustees  chosen  by  the  Corporation,  divided  into  four  classes,  each 
to  serve  four  years.     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  c.v  officio,  who  shall  be  the  Chairman,  the  President,  the  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 
continue  to  serve  as  Trustee  until  the  next  Annual  Meeting  of  the  Corporation,  where- 
upon his  office  as  regular  Trustee  shall  become  vacant  and  be  filled  by  election  by  the 
Corporation  and  he  shall  become  eligible  for  election  as  Trustee  Emeritus  for  life.     The 
Trustees  ex  officio  and  Emeriti  shall  have  all  the  rights  of  the  Trustees,  except  that 
Trustees  Emeriti  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  Chairman  of  the  Board  of  Trustees  who  shall  be  elected 


6  MARINE  BIOLOGICAL  LABORATORY 

annually  and  shall  serve  until  his  successor  is  selected  and  qualified  and  who  shall  also 
preside  at  meetings  of  the  Corporation.  They  shall  elect  a  President  of  the  Corporation 
who  shall  also  be  the  Vice  Chairman  of  the  Board  of  Trustees  and  Vice  Chairman  of 
meetings  of  the  Corporation,  and  who  shall  be  elected  for  a  term  of  five  years  and  shall 
serve  until  his  successor  is  selected  and  qualified,  except  that  such  term  shall  not  run 
beyond  the  Annual  Meeting  of  the  Board  following  his  65th  birthday ;  candidates  over  the 
age  of  65  shall  be  elected  on  an  annual  basis.  They  shall  appoint  a  Director  of  the 
Laboratory  for  a  term  not  to  exceed  five  years,  provided  the  term  shall  not  exceed 
one  year  if  the  candidate  has  attained  the  age  of  65  years  prior  to  the  date  of  the  appoint- 
ment. They  may  choose  such  other  officers  and  agents  as  they  may  think  best.  They 
may  fix  the  compensation  and  define  the  duties  of  all  the  officers  and  agents;  and  may 
remove  them,  or  any  of  them  except  those  chosen  by  the  members,  at  any  time.  They 
may  fill  vacancies  occurring  in  any  manner  in  their  own  number  or  in  any  of  the 
offices.  The  Board  of  Trustees  shall  have  the  power  to  choose  an  Executive  Committee 
from  their  own  number,  and  to  delegate  to  such  Committee  such  of  their  own  powers 
as  they  may  deem  expedient.  They  shall  from  time  to  time  elect  members  to  the 
Corporation  upon  such  terms  and  conditions  as  they  may  think  best. 

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

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

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

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


RESOLUTIONS  ADOPTED  AT  TRUSTEE  MEETING  AUGUST  16, 

1963— EXECUTIVE  COMMITTEE 

I.  RESOLVED: 

(A)  The  Executive  Committee  is  hereby  designated  to  consist  of  ten  members  as 
follows :    ex   officio   members   who   shall   be   the   Chairman    of   the    Board    of   Trustees, 
President,    Director   and   Treasurer;    six   additional    Trustees,    two    of   whom    shall    be 
elected  by  the  Board  of  Trustees  each  year,  to  serve  for  a  three-year  term. 

(B)  The   President   shall   act   as    Chairman   of   the   Executive    Committee   and   the 
Chairman  of  the  Board  of  Trustees  as  Vice  Chairman.     A  majority  of  the  members 
of  the  Executive  Committee  shall  constitute  a  quorum  and  a  majority  of  those  present 
at  any  properly  held  meeting  shall  determine  its  action.     It  shall   meet  at   such  times 
and  places  and  upon  such  notice  and  appoint  such   sub-committees   as   the   Committee 
shall  determine. 

(C)  The  Executive  Committee  shall  have  and  may  exercise  all  the  powers  of  the 
Board  during  the  intervals  between  meetings   of  the   Board  of   Trustees   except   those 
powers  specifically  withheld  from  time  to  time  by  the  Board  or  by  Law. 

(D)  The  Executive  Committee  shall  keep  appropriate  minutes  of  its  meetings,  and 
its  actions  shall  be  reported  to  the  Board  of  Trustees. 


REPORT  OF  THE  DIRECTOR  / 

II.  RESOLVED: 

The  elected  members  of  the  Executive  Committee  shall  be  constituted  as  a  standing 
"Committee  for  the  Nominations  of  Officers,"  responsible  for  making  nominations  at 
the  annual  meeting  of  the  Corporation  and  of  the  Board  of  Trustees,  for  candidates  to 
fill  each  office  as  the  respective  terms  of  office  expire.  (Chairman  of  the  Board,  Presi- 
dent, Director,  Treasurer,  and  Clerk.) 


IV.     REPORT  OF  THE  DIRECTOR 

To :  THE  TRUSTEES  OF  THE  MARINE  BIOLOGICAL  LABORATORY 
Gentlemen : 

I  submit  herewith  the  report  of  the  seventy-eighth  session  of  the  Marine 
Biological  Laboratory. 

1.  Facilities  Developments 

Early  in  June  an  application  was  made  to  the  National  Science  Foundation  for 
a  grant  of  $2,200,000.00  to  match  the  grant  in  the  same  amount  contingently  made 
to  the  Laboratory  by  the  Ford  Foundation.  The  projected  plan  of  the  Laboratory 
calls  for  the  construction  of  a  new  instructional  building,  to  replace  the  present 
outmoded  wooden  frame  buildings,  and  a  new  dining  hall-dormitory. 

A  Building  Committee  was  appointed  which  in  turn  selected  the  firm  of  Peirce, 
Pierce,  and  Luykx  to  develop  plans  for  the  projected  building.  In  order  to  meet 
the  requests  from  the  National  Science  Foundation  for  detailed  plans,  a  number 
of  meetings  were  held  by  the  Building  Committee  with  the  heads  of  the  various 
training  programs  and  the  architects.  A  number  of  plans  were  developed  by  the 
architects  and  finally  one  was  tentatively  adopted  subject  to  modifications  ensuing 
from  engineering  feasibility  and  contractors'  bids. 

During  the  course  of  the  winter,  title  to  Center  Street  was  transferred  from 
the  Town  of  Falmouth  to  the  Laboratory,  increasing  the  area  available  for  the  new 
training  building.  Also,  the  Laboratory  has  acquired  by  purchase  "Re  House,"  the 
second  house  north  of  North  Street  with  frontage  on  the  Eel  Pond,  formerly  the 
property  of  Mrs.  Warren  Vincent.  The  total  area  included  is  7100  square  feet. 

2.  Policy  on  Operation 

During  the  past  several  years  the  Trustees  of  the  Laboratory  have  been  keenly 
interested  in  realizing  the  full  potentialities  of  the  Laboratory  not  only  during  the 
summer  months  but  during  the  rest  of  the  year.  The  prospect  of  a  year-round 
instructional  building  and  heated  student  dormitory  provides  opportunities  for 
broadening  the  scope  of  the  Laboratory  during  the  non-summer  months.  The 
large  number  of  highly  qualified  students  who  cannot  be  accommodated  during  the 
summer  session  prompted  the  Laboratory  to  formulate  policy  encouraging  instruc- 
tion during  the  winter  months. 

In  line  with  the  new  policy  the  Marine  Biological  Laboratory  will  entertain 
proposals  for  the  off-season  use  of  its  facilities  by  other  institutions  offering  teaching 


MARINE  BIOLOGICAL  LABORATORY 

and  research  programs  of  a  high  caliber,  or  scientists  interested  in  similar  programs 
in  their  fields  of  interest.  The  Laboratory  will  sponsor  applications  for  funds  in 
support  of  such  programs  and  will  encourage  participation  by  well  qualified 
individuals  and  groups.  Also,  the  Laboratory  will  continue  to  make  its  facilities 
available  to  qualified  scientists  who  wish  to  conduct  research  or  read  in  the  library 
during  the  non-summer  months. 

3.  Winter  Operation 

During  recent  years  there  has  been  a  continuing  significant  increase  in  the  use 
of  the  facilities  during  the  non-summer  months.  This  current  winter  (1965-1966) 
there  have  been  31  investigators  in  residence  at  the  Laboratory,  supplemented  by 
a  technical  staff  of  the  same  number  (31)  occupying  42  laboratories.  In  addition 
there  have  been  30  investigators  at  the  Laboratory  on  a  transient  basis  to  read  in 
the  library,  collect  certain  seasonal  biological  materials,  or  for  limited  research 
activity. 

The  services  at  the  Laboratory  are  maintained  during  the  non-summer  months. 
The  Supply  Department  must  devote  considerable  time  in  preparation  for  the 
push  of  the  coming  summer,  reconditioning  the  boats,  docks,  floats,  and  gear,  but 
at  the  same  time  maintaining  its  collecting  activities  for  the  resident  investigators 
and  for  the  exploratory  work  of  the  Systematics-Ecology  Program.  Also  during 
this  period,  active  collecting  is  maintained  for  the  supply  of  live  marine  forms  to 
investigators  at  their  home  institutions.  During  this  current  period  over  1000 
shipments  of  material  will  be  made. 

During  the  winter  the  library  operates  as  it  does  during  the  summer  months, 
twenty-four  hours  a  day  for  the  seven  days  of  the  week.  It  serves  the  resident  and 
visiting  investigators  at  the  Laboratory,  the  more  than  135  scientists  at  the  Woods 
Hole  Oceanographic  Institution,  and  the  scientific  personnel  at  the  Laboratory  of 
the  Bureau  of  Commercial  Fisheries. 

The  Apartment  House  is  open  throughout  the  year,  so  is  available  to  both  long- 
term  and  short-term  investigators  and  visitors  at  the  Laboratory  during  the  non- 
summer  months,  as  well  as  visiting  investigators  at  the  Woods  Hole  Oceanograpbic 
Institution. 

4.  Systematics-Ecology  Program 

Plans  have  gone  forward  for  the  new  Laboratory  survey  boat  for  this  program. 
The  initial  Ford  Foundation  Grant  of  $100,000.00  has  been  supplemented  by  addi- 
tional funds  from  the  Grass  Foundation  ($10,000.00)  to  complete  the  outfitting  of 
the  boat.  A  generous  donation  of  some  surplus  equipment  by  the  Oceanographic 
Institution  was  most  welcome. 

The  scuba  diving  program  has  been  expanded  and  now  includes  personnel  in 
both  the  Systematics-Ecology  Program  and  in  the  Supply  Department. 

5.  Committee  on  Organisation 

A  Committee  on  Organization  was  appointed  at  the  end  of  the  summer  (1965). 
under  the  Chairmanship  of  Dr.  Eric  G.  Ball,  to  scrutinize  the  present  administrative 
set-up  of  the  Laboratory  with  a  view  to  making  recommendations  on  modifications 


REPORT  OF  THE  DIRECTOR 

which  will  specify  the  responsibilities  of  the  various  officers  in  relation  to  the 
Executive  Committee  and  the  Board  of  Trustees.  The  Committee  was  also  charged 
to  look  into  the  advisability  of  including  on  the  Board  of  Trustees  a  limited  number 
of  "lay"  trustees,  men  of  public  affairs  interested  in  the  Laboratory  and  its 
operation.  It  is  anticipated  that  the  Committee  will  submit  its  final  report  early 
in  the  summer  (1966). 

6.  Deaths 

This  past  year  the  Laboratory  suffered  irreparable  losses  by  death :  Dr.  Arthur 
K.  Parpart,  President  of  the  Corporation,  who  first  came  to  the  Laboratory  as  a 
graduate  student  and  who,  in  his  mature  years,  has  played  a  prominent  and 
constructive  role  in  the  development  of  the  Laboratory ;  Mr.  James  H.  Wickersham, 
Treasurer  of  the  Corporation,  who  gave  generously  of  his  time  and  talents  in  so 
effectively  managing  the  financial  interests  of  the  Laboratory;  and  Dr.  Ethel  B. 
Harvey,  renowned  for  her  investigations  into  the  embryology  of  Arbacia,  who 
probably  had  worked  more  summers  continuously  in  annual  residence  at  the 
Laboratory  (since  1907)  than  any  other  investigator  in  its  history. 

7.  Personnel  Changes 

At  the  mid-winter  meeting  of  the  Trustees  on  February  12,  1965,  Dr.  Arm- 
strong was  elected  to  continue  as  Director  of  the  Laboratory  until  the  August 
meeting  of  the  Trustees  in  1966. 

Dr.  H.  Burr  Steinbach  was  invited  to  accept  the  Directorship  of  the  Laboratory 
at  the  retirement  of  Dr.  Armstrong,  which  he  has  consented  to  do.  Dr.  Steinbach 
will  bring  to  the  office  an  intimate  knowledge  of  the  history,  traditions  and 
operations  of  the  Laboratory  growing  out  of  an  association  of  over  thirty  years  with 
the  institution.  Also,  he  has  had  a  broad  experience  in  the  administration  of 
scientific  organizations.  At  the  mid-winter  meeting  of  the  Trustees  on  February 
18,  1966,  Dr.  Armstrong  was  elected  President  of  the'  Corporation  to  complete 
the  unexpired  term  of  Dr.  Parpart,  and  Mr.  Alexander  T.  Daignault  was  elected 
Treasurer  of  the  Corporation  to  complete  the  unexpired  term  of  Mr.  James  H. 
Wickersham.  Mr.  Gerard  Swope,  Jr.  expressed  a  desire  to  retire  as  Chairman 
of  the  Board,  but  was  prevailed  on  to  continue  to  serve  until  the  summer  of  1966. 

Two  promotions  have  been  made  in  recognition  of  meritorious  service  to  the 
Laboratory.  Miss  Jane  Fessenden  was  advanced  from  Acting  Librarian  to 
Librarian  and  Mr.  John  J.  Valois  was  appointed  Assistant  Manager  of  the  Supply 
Department. 

1.  MEMORIALS 

ERNST  ALBERT  SCHARRER 
BY  ARNOLD  LAZAROW 

The  life  of  Dr.  Ernst  Albert  Scharrer,  a  leading  neuroendocrinologist  and  head  of  the 
Department  of  Anatomy  at  the  Albert  Einstein  College  of  Medicine,  in  New  York, 
came  to  an  untimely  end  on  April  29th  at  Sarasota,  Florida,  in  a  drowning  accident. 

Dr.  Scharrer  was  born  in  Germany  on  August  1,  1905.     He  was  educated  at  the 


10  MARINE  BIOLOGICAL  LABORATORY 

University  of  Munich  where  he  earned  his  Ph.D.  in  zoology  in  1927,  and  his  medical 
degree  in  1933.     Having  served  as  a  Sterling  Fellow  at  Yale  University  from  1929  to 

1930,  and  as  an  assistant  in  zoology  at  the  University  of  Vienna  from   1930  to   1931, 
he  was  appointed  an  investigator  at  the  Research  Institute  for  Psychiatry  in  Munich  in 

1931.  Two  years  later,  he  was  put  in  charge  of  the  Neurological  Institute  at  the  Uni- 
versity of  Frankfurt-am-Main  where  he  remained  until  1937. 

At  this  time,  however,  Dr.  Scharrer  became  very  concerned  about  the  changing 
political  events  in  his  native  country  and  he  was  worried  about  the  consequences  of 
Hitler's  rise  to  power.  Although  the  Scharrers  were  not  members  of  the  minority 
group,  and  could  have  remained  in  Nazi  Germany  without  fear  of  persecution,  they 
refused  to  become  passive  participants  in  the  horrible  inhumanities  that  were  to  follow. 
Knowing  intuitively  what  was  to  come,  Ernst  and  Berta  Scharrer  decided  to  leave 
Germany.  Receiving  a  Rockefeller  Foundation  fellowship,  he  served  as  a  visiting 
scientist  at  the  University  of  Chicago  during  1937  and  1938  and  at  the  Rockefeller 
Institute  in  New  York  for  the  next  two  years.  In  1940  he  accepted  a  position  as 
Assistant  Professor  of  Anatomy  at  Western  Reserve  University  in  Cleveland.  After 
six  years  there  he  moved  to  the  University  of  Colorado  where  he  served  as  Associate 
Professor  until  1954.  In  1955  he  was  appointed  Professor  and  Chairman  of  Anatomy 
at  the  newly  created  Albert  Einstein  College  of  Medicine.  Included  among  his  many 
outside  activities  was  a  term  of  service  on  the  Morphology  and  Genetics  Study  Section  of 
the  U.  S.  Public  Health  Service  from  1954  to  1959. 

Dr.  Scharrer  spent  his  first  summer  at  the  Marine  Biological  Laboratory,  Woods 
Hole,  in  1937,  and  he  returned  to  this  Laboratory  during  a  dozen  subsequent  summers, 
collecting  material  and  working  as  a  research  investigator.  He  was  elected  a  Trustee 
of  the  Laboratory  in  1962  and  he  is  included  among  the  list  of  distinguished  Friday 
Evening  Lecturers. 

As  a  teacher  and  a  lecturer,  Dr.  Scharrer  was  unexcelled.  His  capacity  for  clear 
presentation,  his  superb  drawing  ability  (being  able  to  create  the  most  intricate  three- 
dimensional  blackboard  illustrations  without  the  use  of  a  single  note),  his  clever  selection 
of  examples  and  use  of  analogies,  his  great  enthusiasm  for  the  subject  matter  and  his 
exquisite  sense  of  drama  all  contributed  to  make  each  of  his  lectures  a  finished  theatrical 
performance  as  well  as  an  artistic  and  educational  experience. 

Dr.  Scharrer  has  made  important  and  imaginative  research  contributions  to  the 
field  of  neuroendocrinology  and  to  our  knowledge  of  the  microscopic  vascular  archi- 
tecture of  the  brain.  In  one  of  his  earliest  papers  published  in  1928  Scharrer  described 
the  presence  of  secretory  droplets  within  .specialized  nerve  cells  in  the  thalamus  of 
Fundulus  hcieroclitus.  Similar  cells  had  been  described  earlier  in  the  spinal  cord  of 
the  skate  by  Speidel  (1919).  On  the  basis  of  his  faith  in  morphologic  observations, 
Scharrer  suggested  that  these  nerve  cells  have  the  capacity  to  secrete  a  hormonal  sub- 
stance. However,  the  concept  of  neurosecretion  was  not  quickly  accepted  and  during 
the  following  decades  Dr.  Scharrer  vigorously  defended  the  thesis  of  neurosecretion 
while  he  continually  extended  his  observations  and  accumulated  more  and  more  con- 
vincing evidence.  Radical  new  ideas  are  slow  to  gain  acceptance  and  most  biologists 
were  reluctant  to  believe  that  nerve  cells  had  a  secretory  function.  In  their  Physiological 
Revicivs  article  entitled  "Neurosecretion"  and  published  in  1945,  the  Scharrers  sum- 
marized the  then-current  evidence  for  neurosecretion;  however,  even  at  that  time  they 
were  still  "lone  voices  in  the  wilderness."  But  in  the  ensuing  years  physiological  and 
biochemical  studies  by  other  investigators  provided  strong  supporting  evidence  for 
neurosecretion  and  as  time  passed  this  concept  was  challenged  less  and  less.  At  present, 
the  idea  of  neurosecretion  is  not  only  widely  accepted,  but  has  become  a  most  fashionable 
field  of  research  endeavor.  During  the  last  decade  there  have  been  increasing  numbers 
of  conferences,  symposia,  and  monographs  devoted  to  this  area. 


REPORT  OF  THE  DIRECTOR  11 

In  a  review  paper  published  in  the  American  Scientist  in  1951  Bargmann  and  Scharrer 
established  the  thesis  that  the  antidiuretic,  oxytocic  and  vasopressor  hormones  are  syn- 
thesized in  the  supraoptic  and  paraventricular  nuclei  of  the  hypothalamus  rather  than 
in  the  posterior  lobe  of  the  pituitary,  as  had  been  generally  believed.  These  hormones 
are  then  transported  in  the  form  of  particulate  droplets  within  and  along  the  axons,  by 
way  of  the  hypothalamo-hypophysial  tract  to  the  neurohypophysis  where  they  are  stored 
and  ultimately  released  into  the  blood. 

Subsequent  studies  by  many  investigators  have  now  established  the  role  of  the  hypo- 
thalamus in  controlling  the  release  of  adrenocorticotropin,  gonadotropins,  thyrotropin 
and  growth  hormone.  Specific  neurohormones  (releasing  factors)  are  synthesized  in 
the  hypothalamus ;  they  are  presumably  transported  along  axons  to  the  median  eminence 
where  they  enter  the  pituitary-portal  circulation.  Upon  reaching  the  adenohypophysis 
they  serve,  for  example,  as  the  adrenocorticotropin  or  thyrotropin  releasing  factors. 
Thus,  the  concept  of  neurosecretion  now  relates  directly  or  indirectly  to  almost  all 
phases  of  endocrine  function. 

It  should  be  emphasized  that  at  the  time  of  their  initial  studies,  the  Scharrers  were 
already  aware  of  the  broad  phylogenetic  significance  of  the  concept  of  neurosecretion, 
having  demonstrated  that  neurosecretory  cells  and  neurosecretory  tracts  were  widely 
distributed  throughout  the  animal  kingdom.  Dr.  Scharrer  used  marine  material  exten- 
sively in  his  investigations  for  he  was  aware  that  marine  forms  often  provided  specialized 
features  that  were  uniquely  adapted  to  the  solution  of  a  particular  problem. 

In  all,  Dr.  Scharrer  was  the  author  or  co-author  of  92  publications  and  these  included 
a  multiplicity  of  original  articles,  many  reviews,  and  a  book  entitled  N  euro  endocrinology, 
published  by  the  Columbia  University  Press  (1963).  Nine  of  these  publications,  includ- 
ing the  book  on  neuroendocrinology,  were  written  jointly  with  his  wife  Berta;  others 
were  written  with  collaborators  including  Wolfgang  Bargmann,  Sanford  L.  Palay 
and  others. 

In  all  of  his  scientific  work,  Dr.  Scharrer  enjoyed  a  very  close  association  with  his 
wife  Berta.  They  met  in  Munich  and  they  were  married  in  1934.  They  were  con- 
stantly together,  both  at  work  and  at  play.  They  formed  a  team  in  which  they  com- 
pletely complemented  each  other,  and  yet  each  pursued  an  independent  area  of  research. 
For  a  time,  they  divided  the  animal  kingdom  between  them,  with  Ernst  Scharrer  con- 
centrating on  vertebrates  and  Berta  Scharrer  studying  invertebrates.  It  is  difficult  to 
think  of  Ernst  without  also  thinking  of  Berta. 

It  is  a  great  tribute  to  Ernst  Scharrer  and  to  Berta's  strength  of  character  that  on 
the  Monday  which  followed  the  tragic  Thursday  in  Florida,  Berta  returned  to  the 
Department  of  Anatomy  at  Einstein  and,  despite  her  great  personal  grief,  assumed  the 
responsibility  of  serving  as  acting  Department  head.  Were  he  here,  Ernst  would  be 
very  proud  of  the  way  Berta  has  carried  out  this  responsibility  and  in  the  way  the 
Department  has  responded. 

I  would  like  to  say  a  few  words  about  Ernst  Scharrer — the  man.  He  was  an 
individual  with  great  moral  strength  and  the  highest  ethical  principles.  He  was  willing 
to  speak  out  against  the  things  he  thought  wrong  and  to  vigorously  defend  the  things 
he  believed  in.  Dr.  Scharrer  had  extremely  high  standards  for  everything  he  did;  he 
demanded  near  perfection  of  himself  and  urged  his  students  and  associates  to  emulate 
these  high  standards.  Yet  he  was  sympathetic  and  understanding  of  the  problems  of 
others.  He  had  a  love  of  life  and  a  zest  for  hard  work.  His  wonderful  sense  of  humor 
always  enlivened  the  discussion  of  any  group.  Ernst  Scharrer  was  one  of  the  rarest 
of  men  for  he  was  a  superb  teacher,  an  imaginative  and  productive  research  investigator, 
and  a  truly  delightful  man.  He  was  a  devoted  husband  to  Berta  and  a  very  warm 
personal  friend  to  many  of  us  here  at  Woods  Hole. 


12  MARINE  BIOLOGICAL  LABORATORY 

2.  THE  STAFF 

EMBRYOLOGY 

I.  INSTRUCTORS 

JAMES  D.  EBERT,  Director,  Department  of  Embryology,  Carnegie  Institution  of  Wash- 
ington, in  charge  of  course 

DONALD  D.  BROWN,  Staff  Member,  Department  of  Embryology,  Carnegie  Institution 
of  Washington 

ALLISON   L.   BURNETT,  Associate   Professor   of   Biology,   Western   Reserve   University 

ROBERT  L.  DEHAAN,  Staff  Member,  Department  of  Embryology,  Carnegie  Institution 
of  Washington 

TOM  HUMPHREYS,  Assistant  Professor  of  Biology,  Massachusetts  Institute  of  Technology 

THOMAS  J.  KING,  Head,  Department  of  Embryology,  Institute  for  Cancer  Research, 
Philadelphia 

JAMES  W.  LASH,  Associate  Professor  of  Anatomy,  University  of  Pennsylvania 

II.  JUNIOR  INSTRUCTOR 
SIDNEY  B.  SIMPSON,  Department  of  Anatomy,  Western  Reserve  University 

III.  LECTURER 
HEWSON  SWIFT,  Professor  of  Zoology,  University  of  Chicago 

IV.  LABORATORY  ASSISTANTS 

C.  B.  KIMMEL,  The  Johns  Hopkins  University 
DAVID  E.  KOHNE,  Purdue  University 

V.  LECTURES 

J.  D.  EBERT  Perspectives  in  developmental  biology 

T.  J.  KING  Analysis  of  early  teleost  development 

T.  J.  KING  Analysis  of  developmental  processes  in  teleosts 

JANE  OPPENHEIMER  Differentiation  of  the  lens  in  Fundulus 

HANS  LAUFER  Nucleocytoplasmic   interactions   during  insect  development 

W.  H.  TELFER  Formation  of  yolk — the  relative  roles  of  ovarian  synthesis 

and  protein  uptake 
E.  ANDERSON  Some  aspects  of  the  fine  structure  of  oocyte  differentiation 

and  vitellogenesis  in  the  roach,  Pcriplaneta  americana 
R.  A.  WALLACE  Biochemical    aspects    of    vertebrate    yolk    formation    and 

structure 

A.  C.  BLACKLER  Experiments  with  amphibian  embryonic  sex  cells 

TOM  HUMPHREYS  The  regeneration  of  sponges  from  dissociated  cells 

TOM  HUMPHREYS  The    molecular    basis    of    species-specific    cell    sorting    in 

sponges 

M.  S.  STEINBERG  Cellular  mechanisms  in  tissue  reconstruction 

M.  S.  STEINBERG  The  cell  surface  in  morphogenesis 

R.  L.  DEHAAN  Cell  contact  interactions :  Organogenesis 

H.  COON  Clonal  stability  of  a  differential  phenotype:  or,  chondrocyte 

dedifferentiation  revisited 


REPORT  OF  THE  DIRECTOR 


13 


J.  W.  SAUNDERS,  JR. 
A.  BURNETT 
A.  BURNETT 
A.  BURNETT 
R.  FLICKINGER 

C.  S.  THORNTON 

ELIZABETH  HAY 

R.  J.  Goss 

T.  W.  LASH 

J.  W.  LASH 

J.  W.  LASH 

HEWSON  SWIFT 

HEWSON  SWIFT 

HARRY  EAGLE 

J.  PAPACONSTANTINOU 

ARON  MOSCONA 

R.  L.  DEHAAN 
R.  L.  DEHAAN 
DAVID  EPE. 

D.  D.  BROWN 

D.  D.  BROWN 

JOHN  GURDON 
T.  J.  KING 

D.  D.  BROWN 
J.  F.  ALBRIGHT 

A.  SlLVERSTEIN 

B.  F.  ARGYRIS 
LAURENS  RUBEN 
LAUREN  s  RUBEN 
ROBERT  GOOD 

*S.  GELFANT 
*Y.  BERWALD 
*S.  SIMPSON 
*L.  SAXEN 

*  Post-course  lectures. 


Epithelial-mesenchymal    interactions    in    limb    development 

Growth  polarity  and  form  regulation  in  hydroids 

Pathways  of  cellular  differentiation  in  hydroids 

A  model  of  cell  differentiation  in  hydroids 

Regeneration  in  planaria 

Some  epidermal  and  neural  factors  in  limb  regeneration 
in  larval  salamanders 

Fine  structure  of  regenerating  limbs 

Compensatory  hypertrophy 

Ascidians  I 

Ascidians  II 

Ascidians  III 

Cytochemical  studies  of  nucleocytoplasmic  interactions 

Nucleic  acids  in  mitochondria  and  chloroplasts 

Biochemical  consequences  of  cellular  interaction  in  culture 

Protein  and  nucleic  acid  changes  in  the  differentiation  of 
lens  cells 

Changes  in  glutamine  synthetase  activity  in  the  neural 
retina  of  the  chick  embryo  /;•;  siiu  and  in  vitro 

Annelids,  molluscs,  echinoderms  I 

Annelids,  molluscs,  echinoderms  II 

Early  biochemical  reactions  of  fertilization 

Biochemistry  of  oogenesis,  fertilization  and  early  develop- 
ment I 

Biochemistry  of  oogenesis,  fertilization  and  early  develop- 
ment II 

Nuclear  transplantation  and  the  control  of  gene  activity 

Developmental  capacity  of  nuclei  of  frog  renal  adeno- 
carcinoma  cells 

Biochemical  consequences  of  nuclear  transplantation 

Competence  of  cells  for  antibody  formation 

The  development  of  immunity  in  the  mammalian  fetus 

Immunological  tolerance 

Post-embryonic  cell  differentiation :  normal  and  neoplastic 

Post-embryonic  induction  in  urodele  limbs 

Ontogeny  and  phylogeny  of  the  immune  response 

The  cell  division  cycle 

Chemical  carcinogenesis  in  vitro 

Aspects  of  appendage  regeneration  in  lizards 

Methods  in  teratology 


PHYSIOLOGY 
I.  CONSULTANTS 

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

ARTHUR  K.  PARPART,  Professor  of  Biology,  Princeton  University 

ALBERT   SZENT-GYORGYI,   Director,   Institute   for   Muscle   Research,   Marine   Biological 

Laboratory 
W.  D.  McELROY,  Director,   McCollum-Pratt  Institute,  The  Johns   Hopkins   University 


14 


MARINE  BIOLOGICAL  LABORATORY 


II.  INSTRUCTORS 

J.  WOODLAND  HASTINGS,  Professor  of  Biochemistry,  University  of   Illinois,   in  charge 

of  course 

EDWARD  A.  ADELBERG,  Professor  of  Microbiology,  Yale  University 
HARLYN  HALVORSON,  Professor  of  Molecular  Biology,  University  of  Wisconsin 
SHINYA  INOUE,  Professor  of  Cytology,  Dartmouth  College 
K.  E.  VAN  HOLDE,  Professor  of  Physical  Chemistry,  University  of  Illinois    (on  leave 

1965) 

FRED  KARUSH,  Professor  of  Microbiology,  University  of  Pennsylvania 
WILLIAM  F.  HARRINGTON,  Professor  of  Biology,  The  Johns  Hopkins  University 
HANS  KORNBERG,  Professor  of  Biochemistry,  Leicester  University,  England 

III.  SPECIAL  LECTURERS 

QUENTIN  H.  GIBSON,  Professor  of  Physiology,  University  of  Pennsylvania 
ANDREW  SZENT-GYORGYI,  Professor  of  Cytology,  Dartmouth  College 
R.  K.  CLAYTON,  C.  F.  Kettering  Laboratory,  Yellow  Springs,  Ohio 


IV.  LABORATORY  ASSISTANTS 


GEORGE  KISSIL,  University  of  Connecticut 
JOHN  CLARK,  Dartmouth  College 


J.  WOODLAND  HASTINGS 

J.  WOODLAND  HASTINGS 
HARLYN  HALVORSON 
HARLYN  HALVORSON 
HARLYN  HALVORSON 
HANS  KORNBERG 
HANS  KORNBERG 
HANS  KORNBERG 
FRED  KARUSH 
FRED  KARUSH 
FRED  KARUSH 
EDWARD  A.  ADELBERG 
EDWARD  A.  ADELBERG 
EDWARD  A.  ADELBERG 
EDWARD  A.  ADELBERG 
SHINYA  INOUE 
SHINYA  INOUE 

SHINYA  INOUE 
W.  F.  HARRINGTON 
W.  F.  HARRINGTON 
W.  F.  HARRINGTON 
QUENTIN  H.  GIBSON 
QUENTIN  H.  GIBSON 
R.  K.  CLAYTON 
R.  K.  CLAYTON 


V.  STAFF  LECTURES 

The  generation  and  utilization  of  excited  states  in  bio- 
luminescent  systems 

Bioluminescent  systems:  II 

The  synthesis  of  protein  in  cell-free  systems 

Ordered  transcription  of  the  genome 

Intracellular  differentiation  in  Bacillus 

Integration  of  metabolism  :  I 

Integration  of  metabolism  :  II 

Integration  of  metabolism  :  III 

The  specific  interaction  of  antibody 

The  nature  of  immunoglobulins 

Biological  aspects  of  the  immune  response 

The  chemical  basis  of  mutation 

Effects  of  mutation  on  protein  synthesis 

Genetic  recombination  in  bacteria  :  I 

Genetic  recombination  in  bacteria  :  II 

Fine  structural  basis  and  physiology  of  mitosis 

The  use  of  polarized  light  for  analysis  of  biological  fine 
structure 

DNA  and  chromosome  arrangement  in  living  sperm 

Stereochemistry  of  polypeptide  chains 

Structure  of  fibrous  proteins  :  I 

Structure  of  fibrous  proteins  :  II 

Hemoglobulin-ligand  reactions 

Mechanisms  of  some  flavoprotein  enzyme  reactions 

Physical  and  photochemical  mechanisms  in  photosynthesis 

The  significance  of  light  emitted  by  photosynthetic  tissues 


REPORT  OF  THE  DIRECTOR 


15 


IRVIN  ISENBERG 
HENRY  MAHLER 
DANIEL  MAZIA 
C.  S.  VESTLING 
CYRUS  LEVINTHAL 
HARRY  GINSBERG 
B.  D.  DAVIS 
W.  P.  JENCKS 
TERU  HAYASHI 
GEORGE  WALD 
PHILIP  HANDLER 
MATTHEW  MESELSON 
DAVID  HOGNESS 
R.  M.  BOCK 
CARL  WOESE 
F.  RITTOSA 

SEYMOUR  COHEN 
ALVIN  NASON 
ALVIN  NASON 
SOL  SPIEGELMAN 

SOL  SPIEGELMAN 
SOL  SPIEGELMAN 
ALBERT  SZENT-GYORGYI 


VI.  INVITED  LECTURES 

Electron  spin  resonance  in  biochemistry 

Properties  of  flavoproteins 

Chemical  resolution  of  chromosomes 

Structural  studies  on  lactic  dehydrogenase 

Computer  construction  and  display  of  molecular  models 

Replication  of  animal  viruses 

Antimicrobial  agents  as  physiological  tools 

Effects  of  solvents  on  protein  structure 

Physiological  and  molecular  aspects  of  muscle  contraction 

The  retinal  basis  of  human  vision 

Enzymatic  mechanisms 

Genetic  recombination 

The  structure  and  function  of  lambda  DNA 

Recent  developments  in  the  study  of  soluble  RNA 

The  genetic  code :  Is  it  really  solved  ? 

The  distribution  of  DNA  complementary  to  ribosomal  and 
soluble  RNA 

Comparative  biochemistry  of  D-arabinose  and  its  nucleosides 

Enzymology  of  inorganic  nitrogen  metabolism  :  I 

Enzymology  of  inorganic  nitrogen  metabolism  :  II 

Communication  between  a  virus  and  host  cell:  A  compari- 
son of  single-stranded  DNA  and  RNA 

Problems  for  replication  for  single-stranded  DNA  and 
RNA  viruses 

Synthesis  of  a  self-propagating  infectious  nucleic  acid  with 
a  purified  enzyme 

Growth 


MARINE  BOTANY 

I.  CONSULTANT 

WILLIAM  RANDOLPH  TAYLOR,  Professor  of  Botany,  University  of  Michigan 

II.  INSTRUCTORS 

WALTER  R.  HERNDON,  Professor  of  Botany,  University  of  Tennessee,  in  charge  of  course 
PHILIP  W.  COOK,  Assistant  Professor  of  Botany,  University  of  Vermont 
H.  WAYNE  NICHOLS,  Associate  Professor  of  Botany,  Washington  University 
FRANK  E.  ROUND,  Lecturer  in  Botany,  University  of  Bristol,  England 
ROBERT  T.  WILCE,  Assistant  Professor  of  Botany,  University  of  Massachusetts 

III.  SPECIAL  LECTURERS 

R.  W.  HOLTON,  Department  of  Botany,  University  of  Tennessee 

I.  M.  LAMB,  Farlow  Herbarium,  Harvard  University 

JOHN  KINGSBURY,  Department  of  Botany,  Cornell  University 

LUIGI  PROVASOLI,  Haskins  Laboratories,  New  York 

FRANCIS  R.  TRAINOR,  Department  of  Botany,  University  of  Connecticut 


16 


MARINE  BIOLOGICAL  LABORATORY 


IV.  LABORATORY  ASSISTANTS 

RUSSELL  G.  RHODES,  University  of  Tennessee 
JEFFERY  S.  PRINCE,  University  of  Massachusetts 

V.  COLLECTOR 
DAVIS  L.  FINDLEY,  University  of  Tennessee 


WALTER  R.  HERNDON 


PHILIP  W. 
PHILIP  W. 
H.  WAYNE 
PHILIP  W. 
H.  WAYNE 
ROBERT  T. 
ROBERT  T. 
WALTER  R. 
WALTER  R. 
PHILIP  W. 
PHILIP  W. 
PHILIP  W. 
PHILIP  W. 
FRANK  E. 
FRANK  E. 
ROBERT  T. 


COOK 
COOK 
NICHOLS 
COOK 
NICHOLS 
WILCE 

WlLCE 

HERNDON 

HERNDON 

COOK 

COOK 

COOK 

COOK 
ROUND 
ROUND 
WILCE 


ROBERT  T.  WILCE 
ROBERT  T.  WILCE 
FRANK  E.  ROUND 
FRANK  E.  ROUND 
FRANK  E.  ROUND 
FRANK  E.  ROUND 
WALTER  R.  HERNDON 
ROBERT  T.  WILCE 
ROBERT  T.  WILCE 
L.  PROVASOLI 
L.  PROVASOLI 
ROBERT  T.  WILCE 
ROBERT  T.  WILCE 
PHILIP  W.  COOK 
H.  WAYNE  NICHOLS 
H.  WAYNE  NICHOLS 
I.  M.  LAMB 
H.  WAYNE  NICHOLS 
H.  WAYNE  NICHOLS 
H.  WAYNE  NICHOLS 
FRANCIS  TRAINOR 


H.  WAYNE  NICHOLS 


VI.  LECTURES 

Marine  plants  and  the  plant  kingdom :  Introduction  to  the 

algae 

Chlorophyceae ;  Volvocales 
Volvocales 

Cultivation  of  marine  and  fresh-water  algae 
Cladophorales 
Ulotrichales 
Ulvales 

Siphonales,  Siphonocladales,  Dasycladales 
Chlorococcales 

Chlorococcales,  Tetrasporales 
Oedogoniales 
Zygnematales  I 
Zygnematales  II 
Zygnematales  III 

Chrysophyta,  introduction  Xanthophyceae 
Chrysophyta,  Chrysophyceae  I 
Phaeophyta,   introduction  to  morphology  and  ecology; 

Ectocarpates 
Phaeophyta  I 
Phaeophyta :  Chordariales 
Chrysophyta,  Chrysophyceae  II 
Chrysophyta,  Bacillariophyceae  I 
Chrysophyta,  Bacillariophyceae  II 
Chrysophyta,  Bacillariophyceae  III 
Euglenophyta 
Phaeophyta  II 
Phaeophyta  III 

Nutrition  and  physiology  of  algae 

Algae  as  food  for  other  organisms,  especially  invertebrates 
Phaeophyta  IV 
Phaeophyta  V 
Cyanophyta 
Rhodophyta  I 
Rhodophyta  II 

Sublittoral  antarctic  benthic  algae 
Rhodophyta  III 
Rhodophyta  IV 
Rhodophyta  V 
Morphogenetic  phenomena   in  green  algae ;  demonstration 

of    zoospore    formation    and    sexual     reproduction     in 

Scenedesmus 
Rhodophyta  VI 


REPORT  OF  THE  DIRECTOR  17 

H.  WAYNE  NICHOLS  Rhodophyta  VII 

WALTER  R.  HERNDON  Charophyta 

H.  WAYNE  NICHOLS  Rhodophyta  VIII 

H.  WAYNE  NICHOLS  Rhodophyta  IX 

FRANK  E.  ROUND  History  of  marine  basins 

JOHN  KINGSBURY  Environment  of  attached  marine  algae;  toxic  algae;  perio- 
dicity in  growth  of  Derbesia-Halicystis 

PHILIP  W.  COOK  Pyrrophyta 

ROBERT  T.  WILCE  Ecology  of  arctic  algae 

R.  W.  HOLTON  Physiology  and  biochemistry  of  blue  green  algae 

INVERTEBRATE  ZOOLOGY 

I.  CONSULTANTS 

FRANK  A.  BROWN,  JR.,  Morrison  Professor  of  Biology,  Northwestern  University 
LIBBIE  H.  HYMAN,  American  Museum  of  Natural  History 
CLARK  P.  READ,  Professor  of  Biology,  Rice  University 
ALFRED  C.  REDFIELD,  Woods  Hole  Oceanographic  Institution 

II.  INSTRUCTORS 

W.  D.  RUSSELL  HUNTER,  Professor  of  Zoology,  Syracuse  University,  in  charge  of  course 
GEORGE  G.  HOLZ,  JR.,  Professor  of  Microbiology,  State  University  of  New  York,  Upstate 

Medical  Center 
NORMAN    MILLOTT,    Professor    of    Zoology,    Bedford    College,    University    of    London, 

England 

IRWIN  W.  SHERMAN,  Assistant  Professor  of  Zoology,  University  of  California,  Riverside 
ALLAHVERDI  FARMANFARMAIAN,  Professor  of  General  Physiology,  Pahlavi  University, 

Shiraz,  Iran 
ERIC  L.  MILLS,  Assistant  Professor  of  Biology,  Queen's  University,  Kingston,  Ontario, 

Canada 

FRANK  M.  FISHER,  JR.,  Assistant  Professor  of  Biology,  Rice  University 
ROBERT  K.  JOSEPHSON,  Associate  Professor  of  Biology,  Western  Reserve  University 

III.  ASSISTANTS 

JOHN  H.  BUSSER,  University  of  Rhode  Island 

W.  BRUCE  HUNTER,  University  of  California,  Santa  Barbara 

IV.  LECTURES 

ROBERT  K.  JOSEPHSON  Cnidaria  I — Introduction  to  the  Cnidaria  and  Ctenophora 

ROBERT  K.  JOSEPHSON  Cnidaria  II — Feeding,  growth,  function  of  the  nematocysts 

ROBERT  K.  JOSEPHSON  Cnidaria  III — Nervous  system  and  behavior 

FRANK  M.  FISHER,  JR.  Turbellaria  and  Trematoda 

FRANK  M.  FISHER,  JR.  Cestoda  and  Rhynchocoela 

FRANK  M.  FISHER,  JR.  Physiological  considerations  of  the  host-parasite  relation- 

ship 

W.  D.  RUSSELL  HUNTER  Mollusca  I — General  molluscan  organization.  Functioning 

of  mantle  cavity  in  Gastropoda 

W.  D.  RUSSELL  HUNTER  Mollusca  II — Gastropoda  (continued).  Mantle  cavity  and 

feeding  mechanisms  in  Bivalvia 


18 


MARINE  BIOLOGICAL  LABORATORY 


W.  D.  RUSSELL  HUNTER 
W.  D.  RUSSELL  HUNTER 

W.  D.  RUSSELL  HUNTER 
W.  D.  RUSSELL  HUNTER 

W.  D.  RUSSELL  HUNTER 
IRWIN  W.  SHERMAN 
IRWIN  W.  SHERMAN 
IRWIN  W.  SHERMAN 
ERIC  L.  MILLS 

IRWIN  W.  SHERMAN 
ERIC  L.  MILLS 

ERIC  L.  MILLS 
ERIC  L.  MILLS 

FRANK  M.  FISHER,  JR. 
FRANK  M.  FISHER,  JR. 
NORMAN  MILLOTT 
NORMAN  MILLOTT 
NORMAN  MILLOTT 
A.  FARMANFARMAIAN 
ERIC  L.  MILLS 
A.  FARMANFARMAIAN 
JAMES  W.  LASH 
GEORGE  G.  HOLZ,  JR. 
GEORGE  G.  HOLZ,  JR. 
GEORGE  G.  HOLZ,  JR. 
GEORGE  G.  HOLZ,  JR. 
NORMAN  MILLOTT 
GEORGE  G.  HOLZ,  JR. 
GEORGE  G.  HOLZ,  JR. 
FREDERIK  B.  BANG 
GEORGE  G.  HOLZ,  JR. 
ROBERT  R.  HESSLER 

W.  D.  RUSSELL  HUNTER 


Short  seminar — Some  problems  of  mechanics  in  molluscs 

Mollusca  III — Adaptations  in  bivalves.    Aspects  of  general 
physiology  of  gastropods  and  bivalves 

Seminar — A  history  of  the  segmented  mollusc 

Mollusca     IV — Functional     morphology     in     Amphineura, 
Cephalopoda  and  minor  groups 

Seminar — The  evolution  of  and  physiological  variation  in 
the  molluscs  of  fresh  waters 

Annelida  I — Introduction:  history,  embryology,  taxonomy, 
general  characteristics,  reproduction 

Annelida     II — Settling     patterns,     regeneration,     feeding 
mechanisms,  hydrostatic  skeleton 

Annelida    III — Respiration,    osmoregulation,   nervous    sys- 
tem and  behavior 

Arthropoda  I — General  features  of  arthropods.     Introduc- 
tion to  crustacean  structure 

Invertebrate  hemoglobins 

Arthropoda   II — Crustacean   structure,   physiology  and  re- 
production 

Arthropoda    III — Crustacean    functional    morphology    and 
evolution 

Arthropoda    IV — Crustacean    functional    morphology    and 
evolution 

Ectoprota — Entoprocta 

Aschelminthes 

Asteroidea 

Development  of  echinoids.    Ophiuroidea 

Holothuroidea.    Crinoidea 

Protochordata  I 

The  biology  of  an  amphipod  crustacean  sibling  species  pair 

Protochordata  II 

Ascidian  metamorphosis 

Porifera 

The  nature  of  the  Protozoa 

Mastigophora  I 

Mastigophora  II 

Seminar — Light  sensitivities   in  echinoderms 

Sarcodina  I 

Sarcodina  II 

Invertebrate  pathology 

Ciliophora 

Miscellaneous    studies    on    an    obscure    crustacean    from 
Nobska  Beach,  Dcrocheilocaris  typicus 

One  approach  to  the  zooplankton 

MARINE  ECOLOGY 
I.  CONSULTANTS 


MELBOURNE  R.  CARRIKER,  Marine  Biological  Laboratory 
BOSTWICK  H.  KETCHUM,  Woods  Hole  Oceanographic  Institution 
EDWIN  T.  MOUL,  Rutgers  University 
JOHN  H.  RYTHER,  Woods  Hole  Oceanographic  Institution 


REPORT  OF  THE  DIRECTOR 


19 


II.  INSTRUCTORS 

W.  ROWLAND  TAYLOR,  Department  of  Oceanography  and  The  Chesapeake  Bay  Institute, 

The  Johns  Hopkins  University,  in  charge  of  course 

DENNIS  J.  CRISP,  Marine  Science  Laboratory,  University  College  of  North  Wales,  U.  K. 
LAWRENCE  B.  SLOBODKIN,  Department  of  Zoology,  University  of  Michigan 
RICHARD  A.  BOOLOOTIAN,  Department  of  Zoology,  University  of  California,  Los  Angeles 
FRANK  E.  ROUND,  Department  of  Botany,  University  of  Bristol,  England  (Joint  appoint- 
ment with  Department  of  Marine  Botany) 
HOWARD  L.  SANDERS,  Woods  Hole  Oceanographic  Institution 

III.  SPECIAL  LECTURERS 

LUIGI  PROVASOLI,  Haskins  Laboratories,  New  York 

PETER  H.  KLOPFER,  Department  of  Zoology,  Duke  University 

IV.  LABORATORY  ASSISTANTS 

MARGARET  C.  LLOYD,  University  of  Michigan 

BARRY  M.  HEATFIELD,  University  of  California,  Los  Angeles 


W.  ROWLAND  TAYLOR 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIX 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
LAWRENCE  B.  SLOBODKIN 
W.  ROWLAND  TAYLOR 
VICTOR  A.  ZULLO 
W.  ROWLAND  TAYLOR 
W.  ROWLAND  TAYLOR 
W.  ROWLAND  TAYLOR 
W.  ROWLAND  TAYLOR 
W.  ROWLAND  TAYLOR 
W.  ROWLAND  TAYLOR 
W.  ROWLAND  TAYLOR 
RICHARD  A.  BOOLOOTIAN 
RICHARD  A.  BOOLOOTIAN 

RICHARD  A.  BOOLOOTIAN 

RICHARD  A.  BOOLOOTIAN 

RICHARD  A.  BOOLOOTIAN 

RICHARD  A.  BOOLOOTIAN 


V.  LECTURES 

Introduction  to  environmental  biology 

Classical  models  of  population  growth  and  competition 

Experimental  analyses  of  population  growth  and  competition 

Intrinsic  rates  of  natural  increase  and  reproductive  value 

Predator-prey  interaction  and  energy  flow 

Evolutionary  strategy 

Species  abundance  distributions  I 

Species  abundance  distributions  II 

Biostatistics  1.    Elementary  concepts  of  errors  and  variance 

Biostatistics  2.    Binomial  and  poisson  distributions 

Biostatistics  3.    Binomial  and  poisson  distributions 

Biostatistics  4.    Normal  distribution  and  variance  analysis 

Biostatistics  5.    Non-parametric  tests 

Zonation  on  rocky  shores 

Systematics  and  ecology 

Chemistry  and  physics  of  sea  water 

Radiant  energy  in  the  marine  environment 

Phytoplankton  organisms  I :  Diatoms 

Phytoplankton  organisms  II :  Diatoms 

Phytoplankton  organisms  III :  Dinoflagellates 

Primary  productivity  I 

Primary  productivity  II 

Aspects  of  coral  atoll  ecology 

Types  of  food  utilization  by  marine  organisms  with  empha- 
sis on  feeding  adaptations 

Arbacia  piinctulata  populations  of  Falmouth  Harbor 

Reproductive  biology  of  marine  organisms :  general  patterns 

Reproductive  cycles  of  marine  organisms 

Reproductive  physiology  of  the  echinoid,  Strongylocentotus 
purpiiratus 


20 


MARINE  BIOLOGICAL  LABORATORY 


RICHARD  A.  BOOLOOTIAN 
RICHARD  A.  BOOLOOTIAN 

V.  G.  TURNER 

L.  PROVASOLI 
L.  PROVASOLI 
FRANK  E.  ROUND 
HOWARD  L.  SANDERS 
DONALD  RHODES 

HOWARD  L.  SANDERS 
HOWARD  L.  SANDERS 

FRANK  E.  ROUND 
HOWARD  L.  SANDERS 

DENNIS  J.  CRISP 

DENNIS  J.  CRISP 

DENNIS  J.  CRISP 

DENNIS  J.  CRISP 

DENNIS  J.  CRISP 

DENNIS  J.  CRISP 

R.    S.    SCHELTEMA 

FRANK  E.  ROUND 
PETER  H.  KLOPFER 
PETER  H.  KLOPFER 

PETER  H.  KLOPFER 
PETER  H.  KLOPFER 
PETER  H.  KLOPFER 
*DENNIS  J.  CRISP 

*  Post-course  lecture. 


Relation  of  nutrition  to  reproduction  in  S.  purpuratus 

The  circulatory  system  of  S.  purpuratus  and  its  relation  to 

nutrition  and  reproduction 
The  effects  of  crowding  on  growth  and  development  of  5". 

drobachiensis 

Culturing  of  algae  in  synthetic  media  I 
Culturing  of  algae  in  synthetic  media  II 
Marine  phytoflagellates  with  scales 
Animal  sediment  relationship 
Biogenic  reworking  of  intertidal  and  subtidal  sediments  of 

the  Cape  Cod  region 
Structure  of  a  soft  bottom  community  and  some  remarks  on 

organic  molecules  as  a  food  source  for  benthic  animals 
Salinity,    hydrography    and    the    distribution    of    estuarine 

animals 

Migration  rhythms  of  intertidal  benthic  diatoms 
Time,  latitude  and  the  structure  of  marine  benthic  com- 
munities and  remarks  on  the  deep-sea  benthos 
The  role  of  pelagic  larvae 

Free-swimming  stages  and  the  problem  of  transition 
The  cyprid,  a  model  settler ;  and  others 
Habitat  selection  I.    Choice  of  deposits 
Habitat  selection   II.     Chemical  inducements  and  a  novel 

chemical  sense 
Territorial  behavior 
Pelagic  larvae  of  the  North  Atlantic 
The  history  of  a  marine  basin 
On  the  classification  of  behavior 
Seminars  in  environmental  biology  on  the  causes  of  tropical 

species  diversity 

Imprinting :  General  introduction 
Imprinting  :  Sexual  selection  in  birds 
Maternal  imprinting  in  mammals 
Ecology  of  marine  larval  settling 


SYSTEMATICS-ECOLOGY  PROGRAM 
THE  STAFF 

Director :  MELBOURNE  R.  CARRIKER 

Resident  Systematist :  VICTOR  A.  ZULLO 

Resident  Ecologist :  ROBERT  H.  PARKER 

Postdoctoral  Fellows  and  Research  Associates:  MARVIN  CANTOR,  JOHN  C.  H.  CARTER, 

MICHAEL  T.  GHISELIN,  DAVID  C.   GRANT,  JACK   B.   PEARCE,   KAY  W.   PETERSEN, 

THOMAS  J.  M.  SCHOPF,  JOSEPH  L.  SIMON,  EDMUND  H.  SMITH,  BARRY  A.  WADE, 

GERALD  E.  WALSH 
Visiting  Investigators  in  Residence:  RICHARD  A.  BOOLOOTIAN,  LOUISE  BUSH,  DUANE 

HOPE,  E.  T.  MOUL,  DONALD  J.  ZINN 
Secretaries  :  KAY  CRAM,  HAZEL  F.  SANTOS 
Artists  :  DIANE  JOHNSON,  RUTH  VON  ARX 
Captain  of  Research  Vessel :  JAMES  P.  W.  OSTERGARD,  JR. 


REPORT  OF  THE  DIRECTOR 


21 


Research  Assistants :  ANDREW  L.  DRISCOLL,  DOUGLAS  EBY,  ROBERT  KAUFMAN,  CAROL 
KOURTZ,  BARRY  MARTIN,  J.  STEWART  NAGLE,  PETER  J.  OLDHAM,  FRANKLYN  OTT, 
PETER  E.  SCHWAMB,  STEWART  SANTOS,  DEHN  SOLOMON,  JUNE  THOMAS,  SUSAN 
TRACY,  VILIA  TURNER,  DIRK  VAN  ZANDT,  ANTHONY  WILLIAMS,  HILARY  M. 
WILLIAMS 


PAUL  GALTSOFF 

EDMUND  SMITH 
ROBERT  F.  GIBBS 
JOHN  ZEIGLER 
GEORGE  HAMPSON 
WOLFGANG  WIESER 
CHARLES  YENTSCH 

ROBERT  CONOVER 

HARRY  F.  RECHER 
ROBERT  F.  SISSON 
LAWRENCE  B.  SLOBODKIN 
WILLIAM  H.  AMOS 

FRANK  E.  ROUND 
DENNIS  J.  CRISP 

VILIA  TURNER 

HORACE  W.  STUNKARD 
ARTHUR  H.  CLARKE 
ERIC  L.  MILLS 


I.    SEMINARS  (winter  not  included) 

Anomalies  and  malformations  in  the  shells  of  Crassostrea 
vir  glide  a 

Review  of  boring-  bivalves 

Progress  report  on  the  Cape  Cod  National  Seashore 

Geology  of  the  Cape  Cod  Region 

The  resurrection  of  Nucula  truncula 

Ecological  approaches  to  the  benthic  meiofauna 

Chlorophyll-phaeophytin  relationships  in  the  marine  plank- 
tonic  environment 

Assimilation  of  organic  matter  by  zooplankton  and  the 
question  of  superfluous  feeding 

Feeding  efficiency  and  herons 

Color  transparencies  of  squid  biology 

Population  dynamics  and  the  escape  reaction  in  Hydra 

Selected  biophotographs  of  Delaware  benthic  and  littoral 
invertebrates 

History  of  marine  basins 

Effects  of  the  cold  winter  of  1962-63  on  British  marine 
fauna 

The  effects  of  diet  and  crowding  on  growth  and  develop- 
ment in  Strongylocentrotus  droebachiensis  larvae 

The  role  of  parasitism  in  animal  ecology  and  systematics 

Fresh-water  mollusks  of  the  American  arctic  water  shed 

The  ecology  of  deep-sea  amphipod  crustaceans  of  New 
England 


THE  LABORATORY  STAFF 
HOMER  P.  SMITH,  General  Manager 


IRVINE  L.  BROADBENT,  Office  Manager 
Miss  JANE  FESSENDEN,  Librarian 
ROBERT  KAHLER,  Superintendent,  Buildings  and 
Grounds 


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

CARL  O.  SCHWEIDENBACK,  Man- 
ager, Supply  Department 


GENERAL  OFFICE 


EDWARD  J.  BENDER 
MRS.  VIVIEN  B.  BROWN 
MRS.  FLORENCE  S.  BUTZ 
MRS.  MARION  C.  CHASE 
MRS.  JANET  S.  CUMMINGS 


MRS.  JUDITH  A.  KECK 
MRS.  ANN  W.  LOOMIS 
MRS.  VIVIAN  I.  MANSON 
Miss  MARGARET  ANN  MORTON 
Miss  DIANE  PIKE 


Miss  KATHERINE  M.  TRACY 


MARINE  BIOLOGICAL  LABORATORY 

MAINTENANCE  OF  BUILDINGS  AND  GROUNDS 

ROBERT  ADAMS  RICHARD  E.  GEGGATT,  JR. 

ELDON  P.  ALLEN  ROBERT  GUNNING 

JOHN  T.  BRADY  DONALD  B.  LEHY 

JAMES  N.  CAREY  RALPH  H.  LEWIS 

BERNARD  F.  CAVANAUGH  RUSSELL  F.  LEWIS 

DANIEL  COSTA  HENRY  F.  POTTER 

MANUEL  P.  DUTRA  FREDERICK  E.  THRASHER 

STANLEY  C.  ELDREDGE  CHARLES  V.  TUTIN 

GARDNER  F.  GAYTON  ROBERT  H.  WALKER,  JR. 

DEPARTMENT  OF  RESEARCH  SERVICE 

GAIL  M.  CAVANAUGH  Miss  MARGARET  E.  SCOTT 

LOWELL  V.  MARTIN  FRANK  E.  SYLVIA 

SUPPLY  DEPARTMENT 

ARNO  J.  BOWDEN  PAUL  SHAVE 

DAVID  H.  GRAHAM  BRUNO  F.  TRAPASSO 

MRS.  ELIZABETH  GREEN  JOHN  J.  VALOIS 

ROBERT  W.  HAMPTON  HALLETT  S.  WAGSTAFF 

ROBERT  O.  LEHY  BRADLEY  WOOD 
Miss  JOYCE  B.  LIMA 

DINING  HALL  AND  HOUSING 

ROBERT  T.  MARTIN,  Manager,  Food  Service 
MRS.  ELIZABETH  KUIL,  Supervisor,  Dining  Room 
MRS.    ELLEN    T.    NICKELSON,    Supervisor,    Dormitories 
ALAN  G.  LUNN,  Supervisor,  Cottage  Colony 

3.  INVESTIGATORS  ;  LALOR  AND  GRASS  FELLOWS  ;  STUDENTS 
Independent  and  Beginning  Investigators,  1965 

ABBOTT,  BERNARD  C.,  Professor  of  Biophysics  and  Physiology,  University  of  Illinois 
ABRAMSON,  HAROLD  A.,  Director  of  Research,  South  Oaks  Research  Foundation,  Inc. 
ADELBERG,  EDWARD  A.,  Professor  of  Microbiology,  Yale  University  School  of  Medicine 
ADELMAN,  WILLIAM  J.,  JR.,  Associate  Professor  of  Physiology,  University  of  Maryland  School 

of  Medicine 

ALLEN,  ROBERT  D.,  Associate  Professor  of  Biology,  Princeton  University 
AMBERSON,  WILLIAM  R.,  Marine  Biological  Laboratory 

AMOS,  WILLIAM  H.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
ARMSTRONG,  PHILIP  B.,  Chairman,  Department  of  Anatomy,  State  University  of  New  York, 

College  of  Medicine  at  Syracuse 

ARNOLD,  JOHN  M.,  Assistant  Professor  of  Zoology  and  Entomology,  Iowa  State  University 
ASHWORTH,  JOHN  MICHAEL,  Lecturer  in  Biochemistry,  Leicester  University 
AUCLAIR,  WALTER,  Assistant  Professor  of  Zoology,  University  of  Cincinnati 
AUERBACH,  ALBERT  A.,  Columbia  University 
AUSTIN,    C.    R.,    Head,    Genetic    and    Developmental    Disorders    Research    Program,    Delta 

Regional  Primate  Research  Center 
BANG,  FREDERIK  B.,  Chairman  and  Professor,  Department  of  Pathobiology,  The  Johns  Hopkins 

University,  School  of  Hygiene  &  Public  Health 
BAYLOR,  MARTHA,  Marine  Biological  Laboratory 


REPORT  OF  THE  DIRECTOR 

BELAMARICH,  FRANK  A.,  Assistant  Professor  of  Biology,  Boston  University 

BELESLIN,  BOGDAN  B.,  Columbia  University 

BELL,  EUGENE,  Associate  Professor  of  Biology,  Massachusetts  Institute  of  Technology 

BENNETT,  M.  V.  L.,  Associate  Professor  of  Neurology,  Columbia  University 

BERRY,  SPENCER  J.,  Assistant  Professor  of  Biology,  Wesleyan  University 

BERSOHN,  R.,  Professor  of  Chemistry,  Columbia  University 

BIGGERS,  JOHN  D.,  King  Ranch  Research  Professor  of  Reproductive  Physiology,  University 

of  Pennsylvania 

BILLIAR,  REINHART  B.,  Research  Fellow,  Harvard  University  Medical  School 
BINSTOCK,  LEONARD,  Electronic  Engineer,  National  Institutes  of  Health 

BLAUSTEIN,  MAUDACAI  P.,  Medical  Research  Officer,  U.  S.  Naval  Medical  Research  Institute 
BOOLOOTIAN,  RICHARD  A.,  Associate  Professor  of  Zoology,  University  of  California,  Los  Angeles 
BOSLER,  ROBERT  B.,  Research  Associate,  Harvard  Medical  School 
BOUCK,  GEORGE  BENJAMIN,  Assistant  Professor  of  Biology,  Yale  University 
BOUSFIELD,  E.  L.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
BRANDT,  PHILIP  W.,  Assistant  Professor  of  Anatomy,  Columbia  University 
BRINLEY,  F.  J.,  JR.,  Assistant  Professor  of  Physiology,  The  Johns  Hopkins  School  of  Medicine 
BROOKS,  AUSTIN  E.,  Research  Associate,  Brown  University 
BROWN,  DONALD  D.,  Staff  Member,  Carnegie  Institution  of  Washington 
BROWN,  FRANK  A.,  JR.,  Morrison  Professor  of  Biology,  Northwestern  University 
BRZIN,  BRONKA,  Research  Assistant,  Institute  of  Microbiology,  Ljubljana,  and  State  University 

of  New  York,  Upstate  Medical  Center 

BRZIN,  MIRO,  Visiting  Assistant  Professor,  University  of  Ljubljana  and  Columbia  University 
BURCH,  HELEN  B.,  Associate  Professor  of  Pharmacology,  Washington  University 
BURNETT,  ALLISON  L.,  Associate  Professor  of  Biology,  Western  Reserve  University 
BUSH,  LOUISE,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
CANTOR,  MARVIN,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
CARRIKER,  MELBOURNE  R.,  Director,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
CARTER,  JOHN  C.  H.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
CASSIDY,  FR.  J.  D.,  Research  Advisor  to  Honors  Science  Program,  Providence  College 
CHENEY,    RALPH    HOLT,    Professor    of    Biology,    Brooklyn    College,    The    City    University    of 

New  York 

CLAFF,  C.  LLOYD,  Treasurer-Director,  Single  Cell  Research  Foundation,  Inc. 
CLARK,  ELOISE  E.,  Assistant  Professor  of  Zoology,  Columbia  University 
CLEMENT,  A.  C.,  Professor  of  Biology,  Emory  University 
COHEN,  LAWRENCE  B.,  Post-Doctoral  Fellow,  Columbia  University 
COLE,  KENNETH  S.,  Chief,  Laboratory  of  Biophysics,  National  Institutes  of  Health 
COLLETT,  THOMAS  STEPHEN,   Post-Doctoral   Research  Assistant,  University   College,   London, 

England 

COLWIN,  ARTHUR  L.,  Professor  of  Biology,  Queens  College  of  The  City  University  of  New  York 
COLWIN,  LAURA  HUNTER,   Lecturer  in   Biology,   Queens   College   of  The   City   University   of 

New  York 

COOK,  PHILIP  WILLIAM,  Assistant  Professor  of  Botany,  University  of  Vermont 
COOPERSTEIN,  SHERWIN  J.,  Professor  of  Anatomy,  University  of  Connecticut 
COPELAND,  D.  EUGENE,  Chairman,  Professor  of  Zoology,  Tulane  University 
COSTELLO,  DONALD  P.,  Kenan  Professor  of  Zoology,  University  of  North  Carolina 
CRISP,  D.  J.,  Director,  Marine  Science  Laboratory,  University  College  of  North  Wales 
CROWELL,  SEARS,  Professor  of  Zoology,  Indiana  University 
DEHAAN,  ROBERT  L.,  Staff  Member,  Carnegie  Institution  of  Washington 
DE  LORENZO,  A.  J.  D.,  Director,  Anatomical  and  Pathological  Research  Laboratories,  The  Johns 

Hopkins  University  School  of  Medicine 

DE  SA,  RICHARD  J.,  Post-Doctoral  Trainee,  University  of  Pennsylvania 
DETTBARN,  WOLF-DIETRICH,  Assistant  Professor  of  Neurology,  Columbia  University,   College 

of  Physicians  and  Surgeons 

DE  VILLAFRANCA,  GEORGE  W.,  Professor  of  Zoology,  Smith  College 
DISCHE,   ZACHARIAS,   Emeritus   Professor   of   Biochemistry,    Columbia   University,    College   of 

Physicians  and  Surgeons 

DUNHAM,  PHILIP  B.,  Assistant  Professor  of  Zoology,  Syracuse  University 
EBERT,  JAMES  D.,  Director,  Department  of  Embryology,  Carnegie  Institution  of  Washington 


24  MARINE  BIOLOGICAL  LABORATORY 

ECKERT,  ROGER,  Assistant  Professor  of  Zoology,  Syracuse  University 

EDDS,  M.  V.,  JR.,  Chairman,  Professor  of  Medical  Science,  Brown  University 

EGYUD,  LASZLO,  Research  Associate,  Institute  for  Muscle  Research 

EHRENSTEIN,  GERARD,  Physicist,  National  Institutes  of  Health 

ELLIS,  RICHARD  A.,  Associate  Professor  of  Biology,  Brown  University 

ERWIN,  JOSEPH,  Assistant  Professor  of  Zoology,  Columbia  University,  Barnard  College 

FARMANFARMAIAN,  A.,  Professor  of  General  Physiology,  Pahlavi  University 

FAUST,   ROBERT   GILBERT,   Assistant   Professor   of   Physiology,   University   of   North    Carolina 

School  of  Medicine 

FINGERMAN,  MILTON,  Professor  of  Zoology,  Tulane  University 
FISHER,  FRANK  M.,  JR.,  Assistant  Professor  of  Biology,  Rice  University 
FISHMAN,  Louis,  Assistant  Research  Professor,  New  York  University  College  of  Dentistry 
FRAENKEL,  GOTTFRIED  S.,  Professor  of  Entomology,  University  of  Illinois 
FREEMAN,  ALAN  R.,  Trainee  Fellow  in  Neurology,  Columbia  University 

FUORTES,  M.  G.  F.,  Head,  Section  on  Neurophysiology,  Ophthalmology  Branch,  National  Insti- 
tute of  Neurological  Diseases  and  Blindness,  National  Institutes  of  Health 
FURSHPAN,    EDWIN    J.,    Assistant    Professor    of    Neurophysiology    and    Neuropharmacology, 

Harvard  Medical  School 

GARCIA,  HORACIO  A.,  Research  Fellow,  Columbia  University 
GELFANT,  SEYMOUR,  Professor  of  Zoology,  Syracuse  University 

GERMAN,  JAMES  L.,  Ill,  Assistant  Professor,  Department  of  Pediatrics,  and  Director,  Labora- 
tory of  Human  Genetics,  Cornell  University  Medical  College 

GIBBINS,  JOHN  RICHARD,  Research  Fellow,  Biological  Laboratories,  Harvard  University 
GILBERT,  DANIEL  L.,  Physiologist,  National  Institutes  of  Health 
GIMENEZ,  MAXIMO,  Visiting  Fellow,  Columbia  University 

GLADE,  RICHARD  W.,  Chairman,  Associate  Professor  of  Zoology,  University  of  Vermont 
GOLDSMITH,  TIMOTHY  H.,  Associate  Professor  of  Biology,  Yale  University 
GORDON,  JEOFFRY,  Grass  Foundation  Fellow,  The  Grass  Foundation 
GRANT,  DAVID  C,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
GRANT,  PHILIP,  Program  Director,  Developmental  Biology,  National  Science  Foundation 
GRANT,  ROBERT  J.,  Research  Associate,  Columbia  University 
GROSCH,  DANIEL  S.,  Professor  of  Genetics,  North  Carolina  State  University 
GROSS,  PAUL  R.,  Associate  Professor  of  Biology,  Brown  University 
GRUNDFEST,  HARRY,  Professor  of  Neurology,  Columbia  University 
GUTTMAN,  RITA,  Associate  Professor  of  Biology,  Brooklyn  College 
HAGINS,  WILLIAM  A.,  Research  Medical  Officer,  National  Institute  of  Arthritis  and  Metabolic 

Diseases,  National  Institutes  of  Health 

HALVORSON,  HARLYN  O.,  Professor  of  Bacteriology,  University  of  Wisconsin 
HARRINGTON,  WILLIAM  F.,  Professor  of  Biology,  The  Johns  Hopkins  University 
HASTINGS,  J.  WOODLAND,  Professor  of  Biochemistry,  University  of  Illinois 
HAYASHI,  TERU,  Chairman  and  Professor  of  Zoology,  Columbia  University 
HEGYELI,  ANDREW  F.,  Research  Associate,  Institute  for  Muscle  Research,   Marine  Biological 

Laboratory 

HENLEY,  CATHERINE,  Research  Associate,  University  of  North  Carolina 
HERNDON,  WALTER  R.,   Professor   of  Botany  and   Associate   Dean,   College   of  Liberal    Arts, 

University  of  Tennessee 

HERVEY,  JOHN  P.,  Senior  Electronics  Engineer,  The  Rockefeller  University 
HESSLER,  ANITA  Y.,  Research  Associate,  Marine  Biological  Laboratory 
HIGGINS,  DON  C.,  Assistant  Professor  of  Medicine,  Yale  University  School  of  Medicine 
HILLE,  BERTIL,  Graduate  Fellow,  The  Rockefeller  University 

HODES,  ROBERT,  Research  Associate  in  Neurophysiology,  The  Mount  Sinai  Hospital 
HOLLAENDER,  ALEXANDER,  Director,  Biology  Division,  Oak  Ridge  National  Laboratory 
HOLZ,  GEORGE  G.,  JR.,  Chairman  and  Professor  of  Microbiology,  State  University  of  New  York, 

Upstate  Medical  Center 

HOPE,  DUANE,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
HOSKIN,  FRANCIS  C.  G.,  Assistant  Professor  of  Neurology,  Columbia  University,  College  of 

Physicians  and  Surgeons 

HUBBARD,  RUTH,  Research  Associate  in  Biology,  Harvard  University 
HUMPHREYS,  TOM,  Assistant  Professor  of  Biology,  Massachusetts  Institute  of  Technology 


REPORT  OF  THE  DIRECTOR  25 

HuNEEUs-Cox,  FRANCISCO,  Research  Associate,  Massachusetts  Institute  of  Technology 
HUNTER,  W.  D.  RUSSELL,  Professor  of  Zoology,  Syracuse  University 
HYMAN,  LIBBIE  H.,  American  Museum  of  Natural  History 

INOUE,  SHINYA,  John  LaPorte  Professor  in  Cytology  and  Chairman  of  the  Department,  Dart- 
mouth Medical  School 
ISENBERG,    IRVIN,    Research    Associate,    Institute    for    Muscle    Research,    Marine    Biological 

Laboratory 
ITO,    SHIZUO,    Assistant    Professor    of   Zoology    and    Physiology,    Kumamoto    University    and 

Columbia  University 
JACKSON,    HAROLD,    Head    of    Experimental    Chemotherapy,    Christie    Hospital,    Manchester, 

England 

JACKSON,  JAMES  A.,  University  of  Connecticut,  The  School  of  Dental  Medicine 
JACOBOWITZ,  DAVID,  Associate,  Department  of  Pharmacology,  University  of  Pennsylvania 
JANOFF,  AARON,  Assistant  Professor  of  Pathology,  New  York  University  School  of  Medicine 
JENCKS,  WILLIAM  P.,  Professor  of  Biochemistry,  Brandeis  University 
JOHNSSON,    RUTH,    Research    Associate,    Institute    for    Muscle    Research,    Marine    Biological 

Laboratory 

JOSEPH  SON,  ROBERT  K.,  Associate  Professor  of  Zoology,  Western  Reserve  University 
KALEY,  GABOR,  Associate  Professor  of  Physiology,  New  York  Medical  College 
KALTENBACH,   JANE   COUFFER,    Associate    Professor    of    Biological    Sciences,    Mount    Holyoke 

College 
KAMINER,  BENJAMIN,  Research  Associate,   Institute  for  Muscle  Research,   Marine  Biological 

Laboratory 

KANE,  ROBERT  E.,  Assistant  Professor  of  Cytology,  Dartmouth  Medical  School 
KARASAKI,  SHUICHI,  Research  Staff,  Putnam  Memorial  Hospital  Institute  for  Medical  Research 
KARUSH,  FRED,  Professor  of  Microbiology,  University  of  Pennsylvania  School  of  Medicine 
KATZ,  GEORGE  M.,  Research  Associate  of  Neurology,  Columbia  University 
KEMPTON,  RUDOLF  T.,  Professor  of  Zoology,  Vassar  College 
KING,   THOMAS   J.,    Senior    Member,    Head,    Department    of    Embryology,    The    Institute    for 

Cancer  Research,  Philadelphia 

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

KLINMAN,  NORMAN  R.,  Post-Doctoral  Fellow,  University  of  Pennsylvania  School  of  Medicine 
KORNBERG,  HANS  LEO,  Head,  Professor  of  Biochemistry,  University  of  Leicester,  England 
KUFFLER,  STEPHEN  W.,  Professor  of  Neurophysiology,  Harvard  University 
LANSING,    ALBERT    I.,    Chairman,    Professor    of    Anatomy    and    Cell    Biology,    University    of 

Pittsburgh 

LASH,  JAMES  W.,  Associate  Professor  of  Anatomy,  University  of  Pennsylvania 
LAZAROW,  ARNOLD,  Head,  Professor  of  Anatomy,  University  of  Minnesota 
LECAR,  HAROLD,  Physicist,  National  Institutes  of  Health 

LERMAN,  SIDNEY,  Associate  Professor  of  Ophthalmology  and  Assistant  Professor  of  Biochem- 
istry, University  of  Rochester  School  of  Medicine  and  Dentistry 
LERNER,  AARON  B.,  Professor  of  Medicine,  Yale  University  School  of  Medicine 
LEVIN,  JACK,  Instructor  of  Internal  Medicine,  The  Johns  Hopkins  University  School  of  Medicine 
LEVINE,  LAWRENCE,  Professor  of  Biochemistry,  Brandeis  University 
LEVINTHAL,  CYRUS,  Professor  of  Biophysics,  Massachusetts  Institute  of  Technology 
LEVY,  MILTON,  Professor  of  Biochemistry,  New  York  University  College  of  Dentistry 
LINDEMANN,  BERND,  Wissenschaftl.  Assistant,  University  of  Saarbrucken,  Germany 
LOCHHEAD,  JOHN  H.,  Professor  of  Zoology,  University  of  Vermont 
LOEWENSTEIN,  WERNER  R.,  Associate  Professor  of  Physiology,  Columbia  University,  College  of 

Physicians  and  Surgeons 

LOPEZ,  ENRIQUE,  Research  Associate,  Columbia  University 
LORAND,  JOYCE  BRUNER,  Research  Associate,  Northwestern  University 
LORAND,  L.,  Professor  of  Chemistry,  Northwestern  University 

LOVE,  WARNER  E.,  Associate  Professor  of  Biophysics,  The  Johns  Hopkins  University 
L0VLIE,  ARNE,  Research  Associate,  University  of  Pittsburgh  and  University  of  Oslo,  Norway 
Lux,  HANS  DIETER,  International  Post-Doctoral  of  the  U.  S.  Public  Health  Service,  National 

Institutes  of  Health 

MAC!NNIS,  AUSTIN  J.,  Assistant  Professor  of  Zoology,  University  of  California,  Los  Angeles 
MACNICHOL,  EDWARD  F.,  JR.,  Professor  of  Biophysics,  The  Johns  Hopkins  University 


26  MARINE  BIOLOGICAL  LABORATORY 

MAHLER,  HENRY  R.,  Professor  of  Chemistry,  Indiana  University 

MARSH,  JULIAN  B.,  Professor  of  Biochemistry,  University  of  Pennsylvania 

MARSLAND,  DOUGLAS,  Research  Professor,  Graduate  School,  New  York  University 

MAUTNER,  HENRY  G.,  Associate  Professor  of  Pharmacology,  Yale  University  School  of  Medicine 

McBRiDE,  ORLANDO  WESLEY,  Research  Associate  of  Biology,  The  Johns  Hopkins  University 

MCGAUGHY,  ROBERT  E.,  Staff  Fellow,  National  Institutes  of  Health 

MELLON,  DEFOREST,  JR.,  Assistant  Professor  of  Biology,  University  of  Virginia 

MENDELSON,   MARTIN,   Assistant   Professor   of   Physiology,    New   York   University    School    of 

Medicine 

METZ,  CHARLES  B.,  Professor,  Institute  of  Molecular  Evolution,  University  of  Miami 
MILKMAN,  ROGER  DAWSON,  Associate  Professor  of  Zoology,  Syracuse  University 
MILLER,  RICHARD  LEE,  Lalor  Fellow,  University  of  Chicago 
MILLOTT,  NORMAN,  Professor  of  Zoology,  Bedford  College,  University  of  London 
MILLS,  ERIC  L.,  Assistant  Professor  of  Biology,  Queen's  University,  Canada 
MONROY,  ALBERTO,  Professor  of  Comparative  Anatomy,  University  of  Palermo,  Italy 
MOORE,  JOHN  W.,  Associate  Professor  of  Physiology,   Chief,   Laboratory  of  Cellular   Neuro- 

physiology,  Duke  University 

MORAN,  JOSEPH  F.,  JR.,  Assistant  Professor  of  Biology,  Russell  Sage  College 
MOSCONA,  A.  A.,  Professor  of  Zoology,  University  of  Chicago 
MOUL,  EDWIN  T.,  Professor  of  Botany,  Rutgers  University 
MULLINS,  L.  J.,  Professor  of  Biophysics,  University  of  Maryland 
NARAHASHI,  TOSHIO,  Assistant  Professor  of  Physiology,  Duke  University 
NASATIR,    MAIMON,   Assistant   Professor   of   Botany,    Assistant    to    Dean,    Pembroke    College, 

Brown  University 

NELSON,  LEONARD,  Associate  Professor  of  Physiology,  Emory  University 
NICHOLLS,  JOHN  G.,  Associate  Professor  of  Physiology,  Yale  University  Medical  School 
NICHOLS,  H.  WAYNE,  Associate  Professor  of  Botany,  Washington  University 
NIMS,  LESLIE  F.,  Senior  Grass  Fellow,  Brookhaven  National  Laboratory 
NOVALES,  RONALD  R.,  Associate  Professor  of  Biological  Sciences,  Northwestern  University 
OCHOA,  MANUEL,  JR.,  Professor  in  Medicine  and  Lalor  Fellow,  Columbia  University,  College 

of  Physicians  and  Surgeons 

OHAD,  ITZHAK,  Research  Associate,  The  Rockefeller  University 
OKAZAKI,  KAYO,  Research  Associate,  University  of  Pennsylvania  School  of  Medicine 
OTSUKA,  MASANORI,  Research  Fellow  in  Neurophysiology  and  Neuropharmacology,  Harvard 

Medical  School 

OZEKI,  MASAHIRO,  Research  Associate  in  Neurology,  Columbia  University 
PALMER,  JOHN  D.,  Assistant  Professor  of  Biology,  New  York  University 
PAOLINI,  PAUL  J.,  JR.,  Grass  Fellow,  University  of  California,  Davis 

PAPPAS,  GEORGE  D.,  Associate  Professor  of  Anatomy,  Columbia  University,  College  of  Physi- 
cians and  Surgeons 

PARKER,  ROBERT  H.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
PARPART,  ARTHUR  K.,  Chairman  and  Professor  of  Biology,  Princeton  University 
PEARCE,  JACK  B.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
PENN,    RICHARD    D.,    Grass    Fellow    in    Neurophysiology,    Columbia    University,    College    of 

Physicians  and  Surgeons 

PERSON,  PHILIP,  Chief,  Special  Dental  Research  Laboratory,  VA  Hospital,  Brooklyn 
PETERSEN,  KAY,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
PHILPOTT,  DELBERT  E.,  Assistant  Professor  of  Biochemistry,  University  of  Colorado  Medical 

School 
PILKINGTON,    THOMAS    R.   E.,    Reader    in   Medicine,    St.    George's    Hospital    Medical    School, 

England,  and  Northwestern  University 

PORTER,  KEITH  R.,  Professor  of  Biology,  Harvard  University 
POTTER,  DAVID  D.,  Assistant  Professor  of  Neurophysiology  and  Neuropharmacology,  Harvard 

Medical  School 

POTTS,  WILLIAM  T.  W.,  Lecturer,  University  of  Birmingham,  England 
PROSSER,  C.  LADD,  Head,  Departments  of  Physiology  and  Biophysics,  University  of  Illinois 
RABIN,  HARVEY,  Assistant  Professor  of  Pathobiology,  The  Johns  Hopkins  University 
READ,  CLARK  P.,  Professor  of  Biology,  Rice  University 
REBHUN,  LIONEL  I.,  Associate  Professor  of  Biology,  Princeton  University 


REPORT  OF  THE  DIRECTOR  27 

REDFIELD,  ALFRED  C,  Woods  Hole  Oceanographic  Institution 

REUBEN,  JOHN  P.,  Assistant  Professor  of  Neurology,  Columbia  University 

REYNOLDS,  GEORGE  T.,  Professor  of  Palmer  Laboratory,  Princeton  University 

RICE,  ROBERT  V.,  Senior  Fellow,  Mellon  Institute 

ROCKSTEIN,  MORRIS,  Professor  of  Physiology,  University  of  Miami  School  of  Medicine 

ROJAS,  EDUARDO  E.,  Visiting  Associate,  National  Institutes  of  Health 

ROSE,  S.  MERYL,  Professor  of  Experimental  Embryology,  Tulane  University 

ROSENBERG,    PHILIP,    Assistant    Professor    of    Neurology,    Columbia    University,    College    of 

Physicians  and  Surgeons 

ROSENKRANZ,  HERBERT,  Assistant  Professor  of  Microbiology,  Columbia  University 
ROSLANSKY,  JOHN  D.,  Research  Associate,  Institute  for  Muscle  Research,  Marine  Biological 

Laboratory 

ROUND,  FRANK  ERIC,  Lecturer,  University  of  Bristol,  England 
RUSHFORTH,  NORMAN  B.,  Assistant  Professor  of  Biology  and  Biostatistics,  Western  Reserve 

University 

RUSTAD,  RONALD  C.,  Associate  Professor  of  Biology,  Western  Reserve  University 
SANDERS,  HOWARD  LAWRENCE,  Associate  Scientist,  Woods  Hole  Oceanographic  Institution 
SATO,  HIDEMI,  Assistant  Professor  of  Cytology,  Dartmouth  Medical  School 
SAUNDERS,  JOHN  W.,  JR.,  Chairman  and  Professor  of  Biology,  Marquette  University 
SAXEN,  LAURI  O.,  Associate  Professor  of  Pathology  and  Senior  Lalor  Fellow,  University  of 

Helsinki,  Finland 
SCHMEER,  SISTER  M.  ROSARII,  Chairman  and  Associate  Professor  of  Biology,  College  of  St. 

Mary  of  the  Springs 

SCHMITT,  FRANCIS  O.,  Professor  of  Biology,  Massachusetts  Institute  of  Technology 
SCHNEIDERMAN,   HOWARD  A.,   Chairman  and   Professor   of   Biology,   Director,    Developmental 

Biology  Center,  Western  Reserve  University 

SCHOPF,  THOMAS  J.  M.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
SCHWARTZ,  TOBIAS  L.,  Trainee  of  Neurology,  Columbia  University 
SCOTT,  GEORGE  TAYLOR,  Chairman  and  Professor  of  Biology,  Oberlin  College 
SENFT,  ALFRED  W.,  Marine  Biological  Laboratory 

SENFT,  JOSEPH  P.,  USPHS  Post-Doctoral  Fellow,  University  of  Maryland  School  of  Medicine 
SHEMIN,  DAVID,  Professor  of  Biochemistry,  Columbia  University 
SHEPRO,  DAVID,   Professor  and  Research   Associate  of  Biology,   Boston  University   Graduate 

School  and  Simmons  College 

SHERMAN,  IRWIN  W.,  Assistant  Professor  of  Zoology,  University  of  California,  Riverside 
SIMON,  JOSEPH  L.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
SIMPSON,  SIDNEY  B.,  JR.,  Assistant  Professor  of  Anatomy,  Western  Reserve  University  Medical 

School 

SINGER,  IRWIN,  Research  Associate,  National  Institutes  of  Health 
SJODIN,  RAYMOND  A.,  Associate  Professor  of  Biophysics,  University  of  Maryland 
SLOBODKIN,  LAWRENCE  B.,  Professor  of  Zoology,  University  of  Michigan 
SMITH,  EDMUND,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
SPEIDEL,  CARL  C.,  Emeritus  Professor  of  Anatomy,  University  of  Virginia 

SPIRTES,  MORRIS  ALBERT,  Clinical  Associate  Professor  of  Pharmacology,  University  of  Pitts- 
burgh Medical  Center 

STEINBACH,  H.  BURR,  Chairman  and  Professor  of  Zoology,  University  of  Chicago 
STEINBERG,  MALCOLM  S.,  Associate  Professor  of  Biology,  The  Johns  Hopkins  University 
STRICKHOLM,  ALFRED,  Assistant  Professor  of  Physiology,  University  of  California  School   of 

Medicine,  San  Francisco 

STRITTMATTER,  PHILIPP,  Associate  Professor  of  Biochemistry,  Washington  University 
STUNKARD,  HORACE  W.,  Research  Associate,  American  Museum  of  Natural  History 
SUSSMAN,  MAURICE,  Professor  of  Biology,  Brandeis  University 

SZABO,  GEORGE,  Assistant  Professor  of  Anatomy  in  Department  of  Dermatology,  at  Massachu- 
setts General  Hospital,  Harvard  Medical  School 

SZENT-GYORGYI,  ALBERT,  Director,  Institute  for  Muscle  Research,  Marine  Biological  Laboratory 
TAKEDA,  KIMIHISA,  Research  Associate,  Columbia  University 

TASAKI,  ICHIJI,  Acting  Chief,  Laboratory  of  Neurobiology,  National  Institutes  of  Health 
TAYLOR,  ROBERT  E.,  Associate  Chief,  Laboratory  of  Biophysics,  National  Institutes  of  Health 
TAYLOR,  WILLIAM  RANDOLPH,  Professor  of  Botany,  University  of  Michigan 


MARINE  BIOLOGICAL  LABORATORY 

TAYLOR,  W.  ROWLAND,  Assistant  Professor  of  Oceanography,  The  Johns  Hopkins  University 

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

TORCH,  REUBEN,  Associate  Professor  of  Zoology,  University  of  Vermont 

TRINKAUS,  J.  P.,  Professor  of  Biology,  Yale  University 

TROLL,  WALTER,  Associate  Professor  of  Environmental  Medicine,  New  York  University  Medical 

Center 

TUBOI,  SYOZO,  Research  Associate,  Columbia  University 

VAN  VUNAKIS,  HELEN,  Associate  Professor  of  Biochemistry,  Brandeis  University 
VILLEE,  CLAUDE  A.,  Andelot  Professor  of  Biological  Chemistry,  Harvard  Medical  School 
VINCENT,  W.  S.,  Associate  Professor  of  Anatomy  and  Cell  Biology,  University  of  Pittsburgh 
WAINIO,  WALTER,  Professor  of  Biochemistry,  Rutgers,  The  State  University  of  New  Jersey 
WALD,  GEORGE,  Professor  of  Biology,  Harvard  University 

WALLACE,  ROBIN  A.,  Research  Associate  of  Biology  Division,  Oak  Ridge  National  Laboratory 
WALSH,  GERALD,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
WARREN,  LEONARD,  Professor  of  Therapeutic  Research,  University  of  Pennsylvania 
WATANABE,  AKIRA,  Consultant,  National  Institutes  of  Health 
WATKINS,  DUDLEY  T.,  Graduate  Student,  Western  Reserve  University 

WEBB,  GEORGE  D.,  Visiting  Fellow,  Columbia  University,  College  of  Physicians  and  Surgeons 
WEBB,    H.    MARGUERITE,    Associate    Professor    of    Biological    Sciences,    Goucher    College    and 

Research  Associate,  Northwestern  University 

WEISS,  LEON,  Associate  Professor  of  Anatomy,  The  Johns  Hopkins  University 
WICHTERMAN,  RALPH,  Professor  of  Biology,  Temple  University 
WIERCINSKI,  FLOYD  S.,  Associate  Professor,  Illinois  Teachers  College  North 
WILCE,  ROBERT  T.,  Assistant  Professor  of  Botany,  University  of  Massachusetts 
WILSON,  WALTER  L.,  Professor  of  Biology,  Oakland  University 
WYTTENBACH,  CHARLES  R.,  Assistant  Professor  of  Anatomy,  University  of  Chicago 
ZELEWSKI,  LEON,  Research  Fellow,  Harvard  University  and  University  of  Gdansk,  Poland 
ZIGMAN,  SEYMOUR,  Professor  of  Biochemistry,  University  of  Rochester 
ZIMMERMAN,  ARTHUR  M.,  Professor  of  Zoology,  University  of  Toronto,  Canada 
ZULLO,  VICTOR  A.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
ZWILLING,  EDGAR,  Professor  of  Biology,  Brandeis  University 

Lalor  Fellows,  1965 

SAXEN,  LAURI  O.,  Senior  Fellow,  University  of  Helsinki,  Finland 

JACKSON,  HAROLD,  Christie  Hospital,  Manchester,  England 

MILLER,  RICHARD  LEE,  University  of  Chicago 

OCHOA,  MANUEL,  JR.,  Columbia  University,  College  of  Physicians  and  Surgeons 

STEINBERG,  MALCOLM  S.,  The  Johns  Hopkins  University 

Grass  Fellows,  1965 

NIMS,  LESLIE  F.,  Forbes  Memorial  Lecturer,  Brookhaven  National  Laboratory 

COLLETT,  THOMAS  STEPHEN,  University  College,  London 

GORDON,  JEOFFRY,  The  Grass  Foundation 

PAOLINI,  PAUL  J.,  JR.,  University  of  California,  Davis 

PENN,  RICHARD  D.,  Columbia  University,  College  of  Physicians  and  Surgeons 

Research  Assistants,  1965 

ADELBERG,  MICHAEL  G.,  University  of  California,  Berkeley 

ANDERSON,  NELS,  Duke  University 

ANTONELLIS,  BLENDA  C,  Western  Reserve  University 

APLEY,  MARTYN  L.,  Syracuse  University 

ARDWIN,  LINDSAY  S.,  Columbia  University 

ARMSTRONG,  SAMUEL  C.,  Upstate  Medical  Center 

ASTERITA,  HARVEY  L.,  New  York  University 

AULT,  KENNETH  A.,  Massachusetts  Institute  of  Technology 


REPORT  OF  THE  DIRECTOR  29 

BABLOUZIAN,  BARKEV  L.,  University  of  Illinois 

BAIRD,  RONALD,  Western  Reserve  University 

BAKER,  ROBERT  F.,  Brown  University 

BALTUS,  ELYANE,  University  of  Brussels 

BAMMAN,  BARBARA  C.,  Princeton  University 

BARNETT,  GERALD,  The  Johns  Hopkins  Medical  School 

BARNHILL,  ROBERT,  Miami  University  at  Ohio 

BARTELS,  EVA,  Columbia  University 

BAUER,  JOHN  J.,  University  of  Miami  School  of  Medicine 

BELL,  ALLEN,  Upstate  Medical  Center 

BENES,  MARY,  Brandeis  University 

BENISEK,  MARY  V.,  University  of  Michigan 

BENNETT,  JUDITH  ANN,  Syracuse  University 

BERKOWITZ,  DANIEL  M.,  New  York  University  Medical  School 

BERKOWITZ,  ELLEN  M.,  New  York  University  Medical  School 

BIKLE,  DANIEL,  Harvard  University 

BILLIAR,  JOANNE,  Harvard  Medical  School 

BOCK,  MARION,  Christie  Hospital,  Manchester,  England 

BODIAN,  HELEN,  Goucher  College 

BOOKMAN,  CHARLES  A.,  Barlow  School  and  Columbia  University,  College  of  Physicians  and 

Surgeons 

BRADLEY,  JOAN  C.,  Drew  University 
BRADY,  FRANCINE,  Syracuse  University 
BURKE,  DONALD  SCOTT,  Western  Reserve  University 
BUSSER,  JOHN  H.,  University  of  Rhode  Island 
CAROL,  JOAN,  Columbia  University 
CAROLAN,  ROBERT  M.,  Dartmouth  Medical  School 
CHAFFEE,  RICHARD  BATES,  JR.,  Syracuse  University 
CLARK,  JOHN  L,  Dartmouth  College 
CLAYBROOK,  MAJORIE  W.,  Columbia  University 
COLLINS,  SANDRA  ELAINE,  Smith  College 
CONOVER,  SHIRLEY,  National  Institutes  of  Health 
CONRAD,  GARY  W.,  Yale  University 
CORFF,  SONDRA,  Western  Reserve  University 
CRAWFORD,  CAROLYN,  Connecticut  College 
DACEY,  JOAN  F.,  Harvard  University 
DAVIDSON,  HAROLD,  National  Institutes  of  Health 
DENGINER,  SUSAN,  College  of  St.  Mary  of  the  Springs 
DOANE,  MARSHALL  G.,  University  of  Maryland 
DONALDSON,  DONALD  JAY,  Tulane  University 
DONLEY,  CLARK,  The  Johns  Hopkins  University 
DRESCHER,  PATRICIA,  Columbia  University 

DRISCOLL,  ANDREW  L.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 
DYRO,  FRANCES  M.,  University  of  Maryland  School  of  Medicine 
EBY,  DOUGLAS,  Boston  University 
EGAR,  MARGARET  W.,  Western  Reserve  University 
EISENBERG,  HENRY,  Columbia  University 
EISENBERG,  JAMES  P.,  Colby  College 
ELDER,  HUGH  Y.,  Glasgow  University,  Scotland 
FASS,  SIMCHA  U.,  Massachusetts  Institute  of  Technology 
FERNANDEZ,  HECTOR  R.,  Yale  University 
FINDLEY,  DAVIS,  University  of  Tennessee 
FISH,  CHERIE  LYN,  Smith  College 
FITZJARRELL,  AUSTIN  T.,  Tulane  University 
FOLDES,  PAUL,  Lehigh  University 
FORAN,  ELIZABETH  H.,  Smith  College 
FRANKLIN,  LUTHER  EDWARD,  University  of  Miami 
FREEMAN,  SALLIE  BOINEAU,  Emory  University 


30  MARINE  BIOLOGICAL  LABORATORY 

FUSARI,  MARGARET  HELENE,  Boston  University 

GARDNER,  GARY,  Oberlin  College 

GATEFF,  ELISABETH,  Western  Reserve  University 

GIORDANO,  VICKI  LYNN,  University  of  Delaware 

GOLDIZEN,  VERNON  C,  Western  Reserve  University 

GOLDMAN,  ROBERT  D.,  Princeton  University 

GOLDSTONE,  ELLEN,  Upstate  Medical  Center 

GOODMAN,  ROSANNE,  The  Johns  Hopkins  University 

GORMAN,  JESSICA  A.,  University  of  California,  Los  Angeles 

GORMAN,  JOHN,  University  of  California,  Los  Angeles 

GRANGER,  RONALD  EUGENE,  The  Johns  Hopkins  University 

GREBE,  STEPHEN  CHARLES,  Oakland  University 

GROSS,  GARY,  Columbia  University,  College  of  Physicians  and  Surgeons 

HABAS,  LINDA  B.,  American  Museum  of  Natural  History 

HACKETT,  PAUL  ROGER,  Western  Reserve  University 

HAINES,  MICHAEL  F.,  Syracuse  University 

HAROSI,  FERENC,  The  Rockefeller  University 

HARRIS,  EDWARD  M.,  Duke  University 

HARTMANN,  JOHN  F.,  University  of  Toronto,  Canada 

HEATFIELD,  BARRY  M.,  University  of  California,  Los  Angeles 

HESS,  MARJORIE  B.,  Dartmouth  Medical  School 

HEYMANN,  PETER  W.,  Washington  University 

HORN,  DIANE,  Stanford  University 

HORNIG,  JOANNA,  Radcliffe  College 

HUMPHREYS,  SUSIE,  Harvard  University 

HUNTER,  WILLIAM  BRUCE,  University  of  California,  Santa  Barbara 

IMLAY,  MARC  JAMES,  Northwestern  University 

ISENBERG,  DAVID,  Deerfield  Academy 

JACOBS,  CAROL  F.,  Brandeis  University 

JACOBSON,  MARCUS,  University  of  Edinburgh 

JOB,  DONALD  D.,  University  of  Illinois 

KAUFMAN,  ROBERT  G.,  Columbia  University 

KEHLENBECK,  EDNA  K.,  Syracuse  University 

KELLY,  ROBERT,  Mellon  Institute 

KEM,  WILLIAM  R.,  University  of  Illinois 

KENNY,  DIANNE,  Simmons  College  and  Boston  University 

KIEN,  MARJA,  Boston  University 

KIMBALL,  FRANCES,  Reed  College 

KIMMEL,  CHARLES  B.,  The  Johns  Hopkins  University 

KISSIL,  GEORGE  WM.,  University  of  Connecticut 

KOHNE,  DAVID,  Purdue  University 

KORNBLITH,  GINA,  Brown  University 

KRATOWICH,  NANCY  ROSALIE,  Columbia  University 

KRAWCHENKO,  JOHN,  Syracuse  University 

LATTMAN,  EATON  E.,  The  Johns  Hopkins  University 

LEFKON,  BRUCE  WARREN,  Columbia  University 

LEITNER,  V.  ENA,  Smith  College 

LENNOX,  EDWIN  S.,  Salk  Institute  for  Biological  Studies 

LENTZ,  JUDITH  P.,  Yale  University 

LENTZ,  THOMAS,  Yale  University 

LIEB,  KATHARINE  H.,  University  of  Illinois 

LIPSKY,  DAVID,  Columbia  University 

LITTNA,  ELIZABETH  M.,  Carnegie  Institution  of  Washington 

LLOYD,  MARGARET  C.,  University  of  Michigan 

LOWE,  LOUISE,  University  of  Toronto,  Canada 

MACDONALD,  VICTOR  W.,  Massachusetts  Institute  of  Technology 

MACDUFF,  MARIE,  New  York  University  Medical  Center 

MAHAR,  CONSTANCE  QUINN,  Syracuse  University 


REPORT  OF  THE  DIRECTOR 

MALCOLM,  DOUGLAS,  University  of  Edinburgh  and  University  of  Pittsburgh 

AIALONY,  PETER  C,  Brown  University 

MATSUMOTO,  YORIMI,  University  of  Illinois 

McDANiEL,  JAMES  S.,  Rice  University 

McGuRN,  ELEANOR  A.,  Western  Reserve  University 

MEISMER,  DONALD  M.,  University  of  Cincinnati 

MERRILL,  CHARLOTTE,  Massachusetts  Institute  of  Technology 

MILLER,  SANDRA  M.,  University  of  Maryland  School  of  Medicine 

MOORAD,  PHILIP  J.,  JR.,  Princeton  University 

MORITZ,  GISELA,  Columbia  University,  College  of  Physicians  and  Surgeons 

MOSCONA,  M.  H.,  University  of  Chicago 

MUNDAY,  JOHN  CLINGMAN,  JR.,  University  of  Illinois 

MUNDAY,  JUDITH  BERRIEN,  University  of  Illinois 

NADOL,  JOSEPH  B.,  JR.,  Harvard  College 

NAGLE,  J.  STEWART,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

NASH,  MARK  S.,  Montgomery  Junior  College 

OLDHAM,  PETER  J.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

ORTIZ,  JOSE  R.,  University  of  Chicago 

OSTERGARD,  JAMES  P.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

OTT,  FRANKLYN,  University  of  Massachusetts 

PAK,  SUNG  KWON,  Princeton  University 

PARKER,  CAROL  ANN,  University  of  Massachusetts 

PHILIPPUS,  PAMELA  L.,  Colorado  College 

PICKERING,  CAROLA,  Oxford  University 

PIKE,  JOHN  DOUGLAS,  Tufts  University 

PITTS,  W.  REID,  JR.,  Harvard  University  Medical  School 

POCOCK,  MARY  WEIR,  University  of  Texas 

POST,  CHARLES  T.,  JR.,  Yale  University 

POTTER,  DIANE,  Columbia  University 

PRINCE,  JEFFREY  S.,  University  of  Massachusetts 

RAAB,  JACOB,  University  of  Chicago 

RAVITZ,  MELVYN  JAY,  University  of  Vermont 

RHODES,  RUSSELL  G.,  University  of  Tennessee 

ROBERTSON,  DOUGLAS  R.,  Upstate  Medical  Center 

ROBERTSON,  LOLA  E.,  American  Museum  of  Natural  History 

ROBINSON,  PEGGY,  The  Johns  Hopkins  University 

ROSE,  FLORENCE  C.,  Tulane  University 

ROSEN,  CHARLES  T.,  University  of  Toronto,  Canada 

ROSENBERG,  MARK  J.,  Amherst  College 

ROSENBERG,  MARTIN,  New  York  University,  Bellevue  Medical  Center 

RUBENSTEIN,  JUDITH,  Columbia  University,  College  of  Physicians  and  Surgeons 

RUFFING,  FAITH  E.,  Western  Reserve  University 

SALTZMAN,  ORAH,  Barnard  College 

SANDLIN,  RONALD  A.,  National  Institutes  of  Health 

SAUL,  DAVID,  The  Johns  Hopkins  University  Medical  School 

SCHINDLER,  GERDA,  New  York  Medical  College 

SCHMIDT,  JOHN  HOWARD,  Marquette  University 

SCHWAMB,  PETER  B.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

SELDIN,  EDWARD  B.,  Harvard  Graduate  School  of  Arts  and  Sciences 

SETTLES,  HARRY  E.,  Tulane  University 

SHRAGER,  PETER  G.,  University  of  California,  Berkeley 

SLOANE,  ELEANOR  MARY,  Mellon  Institute 

SLOANE,  MOLLA  REBECCA,  Wellesley  College 

SMILACK,  JERRY,  The  Johns  Hopkins  University  Medical  School 

SMITH,  BARRY  HAMILTON,  Massachusetts  Institute  of  Technology 

SOLOMAN,  DEHN  E.,  Kalamazoo  College 

SORENSON,  ROBERT  L.,  University  of  Minnesota  Medical  School 

SPECHT,  PHILIP  C.,  Syracuse  University 


32  MARINE  BIOLOGICAL  LABORATORY 

SPENCER,  REBECCA  L.,  Mount  Holyoke  College 

SPIEGELMAN,  MARJORIE  J.,  University  of  Chicago 

SPINDEL,  ROBERT,  Columbia  University,  College  of  Physicians  and  Surgeons 

SPIRTES,  R.  S.,  Reed  College 

SQUIRE,  RICHARD  D.,  North  Carolina  State  University 

STARK,  VIRGINIA  ANN,  Syracuse  University 

STEPHENS,  RAYMOND  E.,  Dartmouth  Medical  School 

STONE,  INGRID,  Columbia  University,  College  of  Physicians  and  Surgeons 

SUDDITH,  ROBERT  L.,  Indiana  University 

SUSSMAN,  JOEL  L.,  Massachusetts  Institute  of  Technology 

SZULMAN,  AARON  E.,  University  of  Pittsburgh  School  of  Medicine 

TAMM,  SIDNEY  L.,  University  of  Chicago 

TANNENBAUM,  ALICE  SUSAN,  University  of  Maryland  School  of  Medicine 

TASAKI,  LYDIA,  National  Institutes  of  Health 

TERMAN,  STANLEY  A.,  Massachusetts  Institute  of  Technology 

THABES,  TAMARA  M.,  University  of  Chicago 

THERRIEN,  EDWARD,  Syracuse  University 

THOMAS,  JUNE  M.,  University  of  California,  Los  Angeles 

TRACER,  CAROLYN,  Princeton  University 

TUCKER,  C.  MICHAEL,  Dartmouth  College 

TURNER,  VILIA,  University  of  California,  Los  Angeles 

TUTTLE,  JOAN,  University  of  Rochester 

VALCOVIC,  LAWRENCE  R.,  North  Carolina  State  University 

VAN  AMBURG,  SHARON  ANN,  Rice  University 

VAN  PRAAG,  DINA,  New  York  University 

VAN  ZANDT,  DIRK,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

WECHSLER,  JAMES  A.,  Yale  University 

WEINER,  BEVERLY,  Harvard  University 

WENGER,  KARLYNN  L.,  Tulane  University 

WERMUTH,  BRUCE,  Yale  University 

WHITE,  ERIC  S.,  Dartmouth  Medical  School 

WILLIAMS,  ANTHONY,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

WILLIAMS,  HILARY  M.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

WONG,  KAI  KONG,  Oberlin  College 

YAMAMOTO,  YOSHIHIRO,  Chiba  University,  Japan,  and  Newcomb  College 

YENTSCH,  ANNE  E.,  Systematics-Ecology  Program,  Marine  Biological  Laboratory 

YUYAMA,  SHUHEI,  Western  Reserve  University 

ZELMAN,  DAVID  ALLEN,  University  of  California,  Berkeley 

ZERKIN,  MILTON,  Columbia  University 

ZOLLINGER,  WILLIAM,  University  of  Pittsburgh 

Library  Readers,  1965 

ALLEN,  M.  JEAN,  Chairman  and  Professor  of  Biology,  Wilson  College 

AMBERSON,  WILLIAM  R.,  Marine  Biological  Laboratory 

ATWOOD,  KIMBALL  CHASE,  Professor  of  Microbiology,  University  of  Illinois 

BALL,  ERIC  G.,  Edward  S.  Wood  Professor  of  Biochemistry,  Harvard  Medical  School 

BERNE,  ROBERT  M.,  Professor  of  Physiology,  Western  Reserve  University 

BODANSKY,  OSCAR,  Chief,  Division  of  Enzymology  and  Metabolism,  Sloan-Kettering  Institute 

for  Cancer  Research 

BRIDGMAN,  ANNA  JOSEPHINE,  Chairman  and  Professor  of  Biology,  Agnes  Scott  College 
BUTLER,  ELMER  G.,  Henry  Fairfield  Osborn  Professor  of  Biology,  Princeton  University 
CARLSON,  ELOF  AXEL,  Assistant  Professor  of  Zoology,  University  of  California,  Los  Angeles 
CARLSON,  FRANCIS  D.,  Professor  of  Biophysics,  The  Johns  Hopkins  University 
CHASE,  AURIN  M.,  Professor  of  Biology,  Princeton  University 
CLARK,  ARNOLD  M.,  Professor  of  Biological  Sciences,  University  of  Delaware 
COHEN,  SEYMOUR  S.,  Chairman  and  Professor  of  Therapeutic  Research,  University  of  Pennsyl- 
vania School  of  Medicine 


REPORT  OF  THE  DIRECTOR 

COLLIER,  J.  R.,  Professor  of  Biology,  Rensselaer  Polytechnic  Institute 

DAVIS,  BERNARD  D.,  Head  of  Department  of  Bacteriology  and  Immunology,  Harvard  Medical 

School 
EISEN,  HERMAN  N.,  Chairman  and  Professor  of  Microbiology,  Washington  University  School 

of  Medicine 

FLESCH,  PETER,  Research  Professor  of  Dermatology,  University  of  Pennsylvania 
GABRIEL,  MORDECAI  L.,  Professor  of  Biology,  Brooklyn  College 
GINSBERG,  HAROLD  S.,  Chairman  and  Professor  of  Microbiology,  University  of  Pennsylvania 

School  of  Medicine 

GORLIN,  RICHARD,  Assistant  Professor  of  Medicine,  Harvard  Medical  School 
GREEN,  JAMES  W.,  Professor  of  Physiology,  Rutgers,  The  State  University 
GUREWICH,  VLADIMIR,  Assistant  Professor  of  Clinical  Medicine,  New  York  College  of  Medicine 
HANDLER,  PHILIP,  James  B.  Duke  Professor  and  Chairman,  Biochemistry,  Duke  University 
HARLEY,  REV.  JAMES  L.,  Associate  Professor  of  Biology,  Georgetown  University 
HAUBRICH,  ROBERT  R.,  Assistant  Professor  of  Biology,  Denison  University 
HURWITZ,  CHARLES,  Chief,  Basic  Science  Laboratory  and  Assistant  Professor  of  Microbiology, 

VA  Hospital,  Albany 

HURWITZ,  JERALD,  Professor  of  Molecular  Biology,  Albert  Einstein  College  of  Medicine 
ISSELBACHER,    KURT   J.,    Chief,    Gastrointestinal    Unit    and    Associate    Professor    of    Medicine, 

Harvard  Medical  School  and  Massachusetts  General  Hospital 

JACOBS,  MERKEL  H.,  Emeritus  Professor  of  Physiology,  University  of  Pennsylvania 
KEOSIAN,  JOHN,  Professor  of  Biology,  Rutgers,  The  State  University 
LEVINE,  R.  P.,  Professor  of  Biology,  Harvard  University 
LOCH  HEAD,  JOHN  H.,  Professor  of  Zoology,  University  of  Vermont 

LONDON,  IRVING  M.,  Chairman  and  Professor  of  Medicine,  Albert  Einstein  College  of  Medicine 
MALKIEL,  SAUL,  Research  Associate,  Children's  Cancer  Research  Foundation,  Inc. 
MARKS,  PAUL  A.,  Associate  Professor,  Columbia  University 
MATEYKO,  G.  M.,  Associate  Professor  of  Biology,  New  York  University 
AlcDoNALD,   SISTER  ELIZABETH    SETON,   Chairman  and  Professor  of  Biology,   College  of  Mt. 

St.  Joseph  on  the  Ohio 

MEISLER,  RICHARD,  Instructor  in  Philosophy,  Antioch  College  and  Columbia  University 
NASON,  ALVIN,  Associate  Director  of  McCollum-Pratt  Institute  and  Professor  of  Biology,  The 

Johns  Hopkins  University 

NOVIKOFF,  ALEX  B.,  Research  Professor,  Albert  Einstein  College  of  Medicine 
NYSTROM,  RICHARD  A.,  Assistant  Professor  of  Biological  Sciences,  University  of  Delaware 
PERLMANN,  GERTRUDE  E.,  Associate  Professor,  The  Rockefeller  University 
READ,  CLARK  P.,  Professor  of  Biology,  Rice  University 
ROTH,  JAY  S.,  Professor  of  Biochemistry,  University  of  Connecticut 
RUSSELL,  HENRY  D.,  Private  Research 

RUTMAN,  ROBERT  J.,  Associate  Professor  of  Chemistry,  University  of  Pennsylvania 
SCHLESINGER,  R.  WALTER,  Chairman,  Department  of  Microbiology  and  Assistant  Dean,  Rutgers 

Medical  School 
SHAPIRO,  HERBERT,  Associate  Member  of  Research  Laboratory,  Albert  Einstein  Medical  Center, 

Philadelphia 

SPIEGEL,  MELVIN,  Associate  Professor  of  Biology,  Dartmouth  College 
SPRAGUE,  JAMES  M.,  Professor  of  Anatomy,  University  of  Pennsylvania 
STEINHARDT,  JACINTO,  Professor,  Georgetown  University 
STURTEVANT,  A.  H.,  Thomas  Hunt  Morgan  Professor  of  Biology,  Emeritus,  California  Institute 

of  Technology 

SWANSON,  CARL  P.,  Professor  of  Biology,  The  Johns  Hopkins  University 
WAKSMAN,  BYRON  H.,  Chairman,  Department  of  Microbiology,  Yale  University 
WHEELER,  GEORGE  E.,  Associate  Professor  of  Biology,  Brooklyn  College 
WIGGANS,  DONALD  S.,  Professor  of  Biochemistry,  University  of  Texas  Southwestern  Medical 

School 

WILSON,  THOMAS  HASTINGS,  Associate  Professor  of  Physiology,  Harvard  Medical  School 
WINTERS,   ROBERT  W.,    Professor   of   Pediatrics,    Career    Scientist,    Health    Research    Council, 

Columbia  University,  College  of  Physicians  and  Surgeons 


34  MARINE  BIOLOGICAL  LABORATORY 

YNTEMA,  CHESTER  L.,  Professor  of  Anatomy,  State  University  of  New  York,  at  Syracuse 
ZACKS,  SUMNER  L,  Neuropathologist,  Pennsylvania  Hospital 
ZORZOLI,  ANITA,  Associate  Professor  of  Physiology,  Vassar  College 

Students,  1965 

All  students  listed  completed  the  formal  course  program,  June  14-July  24.     Asterisk  indi- 
cates students  completing  Post-Course  Research  Program,  July  25-September  4. 

ECOLOGY 

ALLEN,  JAMES  R.,  New  York  University 
*BAILEY,  CLAUDIA  F.,  Oberlin  College 
*BOYER,  JOHN  F.,  University  of  Chicago 

CALABRESE,  ANTHONY,  University  of  Connecticut 

CARRIER,  REV.  YVAN,  Fordham  University 

DUVEL,  WILLIAM  A.,  JR.,  Tufts  University 

GAINES,  ARTHUR  G.,  University  of  Rhode  Island 

GOLDSMITH,  MARIAN  R.,  University  of  Pennsylvania 

HELLER,  SUSAN  P.,  Connecticut  College 

KATONA,  STEVEN  K.,  Harvard  College 
*LANG,  JUDITH  C.,  Yale  University 
*LEVANDOWSKY,  MICHAEL,  Columbia  University 

MALONE,  PHILIP  G.,  Western  Reserve  University 
*McKiBBiNs,  DALE  L.,  University  of  Arizona 
*MILLER,  CHARLES  B.,  Scripps  Institution  of  Oceanography 
*TEITELBAUM,  MAE,  City  College  of  New  York 
*TOLDERLUND,  DOUGLAS  S.,  Columbia  University 

EMBRYOLOGY 

*  ATKINSON,  BURR  G.,  JR.,  University  of  Connecticut 
*BOLENDER,  ROBERT  P.,  Columbia  University 
*CoNE,  MARGARET  V.,  Mount  Holyoke  College 
*CRONIN,  PATRICIA  E.,  Dartmouth  Medical  School 
*DuBRUL,  ERNEST  F.,  Washington  University 
*EISEN,  ARTHUR  Z.,  Harvard  Medical  School 

FUCHSMAN,  LUCY,  Harvard  University 
*GERSHMAN,  HOWARD  S.,  The  Johns  Hopkins  University 
*HAUSCHKA,  PETER  V.,  The  Johns  Hopkins  University 

HERBERT,  THOMAS  J.,  The  Johns  Hopkins  University 
*HOOPER,  ABIGAIL  W.,  Yale  University 
*LARRIVEE,  DENIS  H.,  University  of  California,  Berkeley 
^MACKINTOSH,  FREDERICK  R.,  Massachusetts  Institute  of  Technology 

MARSHALL,  RICHARD  E.,  National  Heart  Institute 
*PEDERSON,  THORU  J.,  Syracuse  University 
*RIDDLE,  BARBARA  J.,  Brandeis  University 
*ROMANOFF,  PHYLLIS  S.,  The  Rockefeller  University 
*SINDELAR,  WILLIAM  F.,  Western  Reserve  University 
*VINSON,  WALTER  C.,  JR.,  Stanford  University 
*WHISNANT,  BETTY  LYNN,  Duke  University 

MARINE  BOTANY 

ANDREWS,  HOLLINGS  T.,  University  of  Kansas 

BARTHOLOMEW,  KAREN  E.,  University  of  California,  Los  Angeles 


REPORT  OF  THE  DIRECTOR  35 


*CONOVER,  CAROL  L.,  Drew  University 
*DABNEY,  MICHAEL  W.,  Seton  Hill  College 
GROSS,  RUDOLPH  E.,  University  of  Maryland 
HALL,  BARBARA  SUE,  University  of  Oregon 
HARGRAVES,  PAUL  E.,  University  of  Rhode  Island 
HILTON,  RICHARD  L.,  JR.,  University  of  Arizona 
HOSTETTER,  HEBER  P.,  Ill,  University  of  Arizona 
*LEMBI,  CAROLE  A.,  University  of  Tennessee 
*LOCKWOOD,  LINDA  G.,  Columbia  University 
LYNN,  RAYMOND  I.,  Indiana  University 
MACKIERNAN,  GAIL  B.,  College  of  William  and  Mary 
*MADSEN,  MARCIA  J.,  University  of  California,  Davis 
MANN,  JAMES  EDWARD,  University  of  Texas 
NALEPA,  THOMAS  F.,  Indiana  University 
*OTT,  FRANKLYN,  University  of  Texas 

SEARS,  JAMES  R.,  University  of  Massachusetts 
*WALDREP,  MARGARET,  University  of  South  Alabama 
*WATSON,  MELVIN  W.,  University  of  Louisville 


PHYSIOLOGY 

*ASCHER,  MICHAEL  S.,  Dartmouth  College  Medical  School 
*BLUMENTHAL,  ALAN  B.,  California  Institute  of  Technology 

BURGESS,  RICHARD  R.,  Harvard  University 
*CAMPBELL,  RICHARD  D.,  The  Rockefeller  University 
*COHN,  CAL,  Cornell  University  Medical  School 
*CRAPO,  LAWRENCE  M.,  Harvard  University 

CUSHMAN,  SAMUEL  W.,  The  Rockefeller  University 
*DUANE,  WARREN,  University  of  Illinois 
*FELDMAN,  JERRY  F.,  Princeton  University 
*GAMOW,  R.  IGOR,  University  of  Colorado  Medical  School 
*HENDRICKSON,  WAYNE  A.,  The  Johns  Hopkins  University 

JARVIS,  DEREK,  University  of  Wisconsin  Medical  School 

KASCHE,  VOLKER,  Brandeis  University 
*LEITER,  EDWARD,  Emory  University 

LESKES,  ANDREA,  The  Rockefeller  University 
*MAKINEN,  MARVIN  W.,  University  of  Pennsylvania 
*MALA\VISTA,  STEPHEN  E.,  Yale  University  School  of  Medicine 
*MEISLER,  MIRIAM  H.,  Ohio  State  University 
*MOYER,  RICHARD  W.,  University  of  California,  Los  Angeles 

MURGOLA,  EMANUEL  J.,  Yale  University 
*PERL,  WILLIAM,  New  York  University  Medical  Center 
*POLLARD,  HARVEY  BRUCE,  University  of  Chicago  Medical  School 
*REINER,  ALBEY  M.,  Harvard  University 

ROTH,  ROBERT,  Brandeis  University 

ROWLAND,  LEWIS  P.,  Columbia  University,  College  of  Physicians  &  Surgeons 

RUBINOW,  SOL  L,  Cornell  University 

*WHITE,  HAROLD  BANCROFT,  III,  Pennsylvania  State  University 
*WIKSELL,  ANDRE  JEAN,  Washington  University 
*WILLIAMS,  JUDITH  A.  O.,  University  of  Illinois 
*WOLFE,  JASON  S.,  University  of  California,  Berkeley 


INVERTEBRATE  ZOOLOGY 

BAST,  SISTER  EILEEN  MARIE,  University  of  Oklahoma 
*BATCHELLER,  RUTHANNE,  University  of  Massachusetts 
*BECK,  ROBERT  MARTIN,  Cornell  University 


36  MARINE  BIOLOGICAL  LABORATORY 

BLAUG,  MAURICE,  University  of  Minnesota 

BRADLEY,  ROSE  MAE  RITA,  Dunbarton  College 

BRIGGS,  ELEANOR  LIVINGSTON,  Columbia  University 
*BURKY,  ALBERT  JOHN,  Syracuse  University 

CAMHI,  JEFFREY,  Harvard  University 

CLARK,  GEORGE  RICHMOND,  California  Institute  of  Technology 

DAWE,  CLYDE  JOHNSON,  National  Institutes  of  Health 
*DELCOMYN,  FRED,  University  of  Oregon 

FOWLER,  SUSAN,  Vassar  College 

FRANKS,  EDWIN  CLARK,  Ohio  State  University 

GEORGE,  STEPHEN  ANTHONY,  The  Johns  Hopkins  University 
*GITTINGER,  JOHN  WILLIAM,  JR.,  Oberlin  College 

HAIN,  MICHAEL  LAWRENCE,  University  of  California,  Davis 
*HATFIELD,  CAROLYN  SUE,  University  of  California,  Berkeley 

KUPFERBERG,  PAUL  LEWIS,  Drew  College 

LARSON,  PRISCILLA  ARLENE,  Yale  University 

LEE,  SUE  YING,  University  of  Illinois 

LIND,  NANCY  KAY,  Harvard  University 

MclvER,  SUSAN  BERTHA,  Washington  State  University 

MURPHY,  NEIL  FRANCIS,  Washington  &  Jefferson  College 

OBERLANDER,  HERBERT,  Western  Reserve  University 

OCHS,  KATHLEEN  FRANK,  Washington  University,  St.  Louis 

PAOLETTI,  ROBERT  ANTHONY,  The  Johns  Hopkins  University 

PHILIPPUS,  PAMELA  LEA,  Colorado  College 

POCOCK,  MARY  Avis  WEIR,  University  of  Texas 

RACEY,  LOUISE  ADELE,  Catholic  University  of  America 
*RADER,  JEANNE  ISABELLE,  Syracuse  University 

ROSENBERG,  MARK  J.,  Amherst  College 

SAMUEL,  GUDISAY,  University  of  Pennsylvania 

SETZLER,  EILEEN  MARIE,  College  of  St.  Mary  of  the  Springs 

SMITH,  DANIEL  PAUL.,  Massachusetts  Institute  of  Technology 
*SPICER,  JEAN  FRANCES,  Pennsylvania  State  University 

THOMASSON,  PATRICIA  ANNE,  University  of  Minnesota 
*TOBIAS,  PETER  STEPHEN,  Oberlin  College 
*TREBATOSKI,  SISTER  MARY  GABRIEL,  University  of  Notre  Dame 

Uzzo,  ANTHONY,  JR.,  C.  W.  Post  College 

WEHMAN,  HENRY  JOSEPH,  The  Johns  Hopkins  University 

4.     FELLOWSHIPS  AND  SCHOLARSHIPS,  1965 

Bio  Club  Scholarship : 

MAE  TEITELBAUM,  Ecology  Course 

Edwin  Linton  Memorial  Endowment  of  the  Washington  and  Jefferson  College : 
NEIL  MURPHY,  Invertebrate  Zoology  Course 

Turtox  Scholarship  Fund : 

MARCIA  MADSEN,  Botany  Course 


5.     TRAINING  PROGRAMS 

FERTILIZATION  AND  GAMETE  PHYSIOLOGY  TRAINING  PROGRAM 
I.  INSTRUCTORS 

CHARLES  B.  METZ,  University  of  Miami,  in  charge  of  program 
C.  R.  AUSTIN,  Tulane  University,  Delta  Regional  Primate  Center 
JOHN  BIGGERS,  University  of  Pennsylvania 


REPORT  OF  THE  DIRECTOR  37 

LUTHER  E.  FRANKLIN,  University  of  Miami 
ALBERTO  MONROY,  University  of  Palermo,  Italy 
LEONARD  NELSON,  Emory  University 

II.  CONSULTANT 

HAROLD  JACKSON,  Christie  Hospital  and  Holt  Radium  Institute,  Manchester,  England 

III.  TRAINEES 

BARROS,  CLAUDIO,  Tulane  University 

BORISY,  GARY  G.,  University  of  Chicago 

BROWN,  GEORGE  G.,  Virginia  Polytechnic  Institute 

CLAYBROOK,  JAMES  R.,  Oregon  Regional  Primate  Research  Center 

FAGAN,  LINDA,  Bryn  Mawr  College 

GREGG,  KENNETH  W.,  Emory  University 

HAND,  GEORGE  S.,  JR.,  University  of  North  Carolina 

HINSCH,  GERTRUDE  W.,  Mount  Union  College 

HOLMAN,  JOSEPHINE  A.,  University  of  New  Hampshire 

LEHRER,  HARRIS  I.,  Brandeis  University 

LONG,  JOHN  A.,  Harvard  University 

RADO,  THOMAS  A.,  Stanford  University 

SCHUETZ,  ALLEN  W.,  University  of  Minnesota 

SINGH,  UDAI  N.,  McGill-Montreal  General  Hospital 

TOBEN,  HOWARD  R.,  University  of  Miami 

WHITTINGHAM,  DAVID  G.,  University  of  Pennsylvania 

IV.  LECTURES 

JOHN  SHAVER  Some  Recent  Work  on  the  Immunobiology  of  Fertilization 

SHELDON  J.  SEGAL  The  Role  of  RNA  in  the  Action  of  Estrogen 

HERBERT  STERN  Biochemical  Studies  of  Gametogenesis  in  Plants 

ROBERT  W.  NOYES  The  Endometrium  and  Fertility 

EVERETT  ANDERSON  Some  Comparative  Aspects  of  the  Fine  Structure  of  Oocytes  during 

Differentiation 

GLENN  W.  SALISBURY       Ageing  Phenomena  in  the  Gamete 
GEORGE  W.  NACE  The    Role    of    Heterosynthesized    and    Autosynthesized    Antigens    in 

Fertilization  in  the  Frog 
A.  ORVILLE  DAHL  Pollen 

WILLIAM  L.  WILLIAMS      Capacitation  and  Decapacitation  of  Rabbit  Sperm 
VINCENT  ALLFREY  Active  and  Inactive  States  of  Chromatin 

HAROLD  JACKSON  Effects  of  Certain  Chemicals  on  Mammalian  Spermatogenesis 

CHARLES  THIBAULT  In    vitro    Fertilization :    A    Technique    in    Mammalian    Reproduction 

Research 
MICHAEL  HARPER  Egg  Transport  within  the  Fallopian  Tube 

NEUROPHYSIOLOGY  TRAINING  PROGRAM 

I.  INSTRUCTORS 

S.  W.  KUFFLER,  Harvard  Medical  School,  in  charge  of  program 
E.  J.  FURSHPAN,  Harvard  Medical  School 
D.  D.  POTTER,  Harvard  Medical  School 

II.  ASSISTANT 

R.  B.  BOSLER,  Harvard  Medical  School 


MARINE  BIOLOGICAL  LABORATORY 

III.  RESEARCH  ASSOCIATE 

J.  G.  NICHOLLS,  Harvard  Medical  School 

IV.  TRAINEES 

DENIS  BAYLOR,  Yale  University 

SniN-Lo  CHUNG,  Harvard  University 

MONROE  W.  COHEN,  McGill  University,  Montreal 

EDWIN  S.  LENNOX,  Salk  Institute  for  Biological  Studies 

CHARLES  R.  MICHAEL,  Harvard  University 

MYRNA  B.  MILLER,  Western  Reserve  University 

STEPHEN  A.  SCHLESINGER,  The  Johns  Hopkins  University 

COMPARATIVE  PHYSIOLOGY  RESEARCH  TRAINING  PROGRAM 

I.  INSTRUCTORS 

C.  LADD  PROSSER,  University  of  Illinois,  in  charge  of  program 

LEWIS  H.  KLEINHOLZ,  Reed  College 

BERNARD  C.  ABBOTT,  University  of  Illinois 

WILLIAM  T.  W.  POTTS,  University  of  Birmingham,  England 

GABOR  KALEY,  New  York  Medical  College 

AARON  JANOFF,  New  York  University  School  of  Medicine 

II.  ASSISTANTS 

DONALD  JOB,  University  of  Illinois 
FRANCES  KIMBALL,  Reed  College 

III.  RESEARCH  ASSOCIATE 

Y.  MATSUMOTO,  University  of  Illinois 

IV.  TRAINEES 

AUGENFELD,  JOHN  M.,  University  of  Oklahoma 
DUNSON,  WILLIAM  A.,  University  of  Michigan 
EVANS,  DAVID  H.,  Stanford  University 
FORD,  ARTHUR  C.,  Rider  College 
HAMBY,  ROBERT  J.,  University  of  Chicago 
LEVIN,  STEPHEN  M.,  New  York  University 
LIEB,  WILLIAM  R.,  University  of  Illinois 
STRATTEN,  WILFORD  P.,  Indiana  University 
DONSHIK,  PETER,  New  York  Medical  College 
SUTTERLIN,  ARNOLD  M.,  University  of  Massachusetts 
TAMAR,  HENRY,  Indiana  State  University 

V.  LECTURES 

CLARK  P.  READ  Physiological  Generalizations  for  Parasitism 

PETER  KLOPFER  On  the  Causes  of  Tropical  Species  Diversity 

JOHN  ANDERSON  Aspects  of  Organ  Regeneration  in  Echinoderms 

DENNIS  J.  CRISP  Ecology  of  Marine  Larval  Settling 

E.  BAYLOR  Meteorology  and  the  Distribution  of  Plankton  Near  the  Surface 

WILLIAM  POTTS  Adaptation  to  Sea  Water  and  Fresh  Water 

FRANK  A.  BROWN,  JR.       Geophysical  Forces  and  Biological  Rhythms 


REPORT  OF  THE  DIRECTOR 


39 


6.  TABULAR  VIEW  OF  ATTENDANCE,  1961-1965 


1961 

INVESTIGATORS — TOTAL 458 

Independent     256 

Library  Readers    49 

Research  Assistants   .  151 


STUDENTS — TOTAL 


Invertebrate  Zoology 


130 
40 


Embryology     21 

Physiology     28 

Botany    19 

Ecology    22 

TRAINEES — TOTAL   

Nerve-Muscle 

Comparative  Physiology   

Fertilization  &  Gamete   

TOTAL  ATTENDANCE   586 

Less  persons  represented  in  two  categories 1 

585 

INSTITUTIONS  REPRESENTED — TOTAL 132 

By    Investigators    107 

By  Students    70 

SCHOOLS  AND  ACADEMIES   REPRESENTED    

By  Investigators   3 

By  Students    0 

FOREIGN  INSTITUTIONS  REPRESENTED  28 

By  Investigators  21 

By  Students  7 


1962 

494 

279 

56 

159 

121 

38 

20 

28 

20 

15 


615 
4 

611 

118 

81 

57 

3 
2 

31 
17 
14 


1963 

490 

261 

51 

178 

124 
40 
20 
28 
20 
16 


1964   1965 


614 

5 

609 

120 

83 

73 

4 
0 

21 

15 
6 


512 

273 

47 

192 

126 

40 

20 

30 

19 

17 

30 

7 

7 

16 

668 
7 

661 

140 

117 

23 

0 
0 

32 

28 

4 


572 

284 

62 

227 

128 

41 

20 

30 

20 

17 

34 

7 

11 

16 

734 

4 

730 

218 

142 

76 

0 
0 

27 

25 

2 


7.  INSTITUTIONS  REPRESENTED,  1965 


American  Museum  of  Natural  History 

Amherst  College 

Antioch  College 

Barlow  School 

Barnard  College 

Boston  University 

Boston  University  School  of  Medicine 

Brandeis  University 

Brookhaven  National  Laboratory 

Brooklyn  College 

Brown  University 

Bryn  Mawr  College 

California  Institute  of  Technology 

Carnegie  Institution  of  Washington 

Catholic  University  of  America 

Children's  Cancer  Research  Foundation,  Inc. 

City  College  of  New  York 

Colby  College 

College  of  Mount  St.  Joseph  on  the  Ohio 

College  of  St.  Mary  of  the  Springs 

College  of  William  and  Mary 

Colorado  College 


Columbia  University 

Columbia  University,  College  of  Physicians  & 
Surgeons 

Connecticut  College 

Cornell  University 

Cornell  University  Medical  College 

Dartmouth  College 

Dartmouth  Medical  School 

Deerfield  Academy 

Delta  Regional  Primate  Research  Center 

Denison  University 

Drew  University 

Duke  University 

Dunbarton  College 

The  Albert  Einstein  College  of  Medicine 

The  Albert  Einstein  Medical  Center,  Phila- 
delphia 

Emory  University 

Fordham  University 

Georgetown  University 

Goldwater  Memorial  Hospital 

Goucher  College 


40 


MARINE  BIOLOGICAL  LABORATORY 


Harvard  College 

Harvard  Medical  School 

Harvard  University 

Haskins  Laboratories 

Illinois  Teachers  College  North 

Indiana  State  University 

Indiana  University 

Institute  for  Muscle  Research 

Iowa  State  University 

The    Johns    Hopkins    School    of    Hygiene    & 

Public  Health 

The  Johns  Hopkins  School  of  Medicine 
The  Johns  Hopkins  University 
Kalamazoo  College 
King  Ranch  Laboratories 
Lehigh  University 
Marquette  University 
Massachusetts  General  Hospital 
Massachusetts  Institute  of  Technology 
Mellon  Institute 
Miami  University  of  Ohio 
Montgomery  Jr.  College 
Mount  Holyoke  College 
The  Mount  Sinai  Hospital 
Mount  Union  College 
National  Cancer  Institute 
National  Heart  Institute 
National    Institute    of   Arthritis    &    Metabolic 

Diseases 

National  Institutes  of  Health 
National  Science  Foundation 
Neurological  Institute 
Newcomb  College 
New  York  College  of  Medicine 
New  York  University 

New  York  University-Bellevue  Medical  Center 
New  York  University  College  of  Dentistry 
New  York  University  Medical  Center 
North  Carolina  State  University 
Northwestern  University 
Oakland  University 
Oak  Ridge  National  Laboratory 
Oberlin  College 
Ohio  State  University 
Oregon  Regional  Primate  Research  Center 
Oxford  University 
Pennsylvania  Hospital 
Pennsylvania  State  College 
Pennsylvania  State  University 
C.  W.  Post  College 
Princeton  University 
Providence  College 
Purdue  University 
Putnam     Memorial     Hospital,     Institute     for 

Medical  Research 
Queens    College    of    the    City    University    of 

New  York 


Radcliffe  College 

Radcliffe  Graduate  Center 

Reed  College 

Rensselaer  Polytechnic  Institute 

Rice  University 

Rider  College 

The  Rockefeller  University 

Russell  Sage  College 

Rutgers  Medical  School 

Rutgers,  The  State  University  of  New  Jersey 

Salk  Institute  for  Biological  Studies 

Agnes  Scott  College 

Scripps  Institution  of  Oceanography 

Seton  Hill  College 

Simmons  College 

Single  Cell  Research  Foundation,  Inc. 

Sloan-Kettering  Institute  for  Cancer  Research 

Smith  College 

South  Oaks  Research  Foundation,  Inc. 

Stanford  University 

State  University  of  New  York  at  Syracuse 

Syracuse  University 

Syracuse  University  Upstate  Medical  Center 

Temple  University 

The  City  University  of  New  York,  Brooklyn 
College 

The  Institute  for  Cancer  Research,  Phila- 
delphia 

Tufts  University 

Tulane  University 

Tulane  University  School  of  Medicine 

U.  S.  Bureau  of  Commercial  Fisheries 

University  of  Arizona 

University  of  California,  Berkeley 

University  of  California,  Davis 

University  of  California,  Los  Angeles 

University  of  California,  Riverside 

University  of  California,  San  Francisco 

University  of  Chicago 

University  of  Chicago  Medical  School 

University  of  Cincinnati 

University  of  Colorado  Medical  School 

University  of  Connecticut 

University  of  Connecticut  School  of  Dental 
Medicine 

University  of  Delaware 

University  of  Illinois 

University  of  Illinois  School  of  Medicine 

University  of  Iowa 

University  of  Kansas 

University  of  Louisville 

University  of  Maryland 

University  of  Maryland  Medical  School 

University  of  Massachusetts 

University  of  Aliami 

University  of  Miami  Institute  of  Molecular 
Evolution 


REPORT  OF  THE  DIRECTOR 


41 


University  of  Miami  School  of  Medicine 

University  of  Michigan 

University  of  Minnesota 

University  of  Minnesota  Medical  School 

University  of  New  Hampshire 

University  of  North  Carolina 

University     of     North     Carolina     School     of 

Medicine 

University  of  Notre  Dame 
University  of  Oklahoma 
University  of  Oregon 
University  of  Pennsylvania 
University  of  Pennsylvania,  Johnson  Research 

Foundation 

University  of  Pennsylvania  School  of  Medicine 
University  of  Pittsburgh 
University  of  Pittsburgh  School  of  Medicine 
University  of  Rhode  Island 
University  of  Rochester  School  of  Medicine  & 

Dentistry 

University  of  South  Alabama 
University  of  South  Florida 


University  of  Tennessee 

University  of  Texas 

University    of    Texas,    Southwestern    Medical 

School 

University  of  Vermont 
University  of  Virginia 
University  of  Wisconsin 
Vassar  College 

Veterans  Administration  Hospital,  Albany 
Veterans  Administration  Hospital,  Brooklyn 
Washington  &  Jefferson  College 
Washington  State  University 
Washington  University 
Washington  University  School  of  Medicine 
Wellesley  College 
Western  Reserve  University 
Western  Reserve  University  School  of  Medicine 
Wilson  College 

Woods  Hole  Oceanographic  Institution 
Yale  University 
Yale  University  School  of  Medicine 


FOREIGN  INSTITUTIONS  REPRESENTED,  1965 


Bedford  College,  University  of  London  University 

Chiba  University,  Japan  University 

Christie  Hospital,  Manchester,  England  University 

Glasgow  University,  Scotland  University 

Kumamoto  University,  Japan  University 

Leicester  University,  England  University 
Marine  Science  Laboratory,  University  College  University 

of  North  Wales  University 

McGill-Montreal  General  Hospital,  Canada  University 

McGill  University,  Canada  University 

Pahlavi  University,  Iran  University 

Queen's  University,  Canada  University 
St.  George's  Hospital  Medical  School,  London  University 
Tokyo  Medical  &  Dental  University 


of  Birmingham,  England 

of  Brussels,  Belgium 

of  Chile 

College,  London 

of  Edinburgh,  Scotland 

of  Gdaush,  Poland 

of  Helsinki,  Finland 

of  Ljubljana,  Poland 

of  Oslo,  Norway 

of  Saarbrucken,  Germany 

of  Sydney,  Australia 

of  Toronto,  Canada 

of  Waterloo,  Canada 


SUPPORTING  INSTITUTIONS,  AGENCIES,  AND  INDIVIDUALS 


Abbott  Laboratories 

Associates  of  the  Marine  Biological  Laboratory 

Atomic  Energy  Commission 

CIBA  Corporation 

The  Commonwealth  Fund 

Josephine  B.  Crane  Foundation 

Dr.  William  D.  Curtis 

The  Ford  Foundation 

Dr.  and  Mrs.  David  W.  Gaiser 

The  Grass  Foundation 

Mr.  and  Mrs.  William  H.  Greer,  Jr. 

Dr.  Ethel  Browne  Harvey 

Mr.  and  Mrs.  George  F.  Jewett,  Jr. 


The  Lalor  Foundation 
Mrs.  Grace  T.  Mast 
Olin  Matheson  Charitable  Trust 
National  Institutes  of  Health 
National  Science  Foundation 
Office  of  Naval  Research 
The  Rockefeller  Foundation 
Schering  Foundation,  Inc. 
Scientific  American,  Inc. 
Gerard  Swope,  Jr. 
The  Upjohn  Company 
Wallace  Laboratories 
James  H.  Wickersham 


42  MARINE  BIOLOGICAL  LABORATORY 

8.  FRIDAY  EVENING  LECTURES,  1965 

July  2 

HOWARD  K.  SCHACHMAN The  Subunit  Structure  of  Proteins  in  Terms  of 

University  of  California,  Berkeley          Their  Functions 

July  8,  Thursday 

LESLIE  F.  NIMS Membranes,  Metabolism  and  Material  Transfer 

Alexander  Forbes  Lecturer  at  the  MBL 
Brookhaven  National  Laboratory 

July  9 

LESLIE  F.  NIMS Galvani,  Volta  and  Bioelectricity 

July  16 

ALEXANDER  HOLLAENDER Studies  in  Radiation  Biology  at  the  Oak  Ridge 

Oak  Ridge  National  Laboratory  National  Laboratory 

July  23 

HOWARD  A.  SCHNEIDERMAN The  Hormonal  Control  of  Insect  Development 

Western  Reserve  University 

July  30 

NORMAN  MILLOTT The  Enigmatic  Echinoids 

Bedford  College,  University  of  London 

August  6 

JOHN  W.  KANWISHER Thermal     and      Respiratory      Physiology     of 

Woods  Hole  Oceanographic  Whales  and  Porpoises 

Institution 

August  13 

LAURI  SAXEN The  Mechanism  of  Kidney  Tubulogenesis 

University  of  Helsinki 

Senior  Lalor  Fellow  at  the  MBL 

August  20 

SHINYA  INOUE    Exploration  of  the  Living  Cell  with  Polarized 

Light 

August  27 

PHILIP  B.  ARMSTRONG Behavior  in  Developing  Embroys 

State  University  of  New  York 
College  of  Medicine  at  Syracuse 

9.  TUESDAY  EVENING  SEMINARS,  1965 

July  13 

GEORGE  SZABO   Suntanning 

M.  PATHAK 

W.  C.  QUEVEDO,  JR. 

W.    S.   VINCENT Ribosome     Synthesis     in     Synchronous    Yeast 

Cultures 
ROBERT  G.  FAUST  Action  of  Bile  Salts  on  ATP-ase  Activity  of 

Mucosal  Homogenates  from  Rat  Jejunum  and 

Ileum 


REPORT  OF  THE  DIRECTOR  43 

July  20 

ARNE  L0VLIE   Photosynthesis   during  the   Cell   Cycle   of  En- 

ijlcna  gracilis 

M.  FINGERMAN   Ncuroendocrine    Control    of    the    Crustacean 

Y.  YAMAMOTO  Hepatopancreas 

D.  E.  PHILPOTT Intracellular  Aggregates  and  Granules  of  Lim- 

P.  PERSON  uliis  Gill  Cartilage 

July  27 

JOHN  D.  PALMER   A  Biological  Rhythm  in  Euglena 

BERXD  LINDEMANN Time-Independent   Negative   Sodium    Conduct- 
ance  in   the   Surface   Structure   of   Frog   Skin 
Epithelium 
RICHARD  L.  MILLER Chemotaxis  (?)  of  Coelenterate  Sperm 

August  3 

BERND  LINDEMANN    Effects  of  Direct  Current  Passing  Through  a 

Model  Epithelial  Cell 
RUTH  JOHNSSON   Chemically  Induced  Orientation  of  the  Growth 

of  Malignant  Cells  in  vitro 

SIDNEY  LERMAN   Properties  of  a  Cryoprotein  in  the  Ocular  Lens 

LEWIS  G.  TILNEY  The  Role  of  Microtubules  in  the  Formation  and 

DOUGLAS  MARSLAND  Maintenance     of    the     Axopodia     of    Actino- 

sphaerium  nudeofihnn 

August  10 

REUBEN  TORCH  The  Effects  of  Actinomycin  on  RNA  Synthesis 

in  the  Brackish-Water  Ciliate,  Tracheloraphis 
sp. 

W.  A.  HAGINS Photon   Statistics  and  Central   Mechanisms  of 

F.  HANSON  Bioluminescence 

J.  B.  BUCK 

ROGER  ECKERT Microsources  of  Luminescence  in  Noctiluca 

GEORGE  REYNOLDS 

RICHARD  CHAFFEE 

J.  W.  HASTINGS Light-induced  Bioluminescence 

Q.  H.  GIBSON 

August  17 

JAMES  W.  LASH   Studies  on  Tunicate  Metamorphosis 

JOHN  R.  REIGART,  II 

DOUGLAS  MARSLAND Synergism  Between  Colchicine  and  High  Pres- 
sure in  Regard  to  Anti-Mitotic  Effects  in  Di- 
viding Eggs  (Lytechinus  variegatus) 

R.  E.  KANE  Identification  and  Isolation  of  the  Mitotic  Ap- 
paratus Protein 

R.  E.  STEPHENS  Characterization  of  the  MA  Protein  and  its 

Subunits 


44  MARINE  BIOLOGICAL  LABORATORY 

10.     MEMBERS  OF  THE  CORPORATION,  1965 
Including  Action  of  1965  Annual  Meeting 

Life  Members 

ADOLPH,  DR.  EDWARD  F.,  University  of  Rochester,  School  of  Medicine  &  Den- 
tistry, Rochester,  New  York 

BRODIE,  MR.  DONALD,  522  Fifth  Avenue,  New  York,  New  York  10018 
COLE,  DR.  ELBERT  C.,  2  Chipman  Park,  Middlebury,  Vermont 
COWDRY,  DR.  E.  V.,  4580  Scott  Avenue,  St.  Louis  10,  Missouri 
CRANE,  MRS.  W.  MURRAY,  820  Fifth  Avenue,  New  York,  New  York  10021 
CURTIS,  DR.  MAYNIE  R.,  Box  8215,  University  Branch,  Coral  Gables,  Florida  33124 
HESS,  DR.  WALTER,  286  North  Fairview  Avenue,  Spartanburg,  South  Carolina 
HISAW,    DR.    F.    L.,    Biological    Laboratories,    Harvard    University,    Cambridge, 

Massachusetts  02138 

IRVING,  DR.  LAURENCE,  University  of  Alaska,  College,  Alaska  99735 
JACOBS,  DR.  M.  H.,  Department  of  Physiology,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania  19104 

LOWTHER,  DR.  FLORENCE,  Barnard  Colege,  New  York,  New  York  10027 
MACDOUGALL,  DR.  MARY  STUART,  Mt.  Vernon  Apartments,  423  Clairmont  Ave- 
nue, Decatur,  Georgia 

MALONE,  DR.  E.  F.,  6610  North  llth  Street,  Philadelphia,  Pennsylvania  19126 
MEANS,  DR.  J.  H.,  15  Chestnut  Street,  Boston,  Massachusetts 
MEDES,  DR.  GRACE,  303  Abington  Avenue,  Philadelphia,  Pennsylvania  19111 
PAYNE,  DR.  FERNANDUS,  Indiana  University,  Bloomington,  Indiana  47405 
PLOUGH,  DR.  H.  H.,  Amherst  College,  Amherst,  Massachusetts  01002 
PORTER,  DR.  H.  C.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania  19104 
SCOTT,  DR.  ERNEST  L.,  Columbia  University,  New  York,  New  York  10027 
SCHRADER,  DR.  SALLY,  Duke  University,  Durham,  North  Carolina  27706 
TURNER,  DR.  C.  L.,  Northwestern  University,  Evanston,  Illinois  60201 
WAITE,  DR.  F.  G.,  144  Locust  Street,  Dover,  New  Hampshire 
WALLACE,  DR.  LOUISE  B.,  359  Lytton  Avenue,  Palo  Alto,  California 
WARREN,  DR.  HERBERT  S.,  2768  Egypt  Road,  Audubon,  Pennsylvania 
WHEDON,  DR.  A.  D.,  21  Lawncrest,  Danbury,  Connecticut 

Regular  Members 

ABBOTT,  DR.  BERNARD  C.,  Department  of  Biophysics  &  Physiology,  University  of 
Illinois,  Urbana,  Illinois  61803 

ABELL,  DR.  RICHARD  G.,  55  East  2nd  Avenue,  New  York,  New  York  10028 

ADELBERG,  DR.  EDWARD  A.,  Department  of  Microbiology,  Yale  University,  New 
Haven,  Connecticut  06515 

ADELMAN,  DR.  WILLIAM  J.,  JR.,  Department  of  Physiology,  University  of  Mary- 
land Medical  School,  Baltimore,  Maryland  21201 

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

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


REPORT  OF  THE  DIRECTOR  45 

ALLEN,  DR.  ROBERT  D.,  Department  of  Biology,  Princeton  University,  Princeton, 
New  Jersey  08540 

ALSCHER,  DR.  RUTH,  Department  of  Physiology,  Manhattanville  College,  Pur- 
chase, New  York 

AMATNIEK,  DR.  ERNEST,  34  Homer  Avenue,  Hastings-on-the-Hudson,  New  York 

AMBERSON,  DR.  WILLIAM  R.,  Katy  Hatch  Road,  Falmouth,  Massachusetts  02540 

ANDERSON,  DR.  J.  M.,  Division  of  Biological  Sciences,  Stimson  Hall,  Cornell  Uni- 
versity, Ithaca,  New  York  14850 

ANDERSON,  DR.  RUBERT  S.,  Medical  Laboratories,  Army  Chemical  Center. 
Maryland 

ARMSTRONG,  DR.  PHILIP  B.,  Department  of  Anatomy,  State  University  of  New 
York,  College  of  Medicine,  Syracuse,  New  York  13210 

ARNOLD,  DR.  JOHN  MILLER,  Department  of  Zoology,  Iowa  State  University,  Ames, 
Iowa  50010 

ARNOLD,  DR.  WILLIAM  D.,  Division  of  Biology,  Oak  Ridge  National  Laboratory, 
Oak  Ridge,  Tennessee  37831 

ASH  WORTH,  DR.  JOHN  MICHAEL,  Department  of  Biology,  Brandeis  University, 
Waltham,  Massachusetts  02154 

ATWOOD,  DR.  KIMBALL  C.,  702  West  Pennsylvania  Avenue,  Urbana,  Illinois 

AUCLAIR,  DR.  WALTER,  Department  of  Biological  Sciences,  University  of  Cin- 
cinnati, Cincinnati,  Ohio  45221 

AUSTIN,  DR.  COLIN  RUSSELL,  Delta  Regional  Primate  Research  Center,  Coving- 
ton,  Louisiana  70433 

AUSTIN,  DR.  MARY  L.,  506^  North  Indiana  Avenue,  Bloomington,  Indiana 

AYERS,  DR.  JOHN  C.,  Department  of  Meteorology  &  Oceanography,  University  of 
Michigan,  Ann  Arbor,  Michigan  48104 

BAITSELL,  DR.  GEORGE  A.,  Department  of  Biology,  Yale  University,  New  Haven, 
Connecticut  06520 

P.ALL,  DR.  ERIC  G.,  Department  of  Biological  Chemistry,  Harvard  Medical  School, 
Boston,  Massachusetts  02115 

BALLARD,  DR.  WILLIAM  W.,  Department  of  Biological  Sciences,  Dartmouth  Col- 
lege, Hanover,  New  Hampshire  03755 

BANG,  DR.  F.  B.,  Department  of  Pathobiology,  The  Johns  Hopkins  University, 
School  of  Hygiene,  Baltimore,  Maryland  21205 

BARD,  DR.  PHILIP,  The  Johns  Hopkins  Medical  School,  Baltimore,  Maryland  21205 

EARTH,  DR.  L.  G.,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 
02543 

BARTH,  DR.  LUCENA,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 
02543 

BARTLETT,  DR.  JAMES  H.,  Department  of  Physics,  University  of  Alabama,  Box 
1921,  University,  Alabama  35486 

BAUER,  DR.  G.  ERIC,  Department  of  Anatomy,  University  of  Minnesota,  Minne- 
apolis, Minnesota  53455 

BAYLOR,  DR.  E.  R.,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  Massa- 
chusetts 02543 

BAYLOR,  DR.  MARTHA  B.,  Marine  Biological  Laboratory,  Woods  Hole,  Massa- 
chusetts 02543 


46  MARINE  BIOLOGICAL  LABORATORY 

BEAMS,  DR.  HAROLD  \Y.,  Department  of  /.oology.  Slate  University  of  Iowa,  Iowa 

City,  Iowa  522  10 
rK.    DR.    L.   V.,    Department    of    rharmacolcgy.    liuliaua    University,    Sehool    of 

Experimental  Medicine,  Bloomington,  Indiana  47405 
K,  PR.  KLINOR  .M..  Black  Mountain.  North  Carolina 
BELL,  DR.  KUGENE,  Department  of  I'.iology,  Massachusetts  Institute  of  Technology, 

Cambridge,  Massacusetts  02139 
BENNETT,  DR.  MICHAEL  V.  L.,  Department  of  Neurology,  Columbia  University, 

College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 
BENNETT,  DR.  MIRIAM  F.,  Department  of  Biology,  Sweet  Briar  College,  Sweet 

Briar,  Virginia  24595 
BERI;.  DR.  WILLIAM  E.,  Department  of  Zoology,  University  of  California,  Berkeley, 

California  94720 
BERMAN.   DR.   MONES,   National   Institutes  of  Health,    Institute  for  Arthritis   & 

Metabolic  Diseases,  Bethesda,  Maryland  20014 
BERNE,  DR.  ROBERT  M.,  Department  of  Physiological  Chemistry,  Western  Reserve 

University.  School  of  Medicine,  Cleveland,  Ohio  44106 
r.KRNHEiMER,  DR.  ALAN   W.,   New   York  University  College  of   Medicine,   New 

York,  New  York  10016 

BERNSTEIN.  DR.  MAURICE,  Department  of  Anatomy,  Wayne  State  University,  Col- 
lege of  Medicine,  Detroit  7,  Michigan 

BERSOHN,  DR.  RICHARD.  Department  of  Chemistry,  959  Havemeyer  Hall.  Colum- 
bia University,  New  York,  New  York  10027 
BERTHOLF,  DR.  LLOYD  M.,  Illinois  Wesleyan   University,   Bloomington.   Illinois 

61701 
BEVKLANDER,  DR.  GERRIT,  University  of  Texas.  Medical  Center,  Dental  Branch, 

Houston,  Texas  77025 
BIGELOW,  DR.  HENRY  B.,  Museum  of  Comparative  Zoology,  Harvard  University. 

Cambridge.  Massachusetts  0213S 
BICCERS.  DR.  JOHN  DENNIS,  Department  of  Reproductive  Physiology,  University 

of  Pennsylvania,   School  of  Veterinary   Medicine,   Philadelphia,   Pennsylvania 

19104 

BISHOP,  DR.  DAVID  W..  Department  of  Embryology,  Carnegie  Institution  of  Wash- 
ington, Baltimore,  Maryland  21210 
BLANC  HARD.  DR.  K.  C,  The  Johns  Hopkins  Medical  School,  Baltimore,  Maryland 

21205 

BLOCK,  DR.  ROBERT,  Adalbertstr.  70,  8  Munich,  13.  Germany 
BLUM,  DR.  HAROLD  F.,  Department  of  Biology,  Princeton  University.  Princeton, 

New  Jersey  08540 
BODANSKY,  DR.   OSCAR.   Department  of  Biochemistry,   Memorial   Cancer   Center. 

444  East  68th  Street.  New  York.  New  York  10021 " 
BODIAN,   DR.   DAVID,   Department  of   Anatomy.   The   Johns   Hopkins   University, 

709  North  Wolfe  Street,  Baltimore.  Mankind  21205 
BOELL,   DR.   EDGAR   J.,    Department   of    Biology,   Yale    University,    New    Haven, 

Connecticut  06520 
BOETTIGER,  DR.  EDWARD  G..  Department  of  Zoology,  University  of  Connecticut, 

Storrs,  Connecticut  06268 


REPORT  OF  THE  DIRECTOR  47 

BOLD,  DR.  HAROLD  C,  Department  of  Botany,  University  of  Texas,  Austin,  Texas 

78712 
BOOLOOTIAN,  DR.  RICHARD  A.,  Department  of  Zoology,  University  of  California, 

Los  Angeles,  California  90024 

BOREI,  DR.  HANS  G.,  Department  of  Zoology,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania  19104 
BOWEX,  DR.  VAUGHAN  T.,  Woods  Hole  Oceanographic  Institution,  Woods  Hole, 

Massachusetts  02543 

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

Decatur,  Georgia 
BRINLEY,  DR.  F.  J.,  JR.,  Department  of  Physiology,  The  Johns  Hopkins  Medical 

School,  Baltimore,  Maryland  21205 

BRONK,  DR.  DETLEV  W.,  The  Rockefeller  University,  New  York,  Xe\v  York  10021 
BROOKS,  DR.   MATILDA  M.,  Department  of  Physiology,  University  of  California, 

Berkeley,  California  94720 
BROWX,  DR.  DUGALD  E.  S.,  Department  of  Zoology,  University  of  Michigan,  Ann 

Arbor,  Michigan  48104 
BRO\VX,   DR.   FRANK  A.,  JR.,   Department  of  Biological   Sciences,   Northwestern 

University,  Evanston,  Illinois  60201 
BROWNELL,  DR.  KATHERINE  A.,  Department  of  Physiology,  Ohio  State  University, 

Columbus,  Ohio  43210 
BUCK,  DR.  JOHN  B.,  Laboratory  of  Physical  Biology,  National  Institutes  of  Health, 

Bethesda,  Maryland  20014 
BULLOCK,    DR.    T.    H.,    Department    of    Zoology,    University    of    California,    Los 

Angeles,  California  90024 
BURBAXCK,  DR.  WILLIAM   D.,  Emory  University,   Box   15134,  Atlanta,  Georgia 

30338 
BURDICK,  DR.  C.  LALOR,  The  Lalor  Foundation,  4400  Lancaster  Pike,  Wilmington, 

Delaware 

BURKEXROAD,  DR.  M.  D.,  3169  Bremerton  Place,  La  Jolla,  California  92037 
BURXETT,  DR.  ALLISON  LEE,  Department  of  Biology,  Western  Reserve  Univer- 
sity, Cleveland,  Ohio  44106 
BUTLER,  DR.  E.  G.,  Department  of  Biology,  Princeton  University,  Princeton,  New 

Jersey  08540 
CAXTOXI,  DR.  GIULLIO,  National  Institutes  of  Health,  Mental  Health,  Bethesda, 

Maryland  20014 

CARLSON,  DR.  FRAXCIS  D.,  Department  of  Biophysics,  The  Johns  Hopkins  Uni- 
versity, Baltimore,  Maryland  21218 

CARPENTER,  DR.  RUSSELL  L.,  Tufts  University,  Medford,  Massachusetts  02155 
CARRIKER,  DR.  MELBOURNE  R.,  Systematics-Ecology  Program,  Marine  Biological 

Laboratory,  Woods  Hole,  Massachusetts  02543 
CASE,  DR.  JAMES,  Department  of  Biology,  University  of  California,  Santa  Barbara, 

California  93 106 
CATTELL,  DR.  McKEEx,  Cornell  University  Medical  College.  1300  York  Avenue. 

New  York,  New  York  10021 


48  MARINE  BIOLOGICAL  LABORATORY 

CHAET,  DR.  ALFRED  B.,  Department  of  Biology,  American  University,  Washing- 
ton, D.  C.  20016 
CHAMBERS,  DR.  EDWARD,  Department  of  Physiology,  University  of  Miami  Medical 

School,  Coral  Gables,  Florida  33124 
CHANG,  DR.  JOSEPH  J.,  Inst.  f.  physikal.  Chemie  an  der  Techn.  Hochscule,  Aachen, 

Germany 
CHASE,  DR.  AURIN  M.,  Department  of  Biology,  Princeton  University,  Princeton, 

New  Jersey  08540 
CHENEY,  DR.  RALPH  H.,  Biological  Laboratory,  Brooklyn  College,  Brooklyn,  New 

York  11210 
CHILD,  DR.  FRANK  M.,  Department  of  Zoology,  University  of  Chicago,  Chicago, 

Illinois  60637 

CLAFF,  DR.  C.  LLOYD,  5  Van  Beal  Road,  Randolph,  Massachusetts  02368 
CLARK,  DR.  A.  M.,  Department  of  Biological  Sciences,  University  of  Delaware, 

Newark,  Delaware  19711 
CLARK,  DR.  ELOISE  E.,  Department  of  Zoology,  Columbia  University,  New  York. 

New  York  10027 
CLARK,  DR.  LEONARD  B.,  Department  of  Biology,  Union   College,   Schenectady, 

New  York  12308 
CLARKE,  DR.  GEORGE  L.,  Biological  Laboratories,  Harvard  University,  Cambridge, 

Massachusetts  02 138 

CLELAND,  DR.  RALPH  E.,  Department  of  Botany,  Indiana  University,  Blooming- 
ton,  Indiana  47405 
CLEMENT,  DR.  A.  C.,  Department  of  Biology,  Emory  University,  Atlanta,  Georgia 

30322 
COHEN,  DR.   SEYMOUR  S.,   Department  of  Therapeutic  Research,  University  of 

Pennsylvania  School  of  Medicine,  Philadelphia,  Pennsylvania  19104 
COLE,  DR.  KENNETH  S.,  Laboratory  of  Biophysics,  National  Institutes  of  Health, 

Bethesda,  Maryland  20014 

COLLETT,  DR.  MARY  E.,  34  Weston  Road,  Wellesley,  Massachusetts  02181 
COLLIER,  DR.  JACK  R.,  Department  of  Biology,  Rensselaer  Fob/technical  Institute, 

Troy,  New' York  12181 

COLTON,  DR.  H.  S.,  Box  699,  Flagstaff,  Arizona 
COLWIN,   DR.   ARTHUR   L.,   Department  of   Biology,   Queens    College,    Flushing, 

New  York  11 367 
COLWIN,  DR.  LAURA  H.,  Department  of  Biology,  Queens  College,  Flushing,  New 

York  11 367 
COOPER,  DR.  KENNETH  W.,  Department  of  Cytology,  Dartmouth  Medical  School, 

Hanover,  New  Hampshire  03755 

COOPERSTEIN,  DR.  SHERWIN  J.,  University  of  Connecticut,  School  of  Dental  Medi- 
cine, Hartford,  Connecticut  06105 
COPELAND,   DR.    D.   EUGENE,    Department   of   Biology,    Tulane   University,    New 

Orleans,  Louisiana  70185 

COPELAND,  DR.  MANTON,  88  Federal  Street,  Brunswick,  Maine  04011 
CORNMAN,    DR.    IVOR,    Department   of   Zoology,   University   of   the   West    Indies, 

Mona,  Kingston,  Jamaica 


REPORT  OF  THE  DIRECTOR  49 

COSTELLO,  DR.  DONALD  P.,  Department  of  Zoology,  University  of  North  Carolina, 

Chapel  Hill,  North  Carolina  27514 
COSTELLO,   DR.   HELEN    MILLER,   Department   of   Zoology,   University   of   North 

Carolina,  Chapel  Hill,  North  Carolina  27514 
CRANE,  MR.  JOHN  O.,  Woods  Hole,  Massachusetts  02543 
CRANE,  DR.  ROBERT  K.,  Department  of  Biochemistry,  The  Chicago  Medical  School, 

2020  West  Ogden  Avenue,  Chicago,  Illinois  60612 
CROASDALE,   DR.    HANNAH    T.,   Dartmouth    College,   Hanover,    New   Hampshire 

03755 

GROUSE,  DR.  HELEN  V.,  Institute  for  Molecular  Biophysics,  Florida  State  Univer- 
sity, Tallahassee,  Florida  32306 
CROWELL,  DR.  SEARS,  Department  of  Zoology,  Indiana  University,  Bloomington, 

Indiana  47405 
CSAPO,  DR.  ARPAD  I.,  Washington  University  School  of  Medicine,  4911  Barnes 

Hospital  Plaza,  St.  Louis,  Missouri  63110 

CURTIS,  DR.  W.  C.,  504  Westmount  Avenue,  Columbia,  Missouri 
DAIGNAULT,  MR.  ALEXANDER  T.,  W.  R.  Grace  &  Company,  7  Hanover  Square, 

New  York,  New  York  10005 

DAN,  DR.  JEAN  CLARK,  Misaki  Biological  Station,  Misaki,  Japan 
DAN,  DR.  KATSUMA,  Misaki  Biological  Station,  Misaki,  Japan 
DANIELLI,    DR.   JAMES    F.,    Department   of    Medicinal    Chemistry,    University   of 

Buffalo  School  of  Pharmacy,  Buffalo,  New  York  14222 
DAVIS,  DR.  BERNARD  D.,  Harvard  Medical  School,  25  Shattuck  Street,  Boston, 

Massachusetts  02 115 
DAWSON,   DR.   A.   B.,    Biological   Laboratories,    Harvard   University,    Cambridge, 

Massachusetts  02138 

DAWSON,  DR.  J.  A.,  129  Violet  Avenue,  Floral  Park,  Long  Island,  New  York 
DEANE,  DR.  HELEN  W.,  Department  of  Anatomy,  The  Albert  Einstein  College  of 

Medicine,  New  York,  New  York  10461 
DEHAAN,  DR.  ROBERT  L.,  Department  of  Embryology,   Carnegie   Institution  of 

Washington,  Baltimore,  Maryland  21210 
DE  LORENZO,  DR.  ANTHONY,  Anatomical  &  Pathological  Research  Laboratories, 

The  Johns  Hopkins  Hospital,  Baltimore,  Maryland  21205 
DETTBARN,  DR.  WOLF-DIETRICH,  Department  of  Neurology,  Columbia  University, 

College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 
DE   VILLAFRANCA,    DR.    GEORGE    W.,    Department    of    Zoology,    Smith    College, 

Northampton,  Massachusetts  01060 
DILLER,  DR.  IRENE  C.,  Institute  for  Cancer  Research,  Fox  Chase,  Philadelphia, 

Pennsylvania  19111 

DILLER,  DR.  WILLIAM  F.,  2417  Fairhill  Avenue,  Glenside,  Pennsylvania 
DODDS,  DR.  G.  S.,  829  Price  Street,  Morgantown,  West  Virginia 
DOLLEY,  DR.  WILLIAM  L.,  Trevillans,  Virginia 
DOOLITTLE,  DR.  R.  F.,  Department  of  Biology,  University  of  California,  La  Jolla, 

California 

DOWBEN,  DR.  ROBERT,  Department  of  Biology,  Massachusetts  Institute  of  Tech- 
nology, Cambridge,  Massachusetts  02139 


50  MARINE  BIOLOGICAL  LABORATORY 

DUNHAM,  DR.  PHILIP  B.,  Department  of  Zoology,  Syracuse  University,  Syracuse, 

New  York  13210 
DURYEE,  DR.  WILLIAM  R.,  George  Washington  University,  2300  K  Street,  N.W., 

Washington,  D.  C. 
EBERT,  DR.  JAMES  DAVID,   Department  of  Embryology,   Carnegie   Institution  of 

Washington,  Baltimore,  Maryland  21210 
ECKERT,  DR.  ROGER  O.,  Department  of  Zoology,  Syracuse  University,  Syracuse, 

New  York  132 10 

EDDS,  DR.  MAC  V.,  JR.,  Department  of  Medical  Science,  Box  G,  Brown  Univer- 
sity, Providence,  Rhode  Island  02912 
EDER,  DR.  HOWARD  A.,  The  Albert  Einstein  College  of  Medicine,  New  York,  New 

York  10461 
EDWARDS,   DR.    CHARLES,   Department  of   Physiology,   University   of   Minnesota, 

Minneapolis,  Minnesota  53455 

EICHEL,  DR.  HERBERT  J.,  Department  of  Biological  Chemistry,  Hahnemann  Medi- 
cal College,  Philadelphia,  Pennsylvania  19102 
EISEN,    DR.    HERMAN,    Department    of    Medicine,    Washington    University,    St. 

Louis,  Missouri  63110 
ELLIOTT,  DR.  ALFRED  M.,  Department  of  Zoology,  University  of  Michigan,  Ann 

Arbor,  Michigan  48104 
ESSNER,  DR.  EDWARD  S.,  Sloan-Kettering  Institute  for  Cancer  Research,  Rye,  New 

York 
EVANS,  DR.  TITUS  C.,  State  University  of  Iowa  College  of  Medicine,  Iowa  City, 

Iowa  52240 
FAILLA,  DR.  P.  M.,  Radiological  Physics  Division,  Argonne  National  Laboratory, 

Argonne,  Illinois  60440 

FARMANFARMAIAN,  DR.  ALLAHVERDI,  Faculty  of  Medicine,  Box  191,  Pahlavi  Uni- 
versity, Shiraz,  Iran 

FAURE-FREMIET,  DR.  EMMANUEL,  College  de  France,  Paris,  France 
FAUST,   DR.   ROBERT   GILBERT,   Department   of   Physiology,   University   of   North 

Carolina  Medical  School,  Chapel  Hill,  North  Carolina  27514 
FAWCETT,  DR.  D.  W.,  Department  of  Anatomy,  Harvard  Medical  School,  Boston, 

Massacusetts  02115 
FERGUSON,  DR.  F.  P.,  National  Institute  of  General  Medical  Sciences,  National 

Institutes  of  Health,  Bethesda,  Maryland  20014 
FERGUSON,  DR.  JAMES  K.  W.,  Connought  Laboratories,  University  of  Toronto, 

Ontario,  Canada 
FIGGE,  DR.  F.  H.  J.,  University  of  Maryland  Medical  School,  Lombard  &  Green 

Streets,  Baltimore,  Maryland  21201 
FINGF.RMAN,   DR.   MILTON,   Department   of  Zoology,   Newcomb   College,   Tulane 

University,  New  Orleans,  Louisiana  70118 
FISCHER,   DR.   ERNST,   Department  of   Physiology,    Medical   College   of  Virginia, 

Richmond,  Virginia 
FISHER,  DR.  FRANK  M.,  JR.,  Department  of  Biology,  Rice  University,  Houston, 

Texas  77001 
FISHER,  DR.  JEANNE  M.,   Department  of  Biochemistry,  University  of  Toronto, 

Toronto,  Canada 


REPORT  OF  THE  DIRECTOR  51 

FISHER,  DR.  KENNETH  O.,  Department  of  Biology,  University  of  Toronto,  To- 
ronto, Canada 

FISHMAN,  DR.  Louis,  218  East  93rd  Street,  Brooklyn,  New  York  11212 

FRAENKEL,  DR.  GOTTFRIED  S.,  Department  of  Entomology,  University  of  Illinois, 
Urbana,  Illinois  61801 

FREYGANG,  DR.  WALTER  H.,  JR.,  6247  29th  Street,  N.W.,  Washington.  D.  C. 

FRIES,  DR.  ERIK  F.  B.,  Box  605,  Woods  Hole,  Massachusetts  02543 

FUORTES,  DR.  MICHAEL  G.  F.,  NINDB,  National  Institutes  of  Health,  Bethesda, 
Maryland  20014 

FURSHPAN,   DR.   EDWIN   J.,   Department   of   Neurophysiology,   Harvard    Medical 
School,  Boston,  Massachusetts  02115 

FURTH,  DR.  JACOB,  99  Fort  Washington  Avenue,  New  York,  New  York  10032 

FYE,  DR.  PAUL  M.,  Woods  Hole  Oceanographic  Institution,  Woods  Hole,  Massa- 
chusetts 02543 

GABRIEL,   DR.   MORDECAI,   Department  of  Biology,   Brooklyn   College,   Brooklyn, 
New  York  11210 

GAFFRON,  DR.  HANS,  Department  of  Biology,  Institute  of  Molecular  Biophysics, 
Tallahassee,  Florida  32306 

GALL,   DR.   JOSEPH   G.,   Department  of   Biology,   Yale  University,   New   Haven, 
Connecticut  06520 

GALTSOFF,  DR.  PAUL  S.,  Bureau  of  Commercial  Fisheries,  Woods  Hole,  Massa- 
chusetts 02543 

GELFANT,  DR.  SEYMOUR,  Department  of  Zoology,  Syracuse  University,  Syracuse, 
New  York  13210 

GERMAN,   DR.   JAMES   LAFAYETTE,    III,    Department   of   Pediatrics   &    Medicine, 
Cornell  University  Medical  School,  New  York,  New  York  10021 

GILBERT,  DR.  DANIEL  L.,  Laboratory  of  Biophysics,  National  Institutes  of  Health, 
Bethesda,  Maryland  20014 

GILMAN,  DR.  LAUREN  C.,  Department  of  Zoology,  University  of  Miami,  Coral 
Gables,  Florida  33124 

GINSBERG,  DR.  HAROLD  S.,  Department  of  Microbiology,  University  of  Pennsyl- 
vania School  of  Medicine,  Philadelphia,  Pennsylvania  19104 

GOLDSMITH,   DR.  TIMOTHY  H.,   Department  of  Biology,  Yale  University,   New 
Haven,  Connecticut  06520 

GOLDSTEIN,   DR.    LESTER,   Department   of   Zoology,   University   of    Pennsylvania, 
Philadelphia,  Pennsylvania  19104 

GOODCHILD,  DR.  CHAUNCEY  G.,  Department  of  Biology,  Emory  University,  Atlanta, 
Georgia  30322 

GOTSCHALL,  DR.  GERTRUDE  Y.,  215  East  68th  Street,  Apt.  9-M,  New  York,  New 
York  10021 

GRAHAM,  DR.  HERBERT  W.,  U.  S.  Fish  &  Wildlife  Service,  Bureau  of  Commercial 
Fisheries,  Woods  Hole,  Massachusetts  02543 

GRAND,  MR.  C.  G.,  Cancer  Institute  of  Miami,  1155  N.  W.  15th  Street,  Miami, 
Florida 

GRANT,  DR.  PHILIP,  National  Science  Foundation,   1800  G  Street,  Washington, 
D.  C.  20550 


52  MARINE  BIOLOGICAL  LABORATORY 

GRAY,  DR.  IRVING  E.,  Department  of  Zoology,  Duke  University,  Durham,  North 

Carolina  27706 
GREEN,   DR.   JAMES  W.,   Department  of   Physiology,   Rutgers   University,    New 

Brunswick,  New  Jersey  08903 

GREEN,  DR.  JONATHAN  PASCAL,  Department  of  Biology,  Brown  University,  Provi- 
dence, Rhode  Island  02912 
GREEN,  DR.  MAURICE,  Department  of  Microbiology,  St.  Louis  University  Medical 

School,  St.  Louis,  Missouri  63103 
GREGG,  DR.  JAMES  H.,  Department  of  Biological  Sciences,  University  of  Florida, 

Gainesville,  Florida  32601 
GREGG,  DR.  JOHN  R.,  Department  of  Zoology,  Duke  University,  Durham,  North 

Carolina  27706 
GREIF,   DR.   ROGER  L.,   Department  of   Physiology,   Cornell  University   Medical 

College,  New  York,  New  York  10021 

GRIFFIN,  DR.  DONALD  F.,  The  Rockefeller  University,  New  York,  New  York  10021 
GROSCH,  DR.  DANIEL  S.,  Department  of  Genetics,  North  Carolina  State  University, 

Raleigh,  North  Carolina  27607 
GROSS,  DR.  PAUL,  Department  of  Biology,  Massachusetts  Institute  of  Technology, 

Cambridge,  Massachusetts  02139 
GRUNDFEST,  DR.  HARRY,  Department  of  Neurology,  Columbia  University,  College 

of  Physicians  &  Surgeons,  New  York,  New  York  10032 
GUTTMAN,  DR.   RITA,   Department  of  Biology,   Brooklyn   College,   Brooklyn    10, 

New  York  112 10 
GWILLIAM,  DR.  G.  F.,  Department  of  Biology,  Reed  College,  Portland,  Oregon 

97202 

HAJDU,  DR.  STEPHEN,  National  Institutes  of  Health,  Bethesda,  Maryland  20014 
HALL,  DR.  FRANK  S.,  Department  of  Physiology,  Duke  University  Medical  School, 

Durham,  North  Carolina  27706 

HALVORSON,  DR.  HARLYN  O.,  Department  of  Bacteriology,  University  of  Wiscon- 
sin, Madison,  Wisconsin  53706 
HAMBURGER,  DR.  VIKTOR,  Department  of  Zoology,  Washington  University,   St. 

Louis,  Missouri  63130 
HAMILTON,   DR.   HOWARD  L.,   Department   of   Biology,   University   of   Virginia, 

Charlottesville,  Virginia  22903 

HANCE,  DR.  ROBERT  T.,  RR  No.  3,  6609  Smith  Road,  Loveland,  Ohio 
HARDING,  DR.  CLIFFORD  V.,  JR.,  Oakland  University,  Rochester,  Michigan  48063 
HARNLY,  DR.   MORRIS  H.,  Washington  Square  College,   New  York  University, 

New  York,  New  York  10003 
HARTLINE,  DR.  H.  KEFFER,  The  Rockefeller  University,  New  York,  New  York 

10021 
HARTMAN,  DR.  FRANK  A.,  Ohio  State  University,  Hamilton  Hall,  Columbus,  Ohio 

43210 
HARTMAN,  DR.   P.  E.,  Department  of  Biology,  The  Johns  Hopkins  University, 

Baltimore,  Maryland  21218 

HARVEY,  DR.  ETHEL  BROWNE,  Marine  Biological  Laboratory,  Woods  Hole,  Massa- 
chusetts 02543 


REPORT  OF  THE  DIRECTOR 

HASTINGS,  DR.  J-  WOODLAND,  Division  of  Biochemistry,  University  of  Illinois, 
Urbana,  Illinois  61803 

HAUSCHKA,  DR.  T.  S.,  Roswell  Park  Memorial  Institute,  666  Elm  Street,  Buffalo 
3,  New  York 

HAXO,  DR.  FRANCIS  T.,  Division  of  Marine  Botany,  Scripps  Institution  of  Oceanog- 
raphy, University  of  California,  La  Jolla,  California  92038 

HAYASHI,  DR.  TERU,  Department  of  Zoology.  Columbia  University,  New  York, 
New  York  10027 

HAYDEN,  DR.  MARGARET  A.,  34  Weston  Road,  Wellesley,  Massachusetts  02181 

HAYWOOD,  DR.  CHARLOTTE,  Box  14,  South  Hadley,  Massachusetts  01075 

HEGYELI,  DR.  ANDREW  F.,  Battelle  Memorial  Institute,  Columbus,  Ohio  43201 

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

HENLEY,  DR.  CATHERINE,  Department  of  Zoology,  University  of  North  Carolina, 
Chapel  Hill,  North  Carolina  27514 

HERNDON,  DR.  WALTER  R.,  Department  of  Botany,  College  of  Liberal  Arts,  Uni- 
versity of  Tennessee,  Knoxville,  Tennessee  37916 

HERVEY,  MR.  JOHN  P.,  Box  735,  Woods  Hole,  Massachusetts  02543 

HESSLER,  DR.  ANITA  Y.,  Marine  Biological  Laboratory,  Woods  Hole,  Massachu- 
setts 02543 

HIATT,  DR.  HOWARD  H.,  Department  of  Medicine,  Harvard  Medical  School,  Bos- 
ton, Massachusetts  02115 

HIBBARD,  DR.  HOPE,  366  Reamer  Place,  Oberlin,  Ohio  44074 

HIRSHFIELD,  DR.  HENRY  I.,  Department  of  Biology,  Washington  Square  Center, 
New  York  University,  New  York,  New  York  10003 

HOADLEY,  DR.  LEIGH,  Biological  Laboratories,  Harvard  University,  Cambridge, 
Massachusetts  02138 

HODES,  DR.  ROBERT,  Department  of  Pediatrics,  The  Mount  Sinai  Hospital,  New 
York,  New  York  10039 

HODGE,  DR.  CHARLES,  IV,  Department  of  Biology,  Temple  University,  Philadelphia, 
Pennsylvania  19122 

HOFFMAN,  DR.  JOSEPH,  Department  of  Physiology,  Yale  University  School  of 
Medicine,  New  Haven,  Connecticut  06515 

HOLLAENDER,  DR.  ALEXANDER,  Biology  Division,  Oak  Ridge  National  Laboratory, 
Oak  Ridge,  Tennessee  37831 

HOLZ,  DR.  GEORGE  G.,  JR.,  Department  of  Microbiology,  State  University  of  New 
York,  Upstate  Medical  College,  Syracuse,  New  York  13210 

HOPKINS,  DR.  HOYT  S.,  59  Heatherdell  Road,  Ardsley,  New  York 

HOSKIN,  DR.  FRANCIS  C.  G.,  Department  of  Neurology,  Columbia  University, 
College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 

HUMPHREYS,  DR.  TOM  DANIEL,  Department  of  Biology,  Massachusetts  Institute  of 
Technology,  Cambridge,  Massachusetts  02139 

HUNTER,  DR.  FRANCIS  R.,  Department  of  Biology,  Centro  Experimental  de 
Estudios  Superiores,  Barquisimeto,  Venezuela 

HUNTER,  DR.  W.  D.  RUSSELL,  Department  of  Zoology,  Syracuse  University, 
Syracuse,  New  York  13210 

HURWITZ,  DR.  CHARLES,  Basic  Science  Research  Laboratory,  VA  Hospital, 
Albany,  New  York 


54  MARINE  BIOLOGICAL  LABORATORY 

HURWITZ,  DR.  J.,  Department  of  Molecular  Biology,  The  Albert  Einstein  College 

of  Medicine,  Bronx,  New  York  10461 
HUTCHENS,   DR.   JOHN    E.,   Department   of   Physiology,   University   of   Chicago, 

Chicago,  Illinois  60637 
HYDE,  DR.  BEAL  B.,  Department  of  Botany,  University  of  Vermont,  Burlington, 

Vermont  05401 

INOUE,  DR.  SHINYA,  Department  of  Cytology,  Dartmouth  Medical  College,  Han- 
over, New  Hampshire  03755 
ISENBERG,    DR.    IRVIN,    Science    Research    Institute,    Oregon    State    University, 

Corvallis,  Oregon  97331 

ISELIN,  MR.  COLUMBUS  O'D.,  Woods  Hole,  Massachusetts  02543 
ISSELBACHER,  DR.  KURT  J.,  Massachusetts  General  Hospital,  Boston,  Massachu- 
setts 021 14 
JANOFF,  DR.  AARON,  Department  of  Pathology,  New  York  University  School  of 

Medicine,  550  First  Avenue,  New  York,  New  York 
JENNER,  DR.  CHARLES  E.,  Department  of  Zoology,  University  of  North  Carolina, 

Chapel  Hill,  North  Carolina  27514 
JOHNSON,  DR.  FRANK  H.,  Department  of  Biology,  Princeton  University,  Princeton, 

New  Jersey  08540 
JONES,  DR.  E.  RUFFIN,  JR.,  Department  of  Biological   Sciences,   University  of 

Florida,  Gainesville,  Florida  32601 
JONES,  DR.  RAYMOND  F.,  Department  of  Biology,  State  University  of  New  York, 

Stony  Brook,  Long  Island,  New  York  11733 
JOSEPHSON,   DR.   R.   K.,   Department  of  Biology,   Western   Reserve  University, 

Cleveland,  Ohio  44106 

KAAN,  DR.  HELEN  W.,  Box  665,  Woods  Hole,  Massachusetts  02543 
RABAT,  DR.  E.  A.,  Neurological  Institute,  Columbia  University,  College  of  Physi- 
cians &  Surgeons,  New  York,  New  York  10032 
KALEY,  DR.  GABOR,  New  York  Medical  College,  Flower  &  Fifth  Avenue  Hospitals, 

5th  Avenue  at  106th  Street,  New  York,  New  York  10029 
KAMINER,   DR.    BENJAMIN,    Institute   for    Muscle   Research,    Marine   Biological 

Laboratory,  Woods  Hole,  Massachusetts  02543 
KANE,   DR.   ROBERT  E.,   Department   of   Cytology,   Dartmouth    Medical    School, 

Hanover,  New  Hampshire  03755 
KARUSH,   DR.    FRED,   Department   of    Microbiology,   University   of   Pennsylvania 

School  of  Medicine,  Philadelphia,  Pennsylvania  19104 
KAUFMAN,  DR.  B.  P.,  Department  of  Zoology,  University  of  Michigan,  Ann  Arbor, 

Michigan  48104 
KEMP,  DR.  NORMAN  E.,  Department  of  Zoology,  University  of  Michigan,  Ann 

Arbor,  Michigan  48104 
KEMPTON,  DR.  RUDOLF  T.,  Department  of  Biology,  Vassar  College,  Poughkeepsie, 

New  York  12601 
KEOSIAN,  DR.  JOHN,  Department  of  Biology,  Rutgers  University,  Newark  2,  New 

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

Hole,  Massachusetts  02543 
KEYNAN,  DR.  ALEXANDER,  Institute  for  Biological  Research,  Ness-Ziona,  Israel 


REPORT  OF  THE  DIRECTOR  55 

KILLE,  DR.  FRANK  R.,  State  Department  of  Education,  Albany  1,  New  York 
KIND,  DR.  C.  ALBERT,  Department  of  Zoology,  University  of  Connecticut,  Storrs, 

Connecticut  06268 

KINDRED,  DR.  JAMES  E.,  2010  Hessian  Road,  Charlottesville,  Virginia 
KING,  DR.  ROBERT  L.,  1229  East  Manhattan  Drive,  Tempe,  Arizona  85281 
KING,  DR.  THOMAS  J.,  The  Institute  for  Cancer  Research,  7701  Burholme  Avenue, 

Philadelphia,  Pennsylvania  19111 
KINGSBURY,  DR.  JOHN   M.,  Department  of  Botany,   Cornell  University,   Ithaca, 

New  York  14850 
KINNE,  DR.  OTTO,  Biologische  Anstalt  Helgoland,  2  Hamburg-Altona,  Palmaille  9, 

Germany 

KISCH,  DR.  BRUNO,  71  Maple  Street,  Brooklyn  25,  New  York 

KLEIN,  DR.  MORTON,  Department  of  Microbiology,  Temple  University,  Philadel- 
phia, Pennsylvania  19122 
KLEINHOLZ,   DR.   LEWIS   H.,   Department   of   Biology,   Reed   College,    Portland, 

Oregon  97202 
KLOTZ,  DR.  I.  M.,  Department  of  Chemistry,  Northwestern  University,  Evanston, 

Illinois  60201 
KOLIN,  DR.  ALEXANDER,  Department  of  Biophysics,  California  Medical  School, 

Los  Angeles,  California  90025 
KORNBERG,  DR.  HANS  LEO,  Department  of  Biochemistry,  University  of  Leicester, 

Leicester,  England 
KORR,  DR.  I.   M.,  Department  of  Physiology,  Kirksville  College  of  Osteopathy, 

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

Illinois  60637 
KRANE,  DR.  STEPHEN  M.,  Massachusetts  General  Hospital,  Boston,  Massachusetts 

02114 
KRASSNER,  DR.  STUART  MITCHELL,  Department  of  Organismic  Biology,  University 

of  California,  Irvine,  California  92650 
KRAUSS,  DR.  ROBERT,  Department  of  Botany,  University  of  Maryland,  Baltimore, 

Maryland 

KREIG,  DR.  WENDELL  J.  S.,  303  East  Chicago  Avenue,  Chicago,  Illinois 
KUFFLER,   DR.   STEPHEN   W.,   Department   of   Pharmacology,   Harvard    Medical 

School,  Boston,  Massachusetts  02115 

KUNITZ,  DR.  MOSES,  The  Rockefeller  University,  New  York,  New  York  10021 
LAMY,  DR.  FRANCOIS,  Department  of  Anatomy,  University  of  Pittsburgh  School  of 

Medicine,  Pittsburgh,  Pennsylvania  15213 
LANCEFIELD,  DR.  D.  E.,  203  Arleigh  Road,  Douglaston  63,  Long  Island,  New 

York 
LANCEFIELD,  DR.  REBECCA  C.,  The  Rockefeller  University,  New  York,  New  York 

10021 

LANDIS,  DR.  E.  M.,  Harvard  Medical  School,  Boston,  Massachusetts  02115 
LANSING,   DR.   ALBERT   I.,   Department   of   Anatomy,   University   of   Pittsburgh 

School  of  Medicine,  Pittsburgh,  Pennsylvania  15213 
LASH,  DR.  JAMES  W.,  Department  of  Anatomy,  University  of  Pennsylvania  School 

of  Medicine,  Philadelphia,  Pennsylvania  19104 


56  MARINE  BIOLOGICAL  LABORATORY 

LAUFER,  DR.  HANS,  Department  of  Zoology  &  Entomology,  University  of  Connecti- 
cut, Storrs,  Connecticut  06268 

LAUFFER,  DR.  MAX  A.,  Department  of  Biophysics,  University  of  Pittsburgh,  Pitts- 
burgh, Pennsylvania  15213 

LAWLER,  DR.  H.  CLAIRE,  Department  of  Biochemistry  &  Neurology,  Columbia 
University,  College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 

LAVIN,  DR.  GEORGE  I.,  6200  Norvo  Road,  Baltimore,  Maryland  21207 

LAZAROW,  DR.  ARNOLD,  Department  of  Anatomy,  University  of  Minnesota  Medical 
School,  Minneapolis,  Minnesota  55455 

LEDERBERG,  DR.  JOSHUA,  Department  of  Genetics,  Stanford  Medical  School,  Palo 
Alto,  California  94305 

LEE,  DR.  RICHARD  E.,  Cornell  University  College  of  Medicine,  New  York,  New 
York  10021 

LEFEVRE,  DR.  PAUL  G.,  University  of  Louisville  School  of  Medicine,  Louisville, 
Kentucky  40208 

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

LEVIN,  DR.  JACK,  Department  of  Medicine,  The  Johns  Hopkins  Hospital,  Balti- 
more, Maryland  21205 

LEVINE,  DR.  RACHMIEL,  Department  of  Medicine,  New  York  Medical  College, 
New  York,  New  York  10029 

LEVY,  DR.  MILTON,  Department  of  Biochemistry,  New  York  University  School  of 
Dentistry,  New  York,  New  York  10010 

LEWIN,  DR.  RALPH  A.,  Scripps  Institution  of  Oceanography,  La  Jolla,  California 
92038 

LEWIS,  DR.  HERMAN  W.,  Genetic  Biology  Program,  National  Science  Foundation, 
Washington,  D.  C.  20550 

LING,  DR.  GILBERT,  307  Berkeley  Road,  Merion,  Pennsylvania 

LITTLE,  DR.  E.  P.,  216  Highland  Street,  West  Newton,  Massachusetts 

LLOYD,  DR.  DAVID  P.  C.,  The  Rockefeller  University,  New  York,  New  York  10021 

LOCH  HEAD,  DR.  JOHN  H.,  Department  of  Zoology,  University  of  Vermont,  Burling- 
ton, Vermont  05401 

LOEB,  DR.  R.  F.,  950  Park  Avenue,  New  York,  New  York  10028 

LOEWENSTEIN,  DR.  WERNER  R.,  Department  of  Physiology,  Columbia  University, 
College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 

LOFTFIELD,   DR.    ROBERT   B.,   Department   of   Biochemistry,    University    of    New 
Mexico  Medical  School,  Albuquerque,  New  Mexico  87106 

LONDON,  DR.  IRVING  M.,  Department  of  Medicine,  The  Albert  Einstein  College  of 
Medicine,  New  York,  New  York  10461 

LORAND,  DR.  LASZLO,  Department  of  Chemistry,  Northwestern  University,  Evan- 
ston,  Illinois  60201 

LOVE,  DR.  WARNER  E.,  1043  Marlau  Drive,  Baltimore,  Maryland  21212 

LUBIN,  DR.   MARTIN,   Department  of   Pharmacology,   Harvard   Medical   School, 
Boston,  Massachusetts  02115 

LYNCH,  DR.  CLARA  J.,  The  Rockefeller  University,  New  York,  New  York  10021 

LYNN,  DR.  W.  GARDNER,  Department  of  Biology,  Catholic  University  of  America, 
Washington,  D.  C.  20017 


REPORT  OF  THE  DIRECTOR  57 

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

New  York  10016 
MAGRUDER,  DR.  SAMUEL  R.,  Department  of  Anatomy,  Tufts  Medical  School,  135 

Harrison  Avenue,  Boston,  Massachusetts 
MAHLER,  DR.  HENRY  R.,  Department  of  Biochemistry,  Indiana  University,  Bloom- 

ington,  Indiana  47405 
MANWELL,    DR.    REGINALD    D.,    Department   of    Zoology,    Syracuse    University, 

Syracuse,  New  York  13210 
MARKS,  DR.  PAUL  A.,  Columbia  University,  College  of  Physicians  &  Surgeons, 

New  York,  New  York  10032 
MARSHAK,    DR.    ALFRED,    Tulane    University    Medical    School,    New    Orleans, 

Louisiana  70112 

MARSLAND,  DR.  DOUGLAS  A.,  48  Church  Street,  Woods  Hole,  Massachusetts  02543 
MARTIN,  DR.  EARL  A.,  682  Rudder  Road,  Naples,  Florida  33940 
MATHEWS,  DR.  SAMUEL  A.,  Thompson  Biological  Laboratory,  Williams  College, 

Williamstown,  Massachusetts  01267 
MAZIA,  DR.  DANIEL,  Department  of  Zoology,  University  of  California,  Berkeley, 

California  94720 
McCANN,  DR.  FRANCES,  Department  of  Physiology,  Dartmouth  Medical  School, 

Hanover,  New  Hampshire  03755 
McCoucH,  DR.  MARGARET  SUMWALT,  University  of  Pennsylvania  Medical  School, 

Philadelphia,  Pennsylvania  19104 
MCDONALD,  SISTER  ELIZABETH  SETON,  Department  of  Biology,  College  of  Mt.  St. 

Joseph  on  the  Ohio,  Mt.  St.  Joseph,  Ohio 

MCDONALD,  DR.  MARGARET  R.,  Waldermar  Medical  Research  Foundation,  Sunny- 
side  Boulevard  &  Waldermar  Road,  Woodbury,  Long  Island,  New  York 
MCELROY,  DR.  WILLIAM  D.,  Department  of  Biology,  The  Johns  Hopkins  Univer- 
sity, Baltimore,  Maryland  21218 
MEINKOTH,  DR.  NORMAN,  Department  of  Biology,  Swarthmore  College,  Swarth- 

more,  Pennsylvania  19081 
MENDELSON,   DR.    MARTIN,    Department   of   Physiology,    New   York   University 

Medical  School,  New  York,  New  York  10016 
METZ,  DR.  C.  B.,  Institute  of  Molecular  Evolution,  University  of  Miami,  Coral 

Gables,  Florida  33124 

METZ,  DR.  CHARLES  W.,  Box  714,  Woods  Hole,  Massachusetts  02543 
MIDDLEBROOK,  DR.  ROBERT,  Dartmouth  Medical  Center,  Hanover,  New  Hampshire 

03755 
MILKMAN,  DR.  ROGER  D.,  Department  of  Zoology,  Syracuse  University,  Syracuse, 

New  York  13210 
MILLER,  DR.  J.  A.,  JR.,  Department  of  Anatomy,  Tulane  University   School  of 

Medicine,  New  Orleans,  Louisiana  70112 
MILNE,  DR.  LORUS  J.,  Department  of  Zoology,  University  of  New  Hampshire, 

Durham,  New  Hampshire  03824 
MILLS,  DR.  ERIC  LEONARD,  Department  of  Biology,  Queen's  University,  Kingston, 

Ontario,  Canada 
MOE,  MR.  HENRY  A.,  Guggenheim  Memorial  Foundation,  551  Fifth  Avenue,  New 

York,  New  York  10017 


58  MARINE  BIOLOGICAL  LABORATORY 

MONROY,  DR.  ALBERTO,  Institute  of  Comparative  Anatomy,  University  of  Palermo, 

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

Durham,  New  Hampshire  03824 

MOORE,  DR.  JOHN  A.,  Department  of  Zoology,  954  Schermerhorn,  Columbia  Uni- 
versity, New  York,  New  York  10027 
MOORE,   DR.   JOHN   W.,   Department   of   Physiology,    Duke   University    Medical 

Center,  Durham,  North  Carolina  27706 
MOORE,  DR.  R.  O.,  Department  of  Biochemistry,  Ohio  State  University,  Columbus, 

Ohio  43210 
MORAN,  DR.  JOSEPH,  Department  of  Biology,  Russell  Sage  College,  Troy,  New 

York 
MORRILL,  DR.  JOHN  B.,  JR.,  Department  of  Biology,  College  of  William  &  Mary, 

Williamsburg,  Virginia  23185 
MOSCONA,  DR.  A.  A.,  Department  of  Zoology,  University  of  Chicago,  Chicago, 

Illinois  60637 
MOUL,  DR.  E.  T.,  Department  of  Biology,  Rutgers  University,  New  Brunswick, 

New  Jersey  08903 

MOUNTAIN,  MRS.  J.  D.,  Charles  Road,  Mt.  Kisco,  New  York 
MULLINS,  DR.  LORIN  J.,  Department  of  Biophysics,  University  of  Maryland  School 

of  Medicine,  Baltimore,  Maryland  21201 
MUSACCHIA,  DR.  XAVIER  J.,  Department  of  Physiology,  University  of  Missouri 

Medical  Center,  Columbia,  Missouri 
NABRIT,  DR.  S.  M.,  Texas  Southern  University,  3201  Wheeler  Avenue,  Houston, 

Texas  77014 

NACE,  DR.  PAUL  FOLEY,  Clapp  Laboratories,  Duxbury,  Massachusetts  02332 
NACHMANSOHN,  DR.  DAVID,  Department  of  Neurology,  Columbia  University,  Col- 
lege of  Physicians  &  Surgeons,  New  York,  New  York  10032 

NASATIR,  DR.  MAIMON,  Division  of  Biological  &  Medical  Sciences,  Brown  Univer- 
sity, Providence,  Rhode  Island  02912 
NASON,  DR.  ALVIN,  McCollum-Pratt  Institute,  The  Johns  Hopkins  University, 

Baltimore,  Maryland  21218 

NAVEZ,  DR.  ALBERT  E.,  206  Churchill's  Lane,  Milton,  Massachusetts  02186 
NELSON,  DR.   LEONARD,  Department  of  Physiology,  Emory  University,   Atlanta, 

Georgia  30322 
NEURATH,  DR.  H.,  Department  of  Biochemistry,  University  of  Washington,  Seattle, 

Washington  98105 

NICOLL,  DR.  PAUL  A.,  Black  Oak  Lodge,  RR  No.  2,  Bloomington,  Indiana 
Niu,  DR.  MAN-CHIANG,  Temple  University,  Philadelphia,  Pennsylvania  19122 
NOVIKOFF,  DR.  ALEX  B.,  Department  of  Pathology,  The  Albert  Einstein  College  of 

Medicine,  New  York,  New  York  10461 
OCHOA,  DR.  SEVERO,  New  York  University  College  of  Medicine,  New  York,  New 

York  10016 
ODUM,   DR.   EUGENE,   Department   of  Zoology,   University  of   Georgia,   Athens, 

Georgia  30602 
OPPENHEIMER,  DR.  JANE  M.,  Department  of  Biology,  Bryn  Mawr  College,  Bryn 

Mawr,  Pennsylvania  19010 


REPORT  OF  THE  DIRECTOR  59 

OSTERHOUT,  DR.  MARION  IRWIN,  450  East  63rd  Street,  New  York,  New  York 

10021 

PACKARD,  DR.  CHARLES,  Woods  Hole,  Massachusetts  02543 
PAGE,  DR.  IRVINE  H.,  Cleveland  Clinic,  Cleveland,  Ohio 
PALMER,  DR.  JOHN  D.,  Department  of  Biology,  New  York  University,  University 

Heights,  New  York  53,  New  York 

PARPART,  DR.  ARTHUR  K.,  Department  of  Biology,  Princeton  University,  Prince- 
ton, New  Jersey  08540 
PASSANO,  DR.  LEONARD  M.,  Department  of  Zoology,  University  of  Wisconsin, 

Madison,  Wisconsin  53706 
PATTEN,  DR.  BRADLEY  M.,  University  of  Michigan,  2500  East  Medical  Building, 

Ann  Arbor,  Michigan  48104 
PERKINS,  DR.  JOHN  F.,  Department  of  Physiology,  University  of  Chicago,  Chicago, 

Illinois  60637 
PERSON,  DR.  PHILIP,  Special  Dental  Research  Program,  VA  Hospital,  Brooklyn 

9,  New  York 
PETTIBONE,  DR.  MARIAN  H.,  Division  of  Marine  Invertebrates,  U.  S.  National 

Museum,  Washington  25,  D.  C. 
PHILPOTT,  DR.  DELBERT  E.,  Department  of  Biochemistry,  University  of  Colorado 

Medical  Center,  Denver  20,  Colorado 

PICK,  DR.  JOSEPH,  Department  of  Anatomy,  New  York  University,  Bellevue  Medi- 
cal Center,  New  York,  New  York  10016 
PIERCE,  DR.  MADELENE  E.,  Department  of  Biology,  Vassar  College,  Poughkeepsie, 

New  York  12601 
POLLISTER,  DR.  A.  W.,  Department  of  Zoology,  Columbia  University,  New  York, 

New  York  10027 

POND,  DR.  SAMUEL  E.,  53  Alexander  Street,  Manchester,  Connecticut 
PORTER,  DR.  KEITH  R.,  Biological  Laboratories,  Harvard  University,  Cambridge, 

Massachusetts  02 138 
POTTER,  DR.  DAVID,  Department  of  Neurophysiology,  Harvard  Medical  School, 

Boston,  Massachusetts  02115 

PROCTOR,  DR.  NATHANIEL,  Department  of  Biology,  Morgan  State  College,  Balti- 
more, Maryland  21212 
PROSSER,   DR.   C.   LADD,   Department  of  Physiology  &   Biophysics,   University   of 

Illinois,  Urbana,  Illinois  61803 
PROVASOLI,  DR.  LUIGI,  Haskins  Laboratories,  305  East  43rd  Street,  New  York, 

New  York  10017 
RABIN,  DR.  HARVEY,  Department  of  Pathobiology,  The  Johns  Hopkins  University, 

Baltimore,  Maryland  21205 
RAMSEY,  DR.  ROBERT  W.,  Department  of  Physiology,  Medical  College  of  Virginia. 

Richmond  19,  Virginia 
RANKIN,  DR.  JOHN  S.,  Department  of  Zoology,  University  of  Connecticut,  Storrs, 

Connecticut  06268 

RANZI,  DR.  SILVIO,  Department  of  Zoology,  University  of  Milan,  Milan,  Italy 
RAPPORT,  DR.  M.,  Department  of  Biochemistry,  The  Albert  Einstein  College  of 

Medicine,  New  York,  New  York  10461 


60  MARINE  BIOLOGICAL  LABORATORY 

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

Foot  of  East  15th  Street,  New  York,  New  York  10009 
RAY,   DR.   CHARLES,   JR.,   Department   of   Biology,   Emory   University,   Atlanta, 

Georgia  30322 
READ,  DR.  CLARK  P.,  Department  of  Biology,  Rice  University,  Houston,  Texas 

77001 
REBHUN,  DR.  LIONEL  I.,  Department  of  Biology,  Princeton  University,  Princeton, 

New  Jersey  08540 
RECKNAGEL,  DR.  R.  O.,  Department  of  Physiology,  Western  Reserve  University, 

Cleveland,  Ohio  44106 

REDFIELD,  DR.  ALFRED  C.,  Woods  Hole,  Massachusetts  02543 
RENN,  DR.  CHARLES  E.,  509  Ames  Hall,  The  Johns  Hopkins  University,  Balti- 
more, Maryland  2 12 18 
REUBEN,  DR.  JOHN  P.,  Department  of  Neurology,  Columbia  University,  College 

of  Physicians  &  Surgeons,  New  York,  New  York  10032 
REZNIKOFF,  DR.  PAUL,  Cornell  University  Medical  School,  1300  York  Avenue, 

New  York,  New  York  10021 

RICH,  DR.  ALEXANDER,  Department  of  Biology,  Massachusetts  Institute  of  Tech- 
nology, Cambridge,  Massachusetts  02139 

RICHARDS,  DR.  A.,  2950  East  Mable  Street,  Tucson,  Arizona 
RICHARDS,  DR.  A.  GLENN,  Department  of  Entomology,  University  of  Minnesota, 

St.  Paul,  Minnesota  55101 

RICHARDS,  DR.  OSCAR  W.,  Research  Center,  American  Optical  Company,  South- 
bridge,  Massachusetts 
ROCKSTEIN,  DR.  MORRIS,  Medical  Research  Building,  1600  N.  W.  10th  Avenue, 

Miami,  Florida 
ROMER,  DR.  ALFRED  S.,  Museum  of  Comparative  Zoology,  Harvard  University, 

Cambridge,  Massachusetts  02138 
RONKIN,   DR.   RAPHAEL   R.,    Department   of   Biological    Sciences,   University   of 

Delaware,  Newark,  Delaware  19711 
ROOT,  DR.  R.  W.,  Department  of  Biology,  College  of  the  City  of  New  York,  New 

York,  New  York  10031 
ROOT,   DR.   W.   S.,   Department  of  Physiology,    Columbia   University,    College   of 

Physicians  &  Surgeons,  New  York,  New  York  10032 
ROSE,  DR.  S.  MERYL,  Department  of  Anatomy,  Tulane  University,  New  Orleans, 

Louisiana  70112 
ROSENBERG,  DR.  EVELYN  K.,  Department  of  Pathology,  New  York  University, 

Bellevue  Medical  Center,  New  York,  New  York  10016 
ROSENBERG,  DR.  PHILIP,  Department  of  Neurology,  Columbia  University,  College 

of  Physicians  &  Surgeons,  New  York,  New  York  10032 
ROSENBLUTH,  Miss  RAJA,  Science  Research  Institute,  Oregon  State  University, 

Corvallis,  Oregon  97331 
ROSENKRANZ,  DR.  HERBERT  S.,  Department  of  Microbiology,  Columbia  University, 

College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 
ROSENTHAL,  DR.  THEODORE  B.,  Department  of  Anatomy,  University  of  Pittsburgh 

Medical  School,  Pittsburgh,  Pennsylvania  15213 


REPORT  OF  THE  DIRECTOR  61 

ROSLANSKY,  DR.  JOHN,  Institute  for  Muscle  Research,  Marine  Biological  Labora- 
tory, Woods  Hole,  Massachusetts  02543 

ROTH,  DR.  JAY  S.,  Department  of  Zoology  &  Entomology,  University  of  Connecti- 
cut, Storrs,  Connecticut  06268 

ROTHENBERG,  DR.  M.  A.,  Dugway  Proving  Ground,  Dugway,  Utah 

RUGH,  DR.  ROBERTS,  Radiological  Research  Laboratory,  Columbia  University, 
College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 

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

RUSTAD,  DR.  RONALD  C.,  Department  of  Radiology,  Western  Reserve  University, 
Cleveland,  Ohio  44106 

RUTMAN,  DR.  ROBERT  J.,  General  Laboratory  Building,  215  South  34th  Street, 
Philadelphia,  Pennsylvania  19104 

RYTHER,  DR.  JOHN  H.,  Woods  Hole  Oceanographic  Institution,  Woods  Hole, 
Massachusetts  02543 

SAGER,  DR.  RUTH,  Department  of  Zoology,  Columbia  University,  New  York, 
New  York  10027 

SANBORN,  DR.  RICHARD  C.,  Department  of  Biological  Sciences,  Purdue  University, 
Lafayette,  Indiana  47907 

SANDERS,  DR.  HOWARD  L.,  Woods  Hole  Oceanographic  Institution,  Woods  Hole, 
Massachusetts  02543 

SATO,  DR.  HIDEMI,  Department  of  Cytology,  Dartmouth  Medical  School,  Hanover, 
New  Hampshire  03755 

SAUNDERS,  DR.  JOHN  W.,  JR.,  Department  of  Biology,  Marquette  University, 
Milwaukee,  Wisconsin  53233 

SAUNDERS,  MR.  LAWRENCE,  West  Washington  Square,  Philadelphia,  Pennsylvania 
19105 

SAZ,  DR.  ARTHUR  KENNETH,  Department  of  Microbiology,  Georgetown  Univer- 
sity, Medical  and  Dental  Schools,  3900  Reservoir  Road,  Washington,  D.  C. 

SCHACHMAN,  DR.  HOWARD  K.,  Department  of  Biochemistry,  University  of  Cali- 
fornia, Berkeley,  California  94720 

SCHARRER,  DR.  BERTA  V.,  Department  of  Anatomy,  The  Albert  Einstein  College 
of  Medicine,  New  York,  New  York  10461 

SCHLESINGER,  DR.  R.  WALTER,  Department  of  Microbiology,  Rutgers  Medical 
School,  New  Brunswick,  New  Jersey  08903 

SCHMEER,  SISTER  M.  ROSARII,  Department  of  Biology,  College  of  St.  Mary  of  the 
Springs,  Columbus,  Ohio  43219 

SCHMIDT,  DR.  L.  H.,  National  Primate  Center,  University  of  California,  Davis, 
California  95616 

SCHMITT,  DR.  FRANCIS  O.,  Department  of  Biology,  Massachusetts  Institute  of 
Technology,  Cambridge,  Massachusetts  02139 

SCHMITT,  DR.  O.  H.,  Department  of  Physics,  University  of  Minnesota,  Minne- 
apolis, Minnesota  55455 

SCHNEIDERMAN,  DR.  HOWARD  A.,  Department  of  Biology,  Western  Reserve  Uni- 
versity, Cleveland,  Ohio  44106 

SCHOLANDER,  DR.  P.  F.,  Scripps  Institution  of  Oceanography,  La  Jolla,  Cali- 
fornia 92038 


62  MARINE  BIOLOGICAL  LABORATORY 

SCHOTTE,  DR.  OSCAR  E.,  Department  of  Biology,  Amherst  College,  Amherst, 
Massachusetts  01002 

SCHRAMM,  DR.  J.  R.,  Department  of  Botany,  Indiana  University,  Bloomington, 
Indiana  47405 

SCHUEL,  DR.  HERBERT,  Department  of  Biology,  Oakland  University,  Rochester, 
Michigan  48063 

SCOTT,  DR.  ALLAN  C,  Colby  College,  Waterville,  Maine  04901 

SCOTT,  DR.  D.  B.  McNAiR,  Lippincott  Building,  25  Locust  Street,  Philadelphia, 
Pennsylvania  19103 

SCOTT,  SISTER  FLORENCE  MARIE,  Seton  Hill  College,  Greensburg,  Pennsylvania 
15601 

SCOTT,  DR.  GEORGE  T.,  Department  of  Biology,  Oberlin  College,  Oberlin,  Ohio 
44074 

SEARS,  DR.  MARY,  Glendon  Road,  Woods  Hole,  Massachusetts  02543 

SELIGER,  DR.  HOWARD  H.,  McCollum-Pratt  Institute,  The  Johns  Hopkins  Uni- 
versity, Baltimore,  Maryland  21218 

SENFT,  DR.  ALFRED  W.,  Marine  Biological  Laboratory,  Woods  Hole,  Massachu- 
setts 02543 

SEVERINGHAUS,  DR.  AURA  E.,  375  West  250th  Street,  New  York  71,  New  York 

SHAPIRO,  DR.  HERBERT,  6025  North  13th  Street,  Philadelphia  41,  Pennsylvania 

SHAVER,  DR.  JOHN  R.,  Department  of  Zoology,  Michigan  State  University,  East 
Lansing,  Machigan  48824 

SHEDLOVSKY,  DR.  THEODORE,  The  Rockefeller  University,  New  York,  New  York 
10021 

SHEMIN,  DR.  DAVID,  Department  of  Biochemistry,  Columbia  University,  College 
of  Physicians  &  Surgeons,  New  York,  New  York  10032 

SHERMAN,  DR.  I.  W.,  Division  of  Life  Sciences,  University  of  California,  River- 
side, California  92502 

SICHEL,  DR.  FERDINAND  J.  M.,  University  of  Vermont,  Burlington,  Vermont 
05401 

SICHEL,  MRS.  F.  J.  M.,  Department  of  Biology,  Trinity  College,  Burlington, 
Vermont  05401  " 

SILVA,  DR.  PAUL,  Department  of  Botany,  University  of  California,  Berkeley, 
California  94720 

SIMMONS,  DR.  JOHN  E.,  JR.,  Department  of  Biology,  Rice  University,  Houston, 
Texas  77001 

SJODIN,  DR.  RAYMOND  ANDREW,  Department  of  Biophysics,  University  of  Mary- 
land School  of  Medicine,  Baltimore,  Maryland  21201 

SLIFER,  DR.  ELEANOR  H.,  308  Lismore  Avenue,  Glenside,  Pennsylvania 

SLOBODKIN,  DR.  LAWRENCE  BASIL,  Department  of  Zoology,  University  of  Michi- 
gan, Ann  Arbor,  Michigan  48104 

SMELSER,  DR.  GEORGE  K.,  Department  of  Anatomy,  Columbia  University,  College 
of  Physicians  &  Surgeons,  New  York,  New  York  10032 

SMITH,  DR.  DIETRICH  C.,  216  Oak  Forest  Avenue,  Catonsville,  Maryland  21228 

SMITH,  MR.  HOMER  P.,  Marine  Biological  Laboratory,  Woods  Hole,  Massachu- 
setts 02543  • 

SMITH,  MR.  PAUL  FERRIS.  Woods  Hole,  Massachusetts  02543 


REPORT  OF  THE  DIRECTOR  63 

SMITH,  DR.  RALPH  I.,  Department  of  Zoology,  University  of  California,  Berkeley, 
California  94720 

SONNEBORN,  DR.  T.  M.,  Department  of  Zoology,  Indiana  University,  Bloomington, 
Indiana  47405 

SONNENBLICK,  DR.  B.  P.,  Rutgers  University,  40  Rector  Street,  Newark  2,  New 
Jersey 

SPECTOR,  DR.  A.,  Howe  Laboratories,  Harvard  Medical  School,  Boston,  Massa- 
chusetts 02115 

SPEIDEL,  DR.  CARL  C,  1875  Field  Road,  Charlottesville,  Virginia  22903 

SPIEGEL,  DR.  MELVIN,  Department  of  Biological  Sciences,  Dartmouth  College, 
Hanover,  New  Hampshire  03755 

SPINDEL,  DR.  WILLIAM,  Department  of  Chemistry,  Yeshiva  University,  Belfer 
Graduate  School  of  Science,  New  York,  New  York  10033 

SPIRTES,  DR.  MORRIS  ALBERT,  VA  Hospital,  Leech  Farm  Road,  Pittsburgh, 
Pennsylvania  15206 

SPRATT,  DR.  NELSON  T.,  Department  of  Zoology,  University  of  Minnesota,  Minne- 
apolis, Minnesota  55455 

SPYROPOULOS,  DR.  C.  S.,  Building  9,  Room  140,  National  Institutes  of  Health, 
Bethesda,  Maryland  20014 

STARR,  DR.  RICHARD  C.,  Department  of  Botany,  Indiana  University,  Bloomington, 
Indiana  47405 

STEINBACH,  DR.  H.  BURR,  Department  of  Zoology,  University  of  Chicago,  Chi- 
cago, Illinois  60637 

STEINBERG,  DR.  MALCOLM  S.,  Department  of  Biology,  The  Johns  Hopkins  Uni- 
versity, Baltimore,  Maryland  21218 

STEINHARDT,  DR.  JACINTO,  Georgetown  University,  Washington,  D.  C.  20007 

STEPHENS,  DR.  GROVER  C.,  Division  of  Biological  Sciences,  University  of  Cali- 
fornia, Irvine,  California  92664 

STETTEN,  DR.  DsWiTT,  Rutgers  University  Medical  School,  New  Brunswick, 
New  Jersey 

STETTEN,  DR.  MARJORIE  R.,  Rutgers  University  Medical  School,  New  Brunswick, 
New  Jersey 

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

STOKEY,  DR.  ALMA  G.,  c/o  Roger  Stokey,  28  Concord  Road,  Wayland,  Massa- 
chusetts 

STONE,  DR.  WILLIAM,  JR.,  Ophthalmic  Plastics  Laboratory,  Massachusetts  Eye 
&  Ear  Infirmary,  Boston,  Massachusetts 

STRAUS,  DR!  W.  L.,  JR.,  Department  of  Anatomy,  The  Johns  Hopkins  University 
Medical  School,  Baltimore,  Maryland  21205 

STREHLER,  DR.  BERNARD  L.,  4115  Westview  Road,  Baltimore,  Maryland  21218 

STRITTMATTER,  DR.  PHILIPP,  Department  of  Biological  Chemistry,  Washington 
University  Medical  School,  St.  Louis,  Missouri  63110 

STUNKARD,  DR.  HORACE  W.,  American  Museum  of  Natural  History,  Central  Park 
West  at  79th  Street,  New  York,  New  York  10024 

STURTEVANT,  DR.  ALFRED  H.,  California  Institute  of  Technology,  Pasadena, 
California  91 109 


64  MARINE  BIOLOGICAL  LABORATORY 

SUDAK,  DR.  FREDERICK  N.,  Department  of  Physiology,  The  Albert  Einstein  Col- 
lege of  Medicine,  New  York,  New  York  10461 

SULKIN,  DR.  S.  EDWARD,  Department  of  Bacteriology,  University  of  Texas, 
Southwestern  Medical  School,  Dallas,  Texas 

SUSSMAN,  DR.  MAURICE,  Department  of  Biology,  Brandeis  University,  Waltham, 
Massachusetts  02154 

SWANSON,  DR.  CARL  PONTIUS,  Department  of  Biology,  The  Johns  Hopkins  Uni- 
versity, Baltimore,  Maryland  21218 

SWOPE,  MR.  GERARD,  JR.,  570  Lexington  Avenue,  New  York,  New  York  10022 

SZABO,  DR.  GEORGE,  Department  of  Dermatology,  Massachusetts  General  Hospital, 
Boston,  Massachusetts  021 14 

SZENT-GYORGYI,  DR.  ALBERT,  Institute  for  Muscle  Research,  Marine  Biological 
Laboratory,  Woods  Hole,  Massachusetts  02543 

SZENT-GYORGYI,  DR.  ANDREW  G.,  Department  of  Cytology,  Dartmouth  Medical 
.School,  Hanover,  New  Hampshire  03755 

TASAKI,  DR.  ICHIJI,  Laboratory  of  Neurobiology,  National  Institutes  of  Health, 
Bethesda,  Maryland  20014 

TAYLOR,  DR.  ROBERT  E.,  Laboratory  of  Biophysics,  National  Institutes  of  Health, 
Bethesda,  Maryland  20014 

TAYLOR,  DR.  WILLIAM  RANDOLPH,  Department  of  Botany,  University  of  Michi- 
gan, Ann  Arbor,  Michigan  48104 

TAYLOR,  DR.  W.  ROWLAND,  Department  of  Oceanography,  The  Johns  Hopkins 
University,  Baltimore,  Maryland  21218 

TE\¥INKEL,  DR.  Lois  E.,  Department  of  Zoology,  Smith  College,  Northampton, 
Massachusetts  01060 

TRACY,  DR.  HENRY  C.,  3595  Mynders  No.  3,  Memphis  11,  Tennessee 

TRACER,  DR.  WILLIAM,  The  Rockefeller  University,  New  York,  New  York  10021 

TRAVIS,  DR.  D.  M.,  Department  of  Pharmacology,  University  of  Florida,  Gaines- 
ville, Florida  32601 

TRINKAUS,  DR.  J.  PHILIP,  Department  of  Biology,  Yale  University,  New  Haven, 
Connecticut  06520 

TROLL,  DR.  WALTER,  Department  of  Industrial  Medicine,  New  York  University 
College  of  Medicine,  New  York,  New  York  10016 

TWEEDELL,  DR.  KEN  vox  S.,  Department  of  Biology,  University  of  Notre  Dame, 
Notre  Dame,  Indiana  46556 

TYLER,  DR.  ALBERT,  Division  of  Biology,  California  Institute  of  Technology, 
Pasadena,  California  91109 

URETZ,  DR.  ROBERT  B.,  Department  of  Biophysics,  University  of  Chicago,  Chicago, 
Illinois  60637 

VAN  HOLDE,  DR.  KENSAL  EDWARD,  Department  of  Chemistry,  University  of 
Illinois,  Urbana,  Illinois  61803 

VILLEE,  DR.  CLAUDE  A.,  Department  of  Biological  Chemistry,  Harvard  Medical 
School,  Boston,  Massachusetts  02115 

VINCENT,  DR.  WALTER  S.,  Department  of  Anatomy,  University  of  Pittsburgh, 
Pittsburgh,  Pennsylvania  15213 

WAINIO,  DR.  W.  W.,  Bureau  of  Biological  Research,  Rutgers  University,  New 
Brunswick,  New  Jersey  08903 


REPORT  OF  THE  DIRECTOR  65 

WALD,    DR.    GEORGE,    Biological    Laboratories,    Harvard    University,    Cambridge, 

Massachusetts  02138 

WARNER,  DR.  ROBERT  C.,  Department  of  Cbemistry,  New  York  University  Col- 
lege of  Medicine,  New  York,  New  York  10016 
WATERMAN,  DR.  T.  H.,  Department  of  Biology,  272  Gibbs  Research  Laboratory, 

Yale  University,  New  Haven,  Connecticut  06520 
WATSON,  DR.  STANLEY  WAYNE,  Woods  Hole  Oceanographic  Institution,  Woods 

Hole,  Massachusetts  02543 
WEBB,   DR.    MARGUERITE,    Department   of    Physiology    &    Bacteriology,    Goucher 

College,  Baltimore,  Maryland  21204 
WEISS,  DR.  LEON  P.,  Department  of  Anatomy,  The  Johns  Hopkins  University 

School  of  Medicine,  Baltimore,  Maryland  21205 

WENRICH,  DR.  D.  H.,  Department  of  Zoology,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania  19104 
WERMAN,   DR.   ROBERT,   Institute   of   Psychiatric   Research,    Indiana   University 

Medical  Center,  1 100  \Vest  Michigan  Street,  Indianapolis  7,  Indiana 
WHITAKER,  DR.  DOUGLAS  M.,  320  Good  Hill  Road,  Kentfield,  California 
WHITE,  DR.  E.  GRACE,  1312  Edgar  Avenue,  Chambersburg,  Pennsylvania 
WHITING,  DR.  ANNA  R.,  535  \Vest  Vanderbilt  Drive,  Oak  Ridge,  Tennessee  37831 
WHITING,  DR.  PHINEAS,  535  West  Vanderbilt  Drive,  Oak  Ridge,  Tennessee  37831 
WICHTERMAN,  DR.  RALPH,  Department  of  Biology,  Temple  University,  Philadel- 
phia, Pennsylvania  19122 

WICKERSHAM,  MR.  JAMES  H.,  791  Park  Avenue,  New  York,  New  York  10021 
WIERCINSKI,   DR.    FLOYD   J.,   Department   of   Biology,    Illinois   Teachers    College 

North,  5500  North  St.  Louis  Avenue,  Chicago,  Illinois  60625 
WIGLEY,  DR.  ROLAND  L.,  U.  S.  Fish  &  Wildlife  Service,  Bureau  of  Commercial 

Fisheries,  Woods  Hole,  Massachusetts  02543 
WILBER,    DR.    C.    G.,    Marine    Laboratories,    University    of    Delaware,    Newark, 

Delaware  197 11 
WILCE,  DR.  ROBERT  THAYER,  Department  of  Botany,  University  of  Massachusetts, 

Amherst,  Massachusetts  01002 
WILLIER,  DR.   B.   H.,   Department   of  Biology,   The   Johns   Hopkins   University, 

Baltimore.  Maryland  21218 
WILSON.  DR.  T-  WALTER,  Department  of  Biology,  Brown  University,  Providence, 

Rhode  Island  02912 
WILSON,  DR.  T.  HASTINGS,  Department  of  Physiology,  Harvard  Medical  School, 

Boston,  Massachusetts  02115 
WILSON,  DR.  WALTER  L.,  Department  of  Biology,  Oakland  University,  Rochester, 

Michigan  48063 
WINTERS,  DR.  ROBERT  WAYNE,  Department  of  Pediatrics,  Columbia  University, 

College  of  Physicians  &  Surgeons,  New  York,  New  York  10032 
WITSCHI.  DR.  EMIL,  UTniversitat  Basel,  Anatomisches  Institut,   Pestalozzistrasse 

20,  Basel,  Switzerland 
WITTENBERG,  DR.  JONATHAN  B.,  Department  of  Physiology  &  Biochemistry,  The 

Albert  Einstein  College  of  Medicine,  New  York,  New  York  10461 
WRIGHT,  DR.  PAUL  A.,  Spaulding  Building,  Department  of  Zoology,  University 

of  New  Hampshire,  Durham,  New  Hampshire  03824 


66 


MARINE  BIOLOGICAL  LABORATORY 


WRINCH,  DR.  DOROTHY,  Department  of  Physics,  Smith  College,  Northampton, 
Massachusetts  01060 

WYTTENBACH,  DR.  CHARLES  R.,  Department  of  Anatomy,  University  of  Chicago, 
Chicago,  Illinois  60637 

YNTEMA,  DR.  C.  L.,  Department  of  Anatomy,  State  University  of  New  York 
College  of  Medicine,  Syracuse,  New  York  13210 

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

ZACKS,  DR.  SUMNER  IRWIN,  The  Pennsylvania  Hospital,  University  of  Pennsyl- 
vania School  of  Medicine,  Philadelphia,  Pennsylvania  19104 

ZIMMERMAN,  DR.  A.  M.,  Department  of  Zoology,  University  of  Toronto,  Toronto 
5,  Ontario,  Canada 

ZINN,  DR.  DONALD  J.,  Department  of  Zoology,  University  of  Rhode  Island,  King- 
ston, Rhode  Island  02881 

ZIRKLE,  DR.  RAYMOND  E.,  Department  of  Radiology,  University  of  Chicago,  Chi- 
cago, Illinois  60637 

ZORZOLI,  DR.  ANITA,  Department  of  Physiology,  Vassar  College,  Poughkeepsie, 
New  York  12601 

ZULLO,  DR.  VICTOR  A.,  Systematics-Ecology  Program,  Marine  Biological  Labora- 
tory, Woods  Hole,  Massachusetts  02543 

ZWEIFACH,  DR.  BENJAMIN,  New  York  University,  Bellevue  Medical  Center, 
New  York,  New  York  10016 

ZWILLING,  DR.  EDGAR,  Department  of  Biology,  Brandeis  University,  Waltham, 
Massachusetts  02 154 

ASSOCIATE  MEMBERS 


ALLEN,   Miss   CAMILLA  K. 
ALTON,   MRS.   BENJAMIN 
ANDRES,  MRS.  WILLIAM 
ARMSTRONG,  MRS.  PHILIP  B. 

AUCLAIR,    DR.    AND    MRS.    WALTER 

BACON,  MR.  AND  MRS.  ROBERT 
BAKALAR,  MR.  AND  MRS.  DAVID 
BALL,  MRS.  ERIC  G. 
BARBOUR,  MRS.  Lucius  H. 
BARROWS,  MRS.  MARY  PRENTICE 
BARTOW,  MR.  AND  MRS.  CLARENCE  W. 
BARTOW,  MRS.  FRANCIS  D. 
BARTOW,  MRS.  PHILIP 
BEALE,  MR.  AND  MRS.  E.  F. 
BELL,  MRS.  ARTHUR  W. 
BIGELOW,  MRS.  ROBERT  P. 
BRADLEY,  DR.  AND  MRS.  CHARLES 
BROWN,  DR.  AND  MRS.  F.  A.,  JR. 
BROWN,  MRS.  THORNTON 
BURDICK,  DR.  C.  LALOR 
BUTLER,  DR.  AND  MRS.  E.  G. 
CAHOON,  MRS.  SAMUEL  T.,  SR. 
CALKINS,  MRS.  GARY  N. 


CALKINS,  MR.  AND  MRS.  G.  N.,  JR. 

CAREY,  Miss  CORNELIA 

CARLTON,  MR.  WINSLOW  G. 

CLAFF,  MRS.  C.  LLOYD 

CLAFF,  MR.  MARK  M. 

CLARK,  MRS.  JAMES  B. 

CLARK,  MRS.  LEROY 

CLARK,  MRS.  ELIOT  R. 

CLARK,  MR.  AND  MRS.  VAN  ALAN 

CLOWES,  MR.  ALLEN  W. 

CLOWES,  DR.  AND  MRS.  GEORGE  H.  A., 

JR. 

COSTELLO,  MRS.  DONALD  P. 

CRAMER,  MR.  AND  MRS.  IAN  D.  W. 

CRANE,  MR.  JOHN 

CRANE,  JOSEPHINE  B.,  FOUNDATION 

CRANE,  Miss  LOUISE 

CRANE,  MRS.  ROBERT 

CRANE,  MR.  STEPHEN 

CRANE,  MRS.  W.  CAREY 

CRANE,  MRS.  W.  MURRAY 

CROCKER,  MR.  AND  MRS.  PETER  J. 

CROSSLEY,  Miss  DOROTHY 


REPORT  OF  THE  DIRECTOR 


67 


CROSSLEY,  MR.  AND  MRS.  ARCHIBALD  M. 
CROWELL,  MR.  AND  MRS.  PRINCE  S. 
CURTIS,  DR.  AND  MRS.  WILLIAM  D. 
DAIGNAULT,  MR.  AND  MRS.  A.  T. 
DANIELS,  MR.  AND  MRS.  B.  G. 
DAY,  MR.  AND  MRS.  POMEROY 
DRAPER,  MRS.  MARY  C. 
DREYER,  MRS.  F.  A. 
DuBois,  DR.  AND  MRS.  A.  B. 
EDDS,  DR.  AND  MRS.  MAC  V.,  JR. 
ELSMITH,  MRS.  DOROTHY  O. 
ENDERS,  MRS.  FREDERICK 
EWING,  MR.  WILLIAM 
FAXON,  DR.  NATHANIEL  W. 
FERGUSON,  MRS.  JAMES  J. 
FIRESTONE,  MR.  AND  MRS.  EDWIN 
FISHER,  MRS.  B.  C. 
FRANCIS,  MR.  LEWIS  H.,  JR. 
GABRIEL,  DR.  AND  MRS.  MORDECAI  L. 
GAISER,  DR.  AND  MR.  DAVID  W. 
GALTSOFF,  MRS.  PAUL  S. 
GAMBLE,  DR.  AND  MRS.  RICHARD  B. 
GARLOCK,  MR.  AND  MRS.  ROBERT 
GlFFORD,  MR.  AND  MRS.  JOHN  A. 

GlLCHRIST,    MR.   AND   MRS.   JOHN   M. 

GILDEA,  DR.  MARGARET  C.  L. 
GILLETTE,  MR.  AND  MRS.  ROBERT  S. 
GLAZEBROOK,  MRS.  JAMES  R. 
GOLDMAN,  DR.  AND  MRS.  ALLEN  S. 
GREEN,  Miss  GLADYS  M. 
GREENE,  MRS.  WILLIAM  C. 
GREER,  MR.  AND  MRS.  WILLIAM  H.,  JR. 
GREIF,  DR.  AND  MRS.  ROGER 
GULESIAN,  MRS.  PAUL  J. 

GUREWICH,    DR.    AND    MRS.    V. 

HAMLEN,  MR.  AND  MRS.  J.  MONROE 
HANDLER,  DR.  AND  MRS.  PHILIP 
HANNA,  MR.  AND  MRS.  THOMAS  C. 
HARRINGTON,  MR.  AND  MRS.  R.  D. 
HARVEY,  DR.  AND  MRS.  E.  NEWTON,  JR. 
HARVEY,  DR.  AND  MRS.  RICHARD 
HERVEY,  MRS.  JOHN  P. 

HlRSCHFELD,   MRS.   NATHAN   B. 

HOPKINS,  MRS.  RALPH  H. 
HOUSTON,  MR.  AND  MRS.  HOWARD  E. 
JEWETT,  MR.  AND  MRS.  G.  F.,  JR. 
JONES,  MR.  AND  MRS.  DEWITT  C.,  JR. 
KAHN,  MR.  AND  MRS.  ERNEST 


KEITH,  MRS.  HAROLD  C. 
KEITH,  MR.  AND  MRS.  J.  R. 
KEOSIAN,  MRS.  JOHN 

KlNNARD,  MR.  AND  MRS.  L.  RlCHARD 
KOLLER,   DR.   AND   MRS.   LEWIS  R. 

LAWRENCE,  MR.  AND  MRS.  MILFORD  R. 
LEMANN,  MRS.  LUCY  BENJAMIN 
LILLIE,  MRS.  KARL  C. 
LOBB,  MR.  AND  MRS.  JOHN 
LOEB,  DR.  AND  MRS.  ROBERT  F. 

LOVELL,    MR.   AND   MRS.    HOLLIS   R. 

MARSLAND,  DR.  AND  MRS.  D.  A. 
MARVIN,  DR.  DOROTHY 
MAST,  MRS.  S.  O. 
MATHER,  MR.  FRANK  J.,  Ill 
MAVOR,  MRS.  JAMES  W. 

McCuSKER,    MR.   AND   MRS.    PAUL  T. 

MCELROY,  DR.  AND  MRS.  W.  D. 

McGlLLICUDDY,  DR.   AND   MRS.  JOHN  J. 

MCKELVY,  MR.  JOHN  E. 

McLANE,   MRS.  HUNTINGTON 

McViTTY,  MRS.  A.  E. 

MEIGS,  MR.  AND  MRS.  ARTHUR 

MEIGS,  DR.  AND  MRS.  J.  WISTER 

MITCHELL,  MRS.  PHILIP 

MIXTER,  MRS.  WILLIAM  JASON 

MOTLEY,  MRS.  THOMAS 

MUELLNER,  DR.  AND  MRS.  S.  RlCHARD 

NEWTON,  Miss  HELEN  K. 

NICHOLS,  MRS.  GEORGE 

THE  AARON  E.  NORMAN  FUND,  INC. 

PACKARD,  MRS.  CHARLES 

PARPART,  MRS.  ARTHUR  K. 

PARK,  MRS.  FRANKLIN  A. 

PARK,  MR.  MALCOLM  S. 

PATTEN,  MRS.  BRADLEY 

PENNINGTON,  Miss  ANNE  H. 

PHILIPPE,  MR.  PIERRE 

PUTNAM,  MR.  AND  MRS.  WILLIAM  A., 

Ill 

REDFIELD,  DR.  AND  MRS.  ALFRED 
REZNIKOFF,  DR.  AND  MRS.  PAUL 
RIGGS,  MR.  AND  MRS.  LAWRENCE,  III 
RIVINUS,  MRS.  F.  M. 
ROGERS,  MRS.  CHARLES  E. 
ROOT,  DR.  AND  MRS.  WALTER  S. 
RUDD,  MRS.  H.  W.  DWIGHT 
SANDS,  Miss  ADELAIDE  G. 


68 


MARINE  BIOLOGICAL  LABORATORY 


SAUNDERS,  MR.  AND  MRS.  LAWRENCE 
SCHWARTZ,  MRS.  VICTOR  B. 
SHIVERICK,  MRS.  ARTHUR 
SINCLAIR,  MR.  AND  MRS.  W.  RICHARD- 
SON 

SMITH,  MRS.  HOMER  P. 
SPEIDEL,  MRS.  CARL  C. 
STONE,  MR.  AND  MRS.  LEO 
STONE,  MRS.  SAMUEL  M. 
STONE,  DR.  AND  MRS.  WILLIAM,  JR. 
STRAUS,  MR.  AND  MRS.  DONALD  B. 
STUNKARD,  MRS.  HORACE 
SWIFT,  MR.  E.  KENT,  JR. 
S  \YOPE,  MR.  DAVID 
SWOPE,  MR.  AND  MRS.  GERARD,  JR. 
SWOPE,  Miss  HENRIETTA  H. 
TOMPKINS,  MR.  AND  MRS.  B.  A. 


WARREN,  DR.  AND  MRS.  SHIELDS 
WEBSTER,  MRS.  EDWIN  S. 
WHITELEY,  Miss  MABEL  W. 
WHITELEY,  MR.  AND  MRS.  GEORGE  C., 

JR. 

WHITING,  DR.  AND  MRS.  PHINEAS  W. 
WHITNEY,  MRS.  GEORGE 
WlCHTERMAN,  MRS.  RALPH 
WlCKERSHAM,  MRS.  JAMES  H. 
WILHELM,  DR.  HAZEL  S. 
WILSON,  MRS.  EDMUND  B. 
WILSON,  DR.  MAY  G. 
WINTERS,  DR.  ROBERT  W. 
WOLFE,  DR.  CHARLES 
WOLFINSOHN,  MRS.  WOLFE 
WRINCH,  DR.  DOROTHY 
YNTEMA,  MRS.  CHESTER  L. 


V.    REPORT  OF  THE  LIBRARIAN 

This  year  the  old  office  section  of  the  Library  was  changed  to  a  small  reading 
reference  room.  Built-in  bookcases  line  the  walls  and  special  racks  hold  the 
week's  receipts  of  journals.  Leather  furniture,  carpet  and  draperies  make  it  a 
comfortable  and  attractive  room.  New  chairs  were  ordered  for  the  large  main 
reading  room  and  plans  were  made  for  8  carrels  and  a  typing  room  on  the  third  floor. 

The  1965  "Serial  Publications"  list  was  published.  In  300  pages  this  book 
lists  the  nearly  3,600  separate  journal  titles  held  by  the  Library.  A  large  number 
of  libraries  have  purchased  this  publication  and  it  has  almost  doubled  our  inter- 
library  loan  requests.  During  the  year  we  received  and  serviced  1,580  requests 
from  colleges  and  universities  throughout  the  country.  We  also  received  many 
requests  from  Germany,  India  and  Japan.  We  made  166  requests  from  other 
libraries  for  the  use  of  investigators  here. 

One  thousand  and  eight  hundred  volumes  were  sent  to  the  bindery  in  1965  and 
we  added  112  new  journals  to  our  collection.  We  made  an  exact  count  of  the 
number  of  volumes  in  the  library  and  our  total  holding  is  now  126,423.  This 
figure  does  not  include  our  235,000  reprints. 

Total  number  of  serial  titles  in  library 3,675 

Number  received  currently 2,025 

On  subscription   840 

On  exchange   899 

On  gift  basis 286 

Total  number  of  reference  books  and  monographs   15,795 

Number  added  in  1965 411 

Received  from  book  exhibits 109 

Total  number  of  reprints   235,663 

Number  added  in  1965   4,312 

Respectfully  submitted, 

JANE  FESSENDEN, 

Librarian 


REPORT  OF  THE  TREASURER  69 

VI.    REPORT  OF  THE  TREASURER 

The  market  value  of  the  general  Endowment  Fund  and  the  Library  Fund  at 
December  31,  1965,  amounted  to  $2,502,903  as  against  book  value  of  $1,261,842. 
This  compares  with  values  of  $2,370,890  and  $1,235,860,  respectively,  at  the  end 
of  the  preceding  year.  The  average  yield  on  the  Securities  was  3.34%  of  the 
market  value  and  6.62%  of  the  book  value.  Uninvested  principal  cash  in  the  above 
accounts  at  the  end  of  the  year  was  $1,878.  Classification  of  the  securities  held  in 
the  Endowment  Fund  appears  in  the  Auditor's  report. 

The  market  value  of  the  pooled  securities  as  of  December  31,  1965,  was  $702,137 
as  compared  with  $580,677  being  the  market  value  as  of  December  31,  1964,  the 
increase  being  the  result  of  addition  of  funds  from  Herbert  W.  Rand  Fellowship, 
Mellon  Foundation  and  Mary  Rogick  Fund.  Uninvested  principal  cash  at  the 
end  of  the  year  was  $13,415.  Book  value  of  securities  in  this  account  at  the 
beginning  of  this  year  was  $562,547  compared  with  $646,802  at  the  close  of  1965. 
The  average  yield  on  market  value  was  2.93%  and  3.18%  on  book  value. 

The  proportionate  interest  in  the  Pool  Fund  Account  of  the  various  Funds  as  of 
December  31,  1965,  is  as  follows: 

Pension  Funds  21.217% 

General  Laboratory  Investment 28.909% 

F.  R.  Lillie  Memorial  Fund   3.151% 

Anonymous  Gift 1 .080% 

Other : 

Bio  Club  Scholarship  Fund 815% 

Rev.  Arsenius  Boyer  Scholarship  Fund 998% 

Gary  N.  Calkins  Fund 937% 

Allen  R.  Memhard  Fund 182% 

Lucretia  Crocker  Fund 3.41 1  % 

E.  G.  Conklin  Fund 576% 

Jewett  Memorial  Fund 303% 

M.  H.  Jacobs  Scholarship  Fund 41 1  % 

Herbert  W.  Rand  Fellowship 22.167% 

Mellon  Foundation 13.760% 

Mary  Rogick  Fund  2.083% 

Donations  from  the  MBL  Associates  for  1965  were  $7,385.00,  as  compared 
with  $4,830.00  for  1964.  Unrestricted  gifts  from  foundations,  societies  and  com- 
panies amounted  to  $53,950. 

During  the  year,  we  administered  the  folowing  grants : 

Investigators  Training            MBL  Institutional 

12NIH  3NIH                      3NIH 

4NSF  2NSF                      2  NSF 

1  Ford  2  ONR 

2  ONR  1 AEC 
1  Commonwealth  1  Ford 
1  Whitehall 

21  ~5  ~9 


70  MARINE  BIOLOGICAL  LABORATORY 

The  rate  of  overhead  on  grants  to  investigators  is  20 %  based  on  the  amount 
expended.  The  overhead  on  these  grants  for  this  year  amounted  to  $94,791  as 
compared  with  $81,239  for  the  preceding  year.  A  new  formula  for  determining  an 
indirect  Cost  Rate  was  introduced  this  year  by  NIH  which  computed  our  rate  of 
overhead  to  be  34.3%  of  direct  cost  and  73.36%  of  salaries  and  wages. 

The  Lillie  Fellowship  Fund  with  a  market  value  of  $158,524  and  a  book  value 
of  $92,887,  as  well  as  the  investment  in  General  Biological  Supply  House  with  a 
book  value  of  $12,700,  is  carried  in  the  Balance  Sheet  item  "Other  Investments." 

The  General  Biological  Supply  House  fiscal  year  ended  June  30,  1965,  and 
had  a  profit  after  taxes  of  $275,080  as  compared  to  $309,651  in  1964,  $241,616  in 

1963,  $302,657  in  1962,  and  $302,851  in  1961. 

During  the  period  covered  by  this  report  the  Marine  Biological  Laboratory 
received  dividends  from  the  General  Biological  Supply  House  of  $63,500  as  against 
$63,500  in  1964,  $42,164  in  1963,  $38,000  in  1962  and  $33,020  in  1961. 

The  following  is  a  statement  of  the  auditors : 

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

We  have  examined  the  balance  sheet  of  Marine  Biological  Laboratory  as  at 
December  31,  1965,  the  related  statement  of  operating  expenditures,  income  and 
current  fund  and  statement  of  funds  for  the  year  then  ended.  Our  examination 
was  made  in  accordance  with  generally  accepted  auditing  standards,  and  accordingly 
included  such  tests  of  the  accounting  records  and  such  other  auditing  procedures  as 
we  considered  necessary  in  the  circumstances.  We  examined  and  have  reported 
on  financial  statements  of  the  Laboratory  for  the  year  ended  December  31,  1965. 

In  our  opinion,  the  accompanying  financial  statements  present  fairly  the  assets, 
liabilities  and  funds  of  Marine  Biological  Laboratory  at  December  31,  1965,  and 

1964,  and  the  results  of  its  operation  for  the  years  then  ended  on  a  consistent  basis. 
The  supplementary  schedules  included  in  this  report  were  obtained  from  the 

Laboratory's  records  in  the  course  of  our  examination  and  in  our  opinion,  are  fairly 
stated  in  all  material  respects  in  relation  to  the  financial  statements,  taken  as  a 
whole. 

Boston,  Massachusetts 

May  2,  1966  LYBRAND,  Ross  BROS.  AND  MONTGOMERY 

ALEXANDER  T.  DAIGNAULT, 

Treasurer 


REPORT  OF  THE  TREASURER  71 

MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEETS 
December  31,  1965  and  1964 


Investments 

1965  1964 

Investments  held  by  Trustee : 

Securities,  at  cost  (approximate  market  quotation  1965— $2,502,903)  $1,261,842    $1,235,860 
Cash     1,878  5,595 


1,263,720  1,241,455 
Investments  of  other  endowment  and  unrestricted  funds : 

Pooled  investments,  at  cost    (approximate  market  quotation   1965— 

$640,065)  less  $5,728  temporary  investment  of  current  fund  cash  522,591  479,344 

Other    investments    119,352  118,888 

Cash     55,608  39,522 

Accounts  receivable  7,384  10,664 


$1,968,655    $1,889,873 


Plant  Assets 

Land,  buildings,  library  and  equipment  (note)    5,481,019      5,136,289 

Less  allowance  for  depreciation  (note)    1,449,145       1,378,887 


4,031,874  3,757,402 

Construction  in  progress   41,253  109,215 

Cash  90,015 

Short-term  investments,  at  cost  50,000  192,360 


$4,213,142    $4,058,977 


Current  Assets 

Cash 35,596  79,637 

Temporary  investment  in  pooled  securities  5,728  5,728 

U.  S.  Treasury  bills,  at  cost  74,210  96,045 

Accounts  receivable  (U.  S.  Government,  1965— $78,108;  1964— $84,793)    .  139,079  138,489 

Inventories  of  supplies  and  Bulletins  35,493  33,401 

Prepaid  insurance  and  other  1,893  6,844 


$  291,999    $   360,144 


72  MARINE  BIOLOGICAL  LABORATORY 

MARINE  BIOLOGICAL  LABORATORY 

BALANCE  SHEETS 
December  31,  1965  and  1964 

Invested  Funds 

1965  1964 

Endownment  funds  given  in  trust  for  benefit  of  the  Marine  Biological 

Laboratory     $1,263,720    $1,241,455 


Endowment  funds  for  awards  and  scholarships : 

Principal    322,135         295,710 

Unexpended  income  24,400  14,289 


346,535  309,999 

Unrestricted  funds  functioning  as  endowment  206,378  206,378 

Retirement  fund   135,952  123,298 

Pooled  investments — accumulated  gain 16,070  8,743 


$1,968,655    $1,889,873 


Plant  Funds 

Funds  expended  for  plant,  less  retirements  5,522,272      5,245,504 

Less  allowance  for  depreciation  charged  thereto  1,449,145       1,378,887 


4,073,127      3,866,617 
Unexpended  plant  funds  140,015         192,360 


$4,213,142    $4,058,977 


Current  Liabilities  and  Funds 

Accounts  payable  and  accrued  expenses 53,563  33,315 

Advance   subscriptions    16,074  10,926 

Unexpended   grants— research    64,094  99,000 

Unexpended  balances  of  gifts  for  designated  purposes 18,772  17,995 

Current    fund    139,496  198,908 


$   291,999    $   360,144 


Note — 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. 


REPORT  OF  THE  TREASURER  73 

MARINE  BIOLOGICAL  LABORATORY 

STATEMENTS  OF  OPERATING  EXPENDITURES,  INCOME  AND  CURRENT  FUND 
Years  Ended  December  31,  1965  and  1964 

Operating  Expenditures 

1965  1964 

Research  and  accessory  services $   294,798  $    293,396 

Instruction     196,509  160,820 

Library  and  publications  (including  book  purchases — 

1965,  $39,356;  1964,  $24,304)    102,595  80,416 

Direct  costs  on  research  grants  514,709  493,890 

Direct  costs  on  institution  support  grants   161,669  150,450 


1,270,280  1,178,972 

Administration  and  general  125,298  112,453 

Plant  operation  and  maintenance  131,885  139,684 

Dormitories  and  dining 193,298  175,696 

Additions  to  plant  from  current  fund 128,448  5,045 


1,849,209      1,611,850 
Less  depreciation  included  in  plant  operation  and  dormitories   and 

dining  above  but  charged  to  plant  funds 71,461  67,437 


1,777,748      1,544,413 


Income 

Research  fees    96,745  88,240 

Accessory  services  (including  sales  of  biological  specimens — 

1965,  $37,586;  1964,  $42,051)   107,204  114,181 

Instruction  fees 28,525  28,470 

Library  fees,  Bulletins,  subscriptions  and  other  53,687  49,393 

Dormitories  and  dining  income 149,754  133,759 

Grants  for  support  of  institutional  activities  : 

Instruction  and  training    198,404  138,540 

Support    services    161,669  150,450 

General    106,445  109,219 

Reimbursements  and  allowances  for  direct  and  indirect  costs  on  specific 

research  grants  592,225  575,129 

Gifts  used  for  current  expenses  66,368  22,135 

Investment  income  used  for  current  expenses 157,310  156,676 


1,718,336      1,566,192 


Excess  current  income  (expenditures)    (59,412)          21,779 

Current  fund  balance  January  1    198,908         177,129 


Current  fund  balance  December  31  $    139,496    $    198,908 


74 


MARINE  BIOLOGICAL  LABORATORY 


MARINE  BIOLOGICAL  LABORATORY 


STATEMENT  OF  FUNDS 


Year  Ended  December  31,  1965 


Balance 

December 

31, 1964 

Invested  funds  $1,889,873 


Unexpended  plant  funds  $    192,360 


Gifts  and  Invest- 
Other  ment 
Receipts  Income 

$     78,513  $169,627 

50,000  7,944 


Used  for  Other  Balance 

Current  Expcndi-  December 

Expenses  turcs  31, 1965 

$    154,291  $  15,067  $1,968,655 


110,289      $    140,015 


Unexpended  research 

grants     $     99,000        1,023,837 

Unexpended  gifts  for 

designated  purposes  $      17,995  72,052 

Current  fund    $    198,908  (59,412)  (1) 

$1,164,990      $177,571 

Gifts  148,477 

Grants  for  research, 

training  and  support  1,023,837 

Appropriated  from 

current  income 

and  other  22,496 

Net  gain  on  sale  of 

securities 29,592 

(1)  Excess  of  current 

expenditures  over 

income     (59,412) 

$1,164,990 

Expended  for  construc- 
tion and  renovation 
of  facilities  

Scholarship  awards   . . . 

Payments  to  pensioners 

Other 


1,058,743 


$     64,094 


66,368  4,907      $     18,772 


$   139,496 


$1,279,402   $130,263 


110,289 

1,101 

14,578 

4,295 

$130,263 


REPORT  OF  THE  TREASURER 


75 


MARINE  BIOLOGICAL  LABORATORY 

SUMMARY  OF  INVESTMENTS 
December  31,  1965 


Cost 


%of 
Total 


Market 
Quotations 


Investment 
%  of       Income 
Total         1965 


Securities  held  by  Trustee  : 
General  endowment  fund : 

U.  S.  Government  securities $       4,993  .5  $       4,973  .2      $     5,008 

Corporate  bonds 657,502  62.5  631,091  30.7  21,432 

Preferred  stocks    54,422  5.2  57,000  2.8  2,300 

Common  stocks    334,791  31.8  1,363,906  66.3  41,384 

1,051,708  logo  2,056,970  100.0  70,124 

General  Educational  Board 
endowment  fund  : 

U.  S.  Government  securities 18,953  9.0  18,405  4.1  1,033 

Other  bonds   115,718  55.1  111,604  25.0  4,515 

Preferred   stocks    15,641  7.4  14,125  3.2  805 

Common  stocks   _  59,822  28.5  301,799  67.7  7,088 

210,134  100.0  445,933  100.0  13,441 

Total  securities  held  by  Trustee  $1,261,842  $2,502,903  $  83,565 

Investments  of  other  endowment  and 

unrestricted  funds : 
Pooled  investments : 

U.  S.  Government  securities 32,984          6.3  31,782          5.0  1,681 

Corporate  bonds 160,391        30.4  153,798        24.0  5,086 

Common  stocks   334,944        63.3          454,485        71.0          13,859 

528,319     TOOO      $   640,065      100.0         20,626 

Less  temporary  investment  of 

current  fund  cash   (5,728)  (246) 

522,591  20,380 

Other  investments : 

U.  S.  Government  securities 27,943  1,128 

Other  bonds    15,029  749 

Preferred  stocks    3,728  130 

Common  stocks   58,887  65,768 

Real  estate    13,765                                                        

119,352  67,775 

Total  investments  of  other 
endowment  and 
unrestricted  funds   $   641,943  88,155 

Total  investment  income  171,720 

Custodian's  fees  charged  thereto (2,093) 

Investments  income  distributed  to  funds  . .  169,627 

Plant  investments  : 

Federal  agency  and  corporate  bonds  . .  $     50,000  7,944 

Current  investments : 
U.  S.  Treasury  bills, 

due  February  3,  1966  $     74,210 

Temporary  investment 

in  pooled  securities  $       5,728  246 

3,019 
$180,590 


CORRELATION  OF  LYSOSOMAL  ACTIVITY  AND 
INGESTION  BY  THE  MANTLE  EPITHELIUM1'2 

GERRIT  BEVELANDER  AND  HIROSHI  NAKAHARA 

Department  of  Histology.  The  University  of  Texas  Dental  Branch,  Houston,  Texas  77025, 
and  The  lieriiiuda  Biological  Station  for  Research 

Despite  the  fact  that  the  structure  and  function  of  the  molluscan  mantle  has 
occupied  the  attention  of  investigators  for  well  over  one  hundred  years  the  role 
of  the  outer  mantle  epithelium  in  the  elahoration  of  the  shell  is  as  yet  poorly  under- 
stood. On  the  basis  of  histochemical  and  radioautographic  studies  (Bevelander 
and  Benzer,  1948;  Bevelander,  1952),  it  was  shown  that  both  mucus  secreted 
by  the  mantle  and  Ca45  derived  from  the  sea  water  environment  were  incorporated 
in  the  shell.  Several  other  authors  (Ojima,  1952;  Wilbur,  1960;  Kado,  1960; 
Tsujii,  1960)  have  also  remarked  on  the  fact  that  mucus  and  other  formed 
substances  play  a  significant  role  in  shell  formation.  It  was  further  shown  by 
Nakahara  (1962a)  that  Ca45  injected  into  the  adductor  muscle  was  incorporated 
into  the  mantle  mucus  which  later  became  an  integral  part  of  the  calcified  nacre. 

The  fine  structure  of  the  mantle  of  Fabulina  was  described  by  Kawaguti  and 
Ikemoto  (1962).  These  authors  contend  that  the  outer  surface  of  the  mantle  is 
made  up  of  three  cell  types.  Mention  is  made  of  the  presence  of  microvilli,  mito- 
chondria, Golgi  bodies,  endoplasmic  reticulum,  but  the  presence  of  lysosomes  is 
not  mentioned.  Furthermore  no  specific  function  of  the  various  cell  types 
described  was  suggested. 

The  above  references,  although  by  no  means  complete  as  to  the  number  of 
published  studies  dealing  with  the  structural  and  functional  aspects  of  the  mantle 
epithelium,  do,  however,  indicate  that  the  chief  interest  and  concern  of  these 
investigations  was  the  identification  of  various  substances  elaborated  and  secreted 
by  the  epithelium  and  their  subsequent  identification  in  the  formed  shell. 

It  was  previously  shown  by  Nakahara  (1962)  that  the  outer  surface  of  the 
mantle  of  Pinctada  jnartensii  ingests  carmine  particles  placed  in  the  extrapallial 
fluid.  In  an  attempt  to  clarify  and  amplify  the  above  observations  we  have 
examined  this  problem  in  more  detail,  utilizing  electron  microscopy,  histochemistry 
as  well  as  experimental  physiological  procedures. 

Briefly  stated,  this  report  deals  with  a  description  of  the  results  obtained 
following  the  procedures  mentioned  above,  by  means  of  which  we  have  demon- 
strated differential  absorption  by  the  outer  surface  epithelium  of  the  mantle.  A 
description  of  the  intracellular  localization  of  acid  phosphatase,  lipids  and  muco- 
polysaccharides  at  the  light  level  and  the  detailed  structure  of  the  ingesting  cell 
follows.  Finally  the  locus  of  ingested  material,  together  with  other  remarks 
concerning  lysosomal  activity  in  the  mantle  cells,  is  described. 

1  This  investigation  was  supported  (in  part)  by  grant  DE-01825-04,  N.I.D.R.,  U.S.P.H.S. 

2  Contribution  No.  385  from  the  Bermuda  Biological  Station. 

76 


INGESTION  BY  THE  MANTLE  EPITHELIUM  77 

MATERIALS  AND  METHODS 

This  study  was  carried  out  on  the  calico  clam,  Macrocallista  niaculata.  Speci- 
mens were  collected  in  Bermuda  waters  and  for  purposes  of  histological  and  E.M. 
studies  the  mantle  was  removed  and  fixed  in  ( 1 )  a  1  %  solution  of  osmium  tetroxide 
in  a  phosphate  buffer  at  a  pH  of  7.4  for  one  hour  at  25°  C.  (2)  Other  specimens 
were  fixed  in  a  6%  solution  of  glutaraldehyde  buffered  in  phosphate  at  pH  7.4  for 
4-6  hours  at  25°  C.  Following  this  treatment  the  tissues  were  repeatedly  washed 
in  phosphate  buffer  and  fixed  in  osmium  tetroxide  for  one  hour  as  described  above. 
Following  fixation,  tissues  were  routinely  dehydrated,  embedded  in  Araldite  and 
were  then  sectioned  for  E.M.  observations  (thin  sections)  and  also  1-2  ^  sections 
which  were  stained  with  a  40%  alcoholic  solution  of  1 : 1000  toluidine  blue  for 
observations  with  the  light  microscope. 

For  the  purposes  of  ascertaining  the  ability  of  the  shell-forming  epithelium  to 
absorb  participate  material  the  following  procedures  were  carried  out:  0.1  cc.  of 
a  2%  filtered  sea  water  suspension  of  finely  ground  carmine  particles  was  injected 
into  the  pallial  space.  Specimens  were  then  placed  in  tanks  furnished  with 
running  sea  water  at  a  temperature  of  25-26°  C.  for  a  period  of  1-3  days.  At  the 
termination  of  these  periods  they  were  removed  from  the  tanks,  the  mantle  was 
excised,  fixed  in  Ca-formol,  dehydrated  and  then  sectioned  for  subsequent  observa- 
tion. This  procedure  was  also  carried  out  with  several  other  specimens  in  which 
a  sea  water-colloidal  gold  (Hartman-Leddon)  mixture  was  injected  in  place  of 
the  carmine.  The  colloidal  gold  mixture  was  prepared  just  prior  to  injection  by 
adding  equal  amounts  of  aliquots  of  colloidal  gold  and  sea  water  concentrated  to 
50%  of  their  original  volumes. 

To  aid  in  the  identification  of  the  ingestion  sites,  three  histochemical  procedures 
were  utilized.  They  consisted  in  testing  the  mantle  epithelium  for  the  presence 
of  acid  phosphatase,  according  to  the  method  of  Gomori  (1950),  in  the  treatment 
of  the  mantle  to  ascertain  the  presence  of  lipids  by  the  Sudan  black  B  method 
(Chiffele  and  Putt,  1950),  and  the  PAS  method  to  demonstrate  the  presence  of 
PAS-positive  reaction  sites. 

OBSERVATIONS  AND  RESULTS 

When  viewed  by  means  of  light  microscopy,  the  cells  of  the  outer  fold  appear 
typically  columnar,  containing  basally  located  nuclei,  folded  cell  membranes, 
numerous  vacuoles  and  a  prominent  cuticular  border  (Fig.  1). 

Examination  of  sections  of  the  mantle  epithelium  following  injection  of  carmine 
into  the  pallial  space  revealed  uptake  of  dye  particles  throughout  the  entire  surface 
of  the  outer  epithelium  of  Macrocallista.  The  most  pronounced  uptake  occurs  in 
the  epithelium  of  the  outer  surface  of  the  outer  fold,  less  pronounced  in  the  thick 
region  of  the  mantle  distal  to  the  base  of  the  outer  fold,  while  the  remainder  of  the 
mantle  epithelium  (thin  portion)  exhibits  still  less  dye  uptake  than  the  other 
regions.  The  localization  of  the  dye  is  observed  as  granules  of  varying  sizes,  in 
some  instances  so  small  as  to  be  hardly  recognized  by  light  microscopy ;  in  others 
the  granules  are  readily  recognizable  at  a  magnification  of  400  X  (compare  Figures 
1  and  2).  In  addition  to  the  outer  (shell)  epithelium,  numerous  amoebocytes  also 
exhibit  marked  uptake  of  the  carmine.  It  should  be  noted  that  the  distribution  of 


78 


GERRIT  BEVELANDER  AND  HIROSHI  NAKAHARA 


FIGURES  1-6. 


INGESTION  BY  THE  MANTLE  EPITHELIUM  79 

the  carmine  particles  observed  in  these  cells  varies  considerably.  One  of  the  factors 
upon  which  this  distribution  is  dependent  is  the  time  allowed  for  ingestion  to  occur. 

Sections  of  the  mantle  examined  following  treatment  for  the  demonstration  of 
acid  phosphatase  show  a  localization  of  this  enzyme  in  the  epithelial  cells  to  be 
similar  in  size  and  distribution  to  the  localization  of  the  carmine  granules  described 
above  (Fig.  3a,  b). 

Examination  of  mantle  epithelium  stained  with  Sudan  black  B  reveals  the 
presence  of  lipids  to  be  rather  widely  distributed,  appearing  as  dark  granules  of 
approximately  the  same  size  and  position  as  those  observed  in  the  epithelium 
exhibiting  ingestion  of  carmine.  Sections  treated  according  to  the  PAS  method 
and  subsequently  digested  with  saliva  exhibit  PAS-positive  granules  in  the  cyto- 
plasm which  correspond  in  size  and  location  to  granules  observed  by  the  other  two 
methods  mentioned  above.  Briefly,  the  localization  of  carmine,  acid  phosphatase, 
lipid  and  PAS-positive  granules  appears  to  be  very  similar  (Figs.  4,  5).  The 
presence  of  acid  phosphatase,  lipids  and  PAS-positive  material  in  granules  occurring 
in  the  cytoplasm  is,  according  to  Novikoff  (1960),  a  criterion  for  the  identification 
of  lysosomes  at  the  level  of  light  microscopy. 

In  the  paragraphs  which  follow  we  shall  describe  the  structure  of  the  epithelial 
cell  as  observed  by  the  electron  microscope.  Although  structural  variations  occur, 
the  example  we  have  chosen  to  illustrate  exhibits  most  of  the  details  which  are 
characteristic  of  these  cells.  At  the  free  surface  one  observes  rather  short  micro- 
villi  which  extend  for  a  considerable  distance  into  the  underlying  cytoplasm. 

A  prominent  feature  of  these  cells  is  the  septate  cell  junction  and  the  very 
pronounced  folding  of  the  cell  membrane.  Mitochondria  are  numerous  and  usually 
occupy  a  position  in  the  distal  half  of  the  cell  (Fig.  7).  In  several  cells  in  this  part 
of  the  mantle,  the  mitochondria  appear  to  be  undergoing  degenerative  changes,  as 
shown  by  the  poorly  defined  cristae  and  outer  membranes.  Ribosomes,  cisternae 
and  glycogen  are  much  less  prominent  than  in  other  regions  of  the  mantle.  The 
Golgi  apparatus,  not  illustrated  in  our  microphotograph,  is  also  characteristic, 
usually  appearing  rather  widely  dispersed  throughout  the  cell.  The  cells  rest 
upon  a  well  defined  basement  membrane  and  from  this  membrane  scattered 
integumental  fibers  arise  and  extend  to  the  free  surface. 

The  presence  of  numerous  micropynocytotic  vesicles  arising  from  the  canaliculi 

Figures  1-5  are  microphotographs  of  epithelium  of  the  mid-portion  of  the  outer  mantle  fold. 

FIGURE  1.  Typical  columnar  epithelium  of  outer  mantle  fold,  showing  cuticle  (microvilli) 
on  surface,  folded  cell  membranes,  numerous  vacuoles  and  prominent  basally  located  nuclei. 
Araldite  section,  stained  with  toluidine  blue.  X  1600. 

FIGURE  2.  Section  from  specimen  injected  with  carmine  and  fixed  24  hours  later.  Note 
distribution  of  fairly  large  dense  granules  indicating  localization  of  carmine  within  the  cell. 
Araldite  section,  unstained.  X  1600. 

FIGURE  3,  a  and  b.  Adjacent  sections  from  specimen  treated  to  show  the  localization  of 
acid  phosphatase  indicated  by  dark  granules.  These  photos  illustrate  the  variation  in  enzymatic 
activity  in  adjacent  parts  of  the  mantle.  <  1600. 

FIGURE  4.  Photograph  of  section  fixed  in  Ca-formol  and  treated  to  show  the  localization 
of  lipids  indicated  by  the  dark  granules  in  the  cytoplasm.  <  1600. 

FIGURE  5.  This  section  was  digested  with  saliva  and  then  treated  to  demonstrate  PAS- 
positive  material.  Note  distribution  of  cytoplasmic  granules.  X  1600. 

FIGURE  6.  Electron  micrograph  of  part  of  cell  showing:  c.j.,  cell  junction;  1,  lysosome ; 
m,  microvilli;  ps,  pallial  space;  p.v.,  pinocytotic  vesicles.  Uranyl  acetate  stain.  X  22,000. 


80 


GERRIT  BEVELANDER  AND  HIROSHI  NAKAHARA 


m 


8 


m 


•.    n 


FIGURES  7-8. 


INGESTION  BY  THE  MANTLE  EPITHELIUM  81 

between  the  bases  of  the  microvilli  arranged  in  linear  arrays  is  a  characteristic 
morphological  feature  of  these  cells  (Fig.  6).  These  arrays  terminate  in  the 
proximity  of  larger  vacuoles  or  lysosomes.  The  lysosomes  vary  in  size,  are 
enclosed  by  a  single  structural  membrane  and  often  contain  electron-dense  particles 
(Fig.  7).  Examination  of  selected  areas  of  cells  injected  with  colloidal  gold 
demonstrates  as  shown  in  Figure  8  that  ingested  participate  matter  comes  to  be 
localized  in  cell  organelles  we  have  identified  as  lysosomes. 

DISCUSSION 

Our  study  of  the  mantle  epithelium  of  Macrocallista  confirms  the  previous 
observations  of  Nakahara  (1962)  for  Pinctada  that  the  epithelium  associated  with 
shell  formation  ingests  participate  matter  derived  from  the  pallial  fluid.  He 
indicated  that  the  ingested  material  came  to  be  localized  in  the  Golgi  region.  Our 
observations  show  that  the  Golgi  apparatus  in  these  cells  is  widely  distributed 
and  accordingly  Nakahara's  observations  in  this  regard  are  essentially  correct. 

Novikoff  (1960)  has  listed  several  examples  illustrating  pynocytosis  as  the 
mechanism  responsible  for  ingestion  and  intracellular  transport  of  substances  which 
do  not  readily  permit  passage  through  the  cell  membrane.  We  have  identified 
pynocytotic  vesicles  indicative  of  cytotic  activity  and  in  addition  have  utilized 
colloidal  gold,  recognized  in  the  electron  microscope,  to  trace  the  pathway  of 
ingested  particles  from  the  cell  surface  to  lysosomes  located  in  various  parts  of 
the  cell.  In  a  similar  study  on  the  segregation  of  ferritin  by  glomerular  epithelia, 
it  was  shown  (Farquhar  and  Palade,  1959),  that  ferritin  particles  accumulate  first 
in  pynocytic  vesicles,  later  in  larger  vesicles  and  finally  in  dense  bodies  or  lysosomes. 
They  assume  that  the  same  pathway  is  followed  by  other  molecules,  especially 
proteins  of  similar  dimensions. 

Our  studies  indicate  that  an  apparently  normal  function  of  the  mantle  cells  is 
the  removal  of  particulate  matter  from  the  pallial  fluid  by  means  of  pynocytotic 
activity.  Studies  currently  in  progress  will  attempt  to  show  in  more  detail  the 
nature  of  the  materials  removed  by  this  method  and  also  whether  the  removal 
of  this  material  is  associated  with  the  mechanism  of  shell  formation. 

SUMMARY 

1.  Ingestion  of  particulate  matter  by  the  outer  mantle  fold  of  the  calico  clam, 
Macrocallista  inaculata,  was  studied.     Following  the  introduction  of  carmine  into 
the  pallial  space,  dye  particles  were  subsequently  localized  in  the  epithelia  of  the 
entire  outer  surface. 

2.  In  an  attempt  to  identify  the  cell  structure  in  which  the  dye  particles  were 
localized,  histochemical  tests  to  identify  acid  phosphatase,   lipids   and  mucopoly- 
saccharides  \vere  employed.     All  of  the  above  methods  gave  a  positive  reaction  at 
the  site  corresponding  to  the  locus  in  which  the  carmine  was  observed. 

FIGURE  7.  Electron  micrograph  of  parts  of  two  adjacent  cells ;  uranyl  acetate  stain, 
X  13,000.  bm,  basement  membrane;  c.j.,  cell  junction;  1,  lysosomes;  m,  microvilli;  mit,  mito- 
chondria ;  n,  nucleus ;  p.v.,  pinocytotic  vesicles ;  tegmental  fiber  designated  by  arrows. 

FIGURE  8.  Electron  micrograph  showing  ingested  particles  of  colloidal  gold  localized  in 
lysosomes ;  X  73,000. 


GERRIT  BEVELANDER  AND  HIROSHI  NAKAHARA 

3.  Additional  experiments  were  carried  out  in  which  colloidal  gold  was  injected 
into  the  pallial  fluid.     Subsequent  examination  of  epithelial  cells  showed  that  the 
colloidal  gold  was  localized   in   organelles   which,   on   the   basis   of   fine-structure 
morphology  and  histochemical  tests,  we  ascertain  to  be  lysosomes. 

4.  Pinocytosis,  occurring  as  a  result  of  the  pinching-off  of  the  bases  of  the 
microvilli   is  a  prominent  activity   of  these  cells.     The   micropinocytotic   vesicles 
arising  by  this  process  apparently  give  rise  to  large  vacuoles  and  lysosomes. 

5.  The  intracellular  mechanism  by  means  of  which   ingestion  by  the  mantle 
cells  occurs  has  not  previously  been  recorded.     The  significance  of  this  activity 
awaits  further  study. 

LITERATURE  CITED 

BEVELANDER,  G.,  1952.     Calcification  in  molluscs.     III.     Intake  and  deposition  of  Ca45  and  P32 

in  relation  to  shell  formation.     Biol.  Bull.,  102:  9-15. 
BEVELANDER,   G.,   AND   P.   BENZER,    1948.     Calcification   in   marine   molluscs.     Biol.   Bull.,    94: 

176-183. 
CHIFFELLE,  T.,  AND  F.  PUTT,  1950.     Propylene  and  ethylene  glycol  as  solvents  for  Sudan  IV 

and  Sudan  black  B.     Stain  Tech.,  26:  51-56. 
FARQUHAR,  M.  G.,  AND  G.   E.   PALADE,   1959.     Segregation  of  ferritin  in  glomerular   protein 

absorption  droplets.     /.  Biophysic.  Biochem.  Cytol.,  7:  297-303. 
GOMORI,  G.,  1950.     An  improved  histochemical  technic  for  acid  phosphatase.     Stain  Tech.,  25: 

81-85. 
KADO,  Y.,  1960.     Studies  on  shell  formation  in  molluscs.     /.  Sci.  Hiroshima   Univ.,  Ser.  B., 

Div.l,  19:  163-210. 
KAWAGUTI,    S.,   AND   N.    IKE^IOTO,    1962.     Electron   microscopy    of   the   mantle    of   a   bivalve, 

Fabulina  nitidnla.     Biol.  J.  Okayaina  Univ.,  8:  21-30. 
NAKAHARA,  H.,  1962a.     Behavior  of  mucous  substance  in  the  mantle  of  Pinctada  martensii  and 

I'iniia  attenuata.     Bull.  Nat.  Pearl  Res.  Lab.,  8:  871-878. 
NAKAHARA,   H.,    1962b.     Observations   on  the   ingestion   of  carmine   particles   by   mantle   and 

pearl-sac  epithelium  of  Pinctada  martensii.    Bull.  Nat.  Pearl  Res.  Lab.,  8:  879-883. 
NOVIKOFF,  A.  B.,  1960.     The  Cell.     Vol.  2.     Academic  Press,  New  York  and  London. 
OJIMA,  Y.,  1952.     Histological  studies  on  the  mantle  of  the  pearl  oyster  Pinctada  martensii. 

Cytologia,  17:  134-143. 
Tsujn,    T.,    1960.     Studies    on    the    mechanism    of    shell    and    pearl    formation    in    Mollusca. 

/.  Fac.  Fish.  Prej.  Univ.  Mie.,  5:  1-70. 
WILBUR,    K.    M.,    1960.     Shell    structure    and    mineralization    in    molluscs.     Calcification    in 

biological  systems.     AAAS  Publ.  No.  64,  Washington,  D.  C,  15-40. 


ADAPTATIONS  TO  TEMPERATURE  IN  TWO  CLOSELY  RELATED 

STRAINS  OF  EUGLENA  GRACILIS  1 

J.  R.  COOK2 

Laboratory  of  Nuclear  Medicine  and  Radiation  Biology,  University  of  California.  Los  ^in/clcs 

A  general  pattern  has  emerged  from  the  many  studies  of  physiological  adapta- 
tions to  temperature  in  protozoans.  Within  the  limits  of  tolerance,  low  tempera- 
tures result  in  reduced  growth  rate  and  increased  cell  size,  the  latter  a  result  of 
increased  amounts  of  practically  all  biochemical  constituents. 

However,  significant  qualitative  differences  between  species  have  also  been 
reported.  Thus,  Johnson  (1962)  found  that  respiratory  activity  in  the  crypto- 
monad  flagellate  Chilomonas  paramcciuin  increased  exponentially  with  tempera- 
ture, while  Buetow  (1963)  showed  that  respiration  in  a  colorless  mutant  of  Englena 
gracilis  var.  bacillaris  increased  in  a  linear  manner  with  temperature.  The  latter 
finding  is  of  particular  interest  as  an  example  of  a  continuously  decreasing  Q10  with 
increasing  temperatures. 

Qualitative  differences  of  this  sort  must  be  an  expression  of  genetic  diversity 
among  flagellates  in  a  most  fundamental  aspect  of  protozoan  physiology.  Because 
of  this,  it  seemed  desirable  to  repeat  Buetow's  work  with  a  wild-type  Euglena. 
This  report  describes  some  physiological  properties  of  two  strains  of  Euglena 
gracilis,  separated  only  by  minor  taxonomic  differences,  as  a  function  of  incubation 
temperature.  These  studies  show  marked  quantitative  but  no  qualitative  differ- 
ences in  temperature  adaptation  between  the  two  strains.  Buetow's  report  of  a 
linear  increase  in  respiratory  activity  with  elevated  temperatures  is  confirmed  when 
exogenous  acetate  is  available,  but  a  markedly  different  pattern  was  observed  in 
endogenous  respiration.  Other  parameters — mass,  protein,  and  RNA — respond 
in  the  expected  manner. 

METHODS 

Original  stocks  of  the  cells  used,  Euglena  gracilis  strain  Z  and  Euglena  gracilis 
var.  bacillaris,  were  obtained  from  Dr.  J.  A.  Gross  and  have  been  maintained 
through  serial  culture  by  the  author.  Growth  rates  of  these  stocks,  measured  under 
the  same  culture  conditions,  have  remained  constant  over  a  period  of  years.  For 
these  studies,  however,  a  single  colony  of  each  strain  was  picked  off  agar,  inoculated 
into  liquid  media,  and  the  resulting  populations  used.  These  two  clonal  popula- 
tions, derived  from  single  cells,  did  not  differ  in  growth  rate  from  the  parent 
populations. 

The  salt  medium  of  Cramer  and  Myers  (1952),  with  sodium  acetate  (25  mM) 
as  sole  carbon  and  energy  source,  was  used  exclusively.  Axenic  cultures  were 

1  Supported  by  Contract  AT  (04-1)   GEN-12  between  the  Atomic  Energy  Commission  and 
the  University  of  California. 

2  Present  address  :  Department  of  Zoology,  University  of  Maine,  Orono,  Maine  04473. 

83 


84  J.  R.  COOK 

grown  in  the  dark  in  cotton-stoppered  erlenmeyer  flasks,  maintained  in  water- 
jacketed  incubators  working  against  an  ambient  temperature  of  10°  C.  Washed  air 
was  flushed  continuously  through  the  incubators. 

Strain  Z  was  examined  at  temperatures  of  15°,  20°,  25°,  29°,  and  34°  C.  E. 
gracilis  var.  bacillaris  grew  well  enough  at  15°  C.,  but  formed  many  clumps  of  cells 
(palmella)  which  made  quantitative  studies  impossible  at  this  temperature;  this 
variety  was  studied  at  17.5°,  20°,  25°,  29°,  and  34°  C.  Temperatures  above  32° 
are  supra-optimal  for  E.  gracilis  and  lead  to  irreversible  bleaching.  At  each 
temperature,  the  cells  were  allowed  to  adapt  through  at  least  15  generations  before 
measurements  were  made.  Population  increase  was  followed  by  periodic  cell 
counts  with  the  Coulter  cell  counter.  The  cells  were  always  harvested  at  a  popula- 
tion density  of  105  cells  per  ml.,  well  below  levels  of  the  stationary  phase. 

At  most  of  these  temperatures,  cells  were  analyzed  in  terms  of  growth  rate, 
mass,  protein  and  RNA  content,  and  respiratory  rates,  the  latter  both  at  the 
temperature  of  incubation  and  also  at  a  test  temperature  of  25°  C.  For  the 
respiration  measurements,  cells  were  harvested  by  gentle  centrifugation  (Buetow, 
1961),  washed  three  times  with  fresh  culture  medium  (without  acetate)  and  made 
up  to  volume  in  this  wash  medium.  Oxygen  consumption  of  the  cell  suspension  was 
followed  with  the  Beckman  oxygen  electrode,  using  a  water- jacketed  reaction  vessel 
with  constant  stirring  of  the  cells  by  a  magnetic  bar ;  with  the  electrode  in  position, 
this  vessel  was  air-tight  and  contained  no  gas  phase.  Depletion  of  oxygen  was 
recorded  graphically,  and  absolute  amounts  of  oxygen  consumed  calibrated  against 
air-  and  nitrogen-flushed  water.  The  temperature  of  the  vessel  was  either  25°  C. 
or  the  incubation  temperature,  held  constant  by  circulating  water  from  a  refrigerated 
bath  through  the  outer  jacket  of  the  reaction  vessel.  Endogenous  respiration  was 
followed  for  20-30  minutes,  after  which  acetate  to  25  mM  was  added  and  respira- 
tion again  followed  for  20-30  minutes.  Over  this  period  of  time,  no  extensive 
precautions  against  bacterial  contamination  were  necessary.  At  the  end  of  such 
a  run,  aliquots  of  the  cell  suspension  were  taken  for  cell  counts,  so  that  the 
respiratory  rates  could  be  referred  to  the  average  cell.  Procedures  for  other 
measurements  have  been  described  previously  (Cook,  1961). 

RESULTS  AND  DISCUSSION 
Growth  rates 

Generation  times  of  the  two  cell  types  at  the  several  temperatures  are  shown 
in  Figure  1.  At  most  temperatures,  the  Z  strain  of  Euglena  exhibits  a  faster 
multiplication  rate  than  var.  bacillaris.  The  degree  of  difference  is  not  constant, 
however;  at  29°  C.  (the  optimum  for  both  cells),  the  generation  time  of  var. 
bacillaris  is  the  greater  by  a  factor  of  about  1.48;  at  20°  C.  only  1.1.  E.  gracilis 
var.  bacillaris  does  not  respond  as  readily  as  strain  Z  to  changes  in  temperature, 
at  least  in  terms  of  multiplication  rate.  Figure  1  also  shows  that  supra-optimal 
temperatures  (34°)  retard  cell  division  more  severely  in  the  Z  strain. 

Cell  mass 

Figure  2  shows  changes  in  total  dry  mass  in  cells  adapted  to  the  various 
temperatures.  Between  20°  and  29°  C.  var.  bacillaris  has  the  larger  mass,  but 


TEMPERATURE  ADAPTATIONS  IN  EUGLENA 


85 


60 


ft  50 

I 

v_x 

LU 
40 


Z 
030 


o: 

UJ20 

m 


10 


10 


15  20  25 

TEMPERATURE 


30 


35 


FIGURE  1.     Generation  times  during  logarithmic  growth  as  a  function  of  incubation  temperature 
in  E.  gracilis  strain  Z  (  •  )  and  var.  bacillaris  (O)- 

this  condition  is  reversed  below  20°  C.  Strain  Z  shows  a  minimum  mass  at  25°, 
but  the  change  in  var.  bacillaris  is  essentially  linear  over  the  temperature  range 
examined. 

Protein  and  RNA 

The  protein  content  of  these  cells  does  not  form  a  constant  fraction  of  cellular 
mass.  As  the  incubation  temperature  is  changed,  the  protein  fraction  ranges 
between  20%  to  30%  of  the  total  mass.  However,  the  protein  content  in  both 
cells  changes  in  the  same  direction  as  total  mass,  being  much  increased  at  lower 
temperatures.  Since  the  polysaccharide  paramylum  will  make  up  most  of  the 
remaining  mass,  it  is  assumed  that  levels  of  paramylum  must  also  change  with 
temperature,  and  in  the  same  direction  as  protein. 


86 


J.  R.  COOK 


-i  3 

LU 

0 
> 

D 
0 

2 

<2 


15 


Protein 


Mass 


20  25 

TEMPERATURE 


30 


FIGURE  2.  Average  cell  mass  (circles),  protein  (squares),  and  RNA  (triangles)  in  E. 
gracilis  strain  Z  (filled  figures)  and  var.  bacillaris  (open  figures)  during  log  growth  at  various 
temperatures.  Ordinate  values  are  in  ^grains  and  for  the  average  cell  should  be  multiplied  by 
10  (RNA),  100  (protein)  and  1000  (total  dry  mass). 


RNA  levels  roughly  parallel  protein  content,  being  minimum  in  both  strains  at 
about  25°  C.  In  E.  gracilis  var.  bacillaris,,  the  RNA  content  is  equal  to  or  slightly 
greater  than  that  of  the  Z  strain  (Fig.  2). 

Respiration 

Figures  3  and  4  summarize  respiratory  characteristics.  At  the  temperature 
of  incubation,  the  respiratory  rate  in  the  presence  of  acetate  is  always  greater  in 
the  Z  strain,  by  an  amount  which  is  nearly  constant  at  all  temperatures  (Fig.  3). 


TEMPERATURE  ADAPTATIONS  IN  EUGLENA 


87 


It  may  be  noted  from  Figure  3  that  these  rates  increase  with  temperature  in  a 
linear,  rather  than  exponential,  manner.  In  the  absence  of  adaptive  changes  in 
respiratory  machinery,  an  exponential  pattern  would  be  expected.  Since  this  was 
not  observed,  it  follows  that  the  respiratory  capacity  of  cells  adapted  to  low  tem- 
perature must  be  greater  than  those  adapted  to  higher  temperatures  (Precht's  type 
1).  That  this  is  the  case  was  demonstrated  by  the  respiratory  rates  at  a  test 
temperature  of  25°  C.  (Fig.  4).  Cells  incubated  at  the  lower  temperatures 
consume  as  much  as  50%  more  oxygen  at  25°  than  do  those  cultured  at  higher 
temperatures,  in  the  presence  of  exogenous  acetate. 

Endogenous  rates  of  respiration  are  essentially  the  same  in  both  strains  of 
Euglena.  It  is  of  interest  to  note  that  these  rates  do  not  change  appreciably  at 
temperatures  below  25°  C.  when  tested  at  the  temperature  of  growth  (Fig.  3). 
Endogenous  respiration  thus  shows  complete  adaption  to  incubation  temperature 
(Precht's  type  2).  There  is  a  slight  increase  in  the  endogenous  rate  above  25°. 
Complete  adaptation  of  this  sort  would  also  give  rise  to  increased  respiratory 
capacity  at  the  lower  temperatures,  to  an  extent  greater  even  than  that  found  in 
the  incomplete  adaptation  in  the  presence  of  exogenous  substrate.  That  this  is 
the  case  is  seen  in  Figure  4.  The  endogenous  consumption  of  oxygen  of  cells 
grown  at  15-17.5°  is  about  twice  as  great  at  25°  as  that  of  cells  grown  at  25°  C. 
It  is  concluded  that  the  endogenous  response  of  Euglena  to  temperature  differs 
qualitatively  from  the  response  in  the  presence  of  exogenous  acetate. 

Rates  of  cellular  processes 

Rate  constants  for  population  expansion  can  be  obtained  from  the  generation 
times  by  use  of  the  familiar  growth  equation,  k  =  In  2/generation  time.  Synthetic 


0 


80 


60 


40 


20 


Acetate 
o 


Endogenous 


15 


20  25 

TEMPERATURE 


30 


FIGURE  3.  Oxygen  consumption  by  E.  gracilis  strain  Z  (filled  circles)  and  var.  bacillaris 
(open  circles)  during  log  growth  at  the  temperature  of  incubation.  The  lower  curves  show 
endogenous  consumption,  and  the  upper  curves  show  consumption  in  the  presence  of  exogenous 
substrate  (acetate).  Qo2  =  /*!.  O2/hr./10°  cells. 


88 


J.  R.  COOK 


80 


CM 

o 


60 


0 


40- 


20 


Acetate 


o 


Endogenous 


15  20  25 

GROWTH      TEMPERATURE 


30 


FIGURE  4.  Rate  of  oxygen  consumption  at  25°  C.  by  E.  gracilis  strain  Z  (solid  circles)  and 
var.  bacillaris  (open  circles)  after  adaptation  to  growth  at  the  temperature  shown  on  the 
abscissa.  Lower  curves,  endogenous  rate ;  upper  curves,  rate  in  the  presence  of  exogenous 
acetate.  Qo2  as  in  Figure  3. 


rates  can  be  estimated  from 


k  = 


M 


1.44  GT, 


(1) 


where  k  is  the  rate  value  (in  amount  synthesized  per  average  cell  per  hour),  GT 
is  the  generation  time,  and  M  is  the  amount  held  by  the  average  cell  in  the  constit- 
uent of  interest  (Cook  and  James,  1964).  Synthetic  rates  for  total  mass,  protein, 
and  RNA  for  both  strains  of  Euglcna  at  the  several  different  temperatures  were 
calculated  from  this  equation.  Figure  5  is  a  logarithmic  plot  of  these  rates.  The 
rate  of  mass  accumulation  by  the  average  cell  increases  exponentially  with  tem- 
perature up  to  29°  ;  this  rate  increases  rather  more  rapidly  in  the  Z  strain  above 
20°  C.  A  more  striking  difference  is  seen  in  the  rates  of  protein  and  RNA  syn- 
thesis. While  these  rates  increase  exponentially  with  temperature  in  both  cell 
types,  the  rate  of  increase  in  the  Z  strain  is  considerably  greater  than  in  var. 
bacillaris.  In  both  strains,  the  rate  of  protein  synthesis  parallels  the  rate  of  RNA 
synthesis.  At  about  20°,  the  two  cell  types  have  equal  rates  of  RNA  and  protein 
synthesis. 

Q10  values 

The  Q10  values  for  these  various  processes  can  be  read  from  the  data  shown  in 
Figure  5.  They  are  listed  in  Table  I.  The  Q10  is  approximately  2  between 
15-17.5°  and  29°  C.  for  the  rates  of  mass,  protein,  and  RNA  accumulation  in  var. 


TEMPERATURE  ADAPTATIONS  IN  EUGLENA 


89 


bacillaris  and  for  mass  accumulation  in  strain  Z ;  the  rates  of  protein  and  RNA 
synthesis  in  strain  Z  have  Q10  values  of  2.9  and  3.9,  respectively. 

The  Q10  for  division  rate  decreases  from  3.3  to  1.3  over  the  range  17.5°-29°  C. 
in  var.  bacillaris  and  drops  to  about  1  between  29°  and  34°  C.  Strain  Z  is  more 
sensitive  to  temperatures  in  terms  of  division  rate,  showing  a  progressive  decrease 
in  Q10  from  5.6  to  1.7  as  the  temperature  is  elevated  from  15°  to  29°  C.  (Table  I). 
Above  29°  C.  the  Q10  is  less  than  unity  in  strain  Z. 

In  the  presence  of  exogenous  acetate,  the  respiratory  Q10  for  strain  Z  is  about 


m 

<   8 

cr 


15 


20  25 

TEMPERATURE 


Mass 


RNA 


Protein 


Respiration 
with  acetate 


Growth 


Endogenous 
Respiration 


30 


35 


FIGURE  5.  Log  plot  of  metabolic  and  synthetic  rates  in  E.  gracilis  var.  bacillaris  (open 
figures)  and  strain  Z  (filled  figures).  Rates  calculated  as  described  in  text.  Ordinate  values 
for  respiration  (/*!.  O2/hr./106  cells,  inverted  triangles)  are  to  be  multiplied  by  10;  for  growth 
rate  (hr."1,  circles)  by  10~2;  for  RNA  (triangles),  protein  (squares),  and  mass  (circles),  all  in 
^gm./cell/hr.,  by  1,  10,  and  10,  respectively. 


90 


J.  R.  COOK 


TABLE  I 
values  for  various  rates  in  two  strains  of  E.  gracilis 


Temperature  range 

1S(17.5)-20 

20-25 

25-29 

29-34 

Cell  strain 

z 

b 

Z 

b 

Z 

b 

Z 

b 

Growth  rate 

5.60 

3.26 

3.93 

2.33 

1.71 

1.32 

.41 

1.05 

Endogenous  respiration 
Respiration  (acetate) 
Mass  increase 

1.00 

1.84 
2.00 

1.00 

1.73 
2.50 

1.15 
1.49 
2.00 

1.00 
1.73 
2.12 

1.28 
1.49 
1.53 

1.16 
1.61 
1.51 

Protein  synthesis 
RNA  synthesis 

2.90 
3.85 

1.74 

2.25 

2.35 
2.64 

1.74 
1.70 

2.35 
2.64 

1.74 
1.70 

1.5  between  20°  and  29°  C,  and  1.8  between  15°  and  20°  C.  Under  comparable 
conditions,  E.  gracilis  var.  bacillaris  shows  an  unchanging  Q10  of  1.7  between 
17.5°  and  29°  C. 

It  will  be  noted  that  respiratory  rates  at  the  temperature  of  incubation  are 
shown  on  a  linear  scale  in  Figure  3,  and  on  a  semi-log  plot  in  Figure  5.  For 
E.  gracilis  var.  bacillaris,  a  straight  line  satisfied  the  experimental  points  in  both 
cases,  which  is  merely  to  say  that  in  a  biological  system  it  is  difficult  to  demonstrate 
whether  a  function  is  linear  or  exponential  when  the  range  of  values  differs  by  no 
more  than  a  factor  of  two.  In  the  present  case,  the  Oo-  at  29°  is  63  and  at  17.5° 
it  is  34. 

The  Q10  for  endogenous  respiration  is  about  1  in  both  strains  at  temperatures 
below  25°,  and  only  slightly  greater  between  25°-29°  (Table  I).  It  was  expected 
that  respiratory  activity  in  the  presence  of  exogenous  substrate  (acetate)  would  be 
greater  than  the  endogenous  level,  since  acetate  should  stimulate  activity  of  the 
Krebs'  cycle  enzymes.  It  was  quite  unexpected  to  find  that  the  degree  of  stimu- 
lation was  not  constant  as  a  function  of  temperature.  The  rate  of  oxygen  con- 
sumption will  be  in  part  a  function  of  the  level  of  respiratory  enzymes,  excluding 
oxidative  reactions  which  are  not  directly  involved  in  respiration.  That  the 
activity  of  these  enzymes  is  adaptively  increased  at  lower  temperatures  is  implied 
by  the  data  for  endogenous  respiration  shown  in  Figure  3.  If  the  stimulatory  role 
of  exogenous  acetate  were  strictly  confined  to  respiratory  activities,  the  Qo2  with 
added  acetate  should  be  reflected  in  a  Q10  nearly  equal  to  that  of  the  endogenous. 
That  this  is  not  the  case  is  strong  presumptive  evidence  for  the  view  that  oxygen 
is  utilized  in  non-respiratory  functions  of  Englena  cultured  on  acetate.  Support- 
ing evidence  of  a  comparative  nature  is  also  suggested  by  the  data  shown  in  Figure 
3:  both  strains  of  Englena  have  essentially  the  same  endogenous  rate  of  oxygen 
consumption,  but  are  stimulated  to  quite  different  levels  of  oxygen  consumption  by 
exogenous  acetate. 

Danforth  and  Wilson  (1961)  have  shown  that  endogenous  respiration  of 
Englena  continues  in  the  presence  of  exogenous  substrate.  The  Qo2  of  Euglena 
adapted  to  growth  and  respiration  on  exogenous  glucose  is  no  greater  than  the 
endogenous  Qo2,  the  latter  having  a  level  equal  to  the  endogenous  rate  of  acetate- 
grown  cells  (Cook  and  Heinrich,  1965).  The  optimal  growth  rate  and  the  cellular 
mass  and  protein  content  are  the  same  when  cultured  on  either  substrate.  These 


TEMPERATURE  ADAPTATIONS  IN  EUGLENA 


91 


data  taken  together  suggest  that  acetate-grown  Euylena  may  consume  oxygen  via 
two  principal  routes:  one  as  terminal  electron  acceptor  in  respiration,  and  the 
other  in  some  non-respiratory  and  non-energy-yielding  reaction (s)  associated  with 
growth  on  acetate. 

Grozi'th  rate  and  synthetic  rate 

Schaechter  et  al.  (1958)  showed  in  the  bacterium  Salmonella  typhiinurmni  that 
mass  and  RNA  levels  were  a  positive  exponential  function  of  the  growth  rate  but 
quite  independent  of  temperature  between  25°  and  37°  C,  when  the  growth  rate  was 
varied  by  culture  on  different  carbon  sources.  Since  temperature  affects  growth 
rate  as  well  as  other  physiological  rates  in  Euglena  with  only  acetate  as  carbon  and 
energy  source,  it  was  of  interest  to  know  whether  the  observed  effects  were  due 
primarily  to  temperature  or  indirectly  to  altered  growth  rates.  The  present  data 
have  been  examined  to  determine  whether  the  biochemical  and  physiological  profiles 
of  Euglena  conform  to  some  general  pattern  of  the  sort  described  by  Schaechter 
et  al.  No  such  pattern  was  found.  Figure  6  shows  mass  and  RNA  as  a  function 


HI 

D 

<   4 

3 


Mass 


RNA 


.01  02  03  04  05          .06 

GROWTH       RATE 


07 


FIGURE  6.  Log  plot  of  mass  and  RNA  content  of  E.  gracilis  strain  Z  (filled  figures)  and 
var.  bacillaris  (open  figures)  as  a  function  of  growth  rate.  Ordinate  values  for  RNA  are  to  be 
multiplied  by  10  and  those  for  mass  by  1000  to  give  the  amount  in 


92 


J.  R.  COOK 


of  growth  rate,  plotted  for  both  E.  gracilis  var.  bacillaris  and  strain  Z.  The  data 
for  protein  are  quite  comparable  (cj.  Fig.  2)  but  are  not  shown  in  Figure  6  for 
purposes  of  clarity. 

Mass  and  RNA  levels  in  Euglena  are  a  negative  exponential  function  of  the 
growth  rate  when  the  latter  is  .05/hr.  or  less,  and  a  positive  function  at  higher 
growth  rates.  In  this  respect  it  is  noted  that  the  growth  rates  of  S.  typhiniurium 
as  studied  by  Schaechter  et  al.  (1958)  were  always  considerably  greater  than  those 
reported  here  for  Euglena. 

It  is  of  interest  to  note  that  a  single  line  satisfies  the  RNA  content  as  a  function 
of  growth  rate  for  both  varieties  of  E.  gracilis.  In  spite  of  the  fact  that  strain  Z 


15 


10 


0 

l_ 

a 


17.5' 


15' 


1.0 


1.5 

RNA 


20 


25 


FIGURE  7.  Relationship  between  the  rates  of  RNA  and  protein  synthesis  in  E.  gracilis 
strain  Z  (filled  circles)  and  var.  bacillaris  (open  circles)  at  various  temperatures  as  indicated. 
Rates  in  yu/ugm./cell/hr. 

and  var.  bacillaris  can  hold  widely  different  values  for  RNA  content  and  growth 
rate  at  a  given  temperature  (cf.  Figs.  1  and  2),  the  conformity  shown  in  Figure  6 
suggests  that  genetic  differences  in  the  two  strains  are  not  yet  great  enough  to 
be  expressed  as  some  divergence  in  the  fundamental  relationships  between  division 
and  synthesis.  This  view  is  strengthened  by  the  comparison  shown  in  Figure  7, 
which  is  a  plot  of  the  rate  of  RNA  synthesis  against  the  rate  of  protein  synthesis 
for  the  two  strains,  when  these  rates  are  varied  by  temperature.  Again,  a  single  line 
satisfies  both  sets  of  data.  The  rate  of  information  translation  is  the  same  in 
both  cell  lines,  at  least  at  a  very  gross  level.  By  exclusion,  it  is  inferred  that  the 
two  strains  may  differ  principally  in  the  rate  of  information  transcription.  While 
DNA  levels  were  not  followed  in  the  present  study,  it  can  safely  be  assumed  that 


TEMPERATURE  ADAPTATIONS  IN  EUGLENA 

the  Q10  for  the  over-all  rate  of  the  DNA  synthesis  will  be  exactly  equal  to  that  of 
the  growth  rate.  While  DNA  replication  in  the  single  cell  is  usually  a  discon- 
tinuous process,  variable  lengths  of  the  S  period  could  very  well  determine 
physiological  and  biochemical  differences  of  the  sort  described  in  this  paper, 
especially  since  DNA  is  presumed  to  be  non-functional  in  RNA  synthesis  during  its 
own  replication  (Prescott  and  Kimball,  1961).  The  possibility  of  ploidy  is  not 
excluded.  It  is  suggested  that  the  more  sluggish  behavior  of  E.  gracilis  var. 
bacillaris  when  compared  to  strain  Z  is  the  result  in  part  of  a  slower  rate  of 
information  transcription. 

SUMMARY 

1.  Certain   biochemical   and   physiological   parameters    in    two    closely    related 
strains  of  Euglena  gracilis  (strain  Z  and  var.  bacillaris)  have  been  examined  after 
adaptation  to  various  incubation  temperatures. 

2.  The  growth  rate  for  the  two  strains  differed  at  all  temperatures,  but  was 
greatest  in  both  at  29°. 

3.  Temperatures  below  optimal  resulted  in  increased  mass,  protein,  and  RNA 
levels.     In  general  E.  gracilis  var.  bacillaris  was  larger  in  all  these  fractions  at  any 
given  temperature. 

4.  Endogenous  respiration  proceeded  at  rates  which  were  essentially  unchanging 
over  the  temperature  range  15°-29°  C.     Both  strains  exhibited  the  same  rate. 

5.  Oxygen    consumption    in    the    presence    of    exogenous    substrate    (sodium 
acetate)  increased  in  a  linear  fashion  with  the  temperature  of  incubation,  rates  in 
strain  Z  being  considerably  greater  than  in  var.  bacillaris. 

6.  Mass,  RNA,  and  protein  content  were  found  to  be  an  exponential  function 
of  the  growth  rate,  with  a  change  in  the  sign  of  the  slope  at  a  growth  rate  of  .05/hr. 
The  rate  of  protein  synthesis  was  a  linear  function  of  the  rate  of  RNA  synthesis 
in  both  strains. 

LITERATURE  CITED 

BUETOW,  D.  E.,  1961.     Variation  of  the  respiration  of  protozoan  cells  with  length  of  centrifuging. 

Anal.  Biochcm.,  2:  242-247. 
BUETOW,    D.    E.,    1963.     Linear    relationship   between   temperature   and    uptake    of   oxygen    in 

Euglena  gracilis.     Nature,  199:  196-197. 
COOK,  J.  R.,  1961.     Euqlena  gracilis  in  synchronous  division.     II.     Biosynthetic  rates  over  the 

life-cycle.    Biol.  Bull.,  121 :  277-289. 
COOK,  J.  R.,  AND  B.  HEINRICH,  1965.     Glucose  vs.  acetate  metabolism  in  Euglena.    J.  Protosool., 

12:  581-583. 
COOK,  J.  R.,  AND  T.  W.  JAMES,  1964.     Age  distribution  of  cells  in  logarithmically  growing  cell 

populations.     In:   Synchrony  in  Cell  Division  and  Growth   (ed.  by  E.  Zeuthen),  John 

Wiley  and  Sons,  Inc.,  New  York. 
CRAMER,    M.,   AND   J.    MYERS,    1952.     Growth   and   photosynthetic    characteristics    of    Euglena 

gracilis.    Archiv.  f.  Mikrobwl..  17:  384-402. 
DANFORTH,  W.  F.,  AND  B.  W.  WILSON,  1961.     The  endogenous  metabolism  of  Euglena  gracilis. 

J.  Gen.  Microbiol,  24:  95-105. 
JOHNSON,  B.  F.,  1962.     Influence  of  temperature  on  the  respiration  and  metabolic  effectiveness 

of  Chilomonas.     Exp.  Cell  Res.,  28:  419-423. 
PRESCOTT,   D.   M.,   AND   R.   F.    KIMBALL,    1961.     Relation   between    RNA,    DNA,   and   protein 

synthesis  in  the  replicating  nucleus  of  Euplotcs.     Proc.  Nat.  Acad.  Sci..  47:  686-693. 
SCHAECHTER,    M.,    O.    MAALE    AND    N.    O.    KJELGAARD,    1958.     Dependency    on    medium    and 

temperature  of  cell  size  and  chemical  composition  during  balanced  growth  of  Salmonella 

typhimurium.     J.  Gen.  Microbiol.,  19:  592-606. 


QUANTITATIVE  ASPECTS  OF  BROWN  ADIPOSE  TISSUE 

THERMOGENESIS  DURING  AROUSAL 

FROM  HIBERNATION1 

JOHN  S.  HAYWARD  2  AND  ERIC  G.  BALL 

Departments  of  Anatomy  and  Biological  Chemistry,  Harvard  Medical  School, 

Boston,  Massachusetts  02115 

Evidence  which  indicates  that  brown  adipose  tissue  may  serve  as  a  specialized 
site  of  heat  production  has  recently  been  reviewed  by  Joel  (1965).  The  relative 
abundance  of  this  tissue  in  hibernators  has  led  a  number  of  workers  to  suggest 
that  brown  adipose  tissue  may  play  an  important  role  in  the  generation  of  heat 
during  arousal  from  hibernation.  There  are,  however,  few  data  available  which 
permit  one  to  assess  the  quantitative  aspects  of  such  a  role  for  this  tissue.  The 
recent  attempts  of  Ball  (1965)  to  calculate  these  quantitative  aspects  from  available 
data  in  the  literature,  involved  many  assumptions  which  only  served  to  emphasize 
the  need  for  more  data.  This  report  describes  one  attempt  to  obtain  such  data  for 
the  big  brown  bat  (Eptcsicus  juscus}  during  its  arousal  from  hibernation.  The 
bat  was  chosen  for  this  study  because  there  is  some  evidence  (Hayward  et  al.,  1965) 
to  indicate  that  hibernating  bats  may  be  the  most  specialized  of  adult  mammals  in 
terms  of  the  magnitude  of  brown  adipose  tissue  thermogenesis. 

A  quantitative  estimate  of  the  contribution  of  heat  by  brown  adipose  tissue 
during  an  arousal  from  hibernation  can  be  obtained  by  comparing  the  oxygen  con- 
sumption of  brown  adipose  tissue  with  the  total  body  oxygen  consumption  during 
the  arousal  process.  Ideally,  the  in  vivo  respiration  of  brown  adipose  tissue  would 
be  measured.  However,  the  small  size  of  the  bats  (about  15  grams)  precludes 
such  an  approach.  As  an  alternative,  we  have  attempted  to  ascertain  the  heat 
production  of  brown  adipose  tissue  from  measurements  of  its  in  vitro  respiration. 
The  respiration  of  liver  and  heart  slices  from  these  bats  has  also  been  determined 
for  comparison  with  brown  adipose  tissue  respiration,  and  to  obtain  a  larger 
estimate  of  the  non-muscular  heat  production. 

The  limitations  involved  in  efforts  to  summate  in  vitro  tissue  respirations  to 
account  for  total  animal  respiration  are  well  known  (von  Bertalanffy  and  Pirozyn- 
ski,  1953).  Such  limitations  have  been  invoked  in  this  study  to  explain  the  failure 
of  our  tissue  respiration  measurements  to  provide  a  satisfactory  estimate  of  the 
quantitative  contribution  of  brown  adipose  tissue  to  arousal  thermogenesis. 

The  technique  of  thermography  has  been  used  to  provide  additional  descriptive 
evidence  of  the  thermogenic  capacity  of  brown  adipose  tissue. 

1  Supported  by  funds  received  from  Life  Insurance  Medical  Research  Fund  and  U.S.P.H.S. 
grants  A-3132,  GM  05611-07  and  GM  05197-08  and  by  Air  Force  Contract  AF  31  (609) -2296. 

-  Present  address :  Department  of  Zoology,  University  of  Alberta,  Edmonton,  Alberta, 
Canada. 

94 


BROWN  ADIPOSE  TISSUE  THERMOGENESIS  95 

METHODS 

Prior  to  experimentation,  the  bats  used  in  this  study  had  been  hibernating 
intermittently  for  a  period  of  approximately  five  months.  Measurement  of  their 
total  oxygen  consumption  during  arousal  from  hibernation  was  accomplished  using 
a  closed-circuit,  volumetric  respirometer.  Exhaled  carbon  dioxide  was  absorbed 
by  soda  lime  and  the  decrease  in  pressure  in  the  closed  system  was  detected  with 
a  sensitive  manometer.  At  two-minute  intervals,  the  pressure  was  restored  to  the 
initial  level  by  allowing  an  accurately-measured  quantity  of  water  to  flow  into 
the  system,  this  measure  being  equal  to  the  oxygen  consumed  in  that  interval.  The 
system  was  designed  to  maintain  normal  atmospheric  gas  concentration  in  the  bat 
chamber  and  to  minimize  errors  due  to  possible  slight  temperature  fluctuations 
in  the  system. 

To  provide  a  criterion  of  stage  of  arousal  from  hibernation,  each  bat  had,  pre- 
viously, a  small  thermocouple  implanted  in  its  interscapular  brown  adipose  tissue. 
During  an  arousal  in  the  respiration  chamber  the  thermocouple  lead  wires  were 
led  from  the  chamber  through  an  air-tight  seal,  enabling  a  continuous  record  of 
brown  adipose  tissue  temperature  to  be  obtained.  All  arousals  were  conducted  at 
the  ambient  temperature  at  which  the  bats  had  been  hibernating  ( 5  °  C. ) . 

Immediately  subsequent  to  each  arousal,  the  bat  was  sacrificed  and  a  weighed 
sample  of  browrn  adipose  tissue  taken  for  the  in  vitro  respiration  measurements. 
The  total  remaining  brown  adipose  tissue,  from  all  locations  in  the  body,  was 
carefully  dissected  and  weighed,  care  being  taken  to  prevent  drying  of  the  tissue 
during  this  procedure.  The  heart  and  liver  from  each  bat  were  also  weighed  and 
several  samples  of  these  tissues  taken  for  respiration  measurements. 

Oxygen  consumption  of  tissue  samples  was  determined  at  37.2°  C.  by  means 
of  the  Warburg  manometric  apparatus.  The  incubation  medium  was  Krebs- 
Henseleit  phosphate  buffer  (Krebs  and  Henseleit,  1932)  modified  to  contain  one- 
half  the  recommended  calcium.  In  some  experiments,  glucose  was  dissolved  in  this 
medium  to  yield  a  concentration  of  1.5  mg./ml.  The  main  compartment  of  the 
vesssels  contained  2.9  ml.  of  medium  and  the  tissue  sample.  The  center  well 
contained  0.2  ml.  of  20%  KOH  and  a  strip  of  filter  paper  to  facilitate  CO2  absorp- 
tion. The  sidearm  contained  0.1  ml.  of  a  catecholamine  solution  dissolved  in  H,,O 
weakly  acidified  with  HC1.  The  gas  phase  was  oxygen.  Brown  adipose  tissue 
was  cut  into  small  pieces  with  a  razor  blade  after  weighing  to  0.1  mg.  on  a  torsion 
balance,  15-20  mg.  of  tissue  being  used  per  vessel.  Liver  and  heart  were  sliced 
with  the  aid  of  a  Stadie-Riggs  tissue  sheer  and  weighed  to  the  nearest  mg.  The 
amount  of  liver  used  per  vessel  was  100-175  mg.  while  heart  samples  weighed 
between  50-100  mg.  In  all  cases,  tissue  samples  were  prepared  and  run  immedi- 
ately after  removal  from  the  bat.  Vessels  were  gassed  with  oxygen  for  10  minutes, 
closed,  and  after  another  five  minutes  for  thermoequilibration,  readings  were  taken 
at  10-minute  intervals  for  40-50  minutes.  The  contents  of  the  sidearm  were  then 
added  and  readings  continued  for  another  50-100  minutes. 

The  epinephrine  used  was  a  sample  of  the  free  base  kindly  supplied  by  Bur- 
roughs-Wellcome  Co.  The  norepinephrine  was  a  bitartrate  preparation  purchased 
from  Calbiochem. 


96 


JOHN  S.  HAYWARD  AND  ERIC  G.  BALL 


Bats  were  prepared  for  thermography  (infrared  radiography)  by  first  shaving 
the  hair  off  the  dorsal  aspect  of  their  bodies.  This  facilitated  the  detection  of 
distinct  differences  in  the  radiation  of  heat  from  the  skin  surfaces,  these  differences 
being  largely  dependent  upon  the  temperatures  of  the  underlying  tissues.  Thermo- 


\ 

E 


z 
o 

0. 

5 
3 
CO 

z 
o 
o 


i 


u_ 
O 


LJ 
* 

a: 


40 


LJ 
O 
< 
QL 
O 

h- 
Z 
LJ 
O 


CO 
LJ 
LJ 

tr 

LJ 
O 


30 


20 


10 


rate  of  oxygen 
consumption 


cumulative   oxygen 
consumption 


brown    fat 
temperature, 


100 


80      o 

t- 
Q. 

5 

Z3 
CO 

60     O 


111 
o 

40     x 
o 


LJ 


20 


O 


10 


20 


30 


40 


50 


MINUTES 


FIGURE  1.  The  typical  patterns  of  oxygen  consumption  and  brown  adipose  tissue  (brown 
fat)  temperature  increase  in  the  bat  during  arousal  from  hibernation.  The  vertical  broken  line 
intersects  the  curves  at  the  time  when  rapid  arousal  is  completed.  Ambient  temperature  during 
the  arousal  was  5°  C. 


BROWN  ADIPOSE  TISSUE  THERMOGENESIS 


97 


grams  are  pictorial   thermal   maps   and  were   obtained   using   a   Barnes   Medical 
Thermograph.3 

RESULTS 
Total  oxygen  consumption  during  arousal 

For  each  bat,  the  rate  of  arousal  from  hibernation  varied  to  a  certain  extent 
with  body  weight,  amount  of  brown  adipose  tissue,  and  apparent  individual  be- 
havior differences.  Amongst  this  variation,  however,  healthy  individuals  of  similar 
body  weight  demonstrated  a  consistent  pattern  of  oxygen  consumption  during 
arousal.  The  data  of  Figure  1  are  an  example  of  one  such  arousal.  The  brown 
adipose  tissue  (brown  fat)  temperature 4  curve  is  included  in  Figure  1  to  aid 
identification  of  the  course  of  arousal.  For  approximately  the  first  15-20  minutes 
of  arousal,  there  is  a  low  and  slowly-increasing  rate  of  oxygen  consumption,  but 

TABLE  I 
Rate  of  oxygen  consumption  of  bat  tissues  at  37.2°  C.  under  various  experimental  conditions 


Tissue 

Treatment 

No.  of 
animals 

No.  of 
measurements 

Mean  rate  of  Oi 
consumption  ±  S.E. 
Gul./lOO  mg.  fresh  tissue/hr.) 

Brown  fat 

Control 

9 

26 

392  ±    42.4 

Epinephrine 

9 

9 

1320  ±    93.9 

Epinephrine  -f  glucose 

6 

6 

1252  ±  107.7 

Liver 

Control 

4 

7 

168  ±      5.0 

Epinephrine 

3 

3 

160  ±    12.6 

Epinephrine  +  glucose 

3 

3 

157  ±      9.3 

Heart 

Control 

2 

4 

119  ±    11.6 

Epinephrine 

2 

2 

117 

Epinephrine  +  glucose 

2 

2 

114 

the  rate  increases  rapidly  and  reaches  high  levels  for  the  20-3 5 -minute  interval. 
After  approximately  34  minutes  (vertical  broken  line),  the  rate  of  oxygen  con- 
sumption begins  to  fall  to  a  lower  level,  coinciding  with  the  approach  of  maximum 
brown  adipose  tissue  temperature.  At  this  point,  the  cumulative  oxygen  con- 
sumption is  near  65  ml. 

The  mean  figures  for  duration  of  arousal  and  total  oxygen  consumption  for 
8  bats  are  36  minutes  and  66.15  ml.,  respectively  (from  Table  II).  Assuming  a  low 
respiratory  quotient  (R.Q.)  of  0.75  for  arousal  (Jansky  and  Hajek,  1961),  this 
oxygen  consumption  represents  312  cal.  of  heat  production,  attributable  to  the 
enthalpy  increase  of  the  body  mass  and  the  heat  loss  during  the  arousal. 

Tissue  respiration 

A  representative  experiment  in  which  the  oxygen  consumptions  of  brown  adipose 
tissue  and  liver  were  measured  is  shown  in  Figure  2.  The  respiratory  rate  of  both 

3  Barnes  Engineering  Company,  Stamford,  Connecticut. 

4  The  temperature  of  interscapular  brown  adipose  tissue  is  typically  about   1°    C.   above 
core  temperature  during  arousal  from  hibernation   (Hayward  et  al.,  1965). 


98 


JOHN  S.  HAYVVARD  AND  ERIC  G.  BALL 


TABLE  II 

Calculation  of  the  percentage  of  the  total  oxygen  consumption  for  arousal  that  is 

attributable  to  brown  fat 


Maximum 

Bat 

Body 
weight 
(g.) 

Duration 
of  arousal 

(min.) 

Weight  of 
brown  fat 
(g.) 

metabolic 
rate  of 
brown  kit 

(yul.   O2/100 

mg./hr.) 

Total  O2 
consumption 
for  arousal 
(ml.) 

Oi  consump- 
tion of 
brown  fat 
for  arousal 
(ml.) 

Percentage  of 
total  O2 
consumption 
by  brown  fat 

A 

15.6 

39 

0.446 

1500 

78.05 

4.35 

5.57 

B 

14.2 

39 

0.453 

1890 

84.16 

5.57 

6.61 

c 

13.3 

44 

0.415 

995 

68.64 

3.03 

4.41 

E 

14.5 

26 

0.432 

1490 

51.88 

2.79 

5.38 

G 

14.8 

22 

0.584 

1390 

52.30 

2.98 

5.69 

H 

13.6 

38 

0.467 

1420 

71.33 

4.20 

5.89 

J 

15.6 

38 

0.643 

1235 

67.44 

5.03 

7.46 

K 

12.7 

42 

0.334 

1070 

55.39 

2.50 

4.52 

Means 

14.3 

36 

0.472 

1374 

66.15 

3.81 

5.69 

tissues  was  unaltered  by  the  addition  of  glucose  to  the  medium.  Addition  of 
epinephrine,  1  /xg./ml.,  caused  a  5QOr/c  increase  in  the  rate  of  oxygen  consumption 
of  brown  adipose  tissue  but  was  without  effect  upon  liver.  In  other  experiments 
in  which  the  action  of  epinephrine  on  brown  adipose  tissue  was  compared  at  concen- 
trations of  0.1,  1.0  and  10  /u,g./ml.,  it  was  found  that  a  maximum  response  was 
obtained  at  1.0  jug./ml.  A  comparison  of  norepinephrine  and  epinephrine  at  con- 


o> 

E 

o 
o 


0> 

o 

"a. 


1200 


1000 


800 


600 


400 


200 


o o 


With     Glucose 
Without     Glucose 


Brown    Adipose    / 
Tissue     / 


Epinephrine 
I  jug/ml 


.-—  ^**  ^r^~ 


Liver   (150) 


30 


60 


90 


Time,    Minutes 


FIGURE  2.  A  representative  experiment  in  which  the  oxygen  consumptions  of  brown  adipose 
tissue  and  liver  of  the  bat  show  the  effects  of  epinephrine  and  glucose  addition.  Numbers  in 
parentheses  are  rates  of  oxygen  consumption  (,ul./100  mg.  fresh  tissue/hr.). 


BROWN  ADIPOSE  TISSUE  THERMOGENESIS 


99 


centrations  of  0.1  /x,g./ml.  also  showed  that  no  significant  difference  in  their  action 
could  be  observed.  Epinephrine  at  a  concentration  of  1  fig./ml.  was  therefore 
employed  routinely  in  the  series  of  experiments  performed. 

A  summary  of  this  series  of  experiments  is  given  in  Table  I.     In  the  absence 


FIGURE  3.  Illustration  of  a  bat  as  positioned  for  thermography,  showing  the  location  of 
the  interscapular  brown  adipose  tissue  and  the  major  temperatures  prevailing  at  the  commence- 
ment of  thermographic  scanning. 

FIGURE  4.  Thermogram  of  the  dorsal  surface  of  a  bat  during  its  arousal  from  hibernation. 
The  higher  the  temperature  and  intensity  of  infrared  radiation  from  the  skin  surface,  the 
brighter  is  the  image. 


100  JOHN  S.  HAYWARD  AND  ERIC  G.  BALL 

of  any  added  stimulation,  the  mean  value  for  the  rate  of  oxygen  consumption  of 
rate  of  brown  adipose  tissue  and  was  without  effect  upon  liver  or  heart  slices.  In 
experiments  not  reported  here,  it  was  found  that  there  was  no  significant  difference 
between  the  respiratory  rate  of  brown  adipose  tissue  sampled  before  and  after 
arousal  from  hibernation. 

brown  adipose  tissue  was  2.33  and  3.29  times  that  of  liver  and  heart,  respectively. 
The  addition  of  epinephrine  caused  an  average  increase  of  350%  in  the  respiratory 

Relative  oxygen  consumption  of  brown  adipose  tissue 

In  Table  II  the  pertinent  data  for  calculating  the  percentage  of  the  total  oxygen 
consumption  for  arousal  that  is  attributable  to  brown  adipose  tissue  are  summarized 
for  each  bat.  Brown  adipose  tissue  averaged  3.30%  of  the  total  body  weight  and, 
based  upon  its  in  vitro  respiration,  utilized  an  average  of  5.69%  of  the  total  oxygen 
consumption.  If  the  oxygen  consumptions  of  liver  and  heart  (averaging  4.31% 
and  1.16%  of  the  total  body  weight,  respectively)  are  included,  a  larger  estimate  of 
the  non-muscular  component  of  heat  production  is  obtained.  Together  with  brown 
adipose  tissue,  the  mean  oxygen  consumption  by  these  tissues  for  a  36-minute 
arousal  would  be  4.55  ml.,  or  6.88%  of  the  total  consumption. 

Thermographic  evidence  of  broztm  adipose  tissue  heat  production 

In  the  bat,  brown  adipose  tissue  is  widely  distributed  between  the  muscles  of 
the  dorsal  thoracic  region,  around  the  neck,  and  surrounding  the  major  vessels 
entering  and  leaving  the  heart.  The  largest  depot  occurs  in  the  interscapular  fossa 
and  extends  to  the  back  of  the  head  and  sides  of  the  neck.  The  tissue  lies  just 
beneath  the  skin  in  the  general  position  illustrated  in  Figure  3.  To  obtain  the  best 
contrast  pattern  on  the  thermogram,  the  following  procedure  was  used.  A 
hibernating  bat  was  stimulated  to  arouse,  and  during  this  process  was  kept  in 
a  cold  environment  of  6°  C.  (42°  F.)  until  the  stage  when  the  brown  adipose  tissue 
temperature  was  approaching  the  ambient  room  temperature  of  22°  C.  (71°  F.). 
It  was  then  removed  from  the  cold  and  held  in  the  scanning  field  of  the  thermo- 
graph. The  wing  membranes  rapidly  equilibrated  with  room  temperature.  Within 
2-3  minutes,  the  skin  temperature  over  the  brown  fat  passed  room  temperature, 
but  the  rest  of  the  body  was  still  cold.  Infrared  scanning  was  commenced  when 
the  major  temperature  areas  were  those  shown  in  Figure  3.  Scanning  took  ap- 
proximately three  minutes,  during  which  time  the  bat  had  to  be  held  immoble. 
The  resulting  thermogram  (Fig.  4)  shows  a  conspicuously-delineated  "hot"  area 
that  coincides  exactly  with  the  shape  of  the  underlying  brown  adipose  tissue. 

DISCUSSION 

An  average  of  only  6.9%  of  the  total  oxygen  consumed  by  the  whole  bat  during 
arousal  from  hibernation  can  be  accounted  for  by  measurements  on  brown  adipose 
tissue,  liver,  and  heart  respiration  in  vitro.  Even  this  value  is  high  since  the 
in  vitro  measurements  were  made  only  at  37°  C.,  a  temperature  approached  by  the 
tissues  only  at  the  end  of  the  arousal  period.  This  is  such  a  surprisingly  low 
percentage  of  the  total  that  it  raises  several  questions. 


BROWN  ADIPOSE  TISSUE  THERMOGENESIS  101 

First,  if  the  data  are  accepted,  one  must  still  account  for  the  remainder  of  the 
oxygen  consumption.  Muscle,  because  of  its  relatively  large  mass,  would  seem  to 
be  the  main  tissue  to  be  considered.  Rough  dissections  of  muscle  from  several 
bats  indicate  the  total  muscle  mass  of  an  average  bat  to  be  about  4  grams.  If,  as 
our  data  would  suggest,  we  attribute  at  least  75%  of  the  total  O2  consumption 
during  a  36-minute  arousal  period  to  muscle,  then  we  obtain  a  rate  of  respiration 

.  0.75  X  66.15  ml.  O,      ^OAC        ^  /        •        TM. 

for  muscle  of  -  —^  -  —  0.345  ml.  O2/g.  mm.     The  corresponding  rate, 

4  g.  X  36  mm. 

as  measured  here,  is  0.224  for  brown  adipose  tissue  and  0.02  for  heart  muscle. 
It  is  difficult  to  reconcile  this  very  high  rate  of  respiration  for  skeletal  muscle  with 
the  indication  that  shivering  is  an  unimportant  feature  of  the  arousal  from  hiberna- 
tion of  the  bat,  since  the  results  of  Hay  ward  and  Lyman  (in  press)  show  that  there 
is  no  difference  in  the  arousal  time  when  bats  are  curarized.  Moreover,  if  muscle 
was  consuming  oxygen  at  this  rate,  it  should  be  producing  over  50%  more  heat 
than  brown  adipose  tissue.  The  thermogram  presented  certainly  does  not  validate 
such  a  conclusion. 

In  considering  possible  reasons  for  this  apparent  discrepancy,  the  reliability 
of  the  measurements  must  be  examined.  For  example,  perhaps  the  measurement  of 
total  oxygen  consumption  is  too  high.  For  this  measurement,  however,  all  con- 
ceivable errors  would  result  in  underestimation  rather  than  overestimation  of  the 
true  rate.  In  addition,  a  theoretical  calculation  of  the  heat  required  to  warm  a 
14.3-g.  bat  from  5°  to  35°  C.  at  an  ambient  temperature  of  5°  C.  verifies  the 
experimental  results.  If  we  assume  an  average  specific  heat  of  0.9  cal./g.°  C.  for 
the  total  tissue  of  the  bat,  then  0.9  cal./g.°  C.  X  14.3  g.  X  30°  C.  "  386  cal.  will 
be  required  for  arousal.  This  value  is  undoubtedly  too  high  since  brown  adipose 
tissue  warms  to  35°  C.  prior  to  all  other  tissues  (Hayward  et  al.,  1965).  Partly 
offsetting  this  consideration,  however,  will  be  the  heat  loss  of  the  environment 
during  arousal.  A  not  unreasonable  value  would  thus  seem  to  be  300  ±  25  calories, 
which  is  comparable  to  the  average  of  312  calories  from  our  experimental  results. 
This  calculation  indicates  that  the  measured  total  oxygen  consumption  is  within  the 
expected  range  and  can  be  acquitted  of  possible  major  error. 

Next,  one  may  question  the  validity  of  the  in  vitro  measurements  of  tissue 
oxygen  consumption.  The  mean  respiratory  rate  of  1374  jul.  O2/100  mg./hr. 
observed  here  for  bat  brown  adipose  tissue  when  stimulated  by  catecholamine  is 
as  high  or  higher  than  that  reported  for  this  tissue  in  other  species.  Joel  (1965) 
gives  an  average  value  of  719  /tl.  O,/100  mg.  fresh  tissue/hr.  for  brown  adipose 
tissue  from  another  hibernator,  the  ground  squirrel  (Citelhis  tridecemlineatus}. 
This  value  was  obtained  in  the  presence  of  1  /xg./ml.  of  norepinephrine  and  was 
raised  to  1260  if  10  ^moles  of  succinate  were  also  present.  In  the  rat,  a  value  of 
725  was  observed  by  Joel  (1965)  when  the  brown  adipose  tissue  was  stimulated  by 
the  addition  of  1  jug./ml.  epinephrine.  Smith  and  Roberts  (1964)  report  values 
of  643  for  brown  adipose  tissue  from  cold-acclimated  rats  and  232  from  normal 
rats.  These  authors  did  not  study  the  effect  of  catecholamine  additions.  The 
rates  found  here  for  heart  and  liver  slices  are  not  greatly  different  from  those 
reported  for  rat  tissues  (Long,  1961,  p.  795). 

Lastly,  one  can  offer  the  explanation  that  the  measured  in  vitro  rates  fall  far 
short  of  those  that  do  occur  in  vivo.  Certainly  the  measured  rate  for  heart  slices 


102  JOHN  S.  HAYWARD  AND  ERIC  G.  BALL 

does  not  reflect  the  rate  exhibited  by  a  heart  actively  beating  in  vivo.  Values  for 
perfused  hearts  are  much  higher  (Fisher  and  Williamson,  1961).  However,  even 
if  the  in  vivo  rates  of  heart  and  liver  respiration  were  10-fold  those  measured  here, 
they  would  still  account  for  only  12%  of  the  total  oxygen  consumption. 

Dynamic  metabolic  conditions  are  characteristic  of  brown  adipose  tissue  during 
arousal  from  hibernation  (Joel,  1965,  p.  84).  We  are  led  to  suspect  that  brown 
adipose  tissue  heat  production  during  arousal  may  be  considerably  greater  than  our 
respiratory  data  would  indicate,  despite  their  high  value,  and  that  the  necessary 
conditions  to  achieve  such  rates  in  vitro  have  not  yet  been  achieved. 


We  appreciate  the  critical  advice  given  by  Dr.  C.  P.  Lyman  on  the  design  and 
conclusions  of  this  study. 

The  use  of  the  Barnes  Thermograph  was  facilitated  by  the  kind  cooperation  of 
the  Department  of  Radiology,  Massachusetts  General  Hospital,  Boston. 

SUMMARY 

The  in  vitro  respiratory  rates  of  brown  adipose,  heart,  and  liver  tissues  were 
studied  in  the  bat  (Eptesicns  fuscus)  to  determine  their  contribution  to  the  heat 
necessary  for  arousal  from  hibernation.  The  mean  oxygen  consumption  of  the 
whole  animal  for  arousal  from  hibernation  was  66.2  ml.  of  which  5.7%  is  estimated 
to  be  utilized  by  brown  adipose  tissue,  and  1.2%  by  heart  and  liver  combined. 
The  maximum  respiratory  rate  of  brown  adipose  tissue  when  stimulated  by  epi- 
nephrine  was  134  /xl.  O2/100  mg.  fresh  tissue/hr.  Despite  this  high  in  vitro 
respiratory  rate,  it  seems  inadequate,  on  the  basis  of  other  evidence,  to  account  for 
the  heat  production  expected  for  brown  adipose  tissue  during  arousal  from  hiberna- 
tion. A  thermogram  of  a  bat  arousing  from  hibernation  is  presented  which  provides 
pictorial  evidence  of  the  large  thermogenic  capacity  of  brown  adipose  tissue.  It  is 
concluded  that  the  conditions  necessary  to  measure  the  maximum  respiratory  rate 
of  brown  adipose  tissue,  such  as  it  occurs  during  arousal  from  hibernation,  have 
not  yet  been  achieved. 

LITERATURE  CITED 

BALL,   E.   G.,    1965.     Some   energy   relationships   in   adipose   tissue.     Ann.   N.    Y.   Acad.   Sci., 

131 :  225-234. 
BERTALANFFY,  L.  VON,  AND  W.  J.   PIROZYNSKI,   1953.     Tissue  respiration,   growth  and  basal 

metabolism.     Biol.  Bull.,  105:  240-256. 
FISHER,  R.  B.,  AND  J.  R.  WILLIAMSON,  1961.     The  effects  of  insulin,  adrenaline  and  nutrients 

on  the  oxygen  uptake  of  the  perfused  rat  heart.     /.  Physiol.,  158:  102-112. 
HAYWARD,  J.  S.,  AND  C.  P.  LYMAN,  (in  press).     Nonshivering  heat  production  during  arousal 

from  hibernation  and  evidence  for  the  contribution  of  brown  fat.     In:   Proceedings  of 

the   III    International    Symposium   on    Natural    Mammalian    Hibernation.     Oliver    and 

Boyd,  Edinburgh. 
HAYWARD,  J.  S.,  C.  P.  LYMAN  AND  C.  R.  TAYLOR,  1965.     The  possible  role  of  brown  fat  as  a 

source  of  heat  during  arousal  from  hibernation.     Ann.  N.  Y.  Acad.  Sci.,  131:  441-446. 
JANSKY,  L.,  AND  I.  HAJEK,  1961.     Thermogenesis  of  the  bat  Myotis  myotis  Borkh.    Physiol. 

Bohemoslov.,  10:  283-289. 


BROWN  ADIPOSE  TISSUE  THERMOGENESIS  103 

JOEL,  C.  D.,  1965.  The  physiological  role  of  brown  adipose  tissue.  In:  Handbook  of  Physiol- 
ogy, Section  5  :  Adipose  Tissue,  ed.  by  A.  E.  Reynold  and  G.  F.  Cahill,  Jr.  American 
Physiological  Society,  Washington,  D.  C.,  pp.  59-85. 

KREBS,  H.  A.,  AND  K.  HENSELEIT,  1932.  Untersuchungen  iiber  die  Harnstoffbildung  im 
Tierkorper.  Z^itschr.  physiol.  Chan.,  210:  33-66. 

LONG,  C.  (editor),  1961.  Biochemists'  Handbook.  Van  Nostrand  Inc.,  Princeton,  New 
Jersey. 

SMITH,  R.  E.,  AND  J.  C.  ROBERTS,  1964.  Thermogenesis  of  brown  adipose  tissue  in  cold- 
acclimated  rats.  Aincr.  J.  Physio!.,  206:  143-148. 


MECHANISM  OF  STARFISH  SPAWNING.     I.    DISTRIBUTION  OF 

ACTIVE  SUBSTANCE  RESPONSIBLE  FOR  MATURATION 

OF  OOCYTES  AND  SHEDDING  OF  GAMETES  1 

HARUO  KANATANI  AND  MIWAKO  OHGURI 

Division  of  Physiology  of  Marine  Organisms,  Ocean  Research  Institute, 
University  of  Tokyo,  Nakano-ku,  Tokyo,  Japan 

Since  Chaet  and  his  co-workers  (Chaet  and  McConnaughy,  1959;  Chaet  and 
Musick,  1960;  Chaet,  1964a)  discovered  that  starfish  (Asterias  forbesi)  can  be 
induced  to  spawn  by  injecting  a  water  extract  of  radial  nerves  into  the  coelomic 
cavity,  some  clue  to  the  elucidation  of  the  mechanism  of  starfish  spawning  has  been 
afforded.  The  active  substance  responsible  for  gamete-shedding,  contained  in 
radial  nerves,  was  reported  to  be  a  polypeptide  with  a  relatively  small  molecular 
weight  (Kanatani  and  Noumura,  1962,  1964;  Chaet,  1964b,  1966).  Further,  the 
nerve  extract  prepared  from  one  species  acts  similarly  in  several  species,  suggesting 
that  the  substances  are  chemically  analogous  among  starfishes  (Hartman  and  Chaet, 
1962;  Noumura  and  Kanatani,  1962;  Chaet,  1964c,  1966),  although  there  may  be 
some  species  differences  in  details ;  for  example,  nerve  extract  of  Asterina  pectini- 
fcra  is  effective  in  inducing  spawning  of  Asterias  amurensis,  but  the  converse  is  not 
true  (Noumura  and  Kanatani,  1962;  Chaet,  1966). 

The  present  investigation  was  carried  out  in  an  attempt  to  determine  the 
following  points  with  regard  to  the  distribution  of  active  substance  in  the  body  of 
the  starfish :  ( 1 )  Whether  or  not  there  exists  any  difference  in  the  quantity  of 
active  substance  between  male  and  female  starfish,  since  males  seem  to  shed  their 
gametes  more  easily  than  females  (Noumura  and  Kanatani,  1962).  (2)  Whether 
or  not  the  active  substance  is  present  only  in  the  radial  nerves.  (3)  How  the  active 
substance  is  quantitatively  distributed  along  the  radial  nerve.  (4)  Whether  the 
active  substance  actually  appears  in  the  body  fluid  of  the  starfish  at  the  time  of 
natural  spawning.  An  attempt  is  also  made  to  discuss  the  mechanism  of  star- 
fish spawning  in  relation  to  the  results  obtained  from  such  investigations. 

MATERIALS  AND  METHODS 

The  material  mainly  used  was  a  common  Japanese  starfish,  Asterias  amurensis, 
during  its  spawning  season.  Asterina  pectinijera  was  also  used  in  some  experi- 
ments dealing  with  comparative  aspects.  These  starfishes  were  obtained  from 
Tokyo  Bay  and  kept  in  crawls  hanging  in  the  sea  or  in  aquaria  supplied  with 
running  sea  water  at  the  Misaki  Marine  Biological  Station. 

In  order  to  obtain  radial  nerves,  the  ambulacral  zone  was  isolated  and  then 
perpendicularly,  but  slightly  diagonally,  cut  with  scissors  into  two  halves  along 

1  Contribution  No.  59  from  The  Ocean  Research  Institute,  University  of  Tokyo. 

104 


MECHANISM  OF  STARFISH  SPAWNING 


105 


the  midline  so  that  the  nerve  could  easily  be  observed.  These  nerves  were  stripped 
off  with  fine  forceps  and  collectd.  The  nerves  were  stored  frozen  and  dried  by 
lyophilization.  This  method  for  obtaining  the  radial  nerves  seems  to  be  more 
simple  than  the  original  method  of  Chaet  (1964a). 

For  obtaining  the  nerve  ring,  a  starfish  was  placed  oral  side  up  in  a  large 
Petri  dish  containing  sea  water,  and  the  oral  spines  and  tube  feet  near  the  mouth 


FIGURE  1.     Nerve  ring  (NR)  and  radial  nerve  (N)  of  Asterias  amitrcnsis. 
illustration  shows  parts  of  nerve  used  in  experiment. 


Lower 


were  carefully  removed  under  a  dissection  microscope  (Fig.  1).     The  nerve  ring 
thus  easily  observed  was  cut  out  with  fine  scissors  and  lyophilized. 

In  an  experiment  comparing  the  activities  of  the  various  parts  of  radial  nerves, 
the  ambulacral  plate  was  cut,  after  removing  the  nerve  ring,  into  four  sections 
as  shown  in  Figure  1,  and  then  the  radial  nerve  of  each  part  was  stripped  off  as 


106  HARUO  KANATANI  AND  MIWAKO  OHGURI 

described  above  and  separately  lyophilized.  The  parts  of  the  radial  nerves  were 
designated  as  N1;  N2,  N3,  and  N4,  from  proximal  to  distal. 

To  make  the  nerve  extract,  a  few  milliliters  of  cold  de-ionized  water  were  added 
to  the  lyophilized  material,  which  was  then  homogenized.  To  the  homogenate, 
de-ionized  water  was  added  to  give  a  concentration  of  4  mg./ml.  of  lyophilized 
nerve  and  the  mixture  was  centrifuged  for  one  hour  at  20,000  g.  An  equal  volume 
of  1  M  sodium  chloride  was  added  to  the  supernatant  to  make  an  isotonic  nerve 
extract,  which  served  as  the  original  extract.  In  preparing  test  solutions,  the 
original  extract  was  diluted  at  various  concentrations  with  sea  water  (successively 
diluted  twice  from  100  /xg./ml.  to  0.8  /xg./ml.,  or  from  200  jug./ml.  to  1.2  /xg./ml.). 
An  extract  of  tube  feet  was  made  by  the  same  procedure :  the  concentrations  of  the 
test  solutions  were  adjusted  from  500  /xg./ml.  of  lyophilized  tube  feet  to  10  /xg./ml. 
by  serial  dilution  with  sea  water.  Extracts  of  body  wall  (ectoderm  of  the  aboral 
surface  of  the  arm),  cardiac  stomach  and  pyloric  caeca  were  made  by  homogenizing 
the  lyophilized  samples  in  sea  water.  (The  body  wall  was  stripped  off  with  fine 
forceps.)  The  homogenates  were  heated  in  a  boiling  water  bath  for  five  minutes 
and  centrifuged.  The  supernatants  were  serially  diluted  with  sea  water.  The 
concentrations  with  adjusted  at  1000-32  jug./ml.  for  body  wall,  5000-312  /xg./ml.  for 
cardiac  stomach  and  50-1.57  mg./ml.  for  pyloric  caeca. 

To  assay  the  capacity  of  the  test  solutions  to  cause  spawning  and  meiosis,  an 
isolated  small  fragment  of  ovary,  about  10  to  12  mm.  in  length,  was  placed  in  a 
small  Petri  dish  containing  4  ml.  of  the  solution  and  observed  (Fig.  2)  (cf.  Chaet. 
Andrews  and  Smith,  1964).  Ovarian  fragments  derived  from  a  single  female 
were  used  in  each  experiment  in  order  to  eliminate  individual  differences  in  reac- 
tivity. These  ovarian  fragments  had  previously  been  rinsed  in  sea  water  for  an 
appropriate  period  until  no  more  eggs  were  released  from  their  cut  surfaces.  In 
•vivo  assays  were  also  performed  by  injecting  the  test  solution  into  the  coelomic 
cavity  of  intact  starfish. 

To  obtain  coelomic  fluid,  a  small  slit  was  made  in  the  aboral  side  of  the  distal 
part  of  an  arm  and  the  coelomic  fluid  was  collected  through  it.  The  coelomic  fluid 
thus  obtained  was  used  immediately  after  centrifugation  for  30  minutes  at  4000 
r.p.m.,  or  stored  in  a  deep  freezer  before  use. 

RESULTS 

Comparison  of  content  of  active  substance  between  males  and  females 

The  radial  nerves  taken  from  each  of  four  male  and  four  female  Astcrias  (arm 
length  10  cm.)  were  separately  homogenized  in  3  ml.  of  de-ionized  water.  The 
homogenate  was  diluted  with  an  equal  volume  of  1  M  sodium  chloride,  and  0.5  ml. 
of  this  nerve  extract  was  injected  into  each  of  five  starfish.  As  shown  in  Table  I, 
the  nerve  extract  obtained  from  male  starfish  was  effective  in  inducing  spawning  of 
females,  and  vice  versa.  After  shedding  the  gametes,  the  gonads  were  very  small 
in  most  of  these  animals.  Isolated  ovaries  placed  in  50  ml.  of  sea  water  containing 
2  ml.  of  these  nerve  extracts  began  to  shed  simultaneously  (after  about  30  minutes), 
regardless  of  the  sex  of  the  nerve  donors.  Control  ovaries,  placed  in  sea  water 
containing  2  ml.  of  0.5  M  sodium  chloride,  failed  to  spawn.  These  preliminary 
experiments  suggested  that  the  spawning  factor  is  the  same  in  the  two  sexes. 


MECHANISM  OF  STARFISH  SPAWNING 


107 


Nerve  extracts  prepared  from  males  were  also  effective  in  inducing  meiosis  of 
oocytes. 

Experiments  were  next  conducted  to  determine  whether  there  exists  any 
difference  between  male  and  female  Astcrias  with  respect  to  the  content  of  shedding 
substance  in  the  radial  nerves,  since  the  males  shed  in  response  to  lower  concentra- 
tions of  injected  nerve  extract  than  do  the  females.  Small  fragments  of  the  ovaries 
taken  from  a  single  female  were  exclusively  used  for  assay  in  each  experiment. 


TABLE  I 

Induction  of  spawning  by  injected  nerve  extracts  obtained  from  male  and  female  Asterias 


Nerve  extracts  obtained  from 

Groups  of 

Male 

Female 

experiments 

Sex 

Time  (min.)* 

Amount  of** 
gametes 

Sex 

Time  (min.) 

Amount  of 
gametes 

Male 

29 

+  +  + 

Female 

39 

+  +  + 

Female 

51 

+  + 

Female 

39 

+  +  + 

1 

Male 

34 

+  +  + 

Female 

43 

+  + 

Female 

51 

+  +  + 

Female 

40 

+  +  + 

Male 

34 

+  + 

Male 

33 

+  + 

Male 

26 

+  +  + 

Male 

26 

+  +  + 

Male 

27 

+  +  + 

Female 

30 

+  +  + 

2 

Male 

28 

+  +  + 

Female 

32 

+  +  + 

Male 

32 

+  +  + 

Male 

35 

+  +  + 

Female 

40 

+  + 

Male 

34 

+  +  + 

Female 

46 

+ 

Male 

28 

+  + 

Male 

33 

+  + 

Male 

34 

+  +  + 

3 

Male 

No  spawning*** 

— 

Female 

29 

+  + 

Male 

40 

+  + 

Female 

37 

+  +  + 

Female 

40 

+  + 

Female 

37 

+  + 

Male 

29 

+  +  + 

Male 

29 

+  +  + 

Female 

35 

+  +  + 

Male 

29 

+  +  + 

4 

Male 

36 

+  +  + 

Female 

32 

+  +  + 

Female 

No  spawning*** 

— 

Female 

49 

+ 

Male 

32 

+  +  + 

Female 

51 

+  +  + 

*  Interval  preceding  discharge  of  gametes. 

:  +  +  +  :  large  amount  of  gametes;   ++:  intermediate  amount  of  gametes;    +:  small 
amount  of  gametes. 

:*  Gonads  poorly  developed,  or  had  already  spawned. 

These  ovarian  fragments  were  placed  in  test  solutions  containing  the  original 
nerve  extract  at  various  concentrations  (0.8-50  /*g.  of  lyophilized  nerve  per  ml.), 
prepared  separately  from  males  and  from  females,  and  their  spawning  reactions 
were  examined  after  one  hour.  Table  II  shows  the  results  of  six  pairs  of  such 
experiments.  These  data  clearly  demonstrate  that  the  content  of  shedding  sub- 
stance in  radial  nerves  is  the  same  in  the  two  sexes. 


108 


HARUO  KANATANI  AND  MIWAKO  OHGURI 


TABLE  II 


In  vitro  assay  to  test  for  sex  difference  in  gamete-shedding  activity  of  nerve  extracts. 
Ovary  fragments  derived  from  one  female  were  used  in  each  pair  of  experiments 


Concentration 

Comparison  of  shedding  induced  by  nerve  extracts  obtained  from  cf  and    9  donor  starfish. 
A-F  :  ovarian  fragments  from  6  different  females. 

of  nerve 

extracts 

(yug./ml. 
lyophilized 

A 

B 

c 

D 

E 

F 

m. 

f.* 

m. 

f. 

m. 

f. 

m. 

f. 

m. 

f. 

m. 

f. 

50 

4. 

4. 

+ 

+ 

4- 

+ 

+ 

4- 

+ 

— 

+ 

4. 

25 

4. 

4- 

4- 

4- 

4- 

+ 

-j- 

4- 

+ 

4- 

4- 

4- 

12.5 

4- 

4- 

+ 

4- 

4- 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

6.3 

4- 

4. 

4- 

4- 

4- 

+ 

+ 

+ 

+ 

4- 

+ 

+ 

3.1 

— 

— 

— 

— 

+ 

+ 

+ 

+ 

— 

— 

4- 

4- 

1.6 

— 

— 

— 

— 

-\- 

+ 

— 

— 

— 

— 

— 

— 

0.8 

— 

— 

— 

— 

— 

— 

— 

— 

— 

— 

—  * 

— 

+  :  positive,  —  :  negative. 
*  Sex  of  nerve  donor. 

Quantitative  distribution  of  active  substance  in  various  parts  of  radial  nerve  and 
in  nerve  ring 

The  results  of  in  vitro  assay  of  the  active  substance  contained  in  the  nerve  ring 
and  various  regions  of  the  radial  nerves  (Fig.  1)  are  shown  in  Table  III.  These 
data  indicate  that  the  active  substance  is  present  in  the  nerve  ring  as  well  as  in  the 
radial  nerves,  and  is  evenly  distributed  in  quantity  per  dry  weight  among  the 
nerve  ring  and  the  various  parts  of  the  radial  nerves. 

The  absolute  amount  of  active  substance  thus  decreases  from  proximal  to  distal 
as  the  size  of  the  cross-section  of  the  radial  nerve  decreases  (Table  IV).  The  ratio 
of  weights  of  lyophilized  nerve  at  the  various  regions  thus  reflects  the  actual  content 
of  the  active  substance  along  the  radial  nerve. 

In  these  in  vitro  experiments,  the  ovarian  fragments  began  to  shed  after  about 
20-25  minutes  (in  some  cases  less  than  20  minutes)  when  they  were  immersed 
in  test  solutions  (4  ml.)  containing  more  than  25  ^g./ml.  of  the  lyophilized  nerve. 

TABLE  III 

Distribution  of  the  gamete-shedding  substance  in  nerve  ring  and  radial  nerves 

(Fig.  1)  of  Asterias 

Minimum  dose  of  nerve  extract  for  induction  of  spawning  within  1  hour  in  ovary  fragments 

(/ig./ml.  lyophilized  nerve) 


Nerve  donors 

A(c?) 

B(cf) 

C(rf) 

D(9) 

E(9) 

F(9) 

Parts  of  nerves 

NR 

12.5 

6.3 

6.3 

6.3 

6.3 

3.1,  6.3* 

N, 

12.5 

6.3 

6.3 

6.3 

6.3 

3.1,  6.3* 

N2 

12.5 

12.5 

6.3 

6.3 

6.3 

3.1,  6.3* 

N3 

6.3 

6.3 

6.3 

6.3 

6.3 

3.1,  6.3* 

N4 

6.3 

12.5 

3.1 

6.3 

6.3 

3.1,  3.1* 

Ovary  from  different  animal. 


MECHANISM  OF  STARFISH  SPAWNING 


109 


In  in  vivo  experiments,  female  starfish  injected  with  1  ml.  of  0.5  M  sodium 
chloride  containing  500  //,g.  of  the  same  lyophilized  nerve  material  usually  began 
spawning  after  about  40  minutes. 

Similar  in  vitro  experiments  on  Asterina  pectinifera  clearly  confirmed  the 
above  results  obtained  in  Asterias;  the  minimum  dose  for  induction  of  spawning 
was  the  same  among  the  various  regions  of  radial  nerves  and  nerve  ring  (10 
ju.g./ml.  of  lyophilized  nerve  in  the  middle  of  June  and  2.5  /ig./ml.  in  the  end  of 
June,  respectively). 

Presence  of  active  substance  in  body  parts  other  than  central  nervous  system 

Since  nervous  tissue  is  plentiful  in  the  tube  feet  (Smith,  1937),  the  gamete- 
shedding  activity  of  a  tube-foot  extract  was  next  examined  in  a  similar  way. 
Table  V  shows  the  results  of  an  in  vitro  assay,  one  hour  after  ovarian  fragments 
were  immersed  in  tube-foot  extracts.  These  data  show  that  the  tube  feet  also 
contain  shedding  substance,  and  that  there  is  little  difference  as  regards  its  content 
between  males  and  females.  The  minimum  doses  for  induction  of  spawning  were 
50  to  100  /j.g.  of  lyophilized  tube  feet  per  ml.,  when  the  experiments  were  conducted 
later  in  the  spawning  season.  When  such  experiments  were  made  earlier  in  the 

TABLE  IV 

Weights  of  various  parts  of  lyophilized  Asterias  nerves  (Fig.  1) 


Region 

NR 

Ni 

N2 

N3 

N< 

Weight*  (mg.) 

2.0  ±  0.1** 

9.0  ±  0.9 

8.3  ±  1.5 

6.7  ±  0.7 

3.6  ±  0.3 

Ratio 

1 

4.5 

4.2 

3.4 

1.8 

*  Average  of  six  animals  (fa.  11  cm.  arm  length)  shown  in  Table  III. 
:*  Standard  deviation. 

season,  the  minimum  doses  for  induction  of  spawning  ranged  between  150  and  200 
/Ag./ml.  Furthermore,  breakdown  of  germinal  vesicles  had  begun  in  most  of 
the  eggs  discharged  from  ovarian  fragments  30  minutes  after  immersion  in  test 
solutions  containing  200  /*.g.  of  lyophilized  tube  feet  per  ml.  These  data  indicate 
that  the  tube  feet  actually  contain  the  active  substance,  although  the  amount  is  small 
as  compared  with  that  in  the  radial  nerves. 

Some  additional  experiments  were  also  carried  out  to  determine  whether  the 
active  substance  exists  in  body  regions  other  than  the  central  nervous  system 
and  the  tube  feet.  When  the  gamete-shedding  activity  of  the  body  wall  was  tested 
in  vitro  with  ovarian  fragments,  it  was  found  that  125  /xg./ml.  or  more  of  the 
lyophilized  material  were  effective.  The  extract  of  the  cardiac  stomach  also 
showed  shedding  activity ;  625  /xg./ml.  or  more  were  effective,  whereas  the  extract 
of  pyloric  caeca  had  no  effect  even  at  a  concentration  of  25  mg./ml. 

Occurrence  of  active  substance  in  coelomic  fluid  at  time  of  natural  spawning 

The  injection  of  coelomic  fluid,  taken  from  an  animal  (Asterias)  before  or  after 
spawning,  into  the  coelomic  cavity  of  another  starfish  with  ripe  gonads,  failed  to 
induce  spawning. 


110 


HARUO  KANATANI  AND  MIWAKO  OHGURI 


2A 


2B 


FIGURE  2.  In  vitro  assay  of  gamete-shedding  substance.  A:  ovarian  fragment  (ca.  12 
mm.)  of  Astcrias  amurensis.  B:  same  ovarian  fragment  discharging  eggs  after  treatment  with 
nerve  extract.  Same  magnification. 

However,  coelomic  fluid  obtained  from  starfish  at  time  of  spawning  showed 
gamete-shedding  activity.  When  coelomic  fluid  was  collected  from  a  starfish 
spawning  naturally  in  the  laboratory  and  at  once  injected  in  toto  into  another  star- 
fish, spawning  occurred  in  some  cases. 

In  vitro  experiments  were  next  performed  in  order  to  verify  more  clearly  the 
presence  of  the  shedding  substance  in  the  coelomic  fluid  of  naturally  spawning 
starfish.  Five  to  20  ml.  of  coelomic  fluid  were  separately  collected  from  each  of  six 


MECHANISM  OF  STARFISH  SPAWNING 


111 


spawning  animals  (three  males  and  three  females).  Small  ovarian  fragments  were 
separately  exposed  to  2.5  ml.  of  such  coelomic  fluid.  Controls  were  exposed  to 
coelomic  fluid  from  six  other  starfish  which  were  not  spawning. 

The  ovarian  fragments  immersed  in  the  control  coelomic  fluid  did  not  shed  their 
eggs  and  the  germinal  vesicles  of  the  ovarian  eggs  remained  intact.  On  the  con- 
trary, spawning  was  induced  within  15  to  20  minutes  in  all  of  the  experimental 
fragments.  The  germinal  vesicles  in  all  the  discharged  eggs  disappeared  within 
30  minutes  after  exposure  of  the  fragments  to  the  coelomic  fluid.  Observations 
after  one  hour  revealed  strongly  shrunken  ovarian  fragments  with  thick  alveolar 
walls,  containing  few,  if  any,  eggs.  These  observations  clearly  indicate  that  the 
active  substance  is  actually  present  in  the  coelomic  fluid  of  a  spawning  starfish. 
When  these  test  fluids  were  filtered  and  ovarian  fragments  taken  from  another 
starfish  were  immersed  in  them,  shedding  began  after  14  to  27  minutes  and  the 
germinal  vesicles  underwent  breakdown  within  30  minutes.  The  degree  of  shed- 
ding reaction  in  these  cases  corresponded  to  that  of  the  ovarian  fragments  exposed 

TABLE  V 

In  vitro  assay  of  the  gamete-shedding  substance  contained  in  tube  feet  of  Asterias 

Minimum  amount  of  tube-foot  extract  necessary  to  induce  spawning  within  1  hour  in  ovarian  fragments 

from  4  females 


Tube  feet 

Tube  feet 

from  males 

Mg./ml. 

lyophilized  tube  feet 

from  females 

Mg./ml. 

lyophilized  tube  feet 

Exp.  No. 

Exp.  No. 

1 

100, 

100, 

100, 

50, 

1 

100, 

100, 

50, 

100, 

2 

100 

100 

100 

100 

2 

100 

100 

100 

50 

3 

100 

100 

150 

— 

3 

100 

100 

100 

100 

4 

100 

100 

100 

100 

4 

150 

100 

50 

50 

5 

50 

100 

50 

50 

5 

50 

100 

50 

50 

6 

50 

100 

100 

— 

6 

50 

100 

50 

~ 

to  the  radial  nerve  extract  containing  12.5  /^g./ml.  or  more  of  lyophilized  material. 
However,  after  the  use  of  the  same  coelomic  fluid  twice  in  this  way,  the  shedding 
activity  decreased  to  some  extent :  ovary  fragments  in  some  experimental  lots 
which  were  immersed  for  the  third  time  began  to  shed  only  after  30  minutes  and 
the  spawning  was  incomplete.  Germinal  vesicles  remained  intact  in  some  of  the 
eggs  within  the  ovary  (10-20%).  The  shedding  reaction  in  the  third  case  seems 
to  correspond  to  the  case  in  which  about  6  ju,g./ml.  of  lyophilized  radial  nerve  were 
used.  This  reduction  in  inducing  capacity  suggests  that  the  active  substance  is 
in  some  way  taken  up  by  the  ovarian  fragments,  or  that  some  inhibitory  substance 
is  released  into  the  experimental  coelomic  fluid  from  the  ovarian  fragments. 

DISCUSSION 

According  to  Unger  (1962),  a  substance  which  causes  shedding  of  sperm  is 
extractable  from  the  radial  nerves  of  male  starfish  only,  in  Asterias  glacialis.  The 
results  of  the  present  study,  however,  clearly  demonstrate  the  presence  of  sperm- 
shedding  substance  in  the  radial  nerves  of  female  as  well  as  of  male  Asterias 


112  HARUO  KANATANI  AND  MIWAKO  OHGURI 

amurcnsis  (Tal)le  I).  The  so-called  egg-shedding  substance  was  also  obtained  from 
male  starfish ;  moreover,  in  vitro  experiments  using  isolated  ovary  fragments  showed 
that  the  content  of  the  active  substance  in  the  radial  nerves  is  equal  in  the  two 
sexes.  Therefore,  the  substance  responsible  for  inducing  the  release  of  sperm 
is  believed  to  be  identical  with  the  substance  which  induces  the  release  of  eggs.  The 
active  substance  was  also  found  to  be  equally  effective  in  inducing  spawning  in  both 
sexes  at  several  steps  in  the  course  of  chemical  purification,  and  could  not  be 
separated  into  two  different  substances  specific  to  males  or  females  (Kanatani  and 
Noumura,  1964;  Kanatani,  unpublished).  Chaet  (1966)  has  recently  reported 
that  the  shedding  substance  is  present  in  Asterias  forbesi  nerves  of  both  sexes,  and 
in  the  same  concentration.  According  to  his  experiments  using  intact  starfish,  shed- 
ding substance  taken  from  nerves  of  either  sex  stimulated  shedding  in  both  sexes. 

With  respect  to  the  location  of  the  gamete-shedding  substance  in  a  single  star- 
fish, Chaet  (1966)  reports  that  hot  (76°  C.)  salt  water  extracts  prepared  from 
various  tissues  other  than  radial  nerve  of  Asterias  forbesi,  including  tube  feet  and 
the  oral  and  aboral  surfaces,  show  no  gamete-shedding  activity.  It  is  quite  possible 
that  his  extraction  procedure  was  inadequate  in  this  case.  As  is  clearly  shown 
in  our  present  results,  the  gamete-shedding  substance  is  not  confined  to  the  nerve 
ring  and  radial  nerve.  For  example,  extracts  of  tube  feet,  body  wall  and  cardiac 
stomach  also  showred  the  shedding  activity.  Considering  the  order  of  the  shedding 
activity  expressed  by  the  extracts  of  various  regions  of  the  body  on  the  one  hand, 
and  the  results  of  the  extensive  study  of  Smith  (1937)  on  the  distribution  of  the 
nervous  system  of  starfish  on  the  other  hand,  the  quantitative  distribution  of 
the  active  substance  in  the  starfish  seems  to  correspond  to  the  quantity  of  nervous 
tissue  present  in  a  given  part  of  the  body.  Within  the  radial  nerve,  Uter  (1966) 
has  reported  that  the  active  gamete-shedding  substance  is  located  only  in  the  most 
aboral  region  of  the  nerve. 

Chaet  (1964c,  1966)  has  suggested  that  the  radial  nerves  also  contain  a 
substance,  "shedhibin,"  which  inhibits  the  action  of  the  gamete-shedding  substance. 
According  to  his  opinion,  natural  control  of  shedding  is  regulated  both  by  the  level 
of  shedding  substance  and  the  presence  (or  absence)  of  shedhibin.  The  nature  of 
shedhibin,  however,  is  still  obscure.  In  this  connection,  attention  must  be  drawn 
to  the  observation  made  in  the  present  study,  that  the  gamete-shedding  substance 
is  present  in  the  coelomic  fluid  only  at  the  time  of  spawning,  even  during  the 
breeding  season.  That  the  shedding  substance  can  act  directly  on  the  gonads  has 
been  clearly  demonstrated  in  our  previous  work,  in  which  the  gamete-shedding 
substance  was  locally  applied  to  an  isolated  ovary  (Kanatani,  1964).  The  ripe, 
distended  gonads,  therefore,  will  readily  respond  to  the  shedding  substance  when 
it  is  released  into  the  coelomic  cavity.  It  thus  appears  highly  probably  that  spawn- 
ing is  controlled  by  some  mechanism  which  introduces  the  gamete-shedding  sub- 
stance into  the  coelomic  cavity  in  which  the  gonads  are  suspended.  The  problems 
remaining  to  be  solved  are :  ( 1 )  from  what  part  of  the  nervous  system  and  through 
what  route  the  shedding  substance  reaches  the  coelomic  cavity;  and  (2)  by  what 
means  such  transport  of  the  substance  is  controlled. 

It  is  unfortunately  difficult  to  determine  the  actual  amount  of  gamete-shedding 
substance  released  into  the  coelomic  cavity,  because  the  substance,  when  released 
into  the  cavity,  seems  to  be  readily  absorbed  by  the  gonads  which  occupy  most  of 


MECHANISM  OF  STARFISH  SPAWNING  113 

the  space  and  present  an  extremely  large  surface  area  because  of  their  complicated 
branching  structure.  In  vitro  experiments  on  the  shedding  activity  of  coelomic 
fluid  carried  out  in  the  present  study  strongly  suggest  that  the  substance  is 
actually  taken  up  by  the  ovarian  fragments.  Evidence  that  Asterias  gonads  can 
absorb  labeled  amino  acids  and  glucose  from  dilute  solution  in  sea  water  and 
coelomic  fluid  (Ferguson,  19641))  supports  this  suggestion. 

In  the  foregoing  considerations  the  gamete-shedding  substance  is  thought  of  as 
being  transported  first  into  the  coelomic  cavity  and  acting  directly,  from  the  outside, 
upon  the  gonads.  However,  it  is  also  possible  that  the  shedding  substance  re- 
sponsible for  natural  spawning  is  released  directly  into  the  gonad,  either  from  the 
genital  sinus  via  the  aboral  coelomic  sinus  or  from  the  nerves  distributed  in  the 
gonad,  with  an  excess  of  the  substance  diffusing  into  the  coelomic  fluid  through  the, 
coelomic  epithelium.  Against  this  possibility,  histological  observation  of  the  ripe, 
distended  ovary  shows  that  the  genital  sinus  is  so  strongly  compressed  as  to  be 
hardly  discernible.  Moreover,  the  strictly  localized  effect  of  externally  applied 
nerve  extract  (Kanatani,  1964)  argues  against  the  existence  of  an  internal  transport 
system  within  the  ovary.  Finally,  the  gonads  are  less  well  innervated  than  other 
organs.  On  the  other  hand,  shrinkage  of  the  gonads  takes  place  in  the  same  way 
after  natural  spawning  as  after  spawning  induced  by  the  presence  of  nerve  extract 
in  the  coelomic  cavity.  Moreover,  the  recent  investigations  of  Ferguson  (1964a, 
1964b)  have  demonstrated  that  the  starfish  coelomic  fluid  plays  an  important  role 
in  the  transport  and  exchange  of  some  substances  between  tissues,  regardless  of 
whether  they  are  located  in  the  coelomic  cavity  or  in  some  other  space. 

It  is  therefore  considered  highly  probable  that  at  the  time  of  spawning  the 
shedding  substance  is  released  into  the  coelomic  fluid  and  taken  up  by  the  gonads, 
where  it  acts  to  bring  about  meiosis  of  oocytes  and  spawning  of  the  gametes. 

Although  it  is  known  that  the  gonad  becomes  more  sensitive  toward  the  end 
of  the  spawning  season,  it  has  not  yet  been  determined  whether  the  shedding 
substance  is  abruptly  released  into  the  coelomic  cavity  at  each  spawning  or  accumu- 
lates little  by  little  until  a  threshold  concentration  is  reached. 


We  wish  to  express  our  gratitude  to  Dr.  J.  C.  Dan  for  her  encouragement  and 
advice,  and  to  Misses  K.  Fujino  and  H.  Shirai  for  their  technical  assistance.  Our 
thanks  are  due  to  the  director  and  the  staff  of  the  Misaki  Marine  Biological  Station 
for  putting  the  research  facilities  of  the  station  at  our  disposal. 

SUMMARY 

1.  The  localization  in  the  starfish  body  of  the  active  substance  responsible  for 
maturation  and  gamete-shedding  was  determined  by  in  vitro  assay,  using  Asterias 
amurensis  and  Asterina  pcctinijcra. 

2.  The  active  substance  was  found  in  the  radial  nerves  at  the  same  concentration 
in  both  male  and  female  starfish,  suggesting  that  the  testis  responds  more  readily 
to  the  action  of  the  substance  than  does  the  ovary.     The  gamete-shedding  substance 
seems  to  be  identical  in  the  two  sexes. 

3.  As  determined  by  the  shedding  reaction  of  isolated  fragments  of  ovaries,  the 
quantity  of  active  substance  (per  dry  weight  of  lyophilized  nerve  materials)   was 
found  to  be  uniform  in  various  parts  of  the  nerve  ring-radial  nerve  system. 


114  HARUO  KANATANI  AND  MIWAKO  OHGURI 

4.  Tube  feet  and  the  body  wall  also  contained  considerable  amounts  of  the 
active  substance,  although  the  content  was  several  times  lower  than  that  of  the 
radial  nerves. 

5.  The  shedding  activity  was  also  detectable  in  the  extracts  of  some  other  regions 
of  the  starfish  in  which  nervous  tissue  is  plentiful :  for  example,  cardiac  stomach. 
However,  the  activity  was  much  less  than  that  of  the  radial  nerves. 

6.  The    active    substance    was    found    in    the    coelomic    fluid    only    when    the 
starfish  were  undergoing  natural  spawning.     The  coelomic  fluid  of  starfish  which 
were  not  spawning  did  not  show  any  shedding  activity,  regardless  of  the  condition 
of  their  gonads. 

7.  The  significance  of  the  appearance  of  active  substance  in  the  coelomic  fluid 
in  relation  to  the  mechanism  of  starfish  spawning  is  discussed. 

LITERATURE  CITED 

CHAET,  A.  B.,  1964a.     A  mechanism  for  obtaining  mature  gametes  from  starfish.     Biol.  Bull., 

126:  8-13. 
CHAET,  A.  B.,  1964b.     The  shedding  substance  activity  of  starfish  nerves.     Texas  Rep.  Biol. 

Med.,  22 :  204. 
CHAET,  A.  B.,  1964c.     Shedding  substance  and  "shedhibin" — from  the  nerves  of  the  starfish, 

Patiria  miniata.    Amcr.  Zool.,  4:  142. 
CHAET,  A.  B.,  1966.     Neurochemical  control  of  gamete  release  in  starfish.     Biol.  Bull.,   130, 

43-58. 
CHAET,  A.  B.,  P.  M.  ANDREWS  AND  R.  H.  SMITH,  1964.      The  shedding  substance  of  starfish 

nerve — its  function  and  micro-assay.     Fed.  Proc.,  23 :  204. 
CHAET,  A.  B.,  AND  R.  A.  McCoNNAUGHY,  1959.     Physiologic  activity  of  nerve  extracts.     Biol. 

Bull,  117:407-408. 
CHAET,  A.  B.,  AND  R.  S.  MUSICK,  1960.     A  method  for  obtaining  gametes  from  Asterias  jorbesi. 

Biol.  Bull,  119:292. 
FERGUSON,   J.,    1964a.     Nutrient   transport   in   starfish.     I.      Properties    of   the   coelomic    fluid. 

Biol.  Bull,  126:  33-53. 
FERGUSON,   J.,    1964b.     Nutrient   transport   in   starfish.     II.     Uptake    of   nutrients    by    isolated 

organs.     Biol.  Bull,  126:  391-406. 
HARTMAN,   H.   B.,   AND   A.    B.    CHAET,    1962.     Gamete   shedding   with   radial    nerve   extracts. 

Fed.  Proc. ,21:  363. 
KANATANI,   H.,   1964.     Spawning  of   starfish :   Action   of  gamete-shedding   substance   obtained 

from  radial  nerves.     Science,  146:  1177-1179. 
KANATANI,  H.,  AND  T.  NOUMURA,  1962.     On  the  nature  of  active  principles  responsible  for 

gamete-shedding  in  the  radial  nerves  of  starfishes.    /.  Fac.  Set.  Univ.  Tok\o,  Scr.  IV , 

9:  403-416. 
KANATANI,  H.,  AND  T.  NOUMURA,  1964.     Separation  of  gamete-shedding  substance  in  starfish 

radial  nerves  by  disc  electrophoresis.     Zool.  Mag.,  73:  65-69. 
NOUMURA,  T.,  AND  H.  KANATANI,  1962.     Induction  of  spawning  by  radial  nerve  extracts  in 

some  starfishes.    J.  Fac.  Sci.  Univ.  Tokyo,  Ser.  IV,  9:  397-402. 
SMITH,   J.   E.,    1937.     On   the   nervous    system   of   the    starfish   Marthasterias   glacialis    (L.). 

Philos.  Trans.  Roy.  Soc.  London,  Ser.  B,  227:  111-173. 
UNGER,  H.,   1962.     Experimented  und  histologische  Untersuchungen  iiber   Wirkfaktoren  aus 

dem  Nervensystem  von  Asterias    (Atarthasterias')    glacialis    (Asteroidea ;    Echinoder- 

mata).      Zool.  Jahrb.  Abt.  Allgcm.  Zool.  Physiol.  Ticre,  69:  481-536. 
UTER,  A.,  1966.     Physiological  location  of  shedding  substance  in  radial  nerve  complex  of  starfish 

(Asterias  forbesi).     Thesis.     The  American  University,  Washington,  D.  C.  (cited  from 

Chaet,  1966). 


AN  ENDOGENOUS  DIURNAL  RHYTHM  OF  BIOLUMINESCENCE 
IN  A  NATURAL  POPULATION  OF  DINOFLAGELLATES  1 

MAHLON  G.  KELLY  AND  STEVEN  KATONA 

Harvard  University,  Cambridge,  Massachusetts  02138,  and  Woods  Hole  Occanographic 
Institution,  Woods  Hole,  Massachusetts  02543 

Several  authors  have  shown  or  suggested  that  dinoflagellates  are  the  major 
source  of  bioluminescence  in  many  surface  regions  of  the  ocean  (Backus,  Clark  and 
Wing,  1965;  Backus,  Yentsch  and  Wing,  1961;  Gold,  1965;  Hardy  and  Kay, 
1964;  Seliger  et  al,  1961,  1962;  Sweeney,  1963;  Yentsch,  Backus  and  Wing,  1964; 
earlier  work  summarized  by  Harvey,  1952,  p.  124).  Hastings  and  Sweeney  (1957, 
1958)  and  Sweeney  and  Hastings  (1957)  have  studied  an  endogenous  diurnal 
rhythm  of  light  production  in  laboratory  cultures  of  the  dinoflagellate  Gonyaulax 
polyedra.  Earlier  work  summarized  by  Harvey  (1952,  p.  128)  has  suggested 
an  endogenous  rhythm  in  flashing,  but  lack  of  dark-adaptation  of  the  observer  makes 
these  reports  questionable.  Harvey  (1952,  p.  129)  reports  a  more  careful  experi- 
ment but  in  an  abnormally  eutrophic  environment.  None  of  these  reports  give 
quantitative  measurements,  and  none  of  these  compare  the  endogenous  influences 
with  the  exogenous  influence  of  light  inhibition. 

An  in  situ  diurnal  rhythm  of  luminescence  within  the  euphotic  zone,  probably 
caused  by  dinoflagellates,  has  been  found  by  Backus  et  al.  (1961)  and  Clarke  and 
Kelly  (1965),  although  this  rhythm  has  not  been  shown  to  be  endogenous.  Other 
workers  have  found  an  in  situ  rhythm  and  concluded  that  it  was  exogenous  in 
origin.  Seliger  et  al.  (1961,  1962)  postulated  that  the  rhythm  was  controlled  by  a 
diurnal  migration  of  the  luminescent  dinoflagellates.  Yentsch  et  al.  (1964)  pointed 
out  that  photo-enhancement  and  photo-inhibition  alone  might  explain  the  amount  of 
bioluminescence  and  that  diurnal  migration  was  not  involved.  Backus  et  al.  (1965) 
found  that  bioluminescent  organisms  in  Eel  Pond  responded  to  the  eclipsing  sun 
much  as  they  normally  respond  to  the  setting  sun,  and  that  their  behavior  from 
mid-eclipse  to  eclipse  end  resembled  dawn  behavior.  They  concluded  that  the 
exogenous  factor  of  changing  light  overrides  such  endogenous  rhythms  as  may 
exist. 

The  purpose  of  the  work  reported  here  was  to  resolve  the  relative  importance  of 
endogenous  and  exogenous  influences  on  the  diurnal  rhythm  of  bioluminescence 
of  a  natural  population  of  phytoplankton  under  controlled  conditions,  and  to 
identify  the  members  of  the  population  responsible  for  the  luminescence  in  a  typical 
inshore  marine  environment. 

METHODS 

Surface  water  was  taken  at  various  times  of  day  from  near  the  entrance  to  Eel 
Pond — a  salt  pond  in  Woods  Hole,  Mass.,  which  is  tidally  flushed  by  water  from 

1  Contribution  No.  1745  from  Woods  Hole  Oceanographic  Institution.  Research  supported 
by  National  Science  Foundation  Grant  2435. 

115 


116  MAHLON  G.  KELLY  AND  STEVEN  KATONA 

the  connecting  harbor,  and  which  has  phytoplankton  populations  similar  to  those 
in  the  harbor.  This  water  was  filtered  through  0.33-mm.  aperture  netting,  and 
placed  in  a  Teflon-lined  15-gallon  steel  drum.  The  contained  organisms  were 
stimulated  by  controlled  air  flow  from  an  aquarium  bubbler  "stone"  placed  near 
the  bottom,  and  bioluminescence  was  measured  using  a  photomultiplier  photometer 
with  logarithmic  output  and  sensitive  to  intensities  as  low  as  10~8  /xw./cm.2.  The 
photometer  window  was  in  the  water  8  cm.  above  the  bubbler,  and  the  output  was 
recorded  on  a  Sanborn  strip-chart  recorder  with  0.01  second  response  time. 
Organisms  were  stimulated  for  40  to  60  seconds  at  various  times  depending  on  the 
particular  experiment  and  flashing  was  recorded  during  stimulation.  Total  flashes 
were  counted  for  the  first  30  seconds  of  stimulation,  and  bioluminescence  expressed 
as  flashes/30  seconds.  This  measurement  was  used  rather  than  total  light  output 
since  amount  of  flashing  is  an  ecologically  more  meaningful  quantity,  and  since  it  was 
impossible  to  know  the  number  of  organisms  subject  to  stimulation.  Stimulation 
provided  sufficient  mixing  to  prevent  stratification  of  the  organisms.  All  experi- 
ments were  performed  in  a  darkroom  at  temperatures  between  20°  and  22°  C. 

EXPERIMENTS 

Three  types  of  experiments  were  performed.  The  first  measured  the  endogen- 
ous luminescence  rhythm  by  recording  luminescence  of  populations  kept  continually 
in  darkness.  The  second  studied  the  recovery  of  ability  to  luminesce  when  popula- 
tions taken  from  normal  daylight  in  the  natural  environment  were  placed  in 
darkness.  The  third  group  of  experiments  measured  the  effects  of  exposure  to 
light  at  various  times  of  day  on  the  luminescence  of  populations  kept  in  darkness. 

In  the  first  experiment,  water  was  collected,  filtered,  and  placed  in  complete 
darkness  in  the  laboratory  just  prior  to  1900,  16  Aug.,  1965,  and  stimulated  flashing 
was  recorded  every  hour  from  1900  until  0300,  20  Aug.,  1965.  Flashes/30  sec.  are 
plotted  against  time  in  Figure  1.  An  endogenous  rhythm  of  flashing  rate  was 
apparent  and  continued  for  three  days,  although  the  maximum  flashing  rate  was 
lower  each  day.  A  similar  experiment  was  performed  between  3  Aug.  and  5  Aug., 
1965,  and  although  the  recording  methods  were  different,  the  results  were  qualita- 
tively the  same.  These  results  are  qualitatively  similar  to  those  found  by  Sweeney 
and  Hastings  (1957)  who  measured  total  light  output  by  cultures  of  Gonyaulax 
polyedra.  The  changes  in  flashing  rate  are  also  similar  to  in  situ  measurements 
made  by  Backus  ct  al.  (1961)  except  that  the  morning  decrease  and  evening 
increase  in  flashing  are  not  as  pronounced  in  the  present  work. 

In  order  to  study  recovery  from  inhibition  due  to  daylight,  two  series  of  experi- 
ments were  performed  in  which  water  was  brought  from  the  surface  of  Eel  Pond 
into  complete  darkness  at  various  times  of  day  (daylight  intensities  from  5  X  104  to 
1  X  105  /uv./cm.2,  measured  with  a  General  Electric  photoelectric  meter).  Flashing 
rates  were  recorded  every  hour  thereafter  until  2300.  The  two  series  gave  similar 
results,  and  the  results  of  the  second  series  and  the  times  of  start  of  dark  exposure 
are  shown  in  Figure  2.  Rates  of  flashing  throughout  the  day  of  organisms  in 
continuous  darkness  are  shown  for  comparison  (results  of  Aug.  30  experiments; 
see  below  and  Figure  3). 

Flashing  rates  in  water  collected  during  daylight  increased  within  two  hours 
to  the  rate  shown  by  a  population  kept  in  darkness  for  the  previous  night,  and 


DiNOFLAGELLATE  Ll'M  INIiSCKNCK  RHYTHM 


117 


then  followed  the  curve  for  that  population.  Flashing  rates  in  water  taken  at 
night  were  initially  much  higher.  Thus,  inhibition  of  flashing  by  daylight  super- 
imposes its  effect  upon  a  daytime  decrease  controlled  by  an  endogenous  rhythm. 
This  is  further  emphasized  in  the  next  group  of  experiments. 

The  third  group  of  experiments  examined  the  effects  of  inhibition  by  exposure 
to  short  periods  of  artificial  light  at  various  times  of  day.  On  three  occasions  water 
was  brought  into  the  darkroom  at  dusk  (2000)  and  the  included  organisms  were 
allowed  to  dark-adapt  until  midnight.  They  were  then  exposed  to  15  minutes  of 
light  every  two  hours  for  24  hours  and  luminescence  was  recorded  15,  30,  45,  60, 
90  and  120  minutes  after  start  of  light  exposure.  Intensities  at  the  surface  of  the 
water,  dates,  and  certain  minor  departures  from  the  described  schedule  are  shown 
in  Figure  3.  The  lower  surface  light  intensity  at  1470  /xw./cm.2  was  provided  by 


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FIGURE  1.  Flashing  rates  during  stimulation  for  30-  to  60-second  duration  recorded  in 
water  collected  at  1900  hr.  16  Aug.  1965  and  kept  continuously  in  the  dark  for  the  period 
shown. 


placing  over  the  barrel  a  bank  of  fluorescent  lamps,  rated  by  the  manufacturer  to 
have  a  spectral  distribution  similar  to  normal  daylight.  The  higher  light  intensity 
of  8820  /Av./cm.2  was  provided  by  an  incandescent  spotlight  which  had  a  different 
spectral  distribution  and  angular  dispersion.  Light  intensity  was  attenuated  by 
about  30%  through  a  60-cm.  water  layer  in  Eel  Pond,  and  probably  by  a  similar 
amount  in  the  barrel.  The  rising  air  bubbles  used  for  stimulation  mixed  the  water 
and  assured  random  dispersal  of  the  organisms  with  uniform  exposure  to  light. 

Results  of  these  experiments  are  shown  in  Figure  3.  The  lower  curves  connect 
the  flashing  rates  after  15  minutes  of  light  exposure,  and  the  upper  the  rates  after 
active  recovery  from  light  inhibition  had  apparently  stopped  (1  hr.  45  min.  after 
exposure).  Although  the  figures  differ  somewhat,  presumably  because  of  popula- 
tion changes,  they  are  all  similar  in  that  they  show  proportionately  greater  inhibition 


118 


MARLON  G.  KELLY  AND  STEVEN  KATONA 


during  daylight  hours.  Since  the  treatment  and  environmental  conditions  were 
the  same  both  day  and  night,  it  may  be  concluded  that  there  is  an  endogenous 
diurnal  rhythm  in  sensitivity  to  light  inhibition.  The  similarity  of  the  flashing  rates 
after  recovery  from  light  inhibition  to  those  of  populations  kept  in  continuous 
darkness  indicates  that  there  is  no  appreciably  long-term  effect  of  light  exposure. 

The  two  intensities  used  are  approximately  equivalent  to  2%  and  12%  of  the 
mid-day  surface  light  intensity  in  Eel  Pond.  Although  the  higher  intensity  of  8820 
juw./cm.2  caused  slightly  greater  inhibition,  the  flashing  was  never  reduced  by  more 
than  f.  Sweeney,  Haxo  and  Hastings  (1959)  noted  that  exposure  of  G.  polyedra 
cultures  to  light  caused  inhibition  of  luminescence  to  varying  degrees,  depending  on 
the  intensity  of  the  light,  and  that  longer  exposure  to  light  altered  the  phase  of  the 
rhythmicity.  They  did  not,  however,  mention  significant  variations  in  sensitivity 
to  inhibition  with  time  of  day.  The  lack  of  a  phase  shift  in  the  present  experiments 
was  probably  due  to  the  relatively  short  exposure  and  low  intensity.  Many 
luminescent  marine  organisms  are  known  to  be  inhibited  by  light  (Harvey,  1952), 
but  only  dinoflagellates  and  euphausids  (Mauchline,  1960)  are  known  to  have 


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FIGURE  2.  Flashing  rates  recorded  using  water  collected  from  the  natural  environment  at 
dates  and  times  shown,  and  placed  immediately  in  darkness.  After  time  "A"  (1600  hr.),  all 
flashing  rates  fell  on  approximately  the  same  curve,  and  only  the  range  of  flashing  rates  is 
shown  by  the  cross-hatched  area. 


DINOFLAGELLATE  LUMINESCENCE  RHYTHM 


119 


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FIGURE  3.  Effect  of  light  inhibition  at  various  times  of  day.  Dates,  times,  and  light 
intensities  as  shown.  Times  of  start  of  light  exposure  for  15  minutes  are  indicated  by  arrows. 
Upper  dashed  line  connects  rates  after  complete  recovery ;  lower  line  connects  rates  after 
light  exposure. 

an  endogenous  rhythm  in  flashing  activity,  and  there  are  no  reports  known  to  us  of 
an  endogenous  variation  in  sensitivity  to  light  inhibition. 

DISCUSSION 

These  experiments  with  natural  populations  brought  into  the  laboratory  attempt 
to  bridge  the  gap  between  the  studies  of  luminescence  in  cultured  dinoflagellates 
made  by  Hastings,  Sweeney,  and  co-workers,  and  the  previous  field  studies  in  which 
rhythms  in  dinoflagellate  luminescence  were  found  (references  in  the  introduction). 
Our  results  indicate  that  the  flashing  rates  of  populations  kept  in  darkness  decrease 
during  daytime  hours,  and  that  the  effect  of  light  in  causing  inhibition  of  flashing  is 
greater  during  daytime.  Both  the  dark-adapted  flashing  rates  and  the  sensitivity 
to  photo-inhibition  are  controlled  by  an  endogenous  diurnal  rhythm. 

Hastings  and  Sweeney  (1958)  found  an  endogenous  rhythm  in  the  effect  of 
periods  of  light  exposure  on  changing  the  phase  of  the  luminescence  rhythm. 
Their  effect  had  a  maximum  sensitivity  during  dark  hours  in  contrast  to  the  varying 
sensitivity  to  light  inhibition  found  here,  which  has  a  maximum  during  daylight. 
It  may  be  inferred  from  this  that  unless  the  experimental  organisms  vary,  different 
mechanisms  are  involved  in  these  two  manifestations  of  light  sensitivity. 

Hastings  and  Sweeney  (1958)  also  found  a  greater  night-day  variation  in  light 
production  than  is  found  here.  This  is  probably  because  they  measured  total 
light  output  rather  than  number  of  flashes.  Since  in  their  experiments  the 
intensity  as  well  as  rate  of  flashing  was  greater  at  night,  total  light  output  increased 
to  a  greater  extent.  This  night-time  increase  in  intensity  was  not  apparent  in 
our  records. 

Because  the  light-inhibition  effect  is  the  most  obvious  with  in  situ  measurements, 
several  of  the  authors  mentioned  in  the  introduction  have  considered  control  of 
flashing  to  be  only  exogenous,  but  this  is  apparently  an  oversimplification.  Yentsch 
et  al.  (1964)  found  that  a  model  involving  only  photo-enhancement  and  photo- 
inhibition  described  the  diurnal  variation,  but  it  appears  that  this  is  useful  only  as 
an  empirical  approximation.  Seliger  ct  al.  (1961,  1962)  have  hypothesized  diurnal 


120  MAHLON  G.  KELLY  AND  STEVEN  KATONA 

migration  as  the  cause  of  variation  in  light  production,  but  this  appears  to  he  neither 
sufficient  nor  necessary  to  explain  the  variations  we  observed. 

Backus  et  al.  (1965)  have  described  the  effect  of  a  solar  eclipse  on  luminescent 
activity  to  be  similar  to  that  of  the  setting  sun,  and  they  concluded  that  the 
exogenous  factor  of  changing  light  overrides  such  endogenous  rhythms  as  may 
exist.  Experiments  described  above,  however,  showed  that  populations  brought 
into  darkness  from  complete  daylight  in  the  natural  environment  increased  their 
flashing  rate  only  by  an  amount  determined  by  the  diurnal  rhythm  and  not  to  a 
night-time  level.  If  the  dark  period  of  the  eclipse  had  been  longer,  recovery  from 
inhibition  might  have  been  complete,  and  it  might  have  become  apparent  that  light- 
inhibition  of  flashing  rates  is  not  the  only  cause  of  the  daytime  decrease  in  flashing 
rate. 

Bode,  DeSa  and  Hastings  (1963)  and  Hastings  and  Key  nan  (1965),  using 
G.  polycdra  cultures,  have  shown  that  normally  more  luciferin  is  produced  at  night 
than  during  the  day.  This  was  inferred  because  if  night-time  flashing  was  inhibited 
by  temperature  or  light,  more  luciferin  could  be  extracted  at  that  time.  Under 
normal  conditions,  however,  night-time  flashing  apparently  utilizes  the  available 
luciferin,  and  more  is  extracted  during  the  day  when  flashing  is  less.  It  thus 
appears  light  inhibition  does  not  affect  substrate  production,  but  rather  acts  upon 
the  stimulus-response  mechanism ;  i.e.,  it  probably  decreases  the  sensitivity  of 
the  cells  to  stimulus.  This  suggests  that  flashing  of  natural  populations  may  be  con- 
trolled both  by  the  availability  of  luciferin  and  by  light-inhibition  of  the  sensitivity 
to  stimulus. 

The  selective  advantage  conferred  upon  a  dinoflagellate  by  its  ability  to  luminesce 
and  to  control  the  amount  of  luminescence  is  undetermined.  McElroy  and  Seliger 
(1962)  have  hypothesized  that  luminescence  first  developed  to  serve  a  biochemical 
function  during  the  early  evolution  of  life.  As  presently  found  in  dinoflagellates, 
however,  the  biochemical  ability  for  luminescence  is  accompanied  by  at  least  three 
mechanisms  that  serve  to  control  the  output  of  light :  ( 1 )  a  sensitivity  to  stimulus 
and  an  associated  effector  system,  (2)  a  mechanism  whereby  sensitivity  to  stimulus 
is  controlled  by  light  inhibition,  and  (3)  an  endogenous  rhythm  in  luciferin  produc- 
tion. Energy  is  required  for  the  production  of  light,  and  it  seems  unlikely  that 
a  complex  energy-requiring  system  such  as  this  would  evolve  and  not  be  lost  in 
such  a  diverse  and  widespread  group  of  organisms  unless  some  selective  advantage 
is  conferred  upon  the  organisms.  More  work  on  the  behavior  and  ecology  of 
luminescence  in  dinoflagellates  is  necessary  to  detect  any  such  advantage. 

Although  many  marine  organisms  are  known  to  be  less  luminescent  during  the 
day  than  at  night  (Harvey,  1952),  the  only  one  other  than  dinoflagellates  which 
has  been  shown  to  have  an  endogenous  rhythm  is  the  euphausid  shrimp  Meganycti- 
pJianes  norvcyica  (M.  Sars)  (Mauchline,  1960).  It  apparently  increases  its  flashing 
rate  at  night  even  after  being  kept  in  the  dark  for  two  days.  Since  the  animal  has 
complex  photophores  with  neural  and  muscular  control,  presumably  luminescence 
is  important  in  its  behavior. 

In  addition  to  endogenous  and  exogenous  influences  on  the  luminescence  of 
species  of  dinoflagellates  within  a  population,  luminescence  in  the  natural  environ- 
ment may  vary  because  the  species  present  change  and  exhibit  different  characteris- 
tics. G.  polyedra  and  Gonyaulax  inonilata  display  similar  endogenous  rhythms, 


DINOFLAGELLATE  LUMINESCENCE  RHYTHM  121 

whereas  Noc til ura  niiliaris  gives  no  indication  of  an  endogenous  rhythm  (Hastings, 
1959).  More  must  he  known  of  the  behavior  of  individuals  and  cultures  of  various 
species  hefore  any  model  can  he  proposed  to  describe  the  behavior  of  a  population 
composed  of  many  species. 

DETERMINATIONS  OF  LUMINESCENT  SPECIES 

In  order  to  determine  which  species  of  dinoflagellates  present  during  the  experi- 
ments were  capable  of  luminescence,  individual  specimens  of  the  species  predominant 
during  August  and  September,  1965,  were  isolated  from  the  plankton  and  tested. 

Tows  were  taken  on  several  afternoons,  using  a  nylon  net  with  35  p.  mesh 
aperture.  Water  passed  through  the  same  net  was  found  to  be  not  luminescent, 
and  it  is  assumed  that  all  luminescent  forms  of  phytoplankton  w^ere  captured. 
Representatives  of  the  dinoflagellates  were  removed  from  the  sample  by  micro- 
pipette,  placed  in  filtered  sea  water,  and  motile  individuals  were  transferred  singly 
from  this  into  0.5  ml.  of  filtered  sea  water  in  test  tubes.  The  organisms  in  tubes 
were  kept  in  the  dark  until  after  2100  hr.  before  they  were  tested,  so  that  potential 
for  luminescence  would  be  high  when  tested,  and  so  that  the  organisms  could 
recover  from  the  isolation  procedure. 

The  tubes  were  placed  in  a  light-tight  holder  in  front  of  the  photometer  that  was 
used  in  the  previous  experiments,  and  air  was  bubbled  through  the  water  to 
stimulate  the  organisms.  After  testing,  the  contents  were  examined  to  determine 
if  the  organisms  were  still  motile,  and  only  those  which  were  motile  or  which  had 
flashed  were  considered  to  have  been  alive  during  testing  and  only  these  are  included 
in  the  results.  The  organisms  were  placed  in  a  drop  of  filtered  sea  water  on  a  slide 
in  a  moist  petri  dish  and  left  overnight.  This  killed  the  organisms  and  often 
resulted  in  a  loss  of  protoplasm  that  simplified  drawing  and  identification. 

The  organisms  tested  were  drawn  with  a  camera  lucida,  and  were  usually  placed 
in  glycerine- jelly  to  facilitate  handling  and  determination  of  plate  structure. 
Drawings  were  then  compared  with  more  thorough  drawings  made  of  specimens 
of  the  same  species  that  were  not  tested,  but  which  were  more  easily  cleared,  stained, 
and  manipulated  without  risk  of  loss. 

Although  cell  counts  of  dinoflagellate  population  density  w7ere  not  made,  it  was 
apparent  that  the  populations  varied  somewhat  from  day  to  day.  Dinoflagellates 
were  greatly  outnumbered  by  diatoms,  but  the  latter  have  never  been  found  to  be 
luminescent  (Sweeney,  1963).  Larger  forms  which  might  have  been  luminescent 
(such  as  copepods  and  ctenophores)  had  been  excluded  by  filtration.  Radiolarians 
may  be  luminescent,  but  were  present  in  very  small  numbers. 

The  results  of  the  tests  are  shown  in  Table  I.  Because  cells  that  had  been  tested 
were  often  difficult  to  recover  for  identification,  only  those  individuals  definitely 
identified  have  been  included  in  the  table.  For  example,  at  least  10  specimens 
of  what  was  tentatively  identified  as  Gonyaulax  digitale  were  tested,  and  most 
were  luminescent,  but  were  not  recovered  after  testing.  Very  few  of  the  tested  G. 
sphiifcra  flashed,  and  few  were  examined  for  motility  after  testing.  Several  speci- 
mens of  Gonyanlax  and  Pcridinimn  believed  to  be  of  different  species  than  those 
identified  were  examined  and  were  luminescent,  but  were  not  identified  owing  to  the 
lack  of  specimens.  These  are  listed  as  spp.  in  Table  I. 


122 


MAHLON  G.  KELLY  AND  STEVEN  KATONA 


Of  the  12  species  and  four  genera  of  dinoflagellates  present  in  the  Eel  Pond 
plankton  during  August  and  September,  1965,  10  species  were  found  to  be 
luminescent.  These  included  the  vast  majority  of  dinoflagellate  individuals  present, 
and  certainly  were  primarily  responsible  for  the  recorded  bioluminescence.  Of  the 
species  found  to  flash,  the  following  have  been  previously  reported  as  luminescent : 
Peridiniiim  conicum  (Sweeney,  1963),  P.  granii  (Ganapati  et  al.,  1959),  and 
Ccratiwn  fusiis  (Lebour,  1925;  Sweeney,  1963).  Sweeney  (1963)  tested  P. 
claudicans  by  a  similar  method  and  found  it  not  to  be  luminescent.  Ceratiitm 
tripos  has  been  reported  by  several  authors  to  be  luminescent  (Sweeney,  1963), 
but  neither  Sweeney  nor  the  present  authors  could  demonstrate  a  luminescence. 


TABLE  I 
Results  of  testing  individual  dinoflagellates  for  bioluminescence 


Species 

Number  of  cells  that  flashed 

Number  of  motile  cells 
that  did  not  flash 

Gonyaulax  digitale 

2 

0 

G.  spinifera  (see  text) 

2 

Several 

Gonyaulax  spp. 

(see  text) 

Glenodinium  lenticula 

0 

10 

Peridiniiim  claudicans 

2 

1 

P.  conicum 

2 

0 

P.  granii 

4 

0 

P.  leonis 

4 

0 

P.  oceanicum 

2 

0 

P.  subinerme  (Var.  punctulatum) 

4 

0 

Peridiniiim  spp. 

(see  text) 

Ceratium  fusus 

2 

3 

C.  linealum 

0 

10 

C.  tripos 

0 

12 

The  present  study  has  therefore  added  6  species  to  the  list  of  dinoflagellates  known 
to  be  luminescent. 

Negative  results  in  tests  such  as  these  must  not  be  considered  conclusive,  since 
an  organism  such  as  P.  claudicans  or  C.  tripos  may  sometimes  flash  and  sometimes 
not.  Thus,  there  appear  to  be  some  species  always  capable  of  luminescence,  some 
that  never  luminesce,  and  others  which  are  capable  of  luminescence  only  under 
certain  conditions. 

TAXONOMY 

No  thorough  taxonomic  work  has  been  done  on  the  armored  dinoflagellates  of 
the  region  directly  south  of  Cape  Cod,  and  identification  must  be  made  with 
reference  to  Lebour  (1925)  and  Schiller  (1937),  who  deal  primarily  with  European 
and  oceanic  forms.  The  species  referred  to  as  Glenodinium  lenticula  (Bergh) 
Schiller,  and  several  species  of  Peridinium  are  in  need  of  revision.  It  is  deemed 
desirable  to  illustrate  and  note  the  characteristics  of  the  five  species  given  below 
so  that  our  identification  will  be  meaningful  in  case  of  future  revision.  The  other 
species  tested  seem  secure  in  their  taxonomic  position, 


DINOFLAGELLATE  LUMINESCENCE  RHYTHM  123 

Glcnodinium  Icnticula  (Bergh)  Schiller  (Fig.  4,  g-j). 

This  species  is  very  variable  and  has  a  lengthy  synonymy  (Schiller,  1937).  The 
form  worked  with  here  varies  considerably  within  the  population.  It  may  have 
four  apical  plates;  i.e.,  a  plate  that  some  authors  have  described  as  the  second 
intercalary  (Schiller,  1937,  p.  104;  Figs.  95,  96)  actually  reaches  the  apical  pore. 
Individual  specimens  may  or  may  not  have  a  small  asymmetrical  intercalary  plate 
between  precingulars  2"  and  3"  and  apicals  2'  and  3'.  Schiller  (1937)  illustrates 
forms  with  and  without  this  plate.  If  this  plate  is  not  present,  there  are  7 
precingulars,  the  third  reaching  further  toward  the  apex  in  place  of  the  asymmetrical 
intercalary ;  if  the  intercalary  is  present,  only  6  precingulars  are  found.  The 
widths  of  the  pre-  and  postcingular  plates  are  very  variable,  and  in  some  cases 
these  plates  are  barely  visible.  The  theca  is  punctate,  the  sutures  are  often  broad 
and  striated,  the  lists  have  very  fine  supporting  spines,  and  the  apical  pore 
may  or  may  not  be  strongly  developed.  Plate  structure  of  the  species  described 
here :  4  apicals,  0  to  1  intercalary,  7  or  6  precingulars,  5  postcingulars,  and  2 
antapicals. 

Peridinium  conicum  (Gran)  Ostenfeld  and  Schmidt  (Fig.  4,  d-f). 

In  the  past  this  species  has  been  confused  with  both  P.  pentagonnm  Gran  and 
P.  leonls  Pavillard,  and  the  differences  between  them  are  slight.  Lebour  (1925, 
p.  Ill)  and  Schiller  (1937,  p.  237)  separate  P.  pentagonnm  from  P.  conicum  on 
the  basis  that  the  former  has  solid  antapical  spines,  and  that  its  right  half  is  larger 
than  its  left,  but  this  is  not  shown  clearly  in  their  figures.  These  characters  are 
nevertheless  sufficient  to  identify  the  species  discussed  here. 

Peridinium  leonis  Pavillard  (Fig.  4,  k-n) 

This  species  is  easily  separated  in  our  samples  from  the  previous  one  although 
earlier  descriptions  (Schiller,  1937,  p.  236)  indicate  a  wide  variation.  It  may  be 
distinguished  from  other  species  described  here  by  the  following  characters:  cell 
dorso-ventrally  flattened,  broad  lists  with  prominent  spines,  girdle  forms  an  acute 
angle  with  cell  axis,  surface  with  reticulations  appearing  striated  on  some  plates, 
first  apical  plate  narrower  than  in  P.  conicum.  Schiller  (1937,  p.  236)  and  Lebour 
(1925,  p.  112)  have  separated  it  from  P.  conicum  on  the  basis  of  its  much  more 
prominent  lateral  sutures,  but  this  is  not  always  evident  in  the  individuals  investi- 
gated here.  The  species  here  corresponds  most  closely  to  those  described  by 
Dangeard  (1927)  and  Klement  (1964),  and  probably  several  similar  species  are 
included  in  P.  leonis  in  the  summary  by  Schiller  (1937). 

Peridinium  granii  Ostenfeld  (Fig.  4,  a-c). 

This  species  is  easily  confused  with  P.  brochii.  The  only  character  separating 
them  is  the  asymmetry  of  the  dorsal  plate  structure,  and  this  is  variable  (Schiller, 
1937,  p.  189).  It  is  easily  separated  from  the  other  species  investigated  here, 
however,  by  the  structure  of  the  first  apical  plate.  The  present  form  corresponds 
most  closely  to  that  illustrated  in  Lebour  (1925,  p.  124).  It  is  characterized  by 
the  structure  of  the  first  apical  plate  and  the  asymmetry  of  the  dorsal  plates. 


124 


MARLON  G.  KELLY  AND  STEVEN  KATONA 


FIGURE   4.     Dinoflagellates    whose    taxonomy    is    discussed    in    text,     a-c,    Peridinium    granii; 
d-f,  Peridinium  conicum;  g-j,  Glenodinium  lenticula;  k-n,  Peridinium  leonis. 


DINOI'LAGKLLATE  LUMINESCENCE  RHYTHM  125 

CONCLUSIONS 

Most  dinoflagellate  species  and  individuals  taken  from  Kel  Pond  during  this 
study  were  luminescent  and  these  were  sufficient  in  abundance  to  explain  all  the 
luminescence  recorded.  This  is  probably  the  case  in  many  marine  environments. 
Macroscopic  organisms  capable  of  luminescence  were  removed  by  nitration,  and  the 
only  microplankton  constituents  capable  of  luminescence  and  present  in  sufficient 
numbers  were  dinoflagellates. 

Dinoflagellate  luminescence  is  commonly  a  cause  of  light  production  in  surface 
regions  of  the  ocean  (Harvey,  1952;  Hastings,  1963)  and  more  knowledge  is  needed 
of  the  luminescent  behavior  of  individuals  and  cultures  of  the  various  species.  The 
effects  of  temperature,  depth  and  other  environmental  conditions  are  unknown. 
Spontaneous  luminescence  without  stimulation  was  observed  in  the  laboratory,  but 
is  very  variable  and  its  extent  in  the  natural  environment  is  not  known.  Much 
work  is  needed  on  the  ecology  of  dinoflagellate  luminescence. 

The  rate  of  luminescent  flashing  of  natural  populations  following  stimulation  is 
greatest  at  night,  is  controlled  by  an  endogenous  diurnal  rhythm,  and  is  inhibited 
by  light.  The  sensitivity  to  light-inhibition  is  also  controlled  by  an  endogenous 
rhythm,  and  is  greatest  during  midday  when  flashing  is  least.  Thus  in  the  natural 
environment,  light-inhibition  and  an  endogenous  rhythm  act  together  in  decreasing 
stimulated  daytime  luminescence. 


The  authors  would  like  to  thank  Dr.  G.  L.  Clarke  of  Harvard  University  and 
Woods  Hole  Oceanographic  Institution  for  his  continued  guidance  and  encourage- 
ment and  for  the  loan  of  equipment  and  facilities,  Dr.  Frank  Round  of  the  Univer- 
sity of  Bristol,  England,  for  his  encouragement  and  assistance  in  the  taxonomic 
work,  and  Drs.  R.  Backus  and  C.  S.  Yentsch  and  various  other  members  of  Woods 
Hole  Oceanographic  Institution  who  have  made  valuable  suggestions  and  criticisms 
throughout  the  work. 

LITERATURE  CITED 

BACKUS,  R.  H.,  R.  C.  CLARK  AND  A.  S.  WING,  1965.     Behavior  of  certain  marine  organisms 

during  the  solar  eclipse  of  July  20,  1963.    Nature,  205:  989-991. 
BACKUS,  R.  H.,  C.  S.  YENTSCH  AND  A.  S.  WING,  1961.     Bioluminescence  in  the  surface  waters 

of  the  sea.     Nature,  192:  518-521. 
BODE,  V.   C.,   R.   DESA   AND  J.   W.    HASTINGS,    1963.     Daily   rhythm   of  luciferin   activity   in 

Gonyaiila.v  polycdra.     Science,  141:  913-915. 
CLARKE,  G.  L.,  AND  L.  R.  BRESLAU,  1960.     Studies  of  luminescent  flashing  in  Phosphorescent 

Bay,    Puerto    Rico,    and    in    the    Gulf   of    Naples    using    a    portable    bathyphotometer. 

Bull.  lust.  Ocean.,  57(1171)  :  1-32. 
CLARKE,  G.  L.,  AND  M.  G.  KELLY,  1965.     Measurements  of  diurnal  changes  in  bioluminescence 

from  the  sea  surface  to  2000  meters  using  a  new  photometric  device.     Limnol.  Occanog., 

10(Suppl.)  :   R54-R66. 
DANGEARD,  P.,  1927.     Phytoplankton  de  la  croisiere  du  S\lvana.    Ann.  Inst.  Oceanog.,  4(8)  : 

285-407. 
GANAPATI,  R.  N.,  D.  G.  V.  PRASADA  RAO  AND  M.  V.  LAKSHMANA  RAO,  1959.     Bioluminescence 

in  Vishakhapafnam  Harbour.     Curr.  Sci.  India,  28:  246-247. 
GOLD,  K.,  1965.     A  note  on  the  distribution  of  luminescent  dinoflagellates  and  water  constituents 

in   Phosphorescent  Bay,   Puerto  Rico.     Ocean   Science  and   Ocean   Engineering,    1965 

Trans,  of  the  Joint  Conf.  and  Exhibits,  1:  77-80. 


126  MAHLON  G.  KELLY  AND  STEVEN  KATONA 

HARDY,   A.   C.,   AND   R.   H.   KAY,    1964.     Experimental    studies   of   plankton   bioluminescence. 

/.  Mar.  Biol.  Assoc.,  44:  435-484. 

HARVEY,  E.  N.,  1952.     Bioluminescence.     Academic  Press,  N.  Y.     632  pp. 
HASTINGS,  J.  W.,  1959.     Unicellular  clocks.     Ann.  Rev.  Microbiol,  13:  297-312. 
HASTINGS,    J.    W.,    AND    A.    KEYNAN,    1965.     Molecular    aspects    of    circadian    systems.     In: 

Orcadian  Clocks,  Proc.  Feldafing  Summer   School,  ed.  Jurgen  Aschoff.     pp.   167-182. 
HASTINGS,  J.  W.,  AND  B.  M.  SWEENEY,  1957.     On  the  mechanism  of  temperature  independence 

in  a  biological  clock.     Proc.  Nat.  Acad.  Set.,  43:  804-811. 
HASTINGS,  J.  W.,  AND  B.  M.  SWEENEY,  1958.     A  persistant  diurnal  rhythm  of  luminescence  'in 

Gonyaulax  polyedra.    Biol.  Bull.,  115:  440-458. 
KLEMENT,   K.   W.,    1964.     Armored   dinoflagellates   of   the   Gulf   of   California.     Bull.   Scripps 

I nst.  Oceanog.,  8(5)  :  347-372. 
LEBOUR,   M.   V.,   1925.     The   Dinoflagellates   of   Northern    Seas.     Mar.    Biol.    Lab.,    Plymouth. 

172  pp. 
MAUCHLINE,  J.,  1960.     The  biology  of  the  Euphausid  crustacean,  Meganyctiphanes  norvegica 

(M.  Sars).     Proc.  Roy.  Soc.  Edinburgh.  Sect.  B.,  67,  Part  2(9)":   141-179. 
MCELROY,  W.  D.,  AND  H.  H.  SELIGER,  1962.     Origin  and  evolution  of  bioluminescence.    In: 

Horizons  in  Biochemistry,  Academic  Press,  Inc.,  New  York,  91-101. 
SCHILLER,  J.,  1937.     Dinoflagellatae  (Peridineae).     In:  Dr.  L.  Rabenhorst's  Kryptogamen-flora. 

Band  X,  Abt.  3,  2  Teil.  pp.  1-589. 
SELIGER,  H.  H.,  W.  G.  FASTIE  AND  W.  D.  MCELROY,  1961.     Bioluminescence  in  Chesapeake  Bay. 

Science,  133:  699-700. 
SEIGER,  H.  H.,  W.  G.  FASTIE,  W.  R.  TAYLOR  AND  W.  D.  MCELROY,  1962.     Bioluminescence  of 

marine  dinoflagellates  I.     An  underwater  photometer  for  day  and  night  measurements. 

/.  Gen.  Physiol.,  45:  1003-1017. 

SWEENEY,  B.  M.,  1963.     Bioluminescent  dinoflagellates.     Biol.  Bull.,  125:  177-181. 
SWEENEY,  B.  M.,  AND  J.  W.  HASTINGS,  1957.     Characteristics  of  the  diurnal  rhythm  of  lumines- 
cence in  Gonyanlanx  polyedra.     J.  Cell.  Comp.  Physiol.,  49:  115-128. 
SWEENEY,  B.  M.,  F.  T.  HAXO  AND  J.  W.  HASTINGS,  1959.     Action  spectra  for  two  effects  of 

light  on  luminescence  in  Gonyaulax  polyedra.    J.  Gen.  Physiol.,  43:  285-299. 
YENTSCH,  C.  S.,  R.  H.  BACKUS  AND  A.  S.  WING,  1964.     Factors  affecting  the  vertical  distribu- 
tion of  bioluminescence  in  the  euphotic  zone.     Limnol.  Oceanog.,  9:  519-524. 


FEEDING  BEHAVIOR  AND  REPRODUCTIVE  CYCLES 
IN  PISASTER  OCHRACEUS1 

KARL  PERRY  MAUZEY 

Department  of  Zoology,  University  of  Washington,  Seattle,  Washington,  and  Friday  Harbor 
Laboratories,  University  of  Washington,  Friday  Harbor,  Washington 

A  number  of  studies  have  shown  the  importance  of  the  relation  between  energy 
intake  and  reproductive  effort  in  higher  organisms.  Lack  (1954)  summarizes  a 
mass  of  data  indicating,  especially  for  birds,  the  dominant  role  played  by  the 
availability  of  food  in  determining  reproductive  strategy.  His  major  conclusion, 
based  on  different  degrees  of  reproductive  success  and  survival,  implicates  a  complex 
interaction  with  the  environment.  Among  invertebrates  the  influence  of  natural 
selection  on  reproductive  patterns  and  processes  is  poorly  understood.  In  the  field, 
the  dependence  of  gamete  production  on  food  has  been  shown  for  copepods  ( Marshall 
and  Orr,  1955),  rotifers  (Edmondson,  1965)  and  for  a  few  other  organisms.  Lab- 
oratory work  is  more  convincing  but  generally  less  applicable.  Experimental  studies 
on  Daphnla  (Richman,  1958),  a  rotifer  (King,  1965),  as  well  as  any  study  showing 
a  relationship  between  rate  of  population  growth  and  food  level,  can  be  thought  of 
as  giving  evidence  concerning  the  general  dependence  of  the  reproductive  perform- 
ance on  the  nutritional  state  of  the  population. 

Among  marine  macro-invertebrates,  a  number  of  studies  have  suggested  such 
dependence,  but  ecological  data  on  feeding  have  been  lacking.  For  example, 
Farmanfarmaian,  Giese,  Boolootian  and  Bennett  (1958)  have  indicated  an  inverse 
relationship  between  the  size  of  the  gonads  and  pyloric  caeca  of  two  carnivorous 
sea-stars,  Pisaster  ochraceus  and  P.  brevispinus.  Pearse  (1965)  suggested  that 
different  populations  of  a  probably  omnivorous  Antarctic  sea-star,  Odontastcr 
validns,  varied  in  reproductive  activity  according  to  local  differences  in  primary 
production.  Boolootian,  Farmanfarmaian  and  Giese  (1962)  have  demonstrated 
reciprocal  relationships  between  genital  and  hepatic  tissue  in  the  abalones  Haliotis 
cracherodli  and  H.  ntfescens,  as  have  Lawrence,  Lawrence  and  Giese  (1965)  for 
the  algivorous  chiton,  Katharina  tunicata.  Most  of  these  authors  have  suggested 
that  the  digestive  glands  are  used  to  stockpile  nutrients  during  the  months  when 
feeding  is  most  efficient.  Later  these  storage  products  are  transferred  to  the 
maturing  gonads.  The  hypothesis  that  changes  in  hepatic  tissues  are  correlated 
with  feeding  can  be  tested  most  feasibly  in  a  carnivorous  species.  Qualitative  and 
cuiantitative  aspects  of  the  nutrition  of  carnivores  are  usually  easier  to  follow  under 
natural  conditions  since  direct  observation  of  ingested  prey  is  possible. 

The  starfish,  Pisaster  ocJiraceus  (hereinafter  referred  to  as  Pisaster  unless 
another  species  is  indicated),  was  chosen  for  this  study  for  several  reasons. 
Pisaster  is  usualy  abundant  in  the  rocky  intertidal  region  of  San  Juan  Island, 

1  This  paper  is  a  condensed  version  of  a  thesis  submitted  to  The  University  of  Washington 
in  partial  fulfillment  of  the  requirements  for  the  degree  of  Master  of  Science. 

127 


128  KARL  PERRY  MAUZEY 

Washington,  as  well  as  on  most  rocky  shores  on  the  West  Coast  of  North  America. 
This,  coupled  with  its  relatively  limited  mobility  and  large  size,  permits  large 
numbers  to  be  obtained  from  repetitive  sampling  of  local,  discrete  populations. 
Feeding  is  easily  observed.  As  reported  by  Feder  (1956,  1959),  Pisastcr  uses 
its  tube  feet  to  force  open  prey,  or  to  wrench  it  off  the  substrate  ;  the  cardiac  stomach 
is  then  everted  onto  the  exposed  soft  tissues.  Consequently,  by  turning  these  star- 
fish over,  the  incidence  of  feeding  and  the  identity  of  the  prey  can  be  observed. 
Pisaster  occupies  the  position  of  "top  predator"  (Paine,  1963)  in  its  community, 
preying  on  the  members  of  several  lower  trophic  levels,  but  with  no  important 
predators  of  its  own  (Paine,  1966).  As  noted  above,  Farmanfarmaian  et  al. 
(1958)  have  demonstrated  inverse  gonad-pyloric  caeca  cycles.  Mauzey  (1963)  has 
previously  noted  that  there  is  also  a  seasonal  feeding  cycle.  The  present  paper 
reports  the  nature  of  the  interrelationships  of  the  organ  and  feeding  cycles  and 
discusses  the  ecological  consequences  and  implications. 

THE  STUDY  AREA 

The  study  was  carried  out  at  Lonesome  Cove,  situated  at  the  northeast  tip  of 
San  Juan  Island  (Latitude.  48°  37'20"  N,  Longitude,  123°  6'30"  W).  The  area  is 
scoured  by  a  strong  tidal  current.  \Yave  action  is  minimal ;  except  for  winter 
storms  there  is  no  more  than  would  be  expected  on  a  medium-sized  lake.  The 
tidal  range  is  from  minus  3.5  feet  to  plus  9.0  feet.  The  temperature  is  at  its 
maximum  of  about  13°  C.  in  July;  the  minimum  of  about  6°  C.  is  reached  in 
January.  Salinity  is  relatively  constant,  varying  from  about  29C/(C  to  31%0. 

The  intertidal  flora  and  fauna  correspond  generally  to  those  described  from 
Vancouver  Island  by  Stephenson  and  Stephenson  (1961;  especially  Brandon 
Island),  although  the  zonation  is  not  as  distinct.  There  is  a  splash  zone  above 
plus  7.0  feet  dominated  by  Littorina  sitkana  and  L.  scutitlota.  Below  this, 
Littorina  is  less  numerous  and  interspersed  among  other  organisms.  There  is  a 
Balanus-Fucus  zone  from  about  3  to  plus  7  feet  and  a  bare  zone,  with  a  few  scattered 
barnacles,  below  this  to  the  0  tide  level.  Below  0,  to  an  indefinite  boundary  several 
feet  below  extreme  lower  low  water,  there  is  an  almost  continuous  covering  of 
brightly  colored  crustose  coralline  algae  (mostly  Lithothainnion  spp.)  and  a  gradual 
increase  in  the  number  and  variety  of  brown  algae,  Laminaria  spp.,  Alaria  valid  a 
and  Nereocystis  luetkeana.  There  is  a  summer  covering  of  several  green  algae 
similar  to  Ulva,  extending  to  plus  2  or  3  feet.  All  the  algae  but  Fucns  are  markedly 
seasonal;  the  low  intertidal  and  subtidal  regions  are  almost  bare  in  the  winter, 
while  the  rocks  are  usually  completely  covered  in  the  summer  months.  There  are 
very  large  populations  of  urchins  (Strongylocentrolus  drobachicnsis  and  S.  fran- 
ciscamts)  that  undoubtedly  account  for  the  algal  disappearance  following  the 
summer's  prolific  growing  season.  These  urchins  are  generally  covered  at  low 
tide,  but  move  up  to  plus  4  feet  to  feed  during  nocturnal  high  tides. 

The  limpet,  Acniaea  persona,  is  prevalent  in  the  upper  part  of  the  Balanus-Fucus 
zone.  A.  digitalis,  A.  f>c!ta  and  A.  scutum  are  abundant  below  the  usual  range  of 
A.  persona  down  to  the  beginning  of  the  coralline  algae  zone.  A.  mitra  occupies 
this  latter  zone,  but  is  never  very  common.  Several  predatory  snails,  Thais 
huncllosa,  Searlcsia  dim  and  occasionally  individuals  of  T.  einaryinata  and  T. 


STARFISH  FEEDING  AND  REPRODUCTION  129 

canaliculate!,  occupy  the  Balatuts-Fucus  zone.  A  set  of  My  til  us  ednlis  occurs  most 
springs  in  this  zone,  but  few  survive  the  summer.  The  chiton,  Katharina  tnnicata, 
is  prevalent  from  the  lower  part  of  the  Balanus-Fucus  zone  well  into  the  coralline 
algae  zone.  Other  chitons,  including  Tonicella  line  at  a  and  several  species  of 
Mopalia,  share  the  same  range,  but  are  much  less  abundant.  There  are  many 
hermit  crabs,  Pagnnts  spp.,  and  shore  crabs,  Hemigrapsus  niidus  and  H. 
oregoncnsis, 

FEEDING  OBSERVATIONS 

Pisastcr  is  one  of  the  most  conspicuous  animals  on  wave-  or  current-swept  rocky 
shores  from  Sitka,  Alaska,  to  Ensenada,  Mexico  (Ricketts  and  Calvin,  1952). 
Most  of  the  population  is  confined  to  the  intertidal  zone.  I  have  only  occasionally 
observed  animals  as  deep  as  30  feet  below-  mean  lower  low  water  (the  zero  of  West 
Coast  Tide  Tables).  Feeler  (1956)  indicates  that  this  is  the  maximum  depth  for 
Pisastcr  in  Central  and  Southern  California. 

Observations  on  feeding  were  made  by  skin-diving  over  the  intertidal  zone  during 
high  tide.  This  phase  of  the  tidal  cycle  was  chosen  because  the  animals  are  then 
at  their  peak  of  foraging  activity  (Mauzey,  unpublished  data).  Each  individual 
was  removed  from  the  substrate  and  its  oral  surface  examined.  An  everted 
stomach  was  taken  as  evidence  of  feeding;  usually  the  prey  could  be  seen  in  the 
folds  of  the  stomach.  These  observations,  together  with  a  size  estimate  of  the 
predator,  were  recorded  on  a  plastic  card.  During  the  first  part  of  the  study,  May, 
1962,  through  July,  1963,  sampling  dives  were  made  twice  monthly.  During  the 
remainder  of  the  study  the  sampling  interval  was  lengthened  to  once  a  month 
because  analysis  of  the  initial  feeding  data  indicated  that  all  trends  discussed  below 
would  be  apparent  with  this  longer  sampling  interval.  From  May,  1962,  through 
April,  1963,  each  dive  was  terminated  after  100  animals  had  been  observed.  After 
April,  1963,  the  entire  sampling  area  was  searched  during  each  dive,  and  all  speci- 
mens of  Pisaster  present  were  counted.  This  change  was  made  because  a  seasonal 
change  in  abundance  was  noticed.  These  samples  indicate  that  the  population 
varies  from  about  100  in  the  winter  to  about  200  in  the  summer.  There  are 
indications  that  this  is  due  in  part  to  more  starfish  being  hidden  in  crevices  and  under 
rocks  in  the  winter,  and  in  part  to  a  seasonal  movement  into  deeper  water. 

The  feeding  results  for  the  entire  period  (22  months)  are  given  in  Figure  1 
which  is  based  on  observations  of  3,820  individuals,  of  which  1,364  individuals 
(35.4%)  were  feeding.  Since  one  sea-star  sometimes  feeds  on  more  than  one  prey 
species  at  a  time,  there  are  1,557  observations  of  feeding  on  particular  prey  species. 
The  category,  Balanus  spp.  includes  predominantly  B.  cariosiis,  but  a  few  small 
B.  glandula  were  also  taken.  The  category  Acniaea  spp.  in  the  feeding  observations 
includes  predominantly  A.  pclta  and  A.  scutum,  but  Pisaster  also  eats  a  few  A. 
digitalis  and  A.  persona  that  are  in  its  range.  Of  all  observations  81%  are  on 
Balanus  spp.,  Acmaea  spp.  and  Mytilits  ediilis;  the  rest  each  account  for  5%  or  less. 

In  an  additional  30  observations  the  stomach  was  everted  but  no  prey  could  be 
found.  These  are  not  included  in  Figure  1.  The  most  likely  explanation  is  that 
these  represent  animals  engaged  in  flagellary-mucous  feeding  on  detritus  (Anderson, 
1960;  Mauzey,  1963;  Pearse,  1965).  An  alternative  explanation,  that  prey  was 
present  but  not  observed,  is  less  likely  since  these  everted  stomachs  were  carefully 
searched,  often  to  the  extent  of  damaging  the  thin-walled  organ. 


130 


KARL  PERRY  MAUZEY 


These  observations  are  very  similar  to  those  of  Feder  (1959)  in  California.  He 
reports  somewhat  greater  feeding  on  barnacles  (Balanus  glandula,  B.  nubilis  and 
Tetraclita  squamosa  rubescens}  (57.0%)  and  on  Mytilus  californianus  (17.0%) 
but  less  feeding  on  Acmaea  spp.  (4.8%)  and  on  chitons  (mostly  Mopalia  muscosa} 
(4.5%).  There  is  a  large  difference  with  respect  to  Katharina  tunicata,  the  chiton 
mainly  eaten  in  the  San  Juan  Island  area.  Only  0.2%  of  his  observations  were  on 
this  species,  as  compared  with  3.8%  of  mine.  In  both  studies,  a  few  organisms 
are  fed  on  heavily,  while  a  large  number  is  eaten  only  occasionally. 

Although  Balanus,  Aciuaea  and  M.  ednlis  are  the  numerically  dominant  prey, 
they  represent  a  much  smaller  percentage  of  the  total  biomass  of  food.  Dry  weight 
versus  length  correlations  were  established  for  the  six  most  prominent  species 

PERCENT 


BALANUS    SPP. 

ACMAEA      SPP. 

MYTILUS     EDULIS 

LITTORINA    SPP. 

KATHARINA   TUNICATA 

THAIS   SPR 

TONICELLA    LINEATA 

MARGARITES    SPP. 

CRABS 

SEARLESIA    DIRA 

MOPALIA     SPP. 

CALLIOSTOMA    COSTATUM 

TUNICATES 

STRONGYLOCENTROTUS    SPP 

OYSTER 

BRACHIOPOD 

AMPHIPOD 

WORM  TUBE 


788 


288 


FIGURE  1.  Percentages  of  Pisastcr  feeding  on  indicated  prey,  summed  over  the  entire  study 
period,  March,  1962,  through  January,  1964.  The  number  observed  feeding  on  each  prey 
category  is  given  at  the  end  of  the  bar. 

consumed  by  Pisaster  (Mauzey,  unpublished  data).  Only  those  parts  of  the  prey 
that  are  actually  eaten  by  Pisaster  were  weighed,  i.e.,  shells,  and  the  girdle  of 
chitons,  were  omitted.  The  size  and  number  of  all  prey  could  not  be  recorded  in 
the  field  due  to  lack  of  time  underwater.  Therefore,  I  estimated  the  average  size 
and  number  of  each  prey  for  all  feedings,  based  on  impressions  gathered  over  the 
entire  period  of  the  study.  This  method  permits  only  a  rough  estimate  of  the 
biomass  ingested  (Table  I).  Research  in  progress  suggests  that  the  seasonal 
pattern  reported  here  is  typical  for  Pisaster  at  Lonesome  Cove.  On  the  basis  of 
dry  weight,  chitons  are  the  most  important  prey ;  Balamis  and  Mytilus  edulis, 
because  of  their  small  size,  are  of  secondary  importance.  The  importance  of 
Acmaea  and  Littorina  is  somewhat  reduced,  and  that  of  Thais  spp.,  a  carnivorous 


STARFISH  FEEDING  AND  REPRODUCTION 


131 


whelk,  is  greatly  increased,  but  it  still  remains  a  small  part  of  Pisaster's  diet. 
Paine  (1966)  has  found  approximately  the  same  reversals  in  importance  on  the 
outer  coast  of  Washington. 

When  these  data  are  observed  with  respect  to  time,  a  definite  feeding  cycle  is 
apparent  in  terms  of  per  cent  feeding,  number  of  individuals  eaten,  and  dry  weight 
ingested.  The  percentages  of  Pisaster  that  were  observed  feeding  in  each  sample, 
and  the  estimated  dry  weight  ingested  are  plotted  in  Figure  2a.  The  calculations  for 


TABLE  I 

Dry  weight  data  for  the  six  most  important  of  Pisaster's  prey.     Since  the  data  are  calculated 

on  the  basis  of  a  common  number  observed  in  each  sample  (100),  the  numbers  in  the  total 

column  do  not  agree  with  Figure  2,  which  is  based  on  the  uncorrected  data.     The  data 

from  the  single  -winter  are  doubled  to  allow  comparison  with  the  data 

from  two  summers 


Prey  organism 

Balanus  spp. 
(B.  cariosus, 
B.  glandula) 

Acmaea  spp. 
(A.  pelta, 
A.  scutum) 

Mytilus 
edulis 

Chitons 
(Katharina, 
Tonicella, 
Mopalia  spp.) 

Littorina  spp. 
(L.  sitchana, 
L.  scululala) 

Thais  spp. 
(T.  lamellosa, 
T.  emargtnaia, 
T.  canali- 
culata) 

Average  size  ingested 

1.60 

2.10 

1.00 

7.50 

0.70 

6.00 

(cm.) 

Dry  weight  of  in- 

0.04 

0.10 

0.01 

2.50 

0.03 

0.60 

gested  size  (gm.) 

Average  number  per 

5 

3 

5 

1 

3 

1 

feeding 

Dry  weight  per 

0.20 

0.30 

0.05 

2.50 

0.10 

0.60 

feeding  (gm.) 

Number  ingested 

Winter  (Dec.  '62- 

20 

18 

10 

48 

2 

4 

Mar.  '63) 

Summer  (June- 

479 

177 

90 

30 

48 

14 

Sept.  '62  &  '63) 

One  year  (May  '62- 

367 

213 

100 

77 

48 

27 

May  '63) 

Total  (Mar.  '62- 

620 

249 

140 

106 

66 

39 

Jan.  '64) 

No  rank 

Winter 

2 

3 

4 

1 

6 

5 

Summer 

1 

2 

3 

5 

4 

6 

One  year 

1 

2 

3 

4 

5 

6 

Total 

1 

2 

3 

4 

5 

6 

Dry  weight  ingested 

\\'i  nter 

4.00 

5.40 

0.50 

120.00 

0.20 

2.40 

Summer 

95.80 

53.10 

4.50 

75.00 

4.80 

8.40 

One  year 

73.40 

63.90 

5.00 

192.50 

4.80 

16.20 

Total 

124.00 

74.70 

7.00 

265.00 

6.60 

23.40 

Weight  rank 

Winter 

3 

2 

5 

1 

6 

4 

Summer 

1 

3 

6 

2 

5 

4 

One  year 

2 

3 

5 

1 

6 

4 

Total 

2 

3 

5 

1 

6 

4 

132  KARL  PERRY  MAUZEY 

the  dry-weight  curve  were  made  with  the  data  of  Tahle  I.  This  introduces  some 
error  since  the  average  size  of  the  shorter-lived  species  changes  during  the  year, 
e.g.,  the  barnacles  are  smaller  in  the  spring  just  after  settling  than  in  the  fall  after 
a  summer  of  growth.  These  small  barnacles  provide  a  large  proportion  of  the 
summer  prey,  but  some  that  are  several  years  old  are  also  eaten.  In  order  to 
compare  the  biomass  of  prey  ingested  on  each  dive,  a  correction  must  be  made 
for  the  different  number  of  starfish  observed  on  different  dives.  In  Figures  2a  and 
b,  observations  are  corrected  to  100  animals  per  dive;  in  Table  I,  to  200  per  month, 
or  two  dives  of  100  animals  each.  Figure  2b  shows  the  number  of  starfish  feeding 
on  various  items ;  starfish  feeding  on  two  prey  species  are  recorded  twice.  Figure  2c 
plots  the  per  cent  of  Pisastcr  feeding  on  a  particular  prey  as  a  percentage  of 
those  feeding. 

Chitons  play  an  unexpectedly  important  role  in  Pisastcr's  nutrition.  Few  are 
ingested  at  any  time,  and,  except  for  a  slight  drop  in  the  summer,  the  rate  at  which 
they  are  eaten  appears  to  be  constant  (Fig.  2b).  In  the  winter  chitons  constitute 
almost  the  sole  food,  while  most  of  the  Pisastcr  individuals  are  feeding  on  other  prey 
in  the  summer.  The  comparatively  large  size  of  chitons  among  Pisaster's  prey, 
and  a  pronounced  seasonal  behavior  pattern  of  the  predator,  account  for  these 
observations.  In  the  summer,  the  sea-stars  are  scattered  singly  over  the  intertidal 
from  about  plus  5  feet  to  minus  2  feet  tide  level,  while  in  winter  from  5  to  25 
starfish  may  clump  together  in  crevices  and  other  protected  areas  between  about 
zero  and  minus  4  feet  tide  level.  The  same  crevices  are  occupied  from  dive  to 
dive,  apparently  by  the  same  starfish,  since  individuals  which  had  distinctive  color 
patterns  were  observed  in  the  same  crevices  for  several  dives.  These  clumped 
animals,  characterized  by  a  low  incidence  of  feeding,  eat  primarily  chitons.  These 
prey,  the  only  ones  that  occur  commonly  below  the  zero  tide  mark,  'form  the  major 
part  of  Pisaster's  winter  diet,  apparently  because  their  grazing  movements  bring 
them  into  the  predator's  winter  clumps. 

Preliminary  observations  suggest  that  Pisastcr  prefers  Mytilus,  and,  in  fact, 
may  have  evolved  in  relation  to  the  dense  populations  of  M.  californianus  on  the 
exposed  coast.  At  Lonesome  Cove,  Pisaster  seems  to  devour  preferentially  indi- 
viduals of  the  species  M.  editlis  within  a  relatively  short  period  after  their  settlement. 
In  areas  like  this,  Pisastcr  must  feed  on  such  large  potential  prey  as  chitons  in 
order  to  sustain  themselves,  although  laboratory  observations  suggest  that  these 
are  eaten  only  with  great  reluctance  in  the  laboratory. 

SEASONAL  CHANGES  IN  GONAD  AND  HEPATIC  TISSUES 

Giese  (1959)  describes  a  method  for  assessing  the  reproductive  cycle  of  an 
animal  by  periodically  determining  a  gonad  index,  a  measure  of  the  ratio  of  gonad 
size  to  body  size.  This  method  assumes  that  in  individuals  large  enough  to  be 
mature,  a  spent  or  immature  gonad  is  small,  and  a  ripe  gonad  is  large.  A  plot  of 
successive  determinations  on  samples  of  a  population  indicates  the  average  reproduc- 
tive state  of  the  population  with  respect  to  time.  Spawning  is  indicated  by  a  sharp 
drop  in  the  gonad  index.  A  similar  method  can  be  applied  to  indicate  the  condition 
of  food-storage  organs. 

Farmanfarmaian  ct  al.  (1958,  p.  356)  have  published  their  procedures  for 
determining  such  gonad  and  hepatic  indices  for  Pisastcr  from  the  Central  California 


STARFISH  FEEDING  AND  REPRODUCTION 


133 


o 

z 

5 
u 


LU 
<_» 

CC 
UJ 

D- 


90 
80 
70- 
60 
50- 
40 
30 
2& 
10 


PERCENT  FEEDING 


___  J 


30 


20 


10 


UJ 


CC 

a. 

Q 

U) 

S 


BALANUS    SPP. 
ACMAEA    SPP. 
MYTILUS    EDULIS 
CHITONS 


o 

o 

z 

Q 

LU 
UJ 

u. 
tt 

UJ 

oo 
co 

Q. 


MA 


1962 


963 


1964 


FIGURE  2.  The  seasonal  feeding  pattern  of  Pisaster.  A.  The  per  cent  of  the  animals 
observed  feeding,  and  an  estimate  of  the  dry  weight  ingested  at  each  sample.  B.  The  number  of 
Pisaster  observed  feeding  on  the  four  most  commonly  eaten  prey.  These  data  are  corrected  to  a 
common  basis  of  100  animals  observed  in  each  sample.  C.  The  data  of  B  plotted  as  a  per  cent 
of  those  feeding,  showing  that  chitons  are  the  only  prey  at  certain  times  of  the  year.  The  legend 
above  B  applies  to  both  B  and  C. 


134  KARL  PERRY  MAUZEY 

coast :  "Ten  specimens  were  gathered  monthly.  Each  specimen  was  weighed  after 
blotting  and  slit  aborally  along  the  arms  towards  the  center  of  the  animal  with  a 
pair  of  scissors.  The  gonads  were  removed  and  the  volume  was  determined  by  its 
displacement  of  a  known  volume  of  water  in  a  graduate  cylinder.  The  ratio  of 
gonad  volume  to  body  weight  X  100  was  taken  as  the  gonad  index.  The  digestive 
gland  was  weighed  and  the  ratio  of  digestive  gland  to  body  weight  X  100  was 
taken  as  the  hepatic  index."  Measurements  of  the  specific  gravity  of  the  gonads 
and  hepatic  caeca  indicated  that  the  specific  gravity  does  not  deviate  significantly 
from  that  of  water,  obviating  the  necessity  of  making  conversion  to  weight.  The 
results  from  this  three-year  study  by  Farmanfarmaian  et  al.  show  a  peak  from  about 
March  through  May,  and  a  rapid  drop  in  May  or  June,  associated  with  gamete 
release.  The  gonad  index  increases  again  in  October  or  November,  and  gradually 
climbs  to  its  spring  peak.  In  one  year  the  peak  was  lower,  and  the  decline  earlier 
and  more  gradual. 

The  hepatic  index  generally  shows  an  inverse  relationship  to  the  gonad  index. 
Farmanfarmaian  et  al.  (loc.  cit.)  suggest  that  this  is  due  to  the  transfer  of  stored 
glycogen,  lipid  and  protein  (shown  to  be  present  in  the  pyloric  caeca  by  Greenfield, 
Giese,  Farmanfarmaian  and  Boolootian,  1958)  to  the  developing  gonad.  They 
further  state  that  this  does  not  seem  to  be  correlated  with  a  seasonal  feeding  cycle. 
However,  starved  individuals  did  show  shrunken  gonads  as  well  as  shrunken  pyloric 
caeca  (cf.  also  Feder,  1956),  indicating  that  if  seasonal  differences  in  the  popula- 
tion's feeding  pattern  do  exist,  they  are  likely  to  have  an  important  effect  on  the 
size  of  these  organs. 

The  method  described  above  was  followed  closely  in  the  present  study,  except 
that  the  volume,  rather  than  the  weight,  of  the  pyloric  caeca  was  used  to  calculate 
the  hepatic  index.  The  substitution  should  not  affect  comparisons,  due  to  the 
closeness  of  the  organ's  specific  gravity  to  1.0.  The  whole  animals  were  drained 
on  paper  toweling  for  about  15  minutes  before  weighing;  the  excised  organs  were 
also  blotted  before  measurement,  for  about  5  minutes.  The  hepatic  index  (Fig.  3) 
rose  during  the  autumn  in  1962  and  1963,  reached  a  plateau  in  March,  1963,  and 
dropped  rapidly  in  June.  As  in  the  earlier  study  by  Farmanfarmaian  et  al.,  the 
female  gonad  index  was  somewhat  higher  than  that  of  the  males. 

Spawning  wras  observed  in  laboratory  tanks  in  early  May  and  again  on  June  7, 
13,  and  14,  1963.  No  spawning  was  observed  in  the  field  that  summer ;  in  other 
summers  I  have  observed  Pisaster  spawning  in  June,  July  and  August.  Moreover, 
Pisaster  gametes  are  often  difficult  to  obtain  for  embryology  classes  during  the 
latter  half  of  the  summer,  whereas  they  are  usually  readily  available  in  the  first 
half  of  the  summer.  The  release  of  most  of  the  gametes  would  seem  to  have 
taken  place  in  the  middle  of  June,  1963,  based  on  the  gonad  index  and  scattered 
laboratory  observations  (as  was  also  the  case  in  1965)  ;  smaller  numbers  are 
released  throughout  the  rest  of  the  summer,  accounting  for  the  continued  slight  fall 
of  the  gonad  index.  In  California  spawning  appears  to  be  more  abrupt,  and  to 
occur  somewhat  earlier.  According  to  Giese  (1959)  Pisaster  in  California  spawns 
from  April  to  May ;  only  occasionally  does  the  spawning  extend  into  June.  The 
gonad  index  there  is  uniformly  low  throughout  the  summer  months. 

As  was  true  in  California,  the  hepatic  index  for  Lonesome  Cove  animals  is 
approximately  inverse  to  the  gonad  index.  An  hepatic  index  minimum  is  reached 


STARFISH  FEEDING  AND  REPRODUCTION 


135 


ORGAN 
INDEX 


20 


15 


10 


GONAD  INDEX 
HEPATIC   INDEX 
INDEX    SUM 
PERCENT    FEEDING 


00 


80 


PERCENT 
FEEDING 


60 


40 


20 


1962 


1963 


j 
1964 


FIGURE  3.  Seasonal  changes  in  the  gonad  and  hepatic  indices,  per  cent  feeding,  and  the 
sum  of  the  gonad  and  hepatic  indices.  Note  that  the  organ  indices  are  approximately  inverse 
to  each  other,  and  that  the  feeding  curve  is  similar  to  that  of  the  hepatic  index,  but  delayed 
about  4  months. 

in  May ;  the  index  rises  all  summer  to  a  peak  in  November,  and  falls  until  the  next 
spring  (Fig.  3).  There  is  no  significant  difference  between  the  male  and  female 
hepatic  index. 

HISTOLOGY 

Specimens  of  the  excised  organs  of  each  starfish  obtained  at  each  sampling  were 
preserved  in  Bouin's  fluid  for  subsequent  histological  observations.  These  provide 
both  general  confirmation  of  the  gross  changes  in  size,  and  some  details  as  to  what 
is  involved  in  these  changes.  The  tissues  were  imbedded  in  paraffin,  sectioned 
at  5  p,  and  stained  with  Harris'  hematoxylin  and  eosin,  Mallory's  triple  stain,  or 
the  periodic  acid-Schiff  routine  (PAS),  counterstained  with  Harris'  hematoxylin 
(Pantin,  1946;  McManus  and  Mawry,  1960).  Salivary  amylase  digestion  was 
used  in  conjunction  with  some  of  the  PAS  material. 

Sections  were  made  of  the  ovaries  of  three  females  from  samples  collected  on 
September  14,  1962  (Fig.  4),  December  25,  1962,  and  April  8,  1963  (Fig.  5). 
The  gonad  indices  of  the  females  were  0.73,  3.59  and  22.91,  respectively;  the  gonad 
indices  of  the  samples  from  which  they  were  taken  were  1.43,  3.38  and  9.47, 
respectively. 

In  September,  the  oocytes  (Fig.  4)  have  diameters  between  10  and  50  /x ;  in 
December,  they  range  from  about  10  /A  to  150  yu,;  in  April  (Fig.  5)  there  are  two 
size  groups,  one  about  150  /*,  and  the  others  less  than  20  /j,.  Below  about  20  p, 
they  are  PAS-negative ;  above  that  size  the  oocytes  become  progressively  more 
and  more  PAS-positive.  The  very  intensely  PAS-positive  oocytes  of  150  ^  are 
probably  "mature,"  although  they  can  only  be  fertilized  if  released  through  the 
oviducts.  Living  eggs  thus  obtained  measure  about  200  /x. ;  the  50  /A  difference  is 


136 


KARL  PERRY  MAUZEY 


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FIGURES  4-5. 


STARFISH  FEEDING  AND  REPRODUCTION  137 

probably  a  fixation  artifact.  The  small,  PAS-negative  oocytes  present  in  April 
are  probably  those  that  will  be  spawned  one  year  hence;  i.e.,  complete  oogenesis 
may  take  more  than  one  year.  However,  since  some  "mature"-sized  oocytes  are 
present  in  September,  December  and  April,  there  could  be  continual,  or  almost 
continual,  production  of  new  oocytes,  with  maturation  taking  perhaps  eight 
months  (April  or  September  to  December  or  May). 

In  the  ovaries  collected  in  September  and  December,  imaginations  of  the  base- 
ment membranes  of  the  germinal  epithelium  produce  ovarian  folds  (Fig.  4),  which 
have  not  been  previously  described  from  asteroid  ovaries.  The  developing  oocytes 
occur  along  the  germinal  epithelium  both  on  and  between  the  folds.  Between  the 
basement  membrane  on  either  side  of  the  folds,  and  between  the  basement  membrane 
and  the  rest  of  the  ovarian  wall  there  is  a  sinus.  This  sinus  has  been  reported 
before  by  several  investigators,  including  Chia  (1964)  in  Leptasterias,  and 
Delavault  (1961)  in  Echinaster  sepositus.  Both  the  basement  membrane  and  the 
coagulated  contents  of  the  sinus  are  strongly  PAS-positive  (see  Fig.  4).  In  the 
mature  ovary,  the  presence  of  many  mature  ova  seems  to  stretch  the  rest  of  the 
ovarian  wall,  and  flatten  out  the  folds.  The  sinus  is  reduced  and  no  longer  PAS- 
positive;  the  basement  membrane  is  still  PAS-positive,  but  appears  much  reduced, 
perhaps  due  to  being  stretched  (see  Fig.  5).  Chia  (1964)  postulates  that  the  sinus 
serves  to  supply  nutrients  to  the  developing  oocytes.  This  hypothesis  is  supported 
by  my  observations  that  (a)  only  mature  gametes  are  free  in  the  lumen,  (b)  this 
space  becomes  reduced  as  a  larger  proportion  of  the  ova  become  mature,  and  (c) 
the  contents  are  PAS-positive  when  the  rate  of  transfer  of  nutrients  to  the  gonads  is 
heaviest. 

Spermatogenesis  has  not  been  followed,  but  some  observations  seem  to  be  in 
general  agreement  with  the  descriptions  of  Cognetti  and  Delavault  (I960)  and 
Pearse  (1965).  Motile  sperm  are  present  in  smears  of  testes  from  at  least  a  few 
specimens  of  Pisastcr  at  all  times  of  the  year.  Testes,  as  well  as  ovaries,  collected 
in  the  spring  tend  spontaneously  to  release  very  large  numbers  of  gametes ;  it  is 
necessary  to  collect  these  shed  gametes  carefully  to  get  an  accurate  gonad  volume 
measurement.  A  preliminary  section  of  the  testes  indicates  the  presence  of  the 
spermatic  papillae  described  by  the  above  authors ;  the  interior  of  these  is  occupied 
by  a  fold  of  basement  membrane  enclosing  a  sinus  whose  contents  are  PAS-positive, 
as  in  the  ovary.  This  does  not  appear  to  be  the  case  in  Odontaster  (Pearse,  1965) 
or  Echinaster  (Cognetti  and  Delavault,  1960). 

Salivary  amylase  treatment  of  sections  of  ovaries  collected  in  December  indicates 
no  detectable  change  in  the  PAS-positive  reaction  of  either  the  gametes  themselves, 
or  the  coagulated  material  within  the  sinuses.  Similar  results  have  been  obtained 
by  Chia  (1964).  This  indicates  that  the  material  is  not  glycogen,  agreeing  with 

FIGURE  4.  Photomicrograph  of  a  Pisaster  ovary  collected  in  September,  1962.  The  tissue 
was  fixed  in  Bouin's  fluid,  imbedded  in  paraffin,  sectioned  at  5  /*,  and  stained  with  PAS  and 
hematoxylin.  The  structure  of  the  ovarian  wall,  the  ovarian  folds,  the  sinus  and  the 
preponderance  of  small  oocytes  are  visible. 

Figure  5.  Photomicrograph  of  a  Pisaster  ovary  collected  in  April,  1963.  Same  technique 
as  in  Figure  4.  The  many  large  oocytes  and  the  few  very  small  ones  should  be  noted. 

Key  to  abbreviations  used  in  Figures  4  and  5 :  C,  coelomic  epithelium ;  G,  germinal 
epithelium  ;  L,  lumen ;  LO,  large  oocytes ;  M,  middle  layer  of  ovarian  wall ;  OP,  distal  end  of 
ovarian  fold ;  S,  sinus ;  SO,  small  oocytes. 


138 


KARL  PERRY  MAUZEY 


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STARFISH  FEEDING  AND  REPRODUCTION  139 

the  observation  of  Greenfield  ct  al.  (1958)  that  glycogen  is  a  minor  constituent  of 
the  gonads  of  Pisaster,  averaging  only  about  0.35%  of  the  dry  weight. 

Sections  of  the  pyloric  caeca  generally  substantiate  the  histology  of  these  organs 
reported  by  Anderson  (1953)  for  Asterias  forbesi,  and  by  Chia  (1964)  for 
Leptasterias  h  exact  is.  There  is  first  an  outer  peritoneal  layer,  then  a  layer  con- 
taining connective,  nervous,  and  muscular  tissue  elements,  and,  finally,  the  epithelial 
layer  of  very  tall,  slender  columnar  cells.  The  latter  is  mostly  responsible  for  the 
thickness  of  the  caecal  wall.  There  are  four  kinds  of  cells  in  this  epithelium:  (1) 
special  current-producers,  (2)  zymogen  or  secretory  cells,  (3)  storage  cells,  and 
(4)  mucous  cells.  Since  the  pyloric  caeca  of  Pisaster  are  larger  and  more  folded 
than  those  of  the  other  starfish  studied,  the  special  current-producers  that  occur 
mainly  on  the  oral  and  aboral  aspects  of  the  central  duct  have  not  been  seen  in  my 
sections  through  the  lobes  of  the  pyloric  caeca.  The  storage  cells  comprise  the 
bulk  of  the  remainder  of  the  epithelial  layer,  with  many  scattered  zymogen  cells, 
and  relatively  few  mucous  cells  (see  Figs.  6  and  7). 

The  location,  staining  qualities  and  seasonal  appearance  of  the  granules  abun- 
dantly present  in  Figure  6  suggest  that  they  represent  nutrients  stored  in  the  pyloric 
caeca  during  the  summer  feeding  period.  These  storage  granules  seem  to  be  within 
the  storage  cells  identified  by  Anderson  (1953)  and  Chia  (1964).  When  stained 
with  Mallory's  triple  stain,  these  granules  can  be  distinguished  as  two  types  which 
form  overlapping  bands  across  the  long  axis  of  the  storage  cells.  The  distal  band 
stains  yellow ;  the  proximal,  wider  band,  blue.  The  proximal  band  is  PAS- 
positive.  In  pyloric  caeca  from  Pisaster  of  low  hepatic  index  (Fig.  7)  there  are 
very  few,  or  no  storage  granules  present.  There  is  a  band  of  nuclei  in  the  middle 
portion  of  the  cells,  presumably  obscured  by  storage  granules  in  Figure  6.  The 
major  morphological  differences  between  Figures  6  and  7  thus  relate  easily  to 
seasonal  patterns  of  transfer  and  storage  of  energy-rich  materials  in  the  hepatic 
caeca. 

DISCUSSION 

The  information  in  the  preceding  sections  on  feeding,  patterns  of  organ  morphol- 
ogy, and  reproductive  cycles  permits  clarification  of  the  relationship  between  these 
phenomena  and  a  discussion  of  the  evolution  of  Pisaster's  seasonal  behavior. 

The  organ  indices  and  the  per  cent  feeding  for  each  sampling  are  plotted 
together  for  comparison  in  Figure  3.  These  curves  indicate  a  functional  relation- 
ship between  the  processes  they  measure.  Food  ingested  during  the  summer  is 
stored  in  the  pyloric  caeca;  investigations  by  Anderson  (1953)  and  Ferguson 

FIGURE  6.  Photomicrograph  of  a  Pisaster  pyloric  caecum  collected  in  September,  1962. 
The  tissue  was  fixed  in  Bouin's,  imbedded  in  paraffin,  sectioned  at  5  n,  and  stained  with 
Mallory's  triple  stain.  Note  the  presence  of  zymogen  cells,  mucous  cells,  and  storage  cells 
filled  with  many  storage  granules.  In  this  and  the  following  figure,  the  epithelial  layer  (E) 
occupies  almost  the  entire  figure. 

FIGURE  7.  Photomicrograph  of  a  Pisaster  pyloric  caecum  collected  in  April,  1963.  Same 
technique  as  in  Figure  6.  Note  the  absence  of  zymogen  granules  and  storage  granules. 

Key  to  the  abbreviations  used  in  Figures  6  and  7 :  B,  brush  border ;  C,  outer  epithelial  layer  ; 
D,  distal  disintegration ;  E,  epithelial  layer ;  L,  lumen ;  M,  middle  layer  of  the  wall  of  the 
pyloric  caecum ;  N,  nuclei  of  cells  of  the  epithelial  layer ;  P,  pigment  layer ;  S,  storage  granules 
in  storage  cells;  U,  mucous  cells;  Z,  zymogen  granules  in  zymogen  cells. 


140  KARL  PERRY  MAUZEY 

(1964a)  strongly  suggest  that  these  are  the  major  storage  organs  of  asteroids. 
There  is  a  2-4  month  interval  between  the  response  maximum  and  minima  of  the  dry 
weight  ingested  (Fig.  2a)  and  the  hepatic  index  curves  (Fig.  3).  This  lag  is 
explained  by  the  fact  that  the  size  of  the  pyloric  caecum  represents  a  temporal 
summation  of  the  excess  of  nutrient  over  metabolic  use.  Reduced  respiratory 
costs  associated  with  declining  water  temperatures  and  lessening  movement  in  the 
fall  must  more  than  offset  the  reduced  caloric  income,  and  the  hepatic  index  con- 
tinues to  rise  even  after  the  maximum  value  of  the  dry  weight  ingested.  In  the 
spring  the  pyloric  caeca  continue  to  decrease  until  feeding  supplies  more  nutrient 
than  is  used. 

The  gonads  begin  to  grow  in  size  in  the  fall,  employing  material  from  the 
pyloric  caeca  accumulated  from  summer  feeding.  Ferguson  (1964a,  1964b)  gives 
experimental  verification  of  nutrient  transfer  from  the  digestive  glands  to  the  other 
tissues  of  starfish  through  the  coelomic  fluid,  even  though  the  concentration  of 
nutrient  in  this  fluid  is  very  low  at  any  one  time.  As  the  gonads  increase  in  size, 
the  removal  of  stored  nutrient  causes  a  decline  in  the  size  of  the  pyloric  caeca. 
Finally,  the  gonad  index  drops  dramatically  when  the  animals  spawn  in  June. 

Pisastcr  probably  spawns  in  the  late  spring,  with  the  effect  that  the  larvae 
are  in  the  plankton  during  the  summer  when  the  larval  food  supply  is  at  its 
greatest  abundance.  The  length  of  time  spent  in  the  plankton  can  be  inferred 
from  a  study  by  Quayle  (1954)  at  Nanaimo,  B.  C.  He  observed  young  starfish 
on  strings  of  oyster  shells  set  out  in  conjunction  with  a  study  of  oyster  settling. 
These  had  been  exposed  for  oyster  settlement  in  August,  1952,  at  which  time  the 
starfish  must  also  have  settled  (Quayle,  1954).  One  can  reasonably  postulate  a 
June  spawning  time  for  these  starfish  for  two  reasons  :  ( 1 )  the  two  known  spawning 
periods  for  Pisastcr  are  very  close :  May,  generally,  for  central  California 
( Farmanfarmaian  ct  al.,  1958)  and  June,  for  the  San  Juan  Island  region,  and 
(2)  the  use  of  the  latter  period  is  justified  by  the  proximity  (50  miles)  of  Nanaimo 
to  my  study  area.  Further,  since  Quayle  reports  a  "heavy  settlement"  of  the  young 
sea-stars,  this  settlement  probably  resulted  from  the  peak  spawning  period  in  June. 
Therefore  the  larval  period  must  last  about  two  months,  from  June  to  August, 
when  the  plankton  is  richest,  the  factor  which  must  ultimately  set  the  timing  of 
Pisaster's  reproductive  cycle. 

The  fundamental  reason  why,  in  Pisaster,  the  volume  of  pyloric  caeca  varies 
seasonally  must  be  related  to  a  pronounced  advantage  of  feeding  during  the  summer. 
If  not,  Pisastcr  could  feed  and  elaborate  gametes  continuously.  To  follow  the 
former  strategy,  some  means  of  energy  storage  is  essential,  this  requisite  being 
met  by  the  pyloric  caecum.  Unless  the  pyloric  caeca  can  serve  this  function,  feeding 
must  be  greatest  at  the  time  of  gonad  growth.  The  loss  of  approximately  10% 
of  the  body  weight  in  gametes  (up  to  23%  in  some  individuals)  must  consume  a 
considerable  proportion  of  the  energy  assimilated  yearly.  On  an  ash-free  dry- 
weight  basis,  these  percentages  are  even  higher  since  calcareous  structural  material 
forms  a  considerable  part  of  the  body  wall.  There  is  no  obvious  reason  why 
Pisaster  could  not  feed  most  heavily  in  the  winter.  A  few  specimens  of  Pisastcr 
are  feeding  in  the  winter  in  the  field,  and  Pisastcr  feeds  all  winter  in  laboratory 
tanks  (1°-H°  C.  warmer  than  in  the  field).  In  addition,  much  of  the  prey  of 
Pisaster  is  perennial  and  therefore  occurs  in  the  intertidal  zone  at  all  seasons. 


STARFISH  FEEDING  AND  REPRODUCTION  141 

However,  Pisaster  feeds  most  heavily  in  the  summer ;  I  will  offer  three  hypotheses 
to  explain  why. 

A.  Physiological  specialisation.     It  could  be  argued  that  Pisaster's  metabolism 
functions  most  efficiently  by  limiting  the  processes  that  occur  within  it  at  any  given 
time.     It  may  be  better  to  either  feed,  and  process  the  components  of  the  food,  or, 
assemble  these  components  into  gametes.     Perhaps  the  two  processes  are  in  some 
way  mutually  inhibitory.     However,  no  other  organisms,  including  starfish,  have 
been  shown  to  profit  from  the  above  mechanism,  and  some  sea-stars  do,  in  fact, 
carry  on  both  processes  simultaneously.     Moreover,  Figure  3  shows  an  increase 
in  feeding  before  spawning. 

B.  Subtle  environmental  cJnuu/cs.     Small  changes  in  a  number  of  environmental 
factors  could  adversely  affect  the  efficiency  of  winter  feeding.     As  a  temperate  sea- 
star,  Pisaster  must  be  able  to  withstand  relatively  high  temperatures,   15-20°   C., 
and,  as  expected,  does  show  a  lowered  metabolic  rate  with  lowered  temperatures 
(Paine,  personal  communication).     Thus,  although  the  annual  temperature  range 
of  sea  water  is  not  very  drastic    (6-13°    C.   at   Lonesome   Cove;    11-15°    C.   at 
Monterey,  California),  the  seasonally  slower  digestion  and  locomotion  may  prevent 
Pisaster  from  effectively  hunting  in  the  intertidal  zone  during  the  limited  period  of  a 
high   tide.     In   addition,   Pisaster  left   exposed   by   the   ebbing   tide   would   more 
likely   be   subjected   to   storms   and   freezing   conditions   in   the   winter.     Pisaster 
seems  resistant  to  heat;  Feder  (1956)  reports  they  can  withstand  exposure  to  the 
summer  sun  for  3-6  hours  and  still  appear  "healthy,  turgid,  and  moist."     The 
higher  summer  feeding  incidence  would  then  result  from  Pisaster  foraging  higher 
in  the  intertidal,  and  hence  encountering  recently-set  Balamis  and  M\tihis.     After 
the  consumption   of  these,   and   the   seasonal   onset   of   less   favorable   conditions, 
Pisaster  migrates  knver  into  the  intertidal  zone.    Quantitatively  less  food,  and  tem- 
perature-inhibited locomotion,  then  produce  the  seasonal  low  in  feeding  intensity. 

C.  Ann    sice    limitation.     Pisaster    is    a    very    hard-bodied    starfish ;    as    an 
inhabitant  of  an  area  regularly  exposed  to  violent  wave  action  and  desiccation,  the 
evolution  of  this  body  strength  is  to  be  expected.     Pisaster  is  too  large  to  protect 
itself  under  rocks  as,  for  example,  Leptasterias,  a  smaller  common  intertidal  starfish, 
usually  does.     This  rigidity  implies  an  approximately  constant  volume. 

Several  arguments  suggest  that  a  medium  size  is  best  for  Pisaster.  Growth 
studies  (Quayle,  1954;  Feder,  1956;  Mauzey,  unpublished  data)  suggest  rapid 
growth  up  to  reproductive  maturity  at  about  150  grams,  slower  thereafter  until 
about  400  grams,  and  very  little  or  none  at  larger  sizes.  Large-sized  starfish 
collected  from  Lonesome  Cove  do  not  seem  to  have  proportionately  larger  gonads, 
suggesting  that  there  is  only  small  gain  in  growing  above  400  grams,  and  that  this 
energy  might  better  be  put  into  each  year's  gamete  production.  Furthermore, 
the  volume,  and  therefore  the  metabolic  demands,  increase  as  the  cube  of  linear 
dimensions  ;  but  the  efficiency  of  hunting,  since  it  seems  to  depend  on  either  contact 
with  the  prey  or  chemoreception,  would  only  increase  with  the  surface  area,  the 
square  of  the  linear  dimension.  The  upper  size  limit  is  probably  not  set  by  the 
maximum  size  a  starfish  of  a  particular  age  can  attain,  but  rather  by  an  interaction 
with  the  size  and  abundance  of  prey  in  any  particular  area.  A  plentiful  supply  of 
large  prey  items  must  be  available  to  meet  the  demands  of  very  large  Pisaster. 

Since  some  coelomic  space  must  be  reserved  for  fluid,  necessary  for  flexibility. 


142  KARL  PERRY  MAUZEY 

whatever  arm  space  is  taken  up  by  pyloric  caeca  cannot  be  used  for  reproduction ; 
the  maximum  number  of  gametes  will  be  released  if  the  pyloric  caeca  are  smallest 
when  the  gonads  are  largest.  Observations  on  lipid  content  in  Pisaster's  organs 
further  suggest  that  space  may  be  a  limiting  factor.  Lipids  represent  about  twice 
the  energy  content  per  unit  of  weight  of  either  proteins  or  carbohydrates.  For 
ovaries,  testes  and  pyloric  caeca,  the  proportion  of  lipid  is  highest  (30%,  18%,  50%, 
respectively)  when  the  organ  involved  is  largest,  and  lowest  (5%,  2%,  30%) 
when  the  organ  is  smallest  (Greenfield  ct  al.,  1958). 

For  the  reasons  given  under  (B)  above,  feeding  might  be  more  efficient  in  the 
summer.  Pisaster  can  reproduce  most  effectively  if  it  does  not  feed  in  the  winter, 
except  minimally  to  replace  metabolic  use ;  calculations  of  the  metabolic  consumption 
of  Pisaster  (Paine,  personal  communication)  indicate  that  the  dry  weight  needed 
for  maintenance  at  winter  temperatures  is  about  equal  to  that  ingested  by  Pisaster 
in  January  and  February  (about  3  grams  per  one  tidal  cycle  per  100  animals). 
According  to  Hypothesis  (C),  the  sum  of  the  gonad  and  hepatic  indices  should  be 
fairly  constant.  This  sum  is  plotted  with  respect  to  time  in  Figure  3.  An  approxi- 
mately constant  level  is  maintained  from  December  through  May.  There  is  an 
abrupt  drop  in  June,  associated  with  spawning,  and  an  eventual  recovery  to  satura- 
tion level  from  July  through  November.  This  cycle  is  inverse  to  the  feeding  curve. 
The  sum  appears  to  be  relatively  constant  at  13%  to  17%,  except  upon  spawning, 
before  feeding  has  had  time  to  build  up  the  pyloric  caeca  again.  Given  a  restricted 
structural  framework,  and  the  great  advantage  of  a  spring  gamete  release,  the 
inverse  gonad-pyloric  caecum  size  is  to  be  expected  in  Pisaster.  Selection  must 
favor  the  greater  number  of  gametes  produced  with  this  strategy. 


My  study  has  benefited  from  the  patience  and  stimulating  discussion  of  Drs. 
Gordon  H.  Orians  and  Robert  T.  Paine.  Dr.  Robert  Fernald  has  been  very  helpful 
in  making  available  the  facilities  of  the  Friday  Harbor  Laboratories  of  the  Univer- 
sity of  Washington.  Financial  support  was  provided  in  part  by  an  NSF  Marine 
Sciences  Training  Grant  to  the  Friday  Harbor  Laboratories. 

Mr.  and  Mrs.  Roy  Durhack,  the  owners  of  the  Lonesome  Cove  Resort,  have 
been  most  gracious  in  allowing  me  to  use  their  property,  and  very  understanding  of 
the  requirements  of  my  research. 

SUMMARY 

1.  Pisaster  ochracens  shows  a  definite  seasonal  feeding  periodicity,  in  terms  of 
per  cent  of  the  population  feeding  at  one  time,  dry  weight  ingested,  and  in  composi- 
tion of  ingested  prey.     Less  than  5%  are  feeding  in  January  and  February;  60% 
to  80%  in  July  and  August.     The  dry  weight  ingested  varies  from  about  3  grams 
per  tidal  cycle  per  100  animals  in  the  winter  to  about  30  grams  in  the  summer 
months.     Chitons  are  the  principal  winter  prey,  while  barnacles  and  limpets  are 
fed  on  most  often  in  the  summer. 

2.  Cyclic  changes  in  gonad  and  pyloric  caeca  size  and  histological  appearance 
characterize  this  species.     The  gonads  are  smallest  in  the  fall,  and  grow  during  the 
winter  to  a  maximum  in  the  late  spring,  when  spawning  occurs.     The  pyloric  caeca 


STARFISH  FEEDING  AND  REPRODUCTION  143 

size-changes  are  approximately  inverse  to  those  of  the  gonads.  Seasonal  histological 
changes  of  the  oocytes,  and  storage  granules  in  the  pyloric  caeca,  are  correlated 
with  the  gross  organ  patterns. 

3.  Two  factors  are  suggested  as  explanations  for  these  cyclic  phenomena, 
(a)  More  favorable  summer  feeding  for  both  the  adult  and  larval  Pisaster  may  have 
led  to  evolution  of  a  storage  function  for  the  pyloric  caeca ;  nutrients  could  then  be 
transferred  to  the  gonads  in  the  winter,  (b)  It  would  seem  evolutionarily  advan- 
tageous to  fill  more  of  the  limited  space  available  in  the  arms  with  gonads  than 
with  pyloric  caeca  in  the  spring,  at  the  time  of  spawning. 

LITERATURE  CITED 

ANDERSON,   J.    M.,    1953.     Structure   and   function    in    the   pyloric   caeca   of   Asterias   forbest. 

Biol.  Bull,  105:  47-61. 
ANDERSON,  J.  M.,  1960.     Histological  studies  on  the  digestive  system  of  a  starfish,  Henricia, 

with  notes  on  Tiedemann's  pouches  in  starfishes.     Biol.  Bull.,  119:  371-398. 
BOOLOOTIAN,  R.  A.,  A.  FARMANFARMAIAN  AND  A.  C.  GIESE,  1962.      On  the  reproductive  cycle 

and  breeding  habits  of  two  Western  species  of  Haliotis.    Biol.  Bull.,  122:  183-193. 
CHIA,  FU-SHIANG,  1964.     The  developmental  and  reproductive  biology  of  a  brooding  starfish, 

Lcptasterias  hcxactis   (Stimpson).     Doctoral  Dissertation,  University  of  Washington. 
COGNETTI,  G.,  AND  R.  DELAVAULT,   1960.     Rccherches  sur  la  sexualite  d'Echinaster  sepositus 

( fichinoderme  Asteride).     fitude   des   glandes   genitales   chez   les   animaux   des   cotes 

de  Livourne.     Cah.  Biol.  Mar.,  1:  421-432. 
DELAVAULT,    R.,    1961.     La    sexualite   chez   Echinaster   sepositus    Gray    du    Golfe    de    Naples. 

Pubbl.  Stas.  Zool.  Napoli,  32:  41-55. 
EDMONDSON,  W.  T.,   1965.     Reproductive  rate  of  planktonic   rotifers  as   related  to  food  and 

temperature   in   nature.     Ecol.  Monog.,   35:   61-111. 

FARMANFARMAIAN,  A.,  A.  C.  GIESE,  R.  A.  BOOLOOTIAN  AND  J.  BENNETT,  1958.     Annual  repro- 
ductive cycles  in  four  species  of  west  coast  starfishes.     /.  E.vp.  Zool.,  138:  355-367. 
FEDER,  H.  M.,  1956.     Natural  history  studies  on  the  starfish,  Pisaster  ochraccus  (Brandt,  1835) 

in  the  Monterey  Bay  area.     Doctoral  Dissertation,   Stanford  University. 
FEDER,  H.  M.,  1959.     The  food  of  the  starfish,  Pisaster  ochraccus,  along  the  California  coast. 

Ecology,  W:  721-724. 
FERGUSON,  J.  C.,  1964a.     Nutrient  transport  in  starfish.     I.     Properties  of  the  coelomic  fluid. 

Biol.  Bull..  126:  33-53. 
FERGUSON,  J.  C.,  1964b.     Nutrient  transport  in  starfish.     II.     Uptake  of  nutrients  by  isolated 

organs.     Biol.  Bull.,  126:  391-406. 

GIESE,  A.  C.,  1959.     Comparative  physiology :   Annual  reproductive  cycles  of  marine  inverte- 
brates.    Ann.  Rev.  Physiology,  21:  547-576. 

GREENFIELD,  L.,  A.  C.  GIESE,  A.  FARMANFARMAIAN  AND  R.  A.  BOOLOOTIAN,  1958.     Cyclic  bio- 
chemical changes  in  several  echinoderms.    J.  Exp.  Zool.,  139:  507-524. 
KING,  C.,  1965.     Food,  age,  and  the  dynamics  of  a  laboratory  population  of  rotifers.     Doctoral 

Dissertation,  University  of  Washington. 

LACK,  DAVID,  1954.     The  Natural  Regulation  of  Animal  Numbers.     Oxford :  Clarendon  Press. 
LAWRENCE,  A.  L.,  J.  M.  LAWRENCE  AND  A.  C.  GIESE,  1965.     Cyclic  variations  in  the  digestive 

gland  and  glandular  oviduct  of  chitons  (Mollusca).     Science,  147:  508-510. 
McMANUs,   J.   F.   A.,  AND  ROBERT   W.   MAWRY,    1960.     Staining   Methods :    Histological   and 

Histochemical.     New  York :  Paul  B.  Hoeber  and  Co. 
MARSHALL,  S.  M.,  AND  A.  P.  ORR,  1955.     The  biology  of  a  marine  copepod,  Calanus  finmarchi- 

cus  (Gunnerus).     Edinburgh:  Oliver  and  Boyd  Ltd. 
MAUZEY,  K.  P.,  1963.     The  feeding  of  the  sea  star,  Pisaster  ochraceus,  near  Friday  Harbor, 

Washington.     Bull.  Ecol.  Soc.  Amcr.,  44:  47. 
PAINE,   R.   T.,    1963.     Trophic   relationships   of  8   sympatric   predatory   gastropods.    Ecology, 

44:63-73. 
PAINE,  R.  T.,  1966.     Food  web  complexity  and  species  diversity.    Amer.  Nat.,  100:  65-75. 


144  KARL  PERRY  MAUZEY 

PANTIN",   C.    F.   A.,    1946.     Notes    on   Microscopical    Techniques   for   Zoologists.     Cambridge : 

Cambridge  University  Press. 
PEARSE,  J.  S.,  1965.     Reproductive  periodicities  in  several  contrasting  populations  of  Odontaster 

validus   Koehler,   a   common   Antarctic   asteroid.     Biology   of   the    Antarctic    Seas    II, 

Anarctic  Research  Series,  5,  39-85. 
QUAYLE,  D.  B.,   1954.     Growth  of  the  purple  seastar.     British  Columbia  Dcpt.  Fish.,   Oyster 

Bull,,  5:  11-13. 
RICHMAN,   S.,   1958.     The  transformation  of  energy  by  Daphnia  pulcx.    Ecol.   Monogr.,  28: 

273-291. 
RICKETTS,  E.  F.,  AND  J.  CALVIN,  1952.      Between  Pacific  Tides.     Third  Ed.,  rev.,  J.  Hedgpeth. 

Stanford  :  Stanford  University  Press. 
STEPHENSON,  T.  A.,  AND  A.  STEPHENSON,  1961.     Life  between  tide  marks  in  North  America. 

IV  A.  Vancouver  Island,  I.  J.  Ecol.,  49:  1-29. 


SEQUELAE    OF    THE    LD/50    X-RAY    EXPOSURE    OF    THE 
PRE-IMPLANTATION  MOUSE  EMBRYO :  DAYS  0.0  TO  5.0  1 

R.  RUGH,  L.  DUHAMEL,  C.  SOMOGYI,  A.  CHANDLER,  W.  R.  COOPER, 

R.  SMITH  AND  G.  STANFORD 

Radiological  Research  Laboratory,  College  of  Physicians  and  Surgeons, 
Columbia  University,  New  York,  N.  Y.  10032 

In  a  recent  study  by  Rugh  and  Wohlfromm  in  1963  it  was  found  that  it  took 
varying  exposures  of  x-rays  to  kill  in  utero  half  of  the  mouse  embryos  of  different 
ages,  and  in  a  still  further  study  by  the  same  investigators  in  1965,  pre-natal 
exposures  and  post-natal  mortality  data  were  presented.  It  was  impossible  to 
establish  the  LD/50/30  of  x-rays  for  the  mouse  embryos  of  the  first  5  days  of 
gestation  (since  they  were  either  killed  in  utero  or  appeared  to  be  unaffected).  It 
was  therefore  decided  to  use  the  LD/50  dose  for  the  embryos  in  utero  and  study 
the  sequelae  exhibited  by  their  survivors.  In  other  words,  doses  from  100  r  to 
350  r  were  used  which  killed  approximately  half  of  the  early  mouse  embryos  in 
utero,  and  those  which  survived  this  radiation  insult  were  examined  during  their 
lifetime  for  evidence  of  permanent  but  tolerable  damage. 

In  a  previous  study  by  Rugh,  Duhamel,  Chandler  and  Varma  in  1964  it  had  been 
shown  that  when  the  mouse  embryos  were  exposed  at  the  various  gestation  ages  to 
the  uniform  dose  of  100  r  x-rays,  there  was  variable  response  with  respect  to 
cataractogenesis.  The  variations  in  response  were  related  to  the  gestation  age  at 
exposure.  It  was  found  that  the  highest  incidence  of  cataract  development,  at  18 
months  of  age,  occurred  when  the  mice  were  x-rayed  at  the  time  of  fertilization 
or  0.0  days.  Such  mice  developed  97%  cataracts  while  the  parallel  controls  showed 
17%  for  the  females  and  13%  for  the  males.  The  difference  between  those  x-rayed 
and  the  controls  represented  at  least  80%,  which  could  be  considered  the  incidence 
of  radiation-induced  cataracts.  There  was  also  a  rather  high  incidence  of  cataracts 
among  those  x-irradiated  during  the  next  several  days  of  gestation,  so  that  it  seemed 
in  order  to  investigate  this  matter  further. 

This  study  includes  effects  of  the  LD/50  x-ray  exposure  of  early  mouse  embryos 
in  terms  of  litter  size ;  sex  ratios  at  birth  and  at  30  days ;  monthly  weight  variations ; 
life  span,  blood,  and  skeletal  changes  and  the  etiology  of  cataracts. 

MATERIALS  AND  METHOD 

White  female  mice  of  the  CF1  strain  were  put  through  a  single  pregnancy,  using 
males  of  the  same  strain  for  mating,  prior  to  their  use  in  this  experiment.  For 
the  experimental  pregnancies  the  females  were  time-mated  for  two  hours  (8-10  AM) 
and  those  with  vaginal  plugs  were  segregated  and  marked  as  to  the  time  of 

1  Based  on  work  performed  under  Contract  AT- (30-1) -2740  for  the  Atomic  Energy  Com- 
mission and  aided  in  part  by  Grants  RH-81  and  RH-97  from  the  Division  of  Radiological 
Health,  Bureau  of  State  Services,  U.  S.  Public  Health  Service. 

145 


146 


RUGH,  ET  AL. 


conception.  Some  were  x-rayed  immediately  (0.0  time)  and  others  on  the  various 
days  from  0.0  to  5.0  at  which  time  implantation  is  in  progress.  Implantation 
actually  hegins  at  4 i days  hut  is  a  continuous  process  for  several  days  prior  to 
placenta  formation.  The  dose  of  x-rays  varied  with  the  gestation  age,  heing 
based  upon  a  prior  study  ( Rugh  and  Wohlfromm,  1963)  in  which  the  dose  which 
would  kill  half  of  the  enihryos  was  determined.  These  doses  are  given  in  Tahle  I 
in  connection  with  litter  size  and  sex  ratios. 

Upon  delivery  the  mice  were  counted,  sexed,  anomalies  recorded,  and  all  were 
given  to  foster  mothers  who  had  not  been  x-rayed  but  who  had  had  simultaneous 
litters.  These  foster  mothers  therefore  provided  normal  post-natal  care  and 
nutrition  until  the  time  of  weaning. 

Those  mice  surviving  at  one  month  constituted  the  initial  group  for  the  selection 
of  mice  for  this  study.  One  hundred  controls  and  50  experimentals  (half  males  and 


TABLE  I 

This  table  gives  the  results  of  exposing  early  mouse  embryos  to  the  previously-determined  LD/50  dose  of 

x-rays,  measured  hi  terms  of  average  Utter  size,  sex  ratios,  and  the  survival  of  the  respective  sexes 

during  their  first  30  days  of  life.     It  demonstrates  a  drastic  reduction  in  initial  litter  size, 

and  (with  the  exception  of  gestation  day  3  exposed  to  140  r)  a  manifold  increase  in 

lethality  during  the  first  30  post-natal  days 


Offspring  at  birth 

Percentage  lost  in  30  days 

Gestation  age 

LD/50 

x-rays 

Litter  size 
average 

Males 

Females 

Males 

Females 

Total 

Controls 

00 

10.3 

66 

78 

3.3% 

3.8% 

3.5% 

0.0  gest.  days 

100  r 

4.6 

41 

37 

17.1 

13.5 

15.4 

0.5  gest.  days 

275  r 

6.7 

45 

48 

11.1 

12.5 

11.8 

1.0  gest.  days 

350  r 

7.6 

56 

50 

19.6 

24.0 

21.7 

2.0  gest.  days 

125  r 

6.1 

44 

42 

11.5 

9.5 

10.4 

3.0  gest.  days 

140  r 

5.9 

40 

55 

2.5 

3.6 

3.1 

4.0  gest.  days 

330  r 

6.5 

48 

73 

37.5 

13.7 

23.1 

5.0  gest.  days 

350  r 

8.1 

70 

69 

20.0 

14.5 

17.9 

Totals  (x-rayed) 

344 

374 

17.0% 

13.0% 

14.8% 

half  females)  were  selected  at  random  from  those  x-irradiated  on  each  gestation 
day.  Each  mouse  was  earmarked  for  permanent  identification  and  was  examined 
and  weighed  each  month  through  31  months,  or  until  none  was  left.  At  2,  6,  9,  12, 
18  and  24  months  the  eyes  of  every  mouse  in  the  series  (2176  eyes)  were  examined 
by  practicing  ophthalmologists  of  this  institution.  The  slit  lamp  was  used  to 
determine  whether  there  were  corneal  opacities  or  incipient  nuclear  or  cortical 
cataracts  in  the  process  of  development.  The  pupils  of  the  eyes  were  dilated  at 
least  one-half  hour  prior  to  the  examination  by  a  drop  of  2.5%  isopto-homatropine, 
used  as  a  mydriatic.  Thus,  with  each  mouse  identified  by  number  and  the  condition 
of  each  eye  recorded,  it  was  possible  to  follow  the  onset  and  the  development  of 
cataracts  in  this  study. 

The  x-ray  facilities  consisted  of  parallel  tubes  in  cross  fire  spaced  at  72  cm. 
each  from  the  center  of  the  gravid  uterus.     The  machine  was  run  at  184  KVP, 


X-IRRADIATION  OF  EARLY  MOUSE  EMBRYO 


147 


30  MA,  with  filters  of  0.28  Cu  and  0.50  Al,  and  having  an  HVL  of  0.6  mm.  Cu. 
The  dose  rate  was  50  r/minule.  The  absorption  from  the  plastic  container  and 
the  scattering  of  radiations  from  the  bodies  of  the  mice  balanced  out  so  that  the 
estimated  dose  absorbed  by  the  embryos  was  very  close  to  the  air  dose  calculated  at 
the  position  of  the  embryos  described  above. 

EXPERIMENTAL  DATA 

The  litter  size  at  birth  gives  a  fair  indication  as  to  the  survival  of  embryos  ex- 
posed to  various  doses  and  the  several  gestational  ages.    However,  an  accurate  lethal 


___  control 
-o-a_oDOdoys  ot  lOOr 
"  275r 

"    350r 


o       i       B       3      10  12      13     14      K>      15     17      IH 


29    30      31 


SURVIVAL  OF  MALE  MICE  :  LD/50  X-RAYS  FROM  0-5  DAYS  POST- FERTILIZATION 

FIGURE  1.  The  dose  of  x-rays  delivered  to  male  mice  on  days  designated  varied  because 
of  the  previous  determination  of  the  lethal  dose  to  approximatly  half  of  such  embryos.  Never- 
theless, the  survival  curve  over  the  31 -month  period  did  not  vary  much  with  the  different 
gestational  age  exposures,  although  most  were  depressed  slightly  below  the  solid  control  line 
of  survival.  The  most  sensitive  gestational  age  was  0.5  day  after  insemination,  at  which  time 
early  embryos  received  275  r  x-rays. 

x-ray  dose  to  half  of  the  embryos  (LD/50)  for  pre-implantation  stages  is  rather 
difficult  to  establish  without  determining  the  number  of  viable  eggs  fertilized.  This 
cannot  be  done  without  sacrifice  of  the  animal.  The  average  implantation  number 
for  this  strain  of  mice  was  found  to  be  between  11  and  12. 

Among  those  mice  which  came  to  term  there  was  a  slightly  higher  number  of 
females  than  of  males,  and  during  the  first  30  days  of  their  lives  \7%  of  the  males 
and  13%  of  the  females  died.  In  all  cases  except  those  x-rayed  at  3.0  days  to  140  r, 


148 


RUGH,  ET  AL. 


deaths  during  these  first  30  days  of  life  far  exceeded  the  record  for  the  controls, 
which  was  3.5%.  Since  this  study  was  based  upon  x-rayed  mice  which  survived 
embryonic  and  fetal  life,  and  also  the  first  30  days  of  post-natal  life,  and  were 
studied  throughout  their  life  span,  the  data  for  weight,  skeletal,  blood,  and  cataract 
changes  relate  only  to  the  hardiest  of  the  mice  exposed  in  utero.  It  must  therefore 
be  presumed  that  death  of  many  mice  deprived  us  of  further  statistical  data  relating 
to  these  physical  variables. 

The  survival  of  mice  selected  at  one  month  of  age  is  shown  for  males  and  females 
separately  in  Figures  1  and  2  during  the  succeeding  30  months.  Note  the  LD/50 
exposures  for  the  various  gestation  days  which  kill  half  of  the  embryos  in  utero. 
These  figures  (1  and  2)  suggest  that  there  is  little  permanent  damage,  in  terms  of 
life-shortening,  when  the  experimental  mice  are  compared  with  the  controls. 
The  controls  are  shown  in  heavy  solid  lines  and  those  of  the  various  gestation  ages 
receiving  different  exposures  are  shown  in  the  other  curves.  The  experimental 
males  did  show  a  slight  reduction  in  survival  value,  while  the  difference  between 
the  sexes  was  not  at  all  pronounced  in  the  controls.  For  the  females  (Fig.  2)  the 
control  curve  cuts  through  the  middle  of  all  of  the  other  experimental  curves, 
indicating  no  effect.  Thus,  it  is  evident  that  those  early  mouse  embryos  which 


14      15      16      17      18 
MONTHS 


19     20 ~      22 23     ~     25     26     27~28  ' 


29    30     31 

SURVIVAL  OF  FEMALE  MICE: LD/50  X-RAYS  FROM  0~5  DAYS   POST-FERTILIZATION 

FIGURE  2.  The  dose  of  x-rays  delivered  to  female  mice  on  gestation  days  designated 
varied  with  the  previously  established  lethal  dose  to  half  of  the  embryos.  Here  again  the 
most  radiosensitive  gestational  age,  with  respect  to  survival,  was  the  embryo  exposed  at  0.5  day 
after  insemination,  with  275  r.  However,  the  deviation  from  the  solid  control  line  was  not 
statistically  significant  for  either  male  or  female  mouse.  In  other  words,  those  mice  which 
survived  x-irradiation  in  utero  tended  to  survive  as  well  as  did  the  controls. 


X-IRRADIATION  OF  EARLY  MOUSE  EMBRYO 


149 


TABLE  II 

Average  weights  in  grams  of  mice  x-rayed  in  ulero  during  early  development 


Day  x-rayed 
LD/50 

Average  weight  in  grams 

2  months 

12  months 

18  months 

24  months 

males 

29.8 

37.2 

36.1 

33.8 

0.0  days 
females 

23.9 

28.3 

31.2 

30.8 

males 

29.1 

35.2 

33.8 

0 

0.5  days 
females 

23.3 

28.6 

29.2 

26.4 

males 

29.4 

37.5 

37.9 

34.8 

1.0  days 
females 

22.7 

30.7 

32.8 

29.8 

males 

30.8 

40.2 

37.1 

33.4 

2.0  days 
females 

23.2 

29.1 

32.7 

26.0 

males 

29.5 

38.6 

36.4 

34.7 

3.0  days 
females 

24.3 

29.2 

32.2 

29.9 

males 

29.7 

36.6 

35.8 

34.5 

4.0  days 
females 

23.3 

30.2 

33.6 

33.7 

males 

29.0 

35.1 

32.7 

30.4 

5.0  days 
females 

23.5 

28.3 

29.2 

25.9 

males 

27.8 

36.2 

36.3 

34.5 

Controls 
females 

22.4 

28.6 

30.7 

29.8 

tolerate  the  x-ray  exposures  used,  and  survive  at  birth,  and  the  first  month  of  life 
thereafter,  can  be  expected  to  survive  almost  as  well  as  do  the  parallel  controls. 

The  monthly  weight  records  for  each  mouse  are  reduced  to  four  periods  (2,  12, 
18  and  24  months)  in  Table  II  below.  It  can  be  seen  that  without  exception, 
among  the  controls  or  the  x-irradiated,  the  females  of  the  same  age  were  lighter 
in  body  weight  than  were  the  parallel  males.  It  is  also  obvious  that  there  was  little, 
if  any,  statistical  difference  between  the  x-irradiated  and  the  control  mice  by  two 
months  of  age.  The  average  weight  for  the  first  month  of  age  is  generally  higher 
for  the  experimental  mice  than  for  the  controls  because  of  the  reduced  litter  size 
of  the  experimentals  and  consequently  more  growing  space  for  the  remaining  mice. 
Thus,  again  it  seems  evident  that  x-irradiation  of  the  pre-implantation  mouse 
embryo,  if  it  survives  the  first  post-natal  month,  will  allow  it  to  be  as  heavy  as 
the  controls. 


150 


RUGH,  ET  AL. 


The  mice  chosen  at  one  month  of  age  for  the  long  term  study  were  radiographed 
at  two  months  in  order  to  determine  whether  there  was  any  evidence  of  permanent 
skeletal  effects.  Fifty  male  and  50  female  controls  were  simultaneously  examined, 
in  the  same  manner  as  the  various  irradiated  groups  (each  comprised  of  25  males 
and  25  females).  By  direct  comparison  of  such  averages  any  contrast  with  the 
controls  is  obvious. 

The  mice  were  not  anesthetized  but  were  fastened  to  a  plastic  board  by  means  of 
adhesive  tape,  and  were  radiographed  at  40  volts,  10  MA,  at  20  inches  distance  from 

TABLE  III 

Skeletal  measurements  by  radiography  at  2  months  of  age  (in  cm.} 


Gest. 
day 

r 

Sex 

Tot.  # 

Skull 

Spine 

Humerus 

Ulna 

Femur 

Tibia 

Lat. 

A.  P. 

Controls 

0 

M 
F 

50 

50 

1.05 
1.04 

1.44 
1.42 

6.11 

5.90 

1.20 
1.15 

1.39 
1.35 

1.47 
1.46 

1.68 
1.67 

0.0 

100 

M 
F 

25 
25 

1.08 
1.09 

1.50 
1.46 

6.45 
6.08 

1.23 
1.19 

1.42 
1.38 

1.52 
1.49 

1.72 
1.71 

0.5 

275 

M 
F 

25 
25 

1.08 
1.07 

1.47 
1.43 

6.21 
6.01 

1.21 
1.16 

1.40 
1.35 

1.51 
1.48 

1.70 
1.66 

1.0 

350 

M 
F 

25 
24 

1.07 
1.05 

1.46 
1.44 

6.25 
5.95 

1.20 
1.14 

1.38 
1.35 

1.50 
1.46 

1.69 
1.68 

2.0 

125 

M 

F 

25 
24 

1.10 

1.08 

1.50 
1.46 

6.22 
6.05 

1.22 
1.18 

1.42 
1.37 

1.53 
1.49 

1.74 
1.69 

3.0 

140 

M 
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25 
25 

1.07 
1.06 

1.47 
1.44 

6.49 
6.28 

1.26 
1.20 

1.43 
1.39 

1.53 
1.52 

1.74 
1.71 

4.0 

350 

M 

F 

23 
25 

1.05 
1.04 

1.46 
1.46 

6.35 
6.18 

1.23 
1.20 

1.40 
1.40 

1.51 
1.53 

1.71 

1.73 

5.0 

350 

M 
F 

25 
24 

1.07 
1.06 

1.47 
1.45 

6.36 
6.23 

1.23 
1.20 

1.42 
1.39 

1.51 
1.52 

1.72 
1.74 

the  x-ray  source,  for  two  seconds,  over  sheet  film.  This  amount  of  x-irradiation 
was  regarded  as  inconsequential  at  two  months  of  age.  The  radiographs  were  of 
sufficient  clarity  as  to  allow  exact  measurements  of  the  skull  (lateral  and  AP)  ; 
spine,  humerus,  ulna,  femur  and  tibia.  Since  the  measurements  were  taken  from 
a  minimum  of  23  x-irradiated  mice,  and  50  controls  of  each  sex,  giving  a  total  of 
345  experimental  and  100  control  radiographs,  the  data  have  statistical  validity. 
Table  III  below  gives  the  average  measurement  in  centimeters  for  both  sexes  of 
each  group  of  experimental  and  control  animals. 

It  can  be  seen  that  the  early  x-irradiation  of  the  mouse  embryo  had  no  adverse 
effect  on  the  skeletal  growth  of  the  survivors,  and  in  fact  there  appeared  to  be  a 
tendency  of  those  x-irradiated  to  be  slightly  larger  than  the  controls.  Again,  it 


X-IRRADIATION  OF  EARLY  MOUSE  EMBRYO  151 

must  be  pointed  out  that  x-irradiated  mice  came  from  depleted  litters  so  that  the 
survivors  had  more  room  in  which  to  grow,  hence  at  birth  and  at  one  or  two 
months  they  would  be  expected  to  be  heavier  and  have  larger  skeletal  parts  than 
did  the  parallel  controls.  It  was  found  by  Rugh,  Duhamel,  Osborne  and  Varma 
in  1964  that  mice  x-rayed  to  100  r  at  12  to  14  days  gestation  showed  serious 
defects  in  skeletal  growth,  and  were  all  somewhat  stunted,  but  these  pre-implanta- 
tion  embryos,  x-rayed  from  100  r  to  350  r,  were  unaffected  with  respect  to  the 
ultimate  skeletal  size. 

Complete  blood  counts  were  made  of  both  the  control  and  x-rayed  mice  at 
2,  6,  9,  12,  18  and  24  months  of  age.  This  included  the  usual  determination  of 
hemoglobin,  white  and  red  cell  counts,  platelets,  and  differentials  based  on  100 
W.B.C.'s.  The  data  do  not  deviate  sufficiently  from  the  controls  to  be  presented  in 
detail ;  suffice  it  to  say  that  the  x-irradiated  mice  tended  to  have  slightly  higher  white 
cell  counts  and  slightly  lower  red  cell  counts  than  the  parallel  controls.  Whatever 
damage  may  have  been  produced  in  the  mice  from  x-irradiation  during  the  first 
five  days  of  gestation  was  rectified  by  the  time  the  survivors  were  two  months  of  age. 

As  all  mice  progressed  in  age  there  was  a  drop  in  the  hemoglobin  and  erythro- 
cyte  counts.  Similarly  a  general  trend  was  observed  in  the  decrease  of  the  number 
of  lymphocytes  and  an  increase  in  the  neutrophils  from  two  to  24  months  of  age. 
There  was  no  evidence  of  leukemia  in  any  of  the  mice  examined.  At  24  months 
four  cases  of  lymphocytosis  were  found,  one  of  which  was  a  control.  The  leukocyte 
counts  varied  from  45,000  to  163,000  in  these  mice,  of  which  an  average  of  89% 
were  lymphocytes,  all  of  a  mature  type.  No  further  histological  examination  was 
provided  for  so  that  the  exact  nature  of  the  lymphocytosis  could  not  be  determined. 
At  18  months  the  highest  leukocyte  count  was  21,800  with  no  comparable  lympho- 
cytosis being  noted. 

A  total  of  1098  examinations  of  mice  are  presented  for  cataracts  (2176  eyes) 
at  2,  12,  18  and  24  months  of  age.  Examinations  were  also  made  at  6,  9  and  29 
months  but  these  data  are  not  included  in  Table  IV.  Three  major  effects 
were  recorded :  corneal  opacities,  which  are  apparently  minor  abrasions  of  the 
cornea  (conjunctiva)  from  which  many  eyes  recover ;  nuclear  sclerosis,  which  is  a 
pre-cataract  condition ;  and  the  distinct  cataract.  The  corneal  opacities  seemed  to 
be  unrelated  to  the  onset  and  development  of  cataracts  while  nuclear  or  even 
cortical  sclerotic  conditions  of  the  lens  almost  always  led  to  cataracts.  Occasionally 
a  corneal  opacity  obscured  a  lens  in  such  a  manner  as  to  make  the  determination 
of  a  cataract  difficult. 

For  the  controls  there  were  50  males  and  50  females  at  the  beginning  of  the 
study,  when  the  mice  were  one  month  of  age.  For  each  of  the  groups  of  mice 
x-irradiated  at  the  various  gestation  days  there  were  selected  25  males  and  25 
females,  also  at  one  month  of  age.  These  original  numbers  dropped  steadily  after 
12  months  of  age  so  that  by  24  months  there  were  very  few  mice  alive,  either  x-rayed 
or  controls  (Figs.  1  and  2).  Thus  the  cataract  data  are  derived  as  percentage  data 
rather  than  actual  numbers,  and  such  percentages  become  less  significant  as  the 
total  number  decreases  (Table  IV). 

The  incidence  of  cataracts  at  18  months  for  the  controls  corresponds  very  well 
with  the  data  of  the  previous  study  by  Rugh,  Duhamel,  Chandler  and  Varma  in 
1964,  but  by  24  months  even  the  controls  showed  an  increasing  incidence  of 


152 


RUGH,  ET  AL. 


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X-IRRADIATION  OF  EARLY  MOUSE  EMBRYO  153 

cataracts  (males  39%  and  females  66%).  This  suggests  that  such  cataracts  are 
truly  senile  cataracts,  but  the  onset  in  this  strain  of  mice  appears  to  be  slower  than 
in  some  other  strains. 

It  appears  that  homozygous  mice  can  have  congenital  cataracts,  while  heterozy- 
gous mice  tend  to  have  normal  vision.  The  onset  of  cataractogenesis  in  different 
strains  may  differ  considerably.  It  is  of  interest  to  note  that  within  any  group  of 
similar  mice,  similarly  irradiated,  cataract  development  is  never  all-or-none;  there 
is  a  great  variation  in  response  (Go wen,  1962). 

Among  the  x-irradiated  mice  surviving  to  18  months,  in  almost  every  set  of 
data  it  is  obvious  that  those  x-rayed  had  a  higher  incidence  of  cataracts  than  did 
the  controls  at  the  same  age.  However,  when  the  incidence  of  cataracts  among  the 
few  survivors  at  24  months  is  determined,  the  range  was  from  0%  to  100%  among 
those  x-rayed,  as  compared  with  an  average  of  53%  for  the  controls.  It  must  be 
recalled  that  mortality  of  many  irradiated  mice  left  only  the  most  hardy  ones  to  be 
included  in  this  study. 

SUMMARY  AND  CONCLUSIONS 

1.  Either  x-rayed  or  control  male  mice  had  average  weights  in  excess  of  the 
females  at  a  comparable  age.     Pre-implantation  mouse  embryos,  subjected  to  x-rays 
and  surviving  for  24  months,  showed  no  gross  adverse  weight  effects  of  the  ex- 
posures.    In  some  instances  those  with  a  radiation  history  were  heavier,  probably 
because  they  came  from  depleted  litters  which  had  more  growing  space  within 
the  uteri. 

2.  Whole  blood  counts  indicated  that  mice  x-irradiated  in  the  pre-implantation 
stage  tended  to  have  slightly  higher  white  cell  counts  and  slightly  lower  red  cell 
counts  than  their  parallel  controls.     Otherwise  any  possible  hematological  damage 
appears  to  have  been  rectified. 

3.  There  were  no  permanent  skeletal  effects  on  mice  x-irradiated  in  utero  during 
the  pre-implantation  stages  of  0.0  to  5.0  days,  as  determined  by  radiographs  of  five 
selected  bones  and  two  skull  measurements  at  2  months  of  age. 

4.  Mice  x-rayed  at  fertilization  or  at  5  days  gestation  showed  almost  as  good 
survival  as  did  the  controls,  but  those  x-rayed  on  days   1,  2,   3   and  4  showed 
slightly  reduced  survivals. 

5.  Corneal  opacities  occur  frequently  in  these  mice.     Their  eyes  appear  to  be 
anesthetized  to  the  participate  material  in  the  bedding.     There  appeared  to  be  no 
direct  relationship   between  corneal   opacities   and   the  development   of  cataracts. 
Many  mice  with  corneal  opacities  ai:  two  months  recovered  normal  corneas  at  a 
later  date. 

6.  Mouse  cataracts  appear  to  arise  as  nuclear  or  cortical  sclerosis  of  the  lens, 
but  those  arising  in  the  nuclear  region  appear  to  be  in  the  majority.     The  ultimate 
cataract,  regardless  of  its  origin,  appeared  to  be  similar  in  its  involvement. 

7.  At  any  test  period  the  percentage  incidence  of  cataracts  among  the  survivors 
was  always  higher  among  those  x-rayed  in  utero  than  among  the  parallel  controls. 

8.  Variations  in  cataractogenesis  existed  between  males  and  females  similarly 
x-rayed,  as  had  also  been  shown  in  the  previous  study  with  the  uniform  exposure 
of  100  r  x-rays.     There  appeared  to  be  a  sex  differential  in  cataractogenesis  of  x-ray 
origin. 


154  RUGH,  ET  AL. 

9.  The  fact  that  cataracts   appeared   earlier   and   to   a  greater   extent   among 
the  x-irradiated  mice  than  among  the  controls  suggests  that  x-rays  may  hasten  the 
onset  of  the  usual  senile  cataracts. 

10.  There  appears  to  be  a  greater  incidence  of  bilateral  as  opposed  to  unilateral 
cataracts,  and  this  seems  to  be  particularly  true  for  the  females.     The  incidence  of 
cataracts  in  one  eye  leading  to  bilateral  cataracts  occurred  more  frequently  in  mice 
x-rayed  at  fertilization  and  in  females  x-rayed  at  1.0  and  5.0  days  gestation.     Thus, 
there  was  no  clear-cut  evidence  of  uni-  leading  to  bilateral  cataract  development 
except  possibly  among  some  potential  females.     The  precursors  of  the  two  eyes  of 
any  mouse  at  these  early  stages  presumably  received  the  same  degree  of  radiation 
insult. 

11.  Since  this  study  is  based  entirely  upon  x-irradiation  of  the  early  mouse 
embryo  from  fertilization  to  5  days  gestation,  and  since  it  has  been  demonstrated 
that  x-rays  during  this  period  do  in  fact  increase  the  incidence  of  cataracts,  it  must 
lie  presumed  that  the  damage  is  clone  to  the  precursors   of  the  lens   since  lens 
development  is  not  initiated  until  about   1 1   days  gestation.     It  is  suggested  that 
the  etiology  of  these  radiation-induced  cataracts  may  be  through  an  interference 
with  the  developmental  process,  originating  with  damage  to  chromosomes  insuffi- 
cient to  be  lethal. 

LITERATURE  CITED 

GOWEN,  J.  W.,  1962.     In:  Methodology  in  Human  Genetics  (W.  J.  Burdette,  ed.),  pp.  191-199. 

Holden-Day,  Inc.,  San  Francisco. 
RUGH,  R.,  AND  M.   WOHLFROMM,    1963.     Can   the  mammalian   embryo  be   killed   by   x-rays? 

/.  Exp.  Zool,  151:  227-244. 
RUGH,    R.,    AND    M.    WOHLFROMM,    1965.     Pre-natal    x-irradiation    and    post-natal    mortality. 

Radiation  Research,  26:  493-506. 
RUGH,  R.,  L.  DUHAMEL,   A.   CHANDLER  AND  A.   VARMA,   1964.     Cataract  development   after 

embryonic  and  fetal  x-irradiation.     Radiation  Research,  22:  519-534. 
RUGH,  R.,  L.  DUHAMEL,  A.  W.  OSBORNE  AND  A.  VARMA,  1964.     Persistent  stunting  following 

fetal  x-irradiation.    Amer.  J.  Anat.,  115:  185-198. 


THE  EFFECT  OF  HYPOPHYSECTOMY  ON  SODIUM  METABOLISM 
OF  THE  GILL  AND  KIDNEY  OF  FUNDULUS  KANSAE  1 

JON  G.  STANLEY  AND  W.  R.  FLEMING 

Zoology  Department,  University  of  Missouri,  Columbia,  Missouri  65202 

If  a  euryhaline  teleost  is  to  maintain  a  reasonably  constant  internal  environment 
when  in  fresh  water  or  in  sea  water,  the  regulatory  mechanisms  operating  in  one 
environment  must  be  capable  of  altered  function  when  the  animal  moves  into  the 
other  environment.  Thus,  the  gill  must  convert  from  a  site  of  ion  uptake  to  one 
of  ion  excretion,  and  the  kidney,  which  functions  primarily  in  excreting  excess 
water  in  dilute  environments,  must  reduce  its  function  to  a  minimum  in  the  other 
situation.  The  degree  and  rate  at  which  a  euryhaline  teleost  can  accomplish  such 
alterations  will  determine,  in  part,  how  rapidly  and  how  successfully  transfers 
from  one  environment  to  the  other  can  be  made. 

It  now  seems  well  established  that  euryhaline  teleosts  can  reduce  urine  flow 
markedly  in  sea  water  (Holmes,  1961;  Stanley  and  Fleming,  1964a,  1964b;  Shar- 
ratt  et  al.,  1964;  Fleming  and  Stanley,  1965),  and  that  the  reduction  is  due  in  part 
to  a  reduction  in  glomerular  filtration  rate  (Holmes  and  McBean,  1963;  Sharratt 
ct  al.,  1964;  Stanley  and  Fleming,  1964a;  Fleming  and  Stanley,  1965),  and  to  an 
increase  in  the  tubular  reabsorption  of  water  (Sharratt  et  al.,  1964;  Fleming  and 
Stanley,  1965).  Further,  rates  of  chloride  and  sodium  flux  have  been  shown  to 
increase  several  times  where  a  euryhaline  teleost  is  moved  from  fresh  water  to  sea 
water  (Mullins,  1950;  Motais,  1961;  Gordon,  1963;  Motais  and  Maetz,  1964, 
1965). 

A  few  reports  of  measurements  comparing  sodium  fluxes  across  the  gill  with 
renal  sodium  loss,  have  appeared  (Maetz,  1963;  Maetz  et  al.,  1964;  Bourquet  et  al., 
1964;  Motais  and  Maetz,  1965),  but  such  measurements  for  a  single  species  in  fresh 
water,  during  the  course  of  adjustment  to  sea  water,  and  after  several  days  in  sea 
water,  have  not,  to  our  knowledge,  been  reported. 

We  wish  here  to  report  the  results  of  such  studies,  and  to  describe  the  effects 
of  hypophysectomy. 

MATERIALS  AND  METHODS 

The  euryhaline  killifish,  Funduhis  kansae,  was  collected  from  a  salt  spring 
"Boonslick"  located  in  Howard  County,  Mo.  The  routine  handling  to  these 
animals,  the  preparation  of  sea  water  (1000  mOsm./kg.)  and  the  techniques  used 
for  hypophysectomy  have  been  described  elsewhere  (Fleming  et  al.,  1964;  Stanley 
and  Fleming,  19641),  1966;  Fleming  and  Stanley,  1965)  and  need  not  be  repeated 
here.  All  experiments  were  carried  out  at  19  ±  1°  C.,  and  only  females  which 

1  Supported  by  a  NSF  Cooperative  Fellowship  to  the  senior  author  and  by  NSF  Grant 
GB-2264. 

155 


156 


JON  G.  STANLEY  AND  W.  R.  FLEMING 


had  been  adapted  to  fresh  water  for  at  least  two  months  were  used.     The  fish 
selected  all  weighed  approximately  2  grams. 

Techniques  for  the  collection  and  sampling  of  urine  have  also  been  described  in 
detail  elsewhere  (Fleming  and  Stanley,  1965)  and  need  only  be  summarized  here. 
Urine  was  collected  in  a  calibrated  polyethylene  cannula  tied  into  the  urogenital 
papilla.  Urine  volumes  were  estimated  by  reading  directly  from  the  calibration 
marks  on  the  collection  cannula.  Figure  1  shows  one  of  10  separate  compartments 
in  an  apparatus  used  to  hold  the  fish  relatively  immobile  during  the  experiment. 
A  constant  flow  of  30  ml.  per  hour  through  each  24-ml.  compartment  was  main- 
tained by  using  a  metering  pump  to  remove  water  and  a  siphon  from  a  constant- 
level  reservoir  to  replace  the  water  removed.  Such  an  arrangement  serves  to 


To 
Collection   Reservoir 


From 
Aerator 


Siphon   From  Constant 
Level  Reservoir 


Catheter 


FIGURE  1.  Cross-section  through  one  of  the  units  used  to  study  the  sodium  metabolism  of 
F.  kansae.  Urine  sodium  is  collected  in  the  catheter ;  that  from  other  sites  is  carried  to  a 
collection  reservoir. 

separate  kidney  and  gill  excretion,  and  to  provide  a  steady  flow  of  water  through 
the  system,  thereby  reducing  the  possibility  that  any  isotope  excreted  by  the  gill 
would  be  recycled.  The.  water  entering  each  compartment  via  the  siphon  was 
aerated,  and  each  compartment  was  provided  with  a  separate  air  line  to  further 
insure  adequate  aeration  and  to  provide  mixing.  The  water  flowing  through  the 
chamber  was  collected  in  a  collection  reservoir. 

As  soon  as  a  cannula  had  been  secured,  each  fish  was  given  an  intraperitoneal 
injection  of  Na22  carried  in  fish  Ringer's.  Each  animal  received  4  microcuries  of 
isotope  carried  in  a  volume  of  7.5  microliters.  A  micrometer-driven  syringe  was 
used  to  control  the  volume  injected. 

At  desired  intervals,  samples  of  urine  and  of  the  fluid  bathing  the  gills  were 


GILL  AND  RENAL  FUNCTION  IN  FUNDULUS  157 

taken.  Urine  was  withdrawn  from  the  collection  cannula  by  carefully  threading 
a  length  of  polyethylene  tube  inside  the  collection  cannula  and  applying  gentle 
suction.  Samples  were  removed  every  three  hours  in  fresh  water  and  every 
six  hours  in  sea  water.  The  entire  quantity  of  urine  produced  for  each  time  period 
was  blown  into  three  milliliters  of  0.02%  Sterox  solution.  The  radioactivity  of 
this  sample  was  determined  by  the  use  of  a  deep  well  scintillation  counter.  The 
total  urine  sodium  was  then  determined  on  the  same  sample  by  flame  photometry 
and  the  specific  activity  of  each  sample  calculated. 

The  collection  reservoir  was  sampled,  the  volume  measured,  and  the  reservoir 
emptied  every  three  hours.  A  3-ml.  sample  was  counted  and  the  total  radioactivity 
lost  via  the  gill  over  the  three-hour  period  determined  by  multiplying  by  one-third 
the  volume  (in  milliliters)  pumped  through  the  chamber.  In  every  case,  the 
counting  error  was  kept  to  within  3%. 

As  mentioned  above,  renal  sodium  loss  was  measured  directly  with  flame 
photometry.  The  extra-renal  (gill)  sodium  loss  for  any  time  interval  was 
determined  by  the  equation : 

Urine  loss  X  Total  gill  counts 

Gill  loss  =  „  .  , — — 

Total  urine  counts 

The  use  of  this  equation  is  based  on  the  assumption  that  the  ratio:  sodium-22/ 
sodium-23,  is  identical  for  sodium  lost  via  the  kidney  and  via  the  gills. 

Three  separate  types  of  experiments  are  reported.  Experiments  la  and  Ib  were 
carried  out  using  fresh-water-adapted  animals  that  were  cannulated,  injected,  and 
placed  into  fresh  water.  Twelve  hours  later,  the  animals  were  switched  to  sea 
water.  Experiments  2a  and  2b  were  carried  out  entirely  in  sea  water,  using 
animals  that  had  been  placed  into  sea  water  8  days  previously.  In  experiments  la 
and  2a,  only  sham-operated  animals  were  used ;  both  sham-operated  and  hypophy- 
sectomized  animals  were  studied  in  experiments  Ib  and  2b.  In  both  sets  of 
experiments,  the  behavior  of  the  sham-operated  animals  was  similar,  and  the 
data  from  these  animals  were  combined.  A  total  of  9  controls  and  6  hypophy- 
sectomized  animals  were  studied  in  fresh  water  and  during  the  initial  course  of 
adjustment  to  sea  water.  Nine  controls  and  seven  hypophysectomized  animals 
were  examined  in  experiments  2a  and  2b. 

Experiment  3  compared  the  rate  of  sodium-22  uptake  of  sham-operated  and 
hypophysectomized  animals  held  in  fresh  water.  A  series  of  8  flasks  were  set  up, 
and  40  ml.  of  tap  water  containing  Na22  were  added  to  each  flask.  The  isotope 
solution  was  such  that  each  initial  sample  provided  approximately  10*  cpm.  Two 
fish  were  weighed  and  placed  into  each  flask.  Enough  additional  solution  was 
added  so  that  the  final  volume  was  exactly  15  times  the  weight  of  the  fish. 
Three-milliliter  samples  were  withdrawn  at  each  sampling  period,  counted,  and 
returned  to  the  flask. 

RESULTS 
Urine  excretion 

The  changes  in  urine  flow  measured  when  F.  kansae  was  transferred  from  a 
dilute  environment  into  sea  water  were  largely  similar  to  those  reported  in  an 
earlier  paper  (Stanley  and  Fleming,  1966)  ;  therefore,  detailed  data  will  not  be 


158 


JON  G.  STANLEY  AND  W.  R.  FLEMING 


given  here.  Immediately  prior  to  transfer,  the  controls  were  excreting  urine  at  a 
rate  of  330  ml./kg./day,  and  the  hypophysectomized  animals  at  a  rate  of  220 
ml. /kg. /day.  Both  groups  reduced  urine  flow  to  approximately  the  same  value, 
i.e.,  20  ml./kg./day,  within  a  few  hours  after  transfer  into  sea  water.  The  same 
levels  of  urine  excretion  were  measured  for  both  groups  after  an  8-day  adaptation 
period  to  the  saline  environment.  One  difference  was  noted  from  the  earlier 


0.7 


0.6 


O) 

O) 

0) 


to 
O 


0.5 


0.4 


I  0.3 

O 
CO 


CO 

S   0.2 

DC 


0.1 


Into 
sea  water 


10  15  20 

Hours    in  Apparatus 


25 


FIGURE  2.  Comparison  of  renal  sodium  loss  of  sham-operated  controls  and  hypophysecto- 
mized F.  kansae  held  in  fresh  water  and  during  the  initial  course  of  adjustment  to  sea  water. 
Data  show  the  mean  ±  S.E.  for  9  control  and  6  hypophysectomized  animals. 


GILL  AND  RENAL  FUNCTION  IN  FUNDULUS 


159 


16 


14 


12 


cr 
0) 


8 


10 


05 

<D      p. 
C£      D 
i 
00 


u 


Into 
sea  water/ 


10  15  2O 

Hours    in  Apparatus 


25 


3O 


FIGURE  3.  Comparison  of  extra-renal  sodium  loss  of  sham-operated  controls  and  hypo- 
physectomized  F.  kansae  in  fresh  water  and  during  the  initial  period  of  adjustment  to  sea  water. 
Data  show  the  mean  ±  S.E.  for  9  control  and  6  hypophysectomized  animals. 


160 


JON  G.  STANLEY  AND  W.  R.  FLEMING 


23 
22 
21 

20 

cr  <ig 
0 

~  18 
3  17 
.5  16 


u 


0) 


15 


03 


£     13 
LJ 


11 


Hyp'ed 


CD 
Q) 


0.1 


10  15  20 

Hours  in   Apparatus 


25 


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cn 

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0) 
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FIGURE  4.  Renal  and  extra-renal  sodium  loss  of  sham-operated  and  hypophysectomized 
F.  kansae  after  an  8-day  exposure  to  sea  water.  Data  show  the  mean  ±  S.E.  for  9  control  and 
7  hypophysectomized  animals. 


GILL  AND  RENAL  FUNCTION  IN  FUNDULUS 


161 


experiments,  i.e.,  the  hypophysectomized  animals  were  able  to  reduce  urine  flow  at 
essentially  the  same  rate  as  the  controls.  Thus,  hypophysectomy  affects  the  rate 
of  urine  excretion  of  F.  kansae  in  fresh  water,  but  not  in  sea  water. 

Renal  sodium  loss 

As  shown  in  Figure  2,  the  renal  sodium  metabolism  of  the  two  groups  differs 
markedly,  both  in  fresh  water  and  in  sea  water.  Thus,  the  mean  renal  sodium  loss 
of  the  control  groups  in  fresh  water  was  0.27  /teq./gm./hr.,  for  the  hypophysec- 
tomized animals  the  mean  figure  was  0.53  /xeq./gm./hr.  Renal  sodium  loss  fell 
to  a  low  figure,  0.04  /xeq./gm./hr.  for  both  groups  shortly  after  transfer  into  sea 
water  and  the  hypophysectomized  animals  remained  low  there-after.  The  control 
groups  showed  a  different  response  in  that  renal  sodium  loss  soon  increased,  and 
by  20  hours  after  transfer  had  reached  a  mean  value  of  0.48  //eq./gm./hr. 

Extra-renal  sodium  loss 

Contrary  to  the  renal  picture,  hypophysectomy  did  not  affect  the  extra-renal 
sodium  loss  of  fish  held  in  fresh  water  ( Fig.  3 ) .  A  marked  difference  was  clearly 
evident,  however,  when  the  two  groups  were  transferred  into  sea  water.  As 
shown  in  Figure  3,  both  groups  showed  a  marked  stimulation  of  sodium  outflux 


10  15  20  25 

Time    in    Hours 


30 


35 


40 


45 


FIGURE  5.  Sodium  uptake  of  sham-operated  and  hypophysectomized  F.  kansae  held  in 
fresh  water.  Data  show  the  counts  per  minute/ml,  absorbed  from  a  medium  containing  10* 
cpm/ml.  and  40  meq.  Na/gm.  of  fish.  Each  point  shows  the  mean  ±  S.E.  of  four  pairs  of  fish. 


162  JON  G.  STANLEY  AND  W.  R.  FLEMING 

after  transfer,  but  the  response  shown  by   the  hypophysectomized   animals   was 
considerably  less  than  that  shown  by  their  controls. 

Sodium  loss  ajter  8  days  in  sea  water 

Figure  4  compares  both  renal  and  extra-renal  sodium  loss  of  control  and 
hypophysectomized  animals  after  an  8-day  adjustment  period  to  sea  water.  Com- 
parisons of  Figures  2,  3,  and  4  show  that  after  8  days  in  sea  water,  both  groups 
of  fish  had  the  same  low  rates  of  renal  sodium  loss.  On  the  other  hand,  extra-renal 
sodium  loss  had  increased  for  both  groups,  with  the  hypophysectomized  animals 
still  showing  somewhat  lower  values. 

Sodium  influx 

Sodium  influx  is  slightly  higher  for  hypophysectomized  animals  than  for  their 
controls  (Fig.  5).  The  disappearance  of  radioactivity  from  the  environment  is 
rapid  at  first  and  then  levels  off,  presumably  because  of  recycling  of  isotope. 
Influx  in  the  controls  for  the  first  5|  hours  was  estimated  by  multiplying  the 
fraction  of  radioactivity  absorbed  (1/6.5)  by  the  sodium  content  of  the  medium 
(40  ju,eq./gm.  of  fish)  to  give  a  value  of  1.1  /xeq./gm./hr.  A  similar  estimate  in 
hypophysectomized  fish  gives  a  value  of  1.25  /ueq./gm./hr. 

DISCUSSION 

As  pointed  out  elsewhere  (Fleming  and  Stanley,  1965),  the  fact  that  F.  kansae 
is  a  small  fish  means  that  a  relatively  large  proportion  of  the  body  surface  consists 
of  water-permeable  surfaces,  i.e.,  gills  and  oral  membranes.  Thus,  a  relatively 
copious  urine  flow,  when  compared  with  data  on  larger  teleosts,  is  not  surprising. 
A  copious  urine,  however  dilute,  could  provide  a  major  site  for  sodium  loss,  and 
such  is  certainly  the  case  for  the  plains  killifish.  Approximately  25%  of  the  total 
sodium  loss  can  be  attributed  to  the  renal  route  when  this  teleost  is  held  in  fresh 
water.  F.  kansae  has  no  difficulty  in  remaining  in  sodium  balance,  however,  and 
our  estimates  of  sodium  influx  balance  well  with  total  sodium  loss. 

According  to  our  estimates,  F.  kansae  turns  over  approximately  1.0  /ueq.  Na+/ 
gm./hr.  in  fresh  water.  This  figure  contrasts  sharply  with  that  estimated  from 
animals  that  had  been  exposed  to  sea  water  for  8  days.  Under  such  conditions, 
renal  sodium  loss  is  negligible  (0.6%  of  the  total),  and  sodium  influx  would 
approximate  outflux,  i.e.,  17  //.eq  ./gm./hr. — a  17-fold  increase  over  the  values 
estimated  in  fresh  water. 

It  is  also  possible  to  estimate  gill  influx  for  the  first  few  hours  after  transfer. 
During  the  first  12  hours  in  sea  water,  gill  outflux  averaged  7  //.eq./gm./hr.  During 
this  same  period,  total  body  sodium  rose  from  62  to  132  meq./kg.  body  weight 
(Stanley  and  Fleming,  1965),  an  average  of  6  ;u.eq./gm./hr.  The  net  increase  in 
body  sodium  plus  the  outflux  provides  an  estimate  of  sodium  influx,  i.e.,  13  ju.eq./ 
gm./hr.  for  the  first  12  hours  in  sea  water,  which  is  slightly  more  than  a  13-fold 
increase  in  sodium  influx  over  the  fresh-water  value. 

Comparisons,  then,  of  the  estimates  of  sodium  influx  show  a  13-fold  increase 
over  the  fresh-water  value  for  the  first  12  hours  in  saline,  and  a  17-fold  increase 


GILL  AND  RENAL  FUNCTION  IN  FUNDULUS  163 

for  those  fish  held  in  sea  water  for  8  days.  Sodium  excretion,  on  the  other  hand, 
was  only  7-fold  higher  during  the  first  12  hours  after  transfer,  in  contrast  to  the 
17-fold  increase  estimated  for  the  8-day  fish.  These  figures  indicate  that  total  body 
sodium  must  rise  following  a  transfer  to  sea  water,  and  indeed  it  does  (Stanley  and 
Fleming,  1965). 

Both  target  organs  respond  promptly  to  the  transfer  into  sea  water,  but  the 
nature  of  the  response  is  somewhat  different.  Thus,  the  kidney  response  was 
diphasic,  sodium  loss  first  falling  from  0.28  ^eq./gm./hr.  to  0.04  ju,eq./gm./hr.,  and 
then  rising  sharply  to  0.49  /xeq./gm./hr.  20  hours  after  entering  the  saline  environ- 
ment. Unfortunately,  the  rapid  loss  of  isotope  in  sea  water  made  it  impractical  to 
continue  these  experiments  for  a  longer  period.  It  seems  not  unlikely  that  this 
figure  would  continue  to  increase,  since  this  teleost  can  excrete  a  blood-hypertonic 
urine  for  a  limited  time  (Stanley  and  Fleming,  1964b;  Fleming  and  Stanley,  1965). 
While  a  figure  of  0.5  jueq./gm./hr.  may  seem  low,  it  is,  nevertheless,  sufficient  to 
remove  nearly  10%  of  the  total  body  sodium  over  a  24-hour  period.  The  low  rate 
of  renal  sodium  loss  in  animals  immediately  after  transfer  and  in  sea  water  for 
eight  days  can  be  ascribed  to  a  low  rate  of  urine  formation. 

It  should  be  pointed  out  that  any  measurement  of  flux  includes  an  error  equal 
to  exchange  diffusion.  In  the  present  experiments,  it  is  possible  to  place  an  upper 
limit  on  the  magnitude  of  this  error.  Exchange  diffusion  should  be  approximately 
equal  for  all  animals  in  sea  water  regardless  of  previous  history.  Exchange 
diffusion  would  then  be  less  than  the  lowest  outflux  measurement,  vis.,  less  than 
5.0  jtieq./gm./hr.  as  measured  for  hypophysectomized  fish  after  initial  adjustment 
to  sea  water  (Fig.  3). 

Previous  experiments  (Stanley  and  Fleming,  1966)  have  suggested  a  negative 
sodium  balance  for  hypophysectomized  F.  kansae  held  in  fresh  water,  i.e.,  such 
animals  had  significantly  less  total-body  sodium  than  did  their  controls.  The  data 
presented  here  suggest  a  negative  sodium  balance  after  hypophysectomy,  and 
ocalize  the  metabolic  fault  at  the  kidney  level.  Thus,  no  differences  in  extra-renal 
sodium  loss  were  observed,  and  the  hypophysectomized  fish  took  up  sodium  at  a 
slightly  higher  rate  than  did  their  controls.  The  increase  in  influx,  however,  is 
not  sufficient  to  compensate  for  renal  loss,  i.e.,  0.54  vs.  0.27  /xeq./gm./hr.  Although 
hypophysectomized  killifish  will  live  for  several  weeks  in  tap-water  without  food,  it 
is  necessary  to  provide  additional  sodium  in  their  diet  if  they  are  to  be  held  for  any 
extended  period.  We  have  held  hypophysectomized  animals  in  fresh  water  for 
several  months  without  difficulty,  by  feeding  a  commercial  fish  chow  supplemented 
several  times  each  week  by  frozen  brine  shrimp. 

A  comparison  of  Figures  3  and  4  suggests  that  hypophysectomy  also  affects  the 
sodium  metabolism  of  the  gill,  at  least  during  the  course  of  initial  adjustment  to 
sea  water,  i.e.,  sodium  outflux  does  not  increase  at  the  rapid  rate  shown  by  the 
control  animals.  After  8  days  in  sea  water,  extra-renal  sodium  loss  is  still  20% 
lower  than  in  controls  (Fig.  4).  Hypophysectomy  also  affects  kidney  function 
during  adjustment  to  sea  water,  i.e.,  hypophysectomized  fish  do  not  produce  hyper- 
tonic  urine  and  do  not  show  any  increase  in  renal  sodium  loss  following  transfer 
(Fig.  2).  Renal  function  is  similar  in  the  two  groups  after  8  days  in  sea  water 
(Fig.  4).  Thus,  hypophysectomized  fish  appear  to  be  less  efficient  in  adjusting 
to  sea  water  because  both  gill  and  kidney  function  are  altered,  but  are  capable  of 


164  JON  G.  STANLEY  AND  W.  R.  FLEMING 

sea-water-adaptation  and  by  8  days  there  are  no  significant  differences  in  renal  or 
extra-renal  sodium  metabolism  between  the  two  groups. 

It  has  long  been  known  that  the  European  eel  (Anquilla  anquilla  L.)  can  survive 
in  fresh  water  after  hypophysectomy  (Fontaine  et  al.,  1949),  and  several  studies 
dealing  with  the  effect  of  such  treatment  on  the  sodium  metabolism  of  this  teleost 
have  appeared  recently  (Chester  Jones  and  Bellamy,  1964;  Leloup-Hatey,  1964; 
Chester  Jones  and  Henderson,  1965;  Chester  Jones  et  al.,  1965).  Contrary  to 
the  data  reported  here  for  F.  kansae,  it  appears  that  the  eel  remains  in  relatively 
close  sodium  balance  after  hypophysectomy,  for  such  animals  can  survive  in  dis- 
tilled water  for  some  time — an  environment  that  the  plains  killifish  cannot  tolerate 
for  more  than  a  few  days  at  best  (Pickford  et  al.,  1966).  The  eel  also  shows  a 
marked  drop  in  urine  flow  after  hypophysectomy  but  urine  sodium  levels  are  not 
affected,  i.e.,  renal  sodium  loss  is  actually  reduced.  In  contrast  to  F.  kansae,  the 
animals  remain  in  sodium  balance  by  reducing  sodium  uptake.  Such  data  do  not 
imply  that  electrolyte  metabolism  has  not  been  affected,  and  it  is  clear  that  such 
is  not  the  case,  for  hypophysectomized  eels  held  in  fresh  water  do  show  a  drop 
in  serum  electrolytes  (Leloup-Hatey,  1964;  Chester  Jones  and  Henderson,  1965; 
Chester  Jones  et  al.,  1965). 

SUMMARY 

1.  Renal  and  extra-renal  sodium  loss  was  measured  for  intact  and  hypophysec- 
tomized Fundulus  kansae  in  fresh  water  and  during  adaptation  to  sea  water. 

2.  In  fresh  water,  urine  was  copious  and  dilute  but  a  major  route  of  sodium  loss. 

3.  Following  transfer  to  sea  water,  urine  flow  was  reduced  and  extra-renal 
sodium  excretion  increased.     Renal  sodium  loss  decreased  (because  of  a  reduction 
in  urine  flow),  then  increased  to  above  fresh  water  values,   then,   after   several 
days  in  sea  water,  returned  to  a  low  value. 

4.  Hypophysectomized  fish  in  fresh  water  had  a  reduced  urine  flow,  an  in- 
creased renal  sodium  loss,  while  extra-renal  sodium  outflux  was  unaffected. 

5.  Following  transfer  to  sea  water,  hypophysectomized  fish  shut-down  urine 
flow  and  although  they  increased  extra-renal  sodium  excretion,  they  did  not  do  so 
as  rapidly  as  controls.     Urine  sodium  loss  was  reduced  and  remained  low. 

LITERATURE  CITED 

BOURQUET,  J.,  B.  LAHLOUH  AND  J.  MAETZ,  1964.  Modifications  experimentales  de  1'equilibre 
hydromineral  et  osmoregulation  chez  Carassius  aitratus.  Gen.  Comp.  Endocrinol.,  4: 
563-576. 

CHESTER  JONES,  L,  AND  D.  BELLAMY,  1964.  Hormonal  mechanisms  in  the  homeostatic  regula- 
tion of  the  vertebrate  body  with  special  reference  to  the  adrenal  cortex.  Chapter  XI 
in  "Homeostasis,"  G.  M.  Hughes,  editor.  Symp.  Soc.  Exp.  Biol,  18:  195-236.  Cam- 
bridge University  Press,  Cambridge. 

CHESTER  JONES,  I.,  AND  I.  W.  HENDERSON,  1955.  Electrolyte  changes  in  the  European  eel 
(Anquilla  anquilla  L.)  /.  Endocrinol.,  32:  111. 

CHESTER  JONES,  I.,  I.  W.  HENDERSON  AND  D.  G.  BUTLER,  1965.  Water  and  electrolyte  flux  in 
european  eel  (Anquilla  anquilla}.  Arch.  Anat.  Micr.  Morph.  Exp.,  54:  453-468. 

FLEMING,  W.  R.,  AND  J.  G.  STANLEY,  1965.  Effects  of  rapid  changes  in  salinity  on  the  renal 
function  of  a  euryhaline  teleost.  Amer.  J.  Physiol.,  209:  1025-1030. 

FLEMING,  W.  R.,  J.  G.  STANLEY  AND  A.  H.  MEIER,  1964.  Seasonal  effects  of  external  calcium, 
estradiol,  and  ACTH  on  the  serum  calcium  and  sodium  levels  of  Fundulus  kansae. 
Gen.  Comp.  Endocrinol.,  4:  61-67. 


GILL  AND  RENAL  FUNCTION  IN  FUNDULUS  165 

FONTAINE,  M.,  O.  CALAMAND  AND  M.  OLIVEREAU,  1949.  Hypophyse  et  euryhalinite  chez 
1'anguille.  C.  R.  Acad.  Sci.,  Paris,  228:  513-514. 

GORDON,  M.  S.,  1963.  Chloride  exchanges  in  rainbow  trout  (Salmo  gairdneri)  adapted  to 
different  salinities.  Biol.  Bull,  124:  45-54. 

HOLMES,  R.  M.,  1961.      Kidney  function  in  migrating  salmonids.     Rep.  Challenger  Soc.,  3:  23. 

HOLMES,  W.  N.,  AND  R.  W.  McBEAN,  1963.  Studies  on  the  glomerular  nitration  rate  of  rain- 
bow trout  (Salmo  gairdneri).  J.  Exp.  Biol.,  40:  335-341. 

LELOUP-HATEY,  J.,  1964.  Corpuscules  de  Stannius  et  equilibre  mineral  chez  1'anguille  (Anguilla 
anguilla  L.).  /.  de  Physiol.,  Paris,  56:  595. 

MAETZ,  J.,  1963.  Physiological  aspects  of  neurohypophysial  function  in  fishes  with  some 
references  to  the  amphibians.  Sym.  Zool.  Soc.  Lond.,  9:  107-140. 

MAETZ,  J.,  J.  BOURQUET  AND  B.  LAHLOUH,  1964.  Urophyse  et  osmoregulation  chez  Carassius 
anratits.  Gen.  Comp.  Endocrinol.,  4:  401-414. 

MOTAIS,  R.,  1961.  Sodium  exchange  in  a  euryhaline  teleost,  Platichthvs  flcsus  flcsits.  C.  R. 
Acad.  Set.,  Paris,  253:  724-726. 

MOTAIS,  R.,  AND  J.  MAETZ,  1964.  Action  des  hormones  neurohypophysaires  sur  les  echanges  de 
sodium  (mesures  a  1'aide  du  radio-sodium  NaLM)  chez  un  teleosteen  euryhalin: 
Platichthys  flcsus  L.  Gen.  Comp.  Endocrinol.,  4:  210-224. 

MOTAIS,  R.,  AND  J.  MAETZ,  1965.  Comparison  des  echanges  de  sodium  chez  un  teleosteen 
euryhalin  (le  flet)  et  un  teleosteen  stenohalin  (le  serran)  en  eau  de  mer.  Importance 
relative  du  tube  digestif  et  de  la  branchie  dans  ces  echanges.  C.  R.  Acad.  Sci.,  Paris, 
261 :  532-535. 

MULLINS,  L.  J.,  1950.  Osmotic  regulation  in  fish  studied  with  radioisotopes.  Acta  Physiol. 
Scand.,21:  303-314. 

PICKFORD,  G.  E.,  P.  K.  T.  PANG,  J.  G.  STANLEY  AND  W.  R.  FLEMING,  1966.  Calcium  and  fresh- 
water survival  in  the  euryhaline  cyprinodonts  Fundulus  kansae  and  Fundulus  hetero- 
clitus.  Comp.  Biochem.  Physiol.,  (in  press). 

SHARRATT,  B.  M.,  I.  CHESTER  JONES  AND  D.  BELLAMY,  1964.  Water  and  electrolyte  composition 
of  the  body  and  renal  function  of  the  eel  (Anguilla  anguilla  L.).  Comp.  Biochem. 
Physiol.,  11:  9-18. 

STANLEY,  J.  G.,  AND  W.  R.  FLEMING,  1964a.  The  effects  of  a  rapid  transfer  from  fresh  water 
to  sea  water  on  the  urine  production  of  Fundulus  kansae.  Amer.  Zool.,  4:  118. 

STANLEY,  J.  G.,  AND  W.  R.  FLEMING,  1964b.  Excretion  of  hypertonic  urine  by  a  teleost. 
Science,  144:  63-64. 

STANLEY,  J.  G.,  AND  W.  R.  FLEMING,  1965.  Sodium  metabolism  in  Fundulus  kansae  in  fresh 
water  and  during  adaptation  to  sea  water.  Amer.  Zool.,  5:  688. 

STANLEY,  J.  G.,  AND  W.  R.  FLEMING,  1966.  Some  responses  of  the  euryhaline  killifish,  Fundu- 
lus kansae,  to  hypophysectomy.  Biol.  Bull.,  130:  430-441. 


THE  EFFECTS  OF  GLYCEROL  AND  OTHER  ORGANIC  SOLUTES 

ON  THE  MOTILITY  AND  RESPIRATION  OF  SOME 

INVERTEBRATE  SPERMATOZOA  1 

H.  BURR  STEINBACH 

Department  of  Zoology,  University  of  Chicago,  Chicago,  Illinois  60637 

Glycerol,  in  high  concentrations  in  aqueous  solution,  has  been  used  for  such 
varied  purposes  as  preparing  ATP-sensitive  contractile  systems  and  as  a  preventive 
of  freeze  damage  to  living  cells.  Added  to  sea  water  in  13%  v/v  concentration, 
some  sperm  of  marine  invertebrates  maintain  a  vibratile  activity  (Steinbach  and 
Dunham,  1961).  Since  there  were  indications  that  the  glycerol  did  penetrate  the 
cells,  it  was  clear  that  the  motile  mechanisms  were  not  completely  impaired  at  a 
total  osmolar  concentration  of  solute  of  at  least  2.5  osmolar. 

The  observations  reported  in  this  paper  were  made  with  the  hope  that  a  study 
of  the  effects  of  other  organic  solutes  might  reveal  clues  about  the  possible  role  of 
water  structure  in  the  motile  mechanism.  Regretfully  it  must  be  admitted  that 
this  hope  was  not  realized  but  the  studies  did  show  certain  physiological  differences 
in  the  actions  of  the  solutes  chosen  and  species  differences  in  the  responses  of  the 
cells. 

MATERIALS  AND  METHODS 

Methods  for  collecting  and  washing  Arbacia  pimctulata  and  Mytilus  edulis 
sperm  have  been  described  (Steinbach  and  Dunham,  1961).  The  effects  of  the 
several  agents  appeared  to  be  uninfluenced  by  the  number  of  washes  of  the  sperm. 
Most  of  the  work  involved  sperm  centrifuged  down  once  from  sea-water  suspension 
and  then  resuspended  in  normal  filtered  sea  water.  All  operations  were  conducted 
at  room  temperature  in  an  air  conditioned  room  23-25  °  C. 

For  studies  of  motility,  appropriate  concentrations  of  the  various  agents  were 
placed  in  small  petri  dishes  and  concentrated  sperm  suspension  added  in  fixed 
amounts.  A  variety  of  methods  for  quantitative  measurement  of  motility  were 
tested  without  success.  The  results  reported  are  hence  subjective  estimates, 
a  —  sign  designated  less  motility  than  the  sea  water,  ±  =  about  the  same  as  controls 
and  +  .  .  .  varying  degrees  of  activation. 

Oxygen  consumption  was  measured  in  Scholander  (1950)  differential  volumeters 
operated  in  a  large  tank  of  water  at  room  temperature.  The  volumeters  were 
shaken  with  a  low  amplitude  oscillatory  movement  at  40  times  per  minute.  No 
extensive  study  was  made  of  effects  of  rate  or  amplitude  of  shaking  other  than 
checking  that  rate  of  volume  decrease  was  a  function  of  the  respiring  mass. 

1  Work  reported  in  this  communication  supported  by  grants  from  U.  S.  Public  Health 
Service  #GM  10542  and  from  the  Wallace  G.  and  Clara  A.  Abbott  Fund  of  the  University 
of  Chicago. 

166 


EFFECTS  OF  GLYCEROL  ON  SPERM  MOTILITY 


167 


In  this  report,  respiration  is  given  as  per  cent  of  that  of  sea  water  controls. 
Most  of  the  published  literature  reports  oxygen  consumption  of  sperm  based  on 
units  of  108  individual  cells.  Since  it  is  not  only  laborious  but  subject  to  consider- 
able variation  to  determine  sperm  numbers  on  each  sample,  we  measured  wet 
y  eight  of  pellet  of  a  centrifuged  sample  of  sperm  suspension  and  referred  oxygen 
consumption  to  units  of  wet  weight  determined  in  that  fashion.  Pellet  weights 
were  determined  by  pipetting  measured  volumes  of  sperm  suspension  into  pre- 
viously weighed  12-ml.  conical  centrifuge  tubes.  The  tubes  were  then  centrifuged 

TABLE  I 

Data  relating  to  some  common  organic  solvents,  their  physical  characteristics, 
ability  to  protect  against  freeze  damage  and  to  activate  Arbacia  sperm, 

motility  or  respiration 


Protection* 
erythrocytes 

Arbacia 
sperm 
activation 

Change 
viscosityb 

HS" 

Surface 
tension"1 

,• 

e' 

v.p.s 

Glycerol 

+  +  + 

±  or  - 

20 

~5/6 

63 

42 

— 

Ethylene  glycol 

+  +  + 

+  + 

13 

4/4 

60? 

2.3 

37 

— 

DM  SO 

+  +  + 

+  +  + 

17 

4/4 

43 

3.9 

49 

0.7 

DMF 

+  +  + 

+  +  + 

17 

3/5 

35 

3.8 

27 

3.7 

DMAC 

+  +  + 

+  +  + 

n.t. 

3/6 

32? 

3.8 

38 

1.3 

Ethyl  alcohol 

±? 

+ 

14 

1/3 

22 

1.7 

24 

+  + 

Methyl  alcohol 

+++? 

n.t. 

6 

2/2 

23 

1.7 

34 

+  + 

Acetone 

— 

n.t. 

7 

0/3 

26 

2.9 

+  + 

H2O 

0 

74 

1.8 

80 

17 

a  Estimated  from  different  sources.  Protection  is  reported  differently  depending  on  the 
freezing  temperature  used.  Cf.  Nash,  1962;  Lovelock,  1954. 

b  Increase  in  viscosity  on  dissolving  1  M/L.  of  substance  in  water.  Approximate  measure- 
ments with  Ostwald  viscosimeter. 

c  Hydrophilic  strength  from  Nash,  1962. 

d  Surface  tension  of  pure  liquids.  Figures  estimated  from  published  tables  and  technical 
literature.  Those  figures  with  question  mark  are  estimated  from  lowering  of  surface  tension  of 
1  M  solutions. 

e  Dipole  moments.     From  technical  literature  and  handbooks. 

f  Dielectric  constants.     From  technical  literature  and  handbooks. 

8  Vapor  pressure.     From  technical  literature  and  handbooks. 

For  the  most  part,  figures  given  above  are  rounded  out  from  more  exact  figures  in  the  source 
material.  For  DMF  and  DMAC,  Dupont  has  a  Review  of  Catalytic  and  Synthetic  Applications 
for  DMF  and  DMAC.  Crown-Zellerbach  publishes  a  technical  review  of  properties  of  DMSO 
(dimethyl  sulfoxide.  Reaction  Medium  and  Reactant). 


15  minutes  at  3,400  rpm.,  10  cm.  radius  to  center  of  tube.  The  supernatants  were 
then  decanted,  the  sides  wiped  dry  with  tissue  and  the  tubes  weighed.  In  some 
instances  dry  weights  were  also  determined. 

The  solutes  are  indicated  in  Table  I.  They  were  chosen  primarily  on  the  basis 
of  previous  studies  of  solutes  conferring  protection  against  freeze  damage  to  cells 
(cf.  Lovelock,  1959;  Nash,  1962).  They  all  fall  into  the  general  category  of 
weakly  protic  (alcohols,  etc.)  or  aprotic  solvents  (cf.  Singer,  1962;  Parker,  1960). 
All  are  polar  compounds  of  relatively  high  dielectric  constants.  All  could  form 


168 


H.  BURR  STEINBACH 


associations  with  water  or  other  molecules  by  accepting  protons  but  would  be  poor 
proton  donors. 

RESULTS 

Tables  I  and  II  summarize  the  major  results  of  observations  on  Arbacia  sperm. 
Arbacia  sperm  show  increased  motility  in  the  presence  of  high  concentrations  of 
DMF,  DMSO,  ethylene  glycol  and  ethyl  alcohol.  At  comparable  concentrations 
glycerol  has  little  effect  or  inhibits,  while  urea,  inorganic  salts  and  hexose  sugars 
(not  shown  in  table)  stop  motility  and  inhibit  respiration.  For  DMF,  the 

TABLE  II 

Relative  rates  of  oxygen  consumption.     Arbacia  sperm;  2  ml.  suspension  per  flask.     Total  dry  weight 

of  sperm  per  flask  on  the  order  of  50-80  mg.     Reagents  added  as  0.25  ml.  fluid  per  flask 

to  give  final  concentrations  noted.     For  each  reagent,  first  value  of  relative  Q02 

/Q02  experimental  X  100\   . 

)  is  for  the  rate  during  the  first  60  minutes. 
\    Qo2  sea  water  control    / 

Figures  in  parentheses  indicate  relative  rates  two  or  more  hours 
later,     -\-or-  symbols  indicate  activity  of  sperm 
(see  text) 


Reagent  added 


Final  cone,  of  reagent  in  sea  water 


0.09 


0.45 


0.9 


DMF 
DMSO 
Ethylene  glycol 

Glycerol 
Urea 


170 

(130) 


125 

(125) 

± 


87 
(57) 


580 
(270) 


225 
(170) 


125 

(125) 


67 


57 
(57) 


340 
(130) 


210 
(150) 


75 


48 
(48) 


optimum  concentration  for  enhancement  of  motility  and  respiration  is  near 
0.5  M  in  sea  water  but  the  effects  are  still  pronounced  in  1  M  concentrations.  In 
2  M  concentrations,  stimulation  is  observed  for  a  short  time  followed  by  irreversible 
depression  of  motility.  Respiration  was  not  measured  at  2  M  concentrations. 

The  figures  in  Table  II  are  given  as  experimental  rates  expressed  as  percentage 
of  Qo2  values  for  sea  water  controls.  For  both  Arbacia  and  Mytilus  sperm  the 
usual  dose  per  flask  was  50-80  mg.  dry  weight  of  sperm.  Under  such  conditions 
Arbacia  sperm  used  oxygen  at  a  rate  of  ca.  2.2  ^l.3  O2/mg.  dry  weight/hour, 
Mytilus,  ca.  1  /xl.3  O2/mg.  dry  weight/hour. 


EFFECTS  OF  GLYCEROL  ON  SPERM  MOTILITY 


169 


Approximately  the  same  series  of  experiments  was  carried  out  with  Mytilus 
sperm  but  the  results  are  not  presented  in  detail  since  all  substances  at  the  lower 
concentrations  had  little  effect  on  either  motility  or  respiration  while  at  the  higher 
concentrations  both  motility  and  respiration  were  depressed.  The  difference 
between  the  responses  of  the  sperm  from  the  different  species  was  clear-cut  and 
invariable  even  though  morphologically  the  two  cell  types  are  rather  similar. 

Respiration  of  Arbacia  sperm  may  continue  at  a  fairly  constant  level  for  hours 
but  more  typically  declines  with  time  (Table  II).  Figure  1  gives  the  results  of 


V) 

z 
o 
o 

CVJ 

o 


80 


70 


60 


50 


. 

5      40 


30 


•=   0  UREA 

0=   0.45   URE.A 
X  =    0.22   UREA 


ttt 


40        160        180 


TIME    (MINUTES) 


FIGURE  1.  Oxygen  consumption  of  Arbacia  sperm  (movement  in  mm.  of  plunger  of 
volumeter)  plotted  against  time.  For  the  first  70  minutes,  sperm  were  in  normal  sea  water. 
At  70  minutes  urea  was  added  (y)  to  three  of  the  volumeters  (lower  curves)  with  DMF,  0.5  M 
final  concentration  (I),  added  to  volumeter  of  upper  curve.  At  140  minutes  DMF  0.5  M 
added  to  volumeters  of  three  lower  curves.  +  indicates  estimated  motility  of  sperm. 

one  run  in  which  additions  of  DMF  (or  sea  water  for  control)  to  urea-treated 
sperm,  stimulated  respiration,  even  made  at  70  minutes  in  the  urea  concentrations 
indicated.  While  no  respiration  experiments  were  carried  out  over  very  long 
time  periods,  the  enhancement  of  motility  by  the  0.5  M  concentrations  of  the  sub- 
stances was  evident  at  least  as  long  as  8  hours  after  the  start  of  the  treatment.  It 
should  be  noted  that  the  stimulating  action  of  DMF,  and  presumably  the  other 
activating  substances,  is  found  with  sperm  held  in  sea  water  until  respiration  has 
dropped  to  a  marked  extent. 


170  H.  BURR  STEINBACH 

Observations,  not  reported  in  detail,  were  made  of  the  effects  of  KC1,  NaCl, 
sucrose  and  glucose  on  motility  and  respiration.  At  0.5  osmolar  in  sea  water  and 
higher  concentrations,  motility  and  respiration  were  irreversibly  depressed  in 
Arbacia  and  motility  in  Mytilus. 

While  an  extensive  study  of  retention  of  fertilizing  capacity  of  sperm  was  not 
attempted,  a  few  tests  showed  that  the  ability  of  Arbacia  sperm  to  activate  Arbacia 
eggs  was  retained  for  several  hours  in  the  stimulating  concentrations  of  DMF. 
There  were,  however,  indications  that  the  sea-water  controls  fared  better  in  this 
respect. 

DISCUSSION 

At  present  there  is  no  good  explanation  for  the  differences  in  the  effects  of 
DMF  and  related  compounds  on  Arbacia  and  on  Mytilus  sperm.  The  morphology 
of  the  two  cell  types  is  rather  similar  and  preliminary  studies  show  no  marked 
effects  of  DMF  on  the  fine  structure  of  either.  A  quick  survey  showed  that  DMF 
in  0.5  M  concentration  also  activated  Phascolosoma  sperm  but  inhibited  the  sperm 
of  Loligo  and  Busycon.  Thus  there  are  clear-cut  comparative  differences  which 
may,  in  the  future,  assist  in  determining  the  nature  of  the  effects  of  the  organic 
substances  on  the  motile  mechanisms  involved. 

Focussing  attention  for  the  moment  on  Arbacia  sperm,  the  cell  type  most 
extensively  studied  here,  there  is  a  parallelism  between  the  stimulating  effects  on 
the  sperm  and  freeze  protection  of  human  erythrocytes.  This  parallelism,  together 
with  some  physical  characteristics  of  the  solvents,  is  noted  in  Table  I. 

On  the  basis  of  Table  I  and  related  data,  the  following  criteria  could  be  listed 
for  either  freeze  protection  or  Arbacia  sperm  activation: 

1.  Substances    effective    are    weakly    protic    polar    compounds    of    fairly    high 
dielectric  constants. 

2.  Substances  interact  with  water  or  form  complexes  in  some  fashion,  as  sug- 
gested by  the  increase  in  viscosities  in  aqueous  solution. 

3.  Substances  in  pure  liquid  form  have  relatively  high  surface  tensions  (ca.  50% 
of  water  or  higher)  and  vapor  pressures  lower  than  water. 

4.  Substances  should  penetrate  cells  readily. 

The  last-named  requirement  of  ready  penetration  places  glycerol  in  a  sort  of 
gray-area  so  far  as  effectiveness  is  concerned.  Glycerol  (and  even  glucose)  is 
known  to  penetrate  some  cells  rapidly,  others  very  slowly.  In  contrast,  there  is 
every  likelihood  that  the  DMF-,  DMSO-type  substances  penetrate  virtually  all 
cell  types  at  rates  comparable  to  that  of  water.  The  requirement  that  substances 
be  not  overly  surface-active  (number  3)  appears  to  distinguish  some  of  the  weakly 
protic  alcohols  that  confer  only  moderate  protection,  followed  by  irreversible  change, 
from  the  most  effective  substances.  The  correlation  between  protective  ability 
towards  freeze  damage  and  hydrophilic  strength  (HS),  as  pointed  out  by  Nash, 
1962,  is  noted  in  Table  I  showing  also  the  characteristic  that  the  substances  of 
high  HS  values  do  not  serve  as  good  protective  agents  if  the  surface  tensions  of 
the  pure  substances  are  low.  While  not  entirely  demonstrated,  it  seems  probable 
that  those  substances  interacting  strongly  with  water  (viscosity  increase,  high  HS 
values,  etc.)  but  which  are  also  surface-active,  are  not  good  protective  agents 


EFFECTS  OF  GLYCEROL  ON  SPERM  MOTILITY  171 

because  of  side  deleterious  effects,  not  because  of  lack  of  effects  similar  to  those 
listed  as  good  protective  agents. 

At  the  present  time  there  appears  to  be  no  single  set  of  characteristics  common 
to  the  various  substances  which  might  explain  their  effects  other  than  the  interac- 
tion with  water.  In  general,  the  reagents  used  might  act  on  biological  systems 
either  by  providing  a  mixed  solvent  of  characteristics  different  from  that  of  water 
or  by  having  specific  chemical  interactions.  At  the  present  state  of  our  knowledge 
it  seems  most  reasonable  to  assume  that  the  reagents  listed  in  Table  I  act  by  altering 
the  solvent  properties  of  the  system. 

AYeakly  protic  and  aprotic  substances,  as  pure  solvents,  do  alter  macromolecular 
structures  (cf.,  Singer,  1962)  although  most  of  the  effects  are  evident  only  in  high 
concentrations  or  in  pure  organic  solvent.  Effects  on  enzymes  show  up  in  general 
only  in  concentrations  somewhat  higher  than  those  used  in  this  paper  (cf. 
Hamaguchi,  1964).  Similarly  antigen-antibody  interactions  appear  little  influenced 
at  concentrations  below  2  M  (Gould  et  al.,  1964).  On  the  other  hand,  ethanol  in 
relatively  low  concentrations  inhibits  ion  transport  in  animal  tissues  (Israel-Jacard 
and  Kalant,  1965)  and  several  solvents  have  a  pronounced  enhancing  effect  on 
relaxation  of  glycerinated  muscle  fibers  (Watanabe  and  Maruyama,  1964).  The 
aprotic  solvents  themselves  (e.g.  DMF,  DMSO)  alter  ionic  mobility  relationships 
markedly  when  used  as  pure  solvents  (cf.  Parker,  1962). 

This  brief  summary  of  representative  effects  of  various  organic  solvents  does 
indicate  that  the  influence  of  the  agents  on  biological  processes  probably  reflects 
rather  delicate  alterations  in  the  functional  machinery  of  the  cell,  rather  than  by 
participating  as  reactants  in  the  metabolic  systems. 

LITERATURE  CITED 

GOULD,  H.  J.,  T.  J.  GILL  AND  H.  W.  KUNZ,  1964.     Studies  on  synthetic  polypeptide  antigens. 

/.  Bin!.  Chan.,  239:  3071-3081. 

HAMAGUCHI,  K..  1964.     Structure  of  Muramidase.     /.  Biochcm.,  55:  333-339.     (Japanese) 
ISRAEL-JACARD,   Y.,   AXD   H.   KALANT,    1965.     Effect   of  ethanol    on   electrolyte   transport   and 

electrogenesis  in  animal  tissues.    /.  Cell.  Comp.  PhysioL,  65:  127-132. 
LOVELOCK,  J.  E.,  1954.     The  protective  action  of  neutral  solutes  against  hemolysis  by  freezing 

and  thawing.     /.  Biochcm..  56:  265-270. 
NASH,  T.,  1962.     The  chemical  constitution  of  compounds  which  protect  erythrocytes  against 

freezing  damage.     /.   Gen.  PhysioL,  46:   167-175. 
PARKER,  A.  S.,  1962.     The  effects  of  solvation  on  the  properties  of  anions  in  dipolar  aprotic 

solvents.     Quart.  Rev.,  16:   163-187. 

SCHOLANDER,  P.  F.,  1950.     Volumetric  plastic  respirometers.     Rev.  Sci.  Instr.,  21:  378-380. 
SINGER,    S.    J.,    1962.     The    properties    of    proteins    in    non-aqueous    solutions.    Adv.    Protein 

Chemistry,  17:  1-69. 
STEINBACH,    H.    B.,    AND    P.    B.    DUNHAM,    1961.     Ionic    gradients    in    some    invertebrate 

spermatozoa.     Bid.  Bull.,  120:  411-419. 
WATANABE,    S.,   AND  K.    MARUYAMA,    1964.     Relaxing   effects   of  formamide   on   glycerinated 

muscle  fibers  and  on  myosin  B  suspension.    Amer.  J.  PhysioL,  207:  809-813. 


UPTAKE  OF  ORGANIC  MATERIAL  BY  AQUATIC  INVERTEBRATES. 

IV.      THE    INFLUENCE    OF    SALINITY    ON    THE    UPTAKE    OF 

AMINO  ACIDS  BY  THE  BRITTLE  STAR,  OPHIACTIS  ARENOSA  1 

GROVER  C.  STEPHENS  AND  RAGHUNATH  A.  VIRKAR 

Department  of  Organismic  Biology,  University  of  California,  Irvine,  California  92664 

The  ability  to  remove  amino  acids  and  other  small  organic  compounds  from 
dilute  solution  is  widespread  among  marine  invertebrates.  Stephens  and  Schinske 

(1961)  reported  examples  from  ten  different  phyla.     This  capacity  has  been  studied 
in  additional  forms  and  has  provided  material  for  a  series  of  reports   (Stephens, 
1962,  1963,  1964;  Stephens  et  al,  1965;  Virkar,  1963).     It  has  been  our  experi- 
ence that  any  soft-bodied  marine  invertebrate  exposed  to  an  amino  acid  such  as 
glycine  or  phenylalanine  at  concentrations  ranging  between  10~5  and  10~6  moles  per 
liter  shows  the  capacity  to  remove  it  from  solution  quite  rapidly.     The  fresh-water 
forms  we  have  examined  remove  amino  acids  from  solution  very  much  more  slowly, 
so   slowly   that   we   have   not   demonstrated    the   occurrence   of   the    process    un- 
ambiguously. 

The  relation  between  external  salinity  and  the  uptake  of  amino  acids  has  proved 
to  be  of  interest.  Stephens  (1964)  showed  that  uptake  of  glycine  in  euryhaline 
nereid  polychaetes  occurred  only  at  moderate  to  high  salinities.  At  lower  salinities, 
uptake  stopped  almost  entirely.  The  salinity  at  which  uptake  ceased  was  closely 
correlated  with  that  at  which  osmoregulation  and  chloride  regulation  began.  The 
data  did  not  permit  firm  conclusions  about  rates  of  uptake  at  intermediate  salinities 
since  they  were  acquired  before  recognizing  the  considerable  capacity  for  adaptation 
in  the  system. 

A  related  matter  of  interest  is  the  regulation  of  the  "free  amino  acid  pool"  in 
marine  invertebrates  in  response  to  changes  in  salinity.  The  tissues  and  body 
fluids  of  most  marine  invertebrates  are  in  osmotic  equilibrium  with  their  environ- 
ment. Numerous  workers  have  reported  large  amounts  of  non-protein  nitrogenous 
substances  in  the  tissues,  of  which  amino  acids  are  the  most  abundant  (see  Awapara, 
1962;  Kittredge  et  al.,  1962).  This  pool  of  amino  acids  is  sufficiently  concentrated 
to  represent  a  major  fraction  of  the  osmotic  concentration  of  the  tissues.  It  has 
been  shown  that  as  salinity  is  decreased,  the  size  of  the  pool  decreases.  Florkin 

(1962)  has  suggested  that  this  behavior  represents  an  osmoregulatory  response  in 
the  sense  that  decreasing  the  size  of  the  free  amino  acid  pool  spares  larger  fluctua- 
tions in  other  cellular  constituents.     This  position  is  supported  by  a  large  number  of 
observations  carried  out  by  Florkin  and  co workers  (reviewed  in  Florkin,  1962),  as 
well  as  observations  by  Potts   (1958),  Shaw   (1958),  and  Lange   (1963,   1964). 
Virkar  (1963,  1965)  has  studied  the  response  of  tissues  of  the  sipunculid  Golfingia 
to  small  changes  in  salinity.     The  change  in  free  amino  acids  in  the  body  wall  which 
is  produced  by  lowering  the  concentration  of  the  ambient  medium  by    10%    is 

1  This  work  was  supported  by  Grant  GM  12889  from  the  USPHS. 

172 


UPTAKE  OF  AMINO  ACIDS  BY  OPHIACTIS  173 

large  enough  to  account  completely  for  the  implied  change  in  intracellular  osmotic 
concentration. 

The  preceding  paragraphs  set  a  context  in  which  several  interesting  questions 
can  he  asked.  What  is  the  source  of  free  ammo  acids  which  form  such  a  surpris- 
ingly concentrated  pool  in  many  marine  organisms?  Awapara  (1962)  argues  it  is 
not  dietary,  on  the  ground  that  there  are  differences  in  animals  found  in  comparable 
habitats,  but  he  does  not  make  positive  suggestions.  By  what  means  is  the  free 
amino  acid  pool  in  the  tissues  decreased  in  response  to  lowered  salinity  ?  We  have 
no  information  whatever  on  this  point.  Does  the  free  amino  acid  pool  contribute 
significantly  to  the  energy  metabolism  of  the  organism  ?  What  is  the  turnover  rate 
of  individual  constituent  acids  in  the  pool  ? 

The  earlier  work  in  our  laboratory  to  which  we  have  alluded  is  based  on  sup- 
plying uniformly  labelled  compounds  to  marine  invertebrates  in  very  dilute  solution 
in  the  ambient  medium.  This  provides  a  technique  for  labelling  specific  constituents 
of  the  free  amino  acid  pool  at  will.  The  work  to  be  reported  uses  this  technique 
to  provide  data  relevant  to  the  questions  raised  above  concerning  the  role  of  free 
amino  acids  in  marine  organisms.  A  portion  of  this  work  has  appeared  in  abstract 
form  (Stephens  and  Virkar,  1965). 

MATERIAL  AND  METHODS 

Ophiactis  arenosa  is  a  small  brittle  star  which  lives  in  close  association  with 
several  sponges  found  on  floating  docks  and  on  pilings.  Animals  were  collected 
as  required  from  Newport  Bay  south  of  Los  Angeles.  Masses  of  sponge  were 
brought  into  the  laboratory  and  kept  in  sea  water.  The  brittle  stars  emerged  on  the 
surface  of  the  sponge  mass  in  about  an  hour  and  were  placed  in  sea  water  in  dish 
pans.  Several  hundred  animals  were  kept  in  a  single  pan  in  an  incubator  at  a 
temperature  of  15-16°  C.  Observations  were  carried  out  at  room  temperature 
(about  21°  C.).  Individuals  used  in  the  observations  reported  were  selected  in 
the  size  range  of  10  to  30  mg.  wet  weight  except  for  the  data  concerning  the 
relation  between  weight  and  rate  of  uptake  of  glycine. 

These  animals  may  be  exposed  to  moderate  salinity  variations  in  their  normal 
environment  but  presumably  do  not  suffer  rapid  changes.  However,  they  proved 
capable  of  surviving  a  direct  change  from  full-strength  sea  water  to  60%  sea  water. 
Acclimation  to  60%  sea  water  was  necessary  for  survival  at  50%  sea  water.  One 
set  of  observations  is  based  on  the  responses  of  organisms  transferred  abruptly  to 
60%  sea  water.  Aside  from  this,  animals  were  allowed  to  adapt  by  placing  them 
one  day  at  90%,  80%,  and  70%  sea  water  successively.  They  were  kept  for  two 
days  at  60%  and  50%  sea  water.  Observations  were  undertaken  after  all  animals 
had  been  acclimated  in  this  fashion.  Dilutions  of  sea  water  were  prepared  with 
distilled  water.  The  salinity  of  the  sea  water  stock  was  33.08/fc. 

Water  content  was  determined  by  weighing  individuals  after  drying  on  filter 
paper  and  reweighing  them  after  approximately  24  hours  at  110°  C.  Amino  acid 
determinations  were  carried  out  by  measuring  ninhydrin-positive  material  using 
extracts  in  cold  80%  ethanol.  We  used  a  technique  described  by  Clark  (1964)  and 
are  indebted  to  her  for  earlier  personal  communication  of  the  method.  The  proced- 
ure was  calibrated  periodically  using  glycine  standards,  and  such  standards  were 
determined  routinely  with  unknown  samples.  The  ninhydrin-positive  material 


174  GROVER  C.  STEPHENS  AND  RAGHUNATH  A.  VIRKAR 

in  the  extracts  is  treated  as  free  amino  acid  and  concentrations  expressed  as  milli- 
moles  amino  acid  per  kg.  body  water. 

Uptake  of  amino  acids  was  measured  by  supplying  randomly  labelled  glycine-C1* 
or  /-isomers  of  the  other  amino  acids  employed.  Concentrations  greater  than 
10"6  moles  per  liter  were  obtained  by  adding  unlabelled  amino  acid.  Determinations 
of  radioactivity  were  made  using  a  thin-window  gas  flow  detector  system.  Animals 
were  exposed  to  sea  water  solutions  of  labelled  amino  acids  for  a  predetermined 
time.  Initial  and  final  radioactivity  in  the  sea  water  was  determined.  Each 
individual  was  extracted  for  24  hours  in  2.0  ml.  of  80%  ethanol.  Five-tenths-ml. 
samples  of  this  extract  were  evaporated  on  planchets  and  counted.  Each  individual 
was  then  ground  in  2.0  ml.  of  distilled  water  and  0.5-ml.  samples  of  the  brei 
evaporated  on  planchets.  All  data  presented  have  been  corrected  for  background 
and  sample  thickness. 

Care  was  taken  to  insure  that  the  data  collected  in  one  particular  set  of  obser- 
vations would  be  internally  comparable  by  preparing  labelled  solutions  from  a 
single  stock  to  the  same  final  concentration.  Thus  no  corrections  for  small  differ- 
ences in  ambient  radioactivity  were  required. 

Descending  paper  chromatograms  were  prepared  using  w-butanol-acetic  acid- 
water  (120:30:50)  followed  by  phenol-water  (80%  by  weight)  as  described  by 
Smith  (1960).  One-dimension  descending  chromatograms  were  also  prepared 
using  w-butanol-acetic  acid-water.  Autoradiographs  were  prepared  by  exposing 
Kodak  No-Screen  x-ray  film  to  the  chromatograms  for  a  seven-day  period. 

RESULTS 

When  exposed  to  a  solution  of  glycine-C14,  uptake  of  the  radioactive  label  is 
rapid  and  approximately  linear  for  at  least  30  minutes.  Under  normal  circum- 
stances, the  greater  part  of  the  radioactivity  is  in  the  alcohol-soluble  fraction  while 
only  a  small  percentage  of  the  total  is  found  in  the  brei.  At  the  end  of  a  30-minute 
exposure,  the  ratio  of  alcohol-soluble  to  alcohol-insoluble  radioactivity  is  of  the 
order  of  30: 1. 

A  one-dimensional  chromatogram  of  the  alcohol  extract  of  Ophiactis  shows 
several  ninhydrin-positive  spots.  The  most  prominent  have  Rf  values  which  agree 
with  those  of  glycine,  alanine,  taurine,  and  threonine.  In  both  one-  and  two- 
dimensional  chromatograms,  autoradiographs  show  radioactivity  in  the  region 
identified  as  glycine.  Hence,  it  appears  that  the  great  bulk  of  the  radioactivity  is 
still  in  the  form  in  which  it  was  supplied.  This  was  also  true  of  an  alcohol  extract 
prepared  from  animals  which  had  been  sacrificed  24  hours  after  a  30-minute 
exposure  to  labelled  glycine. 

A  number  of  experiments  were  performed  using  a  larger  brittle  star,  Ophionereis 
annnlata.  The  animals  were  induced  to  autotomize  their  arms,  and  uptake  of 
glycine  by  the  isolated  arms  was  measured.  With  suitable  corrections,  it  appears 
that  this  preparation  is  about  as  effective  as  the  whole  animal,  at  least  for  three  or 
four  hours.  Consequently,  it  is  likely  that  the  gut  is  not  involved  in  any  extensive 
way  in  this  uptake.  This  would  agree  with  previous  reports  (Stephens,  1962, 
1963,  1964)  for  other  invertebrates,  and  very  probably  applies  to  Ophiactis  as  well. 

Observations  wrere  undertaken  to  relate  uptake  of  labelled  glycine  to  the  weight 
of  the  animals.  When  the  log  of  uptake  was  plotted  against  the  log  of  wet  weight, 


UPTAKE  OF  AMINO  ACIDS  BY  OPHIACTIS 


175 


a  regression  line  of  slope  0.545  was  calculated  by  the  least  squares  method.  Rather 
than  correcting  for  weight  based  on  the  exponential  relation  this  implies,  weights 
of  the  animals  employed  for  subsequent  observations  were  kept  as  closely  comparable 
as  possible  and  within  the  range  of  10  to  30  milligrams.  Uptake  is  expressed  as 
cpm./mg.  for  animals  in  this  range. 

Observations  relating  uptake  to  ambient  concentration  of  amino  acid  were  car- 
ried out  using  glycine,  valine,  alanine,  and  arginine.  When  the  data  were  plotted 
as  the  reciprocal  of  concentration  against  the  reciprocal  of  uptake,  the  straight 
line  which  was  expected  was  not  obtained.  Uptake  was  systematically  too  high  at 
high  concentrations  for  all  of  the  amino  acids  used.  A  more  extended  series  of 


K        «H 


4    - 


en 

UJ 


o      3 
^ 


LJ 


O 

o 

_J 


o  o 


LOG     GLYCINE    CONCENTRATION      (MOLES/l-xlO*) 

FIGURE  1.     Rate  of  glycine  uptake  by  Ophiactis  as  a  function  of  ambient  concentration  of 
glycine.     Each  point  represents  the  average  value  for  ten  or  more  individuals. 


concentrations  of  glycine  was  used,  covering  the  concentration  range  from  2.6  X  10~s 
to  10"2  moles  per  liter.  The  data  are  presented  in  Figure  1.  It  will  be  noted  that 
the  accumulation  system  is  not  saturated  at  the  highest  concentration  used.  This 
differs  from  the  relation  reported  for  Fungia,  Clymenella,  Nereis  sp.,  Golfingia, 
and  a  number  of  other  invertebrates  which  show  a  definite  maximum  rate  of  ac- 
cumulation (Stephens,  1962,  1963,  1964;  Virkar,  1963,  and  unpublished  observa- 
tions). At  the  end  of  a  30-minute  exposure  to  the  lowest  concentration  employed 
(2.6  X  10~8  M),  about  64  times  as  much  labelled  carbon  per  kilogram  of  water  was 
found  in  the  alcohol-solution  fraction  of  Ophiactis  as  was  present  in  the  ambient 
solution.  The  lower  limit  in  concentration  was  imposed  by  the  specific  activity 


176 


GROVER  C.  STEPHENS  AND  RAGHUNATH  A.  VIRKAR 


TABLE  I 

Average  radioactivity  in  the  alcohol- soluble  and  alcohol-insoluble  fraction  of  Ophiactis  at  various  times 
after  a  30-minute  exposure  to  glycine-Cu  (  U.L.).     The  data  are  presented  as  cpm./O.S  ml. 
extract  divided  by  weight  in  mg.  Standard  deviations  are  included;  n  is  10  for 

all  groups 


Time 
(hours) 

(cpm./mg.) 
alcohol-soluble 

(cpm./mg.) 
alcohol-insoluble 

0 

117  ±32 

2.7  ±  0.7 

0.5 

108  ±  24 

3.6  ±  0.9 

1 

106  ±  24 

4.7  ±  1.2 

2 

111  ±28 

5.4  ±  0.9 

4 

106  ±  24 

9.7  ±2.3 

6 

86  ±  25 

11.5  ±  2.7 

24 

45  ±  7 

24.2  ±  4.4 

of  the  labelled  glycine  and  does  not  reflect  a  limitation  of  the  physiological  system 
involved.  As  is  the  case  in  forms  previously  examined,  this  accumulation  system 
is  essentially  one  way ;  no  significant  exchange  of  labelled  material  for  unlabelled 
amino  acid  in  the  ambient  medium  was  obtained. 

When  animals  were  exposed  to  glycine-C14  for  30  minutes  and  then  allowed  to 
remain  in  sea  water  for  various  periods  subsequent  to  this  exposure,  there  was  a 
gradual  increase  in  radioactivity  in  the  alcohol-insoluble  fraction  of  the  animal. 
Table  I  lists  the  alcohol-soluble  and  alcohol-insoluble  radioactivity  at  various  times 
after  a  30-minute  exposure  to  labelled  glycine.  It  is  apparent  that  total  radioactivity 
decreases  with  time  although  the  alcohol-insoluble  fraction  increases  in  absolute 
level  and  not  merely  as  a  ratio. 

At  least  a  portion  of  the  radioactivity  which  is  lost  from  the  system  represents 
C14-labelled  carbon  dioxide.  Water  in  which  brittle  stars  have  been  placed  for  24 
hours  after  an  exposure  to  labelled  glycine  shows  some  radioactivity.  This 
disappears  on  acidification  and  can  be  trapped  on  alkali  in  a  Conway  diffusion  flask. 
Table  II  presents  a  balance  sheet  accounting  for  92%  of  the  radioactivity  initially 
present  in  the  system.  A  later  experiment  showed  that  CO2  was  lost  to  the 
atmosphere  before  acidifying.  About  three-quarters  of  the  radioactive  CO2  was 
trapped  on  alkali  before  the  sea  water  was  acidified.  The  measurements  presented 
in  Table  II  are  thus  systematically  low.  The  estimated  correction  for  loss  to  the 
atmosphere  (the  parenthetical  figures  in  the  table)  is  probably  too  large  since 

TABLE  II 

Assimilation  of  glycine- Cu  by  Ophiactis  during  24  hours  following  a  30-minute  exposure. 

Radioactivity  is  expressed  as  counts  per  minute  per  milligram.     The  parenthetical 

figures  include  an  estimate  for  Cu  Oz  lost  to  the  atmosphere  based  on 

separate  measurements;  n  =  10  for  all  groups 


Time 

Alcohol  extract 

Brei 

Medium 

Total 

0 

573  ±41 

10.4  ±  1.9 



583 

24  hrs. 

360  ±  67 

150  ±  27 

27 

537 

(114) 

(624) 

UPTAKE  OF  AMINO  ACIDS  BY  OPHIACTIS 


177 


the  free  surface  was  not  comparable  in  the  two  situations,  but  some  additional  CO2 
was  certainly  evolved. 

A  series  of  observations  was  undertaken  to  explore  the  effect  of  various 
inhibitors  on  accumulation  of  glycine  by  Ophiactis  and  its  assimilation  into  alcohol- 
insoluble  compounds.  In  each  case,  the  animals  were  kept  in  solutions  containing 
the  inhibitor  for  one  hour  prior  to  exposure  to  labelled  glycine  in  the  presence  of 
the  inhibitor.  Inhibitor  concentrations  of  10"3,  10~4,  and  10~5  M  were  employed 
(except  for  N2  where  the  animals  were  kept  in  nitrogen-saturated  sea  water). 
The  data  are  summarized  in  Table  III. 

It  is  apparent  that  both  accumulation  and  assimilation  can  be  inhibited  by  a 
variety  of  agents.  There  is  no  reason  to  think  that  any  of  the  inhibitors  used  has 
a  specific  effect  on  the  system  mediating  accumulation.  Both  processes  are  sensi- 
tive to  all  of  the  compounds  used.  Assimilation  seems  to  be  reduced  more  than 
uptake  in  almost  all  cases.  The  inhibition  noted  probably  reflects  no  more  than 

TABLE  III 

Percentage  oj  inhibition  of  accumulation  and  assimilation  of  glycine  by  the  agents  listed  at  the 

concentrations  indicated.     Glycine  concentration  was  approximately  10~6  nicies  per  liter. 

All  differences  are  significant  at  the  1%  level 


\Concentration 

10-3 

10-4 

10-6 

Inhibitor\ 

Alcohol 

Brei 

Alcohol 

Brei 

Alcohol 

Brei 

KCN 

33% 

66% 

.  * 

58% 

— 

39% 

2,4-DNP 

47% 

83% 

26% 

* 

.  * 

* 

IAA 

36% 

74% 

— 

47% 

•  — 

45% 

N3- 

— 

25% 

— 

— 

•  — 

•  — 

N4 

43% 

54% 

*  Difference  at  the  5%  level  of  significance. 
|  Nitrogen-saturated  sea  water. 

the  general  dependence  of  the  organism  on  oxidative  metabolism.  It  may  be  noted 
that  these  animals  do  not  survive  for  more  than  a  few  hours  in  nitrogen-saturated 
sea  water.  The  responses  of  Ophiactis  contrast  with  the  insensitivity  to  various  in- 
hibitors reported  for  Clymenella  and  the  latter's  capacity  to  tolerate  long  periods 
without  oxygen  (Stephens,  1963). 

The  water  content  of  animals  acclimated  to  salinities  between  100%  sea  water 
and  50%  sea  water  was  determined.  Measurements  were  also  made  of  the 
alcohol-soluble  ninhydrin-positive  material.  These  data  are  presented  in  Figure  2. 
The  ninhydrin-positive  material  is  treated  as  free  amino  acid.  Ten  amino  acids 
were  tentatively  identified  from  chromatograms  on  the  basis  of  their  Rf  values.  In 
view  of  the  reports  of  taurine  in  echinoderms  (Kittredge  et  al.,  1962),  the  o-phthal- 
aldehyde  color  reaction  (Smith,  1960)  was  used  to  check  for  its  presence  in  chroma- 
tograms. It  was  shown  to  be  present  by  this  criterion.  However,  the  largest 
single  constituent  in  the  amino  acid  pool  was  glycine. 

It  is  clear  that  decreasing  salinity  is  correlated  with  a  decrease  in  ninhydrin- 
positive  material  in  alcohol  extracts.  Uptake  of  glycine  and  of  valine  was  observed 


178 


(iKOVKK  C.  STKl'IIHXS  AND  RAGHUNATH  A.  VIRKAR 


at  \arious  salinities,  and  assimilation  into  alcohol-insoluble  material  also  determined. 
The  measurements  of  radioactivity  were  made  on  alcohol  extracts  and  breis  prepared 
after  a  30-minute  exposure  to  labelled  material  and  a  brief  rinse  in  sea  water. 
Figure  3  summari/es  the  results  for  a  typical  set  of  observations  employing  glycine. 
The  initial  increase  in  the  rate  of  accumulation  as  slightly  lower  salinities  are  en- 
countered by  the  organism  is  a  constant  feature  of  our  observations  of  this  kind. 
\Ye  also  consistently  find  an  increase  in  the  percentage  of  labelled  material 


o 
o 


LJ 
LU 
GC 


220 


200 


180 


160 


140 


120 


100 


CD 

60%      iu 


LU 

50%      H 

z 

UJ 


o 
o 


40% 


tr 

UJ 


100    90      80      70      60      50 
SALINITY      (%  SEA    WATER) 

2.  Total  free  amino  acids  (expressed  as  millimoles  per  kilogram  body  water) 
and  water  content  of  Ofhiactis  as  a  function  of  external  salinity.  Vertical  bars  represent 
standard  deviations  for  amino  acid  values,  dashed  line  the  water  content ;  n  is  10  for  amino 
acid  determinations,  5  for  water  content. 


accumulated  which  is  assimilated  into  an  alcohol-insoluble  form.  Observations 
with  valine  are  similar.  However,  although  the  ratio  of  C14  assimilated  increases 
\vith  decreasing  salinity,  the  absolute  rate  at  which  C14  appears  in  breis  prepared 
after  exposure  at  low  salinities  (50%,  60%)  m<'iy  decline.  In  any  case,  the 
specific  stimulation  of  the  assimilation  of  labelled  carbon  into  the  alcohol-insoluble 
fraction,  concomitant  with  decreased  uptake,  contrasts  with  the  effect  of  all 
inhibitors  studied. 


UITAK1-:  OK  AMINO  ACIDS  BY  OPHIACTIS 


179 


A  set  of  observations  was  carried  out  to  study  the  time  course  of  the  change 
in  level  of  the  free  amino  acid  pool  as  well  as  the  time  course  of  the  changes  in 
accumulation  and  assimilation.  Animals  were  transferred  from  100%  sea  water 
to  60%  sea  water.  Groups  of  ten  animals  were  exposed  to  glycine-C14  and  sacri- 
ficed at  intervals  for  ten  days  following  the  transfer.  The  radioactivity  of  the 


(40)160 


(35)140  - 


(30)120  - 


(25)100 


tr 


(20)80 


(15)60 


o      (10)40 


(5)20 


I                            1 

100        90 
SALINITY 

i                    r"               "i 

80         70         60 

(  %    SEA    WATER  ) 

50 

FIGURE  3.  Radioactivity  (expressed  as  counts  per  minute  per  milligram  in  0.5  nil. 
extract  or  brei)  recovered  in  the  alcohol-soluble  (solid  curve)  and  alcohol-  insoluble  (dashed 
curve,  parenthetical  figures  on  ordinate)  fractions  of  Ophiactis  adapted  to  various  salinities. 
The  animals  were  exposed  to  glycine-C14  in  their  respective  media  fur  30  minutes ;  n  is  10  in 
all  cases.  Vertical  bars  represent  standard  deviations. 


alcohol-soluble  and  the  alcohol-insoluble  fractions  was  determined  for  each  animal. 
Measurements  were  made  of  the  free  amino  acid  pool  in  each  group.  A  control 
group  which  was  kept  in  100%  sea  water  under  otherwise  comparable  conditions 
was  sampled  during  the  same  period.  Figure  4  presents  the  change  in  the  free 
amino  acid  pool  during  the  ten-day  period  for  the  two  groups.  Figure  5  presents 


180 


GROVER  C.  STEPHENS  AND  RAGHUNATH  A.  VIRKAR 


the  percentage  of  the  total  radioactivity  which  was  found  in  the  alcohol-insoluble 
fraction  of  the  animals.  It  will  be  noted  that  the  response  of  the  free  amino  acid 
pool  is  rather  slow.  This  is  also  true  with  respect  to  the  stimulation  of  incorpora- 
tion of  radioactivity  into  alcohol-insoluble  compounds. 

DISCUSSION 

Like  almost  all  other  marine  invertebrates  which  have  been  examined,  Ophiactis 
is  capable  of  removing  amino  acids  from  extremely  dilute  solution  in  the  surround- 
ing sea  water.  The  failure  to  obtain  a  definite  maximum  velocity  of  uptake 
contrasts  with  results  which  have  been  reported  previously.  The  continued  increase 
in  rate  which  was  observed  over  an  ambient  concentration  range  covering  six  orders 
of  magnitude  suggests  a  diffusion  process.  However,  we  will  note  later  that  the 
"free"  glycine  pool  exceeds  even  the  highest  of  the  external  concentrations  employed 
by  a  factor  of  ten.  It  should  be  reemphasized  that  there  is  an  overall  accumulation 
of  amino  acids,  that  amino  acids  do  not  freely  exchange  with  the  medium,  and  that 
the  concentration  of  amino  acids  in  the  normal  habitat  of  these  organisms  must  be 
very  low  compared  to  the  intracellular  pool. 

Little  is  known  about  the  feeding  habits  of  this  particular  animal.  It  is  also 
difficult  to  defend  any  very  specific  comments  about  the  possible  contribution  that 
might  be  made  by  dissolved  organic  compounds.  The  close  association  of  Ophiactis 


0 
O 


200   - 


100   - 


UJ 

ui 


.00-0 


8 


10 


TIME    (DAYS) 


FIGURE  4.  Time  course  of  change  in  free  amino  acid  concentration  of  Ophiactis.  Concen- 
tration is  expressed  as  millimoles  per  kilogram  body  water.  Animals  were  transferred  from 
100%  sea  water  to  60%  sea  water  at  zero  time.  Open  circles,  60  %  sea  water;  solid  circles, 
controls  in  100%  sea  water.  Each  point  is  average  of  ten  animals. 


UPTAKE  OF  AMINO  ACIDS  BY  OPHIACTIS 


181 


O 
< 
O 

Q 

< 

o: 

LJ 

_J 

m 


o 

CO 


o 

X 
O 
O 


40 


30 


20 


10 


8 


10 


TIME     (DAYS) 


FIGURE  5.  Radioactivity  in  the  alcohol-insoluble  fraction  expressed  as  percentage  of  total 
radioactivity  in  Ophiacfis  exposed  to  glycine-C"  for  30  minutes  at  various  periods  of  time 
following  transfer  to  60%  sea  water.  In  every  case,  ten  animals  each  in  60%  and  100% 
sea  water  were  used.  Open  circles,  60%  sea  water;  solid  circles,  100%  sea  water. 

with  mussel  beds  and  with  sponges  involves  a  microhabitat  concerning  which  we 
have  no  chemical  information  whatever.  Nonetheless,  it  is  worth  looking  at  the 
relation  between  the  observed  rate  of  amino  acid  accumulation  and  the  metabolic 
needs  of  the  animal. 

Measurements  of  the  oxygen  consumption  of  animals  in  the  size  range  of  10  to 
30  milligrams  wet  weight,  using  a  Gilson  respirometer,  gave  approximately 
0.147  ml.  O2/gm./hr.  This  is  roughly  equivalent  to  147  micrograms  of  glycine/ 
gm./hr.  Our  measurements  indicate  that  the  animals  can  obtain  64  micro- 
grams/gm./hr.  at  an  ambient  concentration  of  10  micromoles  glycine  per  liter 
(0.75  mg./l.).  This  is  roughly  the  concentration  of  glycine  measured  in  the 
interstitial  water  of  mud  flats  (Stephens,  1963)  and  in  inshore  water  samples 
(Belser,  1959,  1963).  This  represents  43%  of  the  organic  material  necessary 
to  support  the  observed  oxygen  consumption.  Since  we  have  no  information 
concerning  conditions  in  the  immediate  environment  of  these  brittle  stars,  we  can 
only  note  that  very  modest  ambient  concentrations  would  permit  this  pathway  to 
make  a  significant  contribution  to  the  energy  needs  of  the  organism. 

The  production  of  C14(X  indicates  that  the  amino  acids  entering  the  organism 
are  available  for  oxidation.  Assimilation  of  labelled  carbon  into  the  alcohol- 
insoluble  fraction  implies  that  this  material  contributes  to  synthesis  path\vays. 
A  rough  estimate  concerning  the  relative  magnitude  of  the  contribution  of  the 


182  C.ROYKK  C  STKIMIFNS   AXP  KAlMIUNATII  A.  VIRKAR 

aniino  acid  pool  to  energ\  metabolism  can  he  made.  Although  complete  quanti- 
tative information  concerning  the  individual  amino  acids  in  alcohol  extracts  is  not 
available,  rough  estimates  were  made  by  comparing  ehromatograms  with  controlled 
chromatograms  of  known  amounts  of  glyeine  and  tanrine.  An  estimate  of  0.1  mole 
of  gKcine  per  kilogram  of  body  water  is  reasonable  for  normal  salinities.  At  least 
5'  ,  of  the  labelled  glycine  which  is  accumulated  appears  as  carbon  dioxide  in  our 
ol  -cnations  (Table  II).  This  implies  that  this  percentage  of  the  pool  has  been 
oxidi/ed.  This  figure  may  be  higher  if  one  assumes  that  the  labelled  carbon  which 
disappears  from  the  system  in  the  observations  summarixed  in  Table  T  represents 
oxidixed  material.  The  figure  would  then  rise  as  high  as  40c/c.  The  glycine  pool 
represents  about  7.5  mg.  g.  body  water  or  about  3  nig.  gm.  wet  weight  at  normal 
salinities,  llence  0.15  to  1.2  mg.  of  glycine  enters  oxidation  pathways.  The 
daily  requirement  on  the  basis  of  O2  consumption  is  about  3.5  mg.  Hence  glycine 
miclit  account  for  approximately  4c/c  to  34^r  of  this  requirement.  Although  the 
estimate  is  rough,  it  probably  brackets  the  typical  contribution  of  the  glycine  pool 
to  energy  metabolism  and  indicates  that  it  is  ancillary  and  not  the  primary  energy 
source. 

\Ye  can  estimate  major  outputs  which  influence  the  size  of  the  glycine  pool 
in  these  animals.  Energy  metabolism  drains  0.15  to  1.2  mg.  of  glycine  per  gram 
wet  weight  from  the  pool  per  day.  Assimilation  into  alcohol-insoluble  compounds 
removes  about  12r('  of  the  pool  per  day  (Table  I)  or  about  0.36  mg.  per  gram  wet 
weight.  We  can  ask  whether  rates  of  uptake  measured  in  these  animals  could 
contribute  to  maintaining  the  pool  in  the  face  of  these  deficits.  Again,  we  must 
simply  assume  some  reasonable  ambient  concentration  failing  direct  information. 
If  we  accept  the  figure  of  10  micromoles  per  liter  suggested  above,  the  input  to 
the  pool  amounts  to  slightly  more  than  1.5  mg.  glvcine  per  gram  wet  weight  per 
day.  The  fact  that  this  figure  balances  the  losses  indicated  so  closely  is  of  course 
gratuitous.  However,  one  may  suggest  that  the  uptake  of  amino  acids  from  the 
ambient  medium  is  potentially  capable  of  maintaining  the  size  of  the  free  amino  acid 
pool  and  supplying  the  known  drains  on  that  pool.  This  suggestion  rests  on  the 
assumption  that  the  modest  amounts  of  free  amino  acid  stipulated  occur  in  the 
specialized  habitat  of  this  organism. 

In  common  with  many  other  marine  invertebrates,  Ophiactis  responds  to  a  reduc- 
tion of  salinity  by  a  decrease  in  the  pool  of  alcohol-soluble  ninhydrin-positive 
materials.  If  allowance  is  made  for  the  increased  water  content  of  the  organisms, 
the  pool  decreases  to  about  83 rr  of  its  normal  size  in  70%  sea  water.  Further 
apparent  decreases  a:  60'  !  and  50ro  sea  water  are  produced  almost  entirely  by 
the  increase  in  water  content  of  the  organisms.  It  should  be  noted  that  the 
present  data  are  not  comparable  to  those  reported  by  Yirkar  (  1°63.  l0^?")  in  which 
a  large  initial  response  of  the  free  amino  acid  pool  was  noted.  It  was  possible 
to  distinguish  between  intracellular  fluid  and  coelomic  fluid  in  the  case  of  Golfingia. 
Since  no  such  distinction  was  made  in  Opliiactis.  one  cannot  directly  assess  the 
effectixeness  of  the  reduction  of  the  free  aniino  acid  pool  in  the  latter  as  an  osmo- 
regulatory  response.  However,  there  is  no  inconsistency  in  the  two  reports. 

Our  observations  indicate  that  the  effect  of  a  small  decrease  in  salinity  is  an 
increase  in  both  the  rate  of  accumulation  and  the  rate  of  assimilation  of  amino  acids. 
As  salinity  is  reduced  further,  the  rate  of  accumulation  drops  but  assimilation 


UPTAKE  OF  AMINO  ACIDS  BY  OPHIACTIS  183 

increases  in  rate.  At  50%  sea  water,  the  rate  of  uptake  of  glycine  is  about  half 
its  value  at  normal  salinities  but  the  rate  of  assimilation  has  increased  by  a  factor 
of  seven.  If  lowering  the  salinity  merely  interfered  with  the  energy  metabolism 
of  the  organisms,  we  would  expect  a  decrease  in  the  rate  of  assimilation  as  was  the 
case  with  all  the  metabolic  inhibitors  employed. 

There  are  two  major  ways  in  which  one  might  account  for  the  increase  in 
rate  of  assimilation  at  reduced  salinities.  They  are  not  mutually  exclusive.  As 
the  size  of  the  pool  decreases,  there  would  be  an  apparent  increase  in  .the'  rate  of 
assimilation  in  our  experiments  because  of  the  increased  specific  activity  of  labelled 
glycine  in  the  pool.  This  may  be  an  element  in  the  observed  response.  It  does  not 
seem  to  be  a  sufficient  explanation.  The  total  decrease  in  alcohol-soluble  amino 
acids  amounts  to  about  50%  ;  the  apparent  increase  in  rate  of  assimilation  of  glycine 
is  about  700%.  Although  previous  reports  have  indicated  that  particular  amino 
acids  such  as  glycine  and  alanine  may  be  disproportionately  involved  in  responses 
to  salinity  change,  chromatography  does  not  indicate  any  change  in  concentration 
of  glycine  of  this  magnitude  in  our  animals.  Hence,  the  specious  increase  in  assimi- 
lation rate  which  would  be  produced  by  the  decrease  in  si/e  of  the  pool  is  probably 
not  the  most  important  effect  reflected  by  our  data. 

The  other  possibility  is  that  a  decrease  in  salinity  may  produce  an  increase  in 
the  rate  of  incorporation  of  free  amino  acids  into  polypeptide  and  that  this  is  the 
cause  of  the  decrease  in  size  of  the  pool.  This  seems  reasonable.  Incorporation 
of  the  free  amino  acids  into  an  osmotically  inactive  pool  would  be  the  most 
economical  way  to  reduce  the  pool.  It  it  apparent  from  the  magnitude  of  the 
increase  in  rate  of  assimilation  that  this  could  provide  a  rapid  adjustment.  At 
normal  salinities  about  3%  to  5%  of  the  pool  is  assimilated  in  a  30-minute  period. 
In  50%  sea  water,  about  36%  of  the  pool  is  assimilated  in  the  same  time. 

There  is  an  apparent  discrepancy  between  the  time  course  of  the  change  in  the 
free  amino  acid  concentration  of  Ophiactis  (Fig.  4)  and  the  time  course  of 
stimulation  of  incorporation  of  radioactivity  into  the  alcohol-insoluble  fraction  of 
the  animals  (Fig.  5).  The  drop  in  the  size  of  the  pool  follows  this  stimulation  by 
as  much  as  a  day.  A  decrease  in  salinity  must  also  produce  an  increase  in  the 
rate  at  which  glycine  is  returned  to  the  pool,  so  that  it  is  more  accurate  to  think  of 
the  salinity  stress  as  stimulating  both  the  exit  from  and  the  entry  into  the  pool. 
The  acclimation  in  turnover  rate  which  is  observed  after  several  days  in  60% 
sea  water  is  accompanied  by  a  maintained  lower  level  of  free  amino  acids.  Pre- 
sumably a  new  steady-state  balance  between  inputs  and  outputs  is  reflected  in 
the  data.  This  slow  change  is  reminiscent  of  the  slow  acclimation  to  increased 
salinity  of  the  accumulation  system  in  Nereis  limnicola  (Stephens,  1964).  A 
detailed  explanation  of  the  decline  in  alcohol-insoluble  radioactivity  after  prolonged 
exposure  to  reduced  salinity  awaits  further  work. 

Since  labelled  carbon  dioxide  is  produced,  metabolism  of  the  free  amino  acids 
cannot  be  limited  to  the  shuttling  back  and  forth  from  polypeptides  to  the  pool  which 
the  autoradiographs  might  suggest.  The  specific  activity  of  the  glycine  pool  is 
very  low  despite  considerable  radioactivity  in  the  alcohol-soluble  fraction  because 
of  the  remarkable  concentration  of  the  free  amino  acid  pool.  It  is  probably  this 
which  accounts  for  the  failure  to  note  intermediate  compounds  in  oxidative  pathways 
by  autoradiography. 


184  GROVER  C.  STEPHENS  AND  RAGHUNATH  A.  VIRKAR 

The  pattern  of  regulation  in  Ophiactis  which  is  suggested  hy  these  observations 
depends  on  regulation  of  the  rate  of  synthesis  and  breakdown  of  polypeptide  in 
response  to  a  change  in  salinity.  It  is  surprising  to  find  that  the  initiation  of  these 
changes  is  as  slow  as  it  seems  to  be.  The  fact  that  24  to  48  hours  are  required 
to  produce  a  reduction  in  free  amino  acids  in  response  to  a  sharp  challenge  suggests 
that  the  initial  survival  of  the  animal  depends  on  its  capacity  to  accommodate  to 
drastic  change.  The  great  diversity  of  osmoregulatory  mechanisms  which  have 
been  described  with  regard  to  inorganic  ion  regulation  make  it  very  risky  to  attempt 
to  generalize  from  the  work  we  are  reporting.  We  contemplate  studies  of  other 
euryhaline  animals  to  provide  comparative  data. 

SUMMARY 

1 .  The  brittle  star  Ophiactis  arenosa  shows  uptake  of  C14-labelled  glycine,  valine, 
alanine  and  arginine  from  dilute  solution.     The  process  is  linear  with  time  for  at 
least  30  minutes. 

2.  The  bulk  of  the  radioactivity  accumulated  during  a  30-minute  exposure  to 
glycine-C14  remains  in  alcohol-soluble  form.     Autoradiography  reveals  the  radio- 
activity to  be  associated  with  glycine. 

3.  If  the  animals  are  allowed  to  remain  in  sea  water  following  such  an  exposure, 
there  is  a  gradual  assimilation  of  the  label  into  alcohol-insoluble  compounds.     Some 
radioactivity  appears  as  C14O2,  implying  oxidation  of  the  amino  acid. 

4.  A  double-reciprocal  plot  of  concentration  against  rate  of  uptake  does  not 
3,'ive  a  straight  line.     Even   at  ambient   concentrations   as   high   as    10~2   M,   the 
accumulation  system  apparently  is  not  saturated. 

5.  Common  metabolic  inhibitors  decrease  the  rate  of  both  accumulation  and 
assimilation. 

6.  The  free  amino  acid  pool  of  Ophiactis  in  100%  sea  water  is  of  the  order  of 
200  inM/kg.  body  water.     In  animals  subjected  to  reduced  salinities,  there  is  a 
decrease  in  the  size  of  the  pool  corresponding  to  the  degree  of  dilution  of  the 
medium. 

7.  One-  and  two-dimensional  chromatograms  of  alcohol  extracts  of  the  animals 
show  several  ninhydrin-positive  spots,  of  which  glycine,  alanine,  threonine,   and 
taurine  are  most  prominent. 

8.  When  animals  maintained  at  reduced  salinities  are  exposed  to  labelled  glycine 
or  valine,  the  response  to  modest  decrease  in  salinity  is  a  stimulation  of  uptake. 
As  salinity  is  decreased  further,  there  is  a  decrease  in  the  rate  of  accumulation. 
In  all  cases,  however,  there  is  a  marked  increase  in  the  rate  of  assimilation  of  the 
accumulated  material  into  alcohol-insoluble  compounds. 

9.  The  response  to  reduced  salinity,  with  respect  to  both  the  size  of  the  free 
amino  acid  pool  and  the  incorporation  of  the  label  into  alcohol-insoluble  fraction,  is 
slow,  occurring  over  a  period  of  several  days. 

10.  The  significance  of  the  results  is  discussed  in  terms  of  the  energy  relations 
of  the  animals,  and  the  functions  of  the  free  amino  acid  pool. 

LITERATURE  CITED 

AWAPARA,  J.,  1962.  Free  amino  acids  in  invertebrates  :  a  comparative  study  of  their  distribu- 
tion and  metabolism.  In:  Holden,  J.  T.,  Ed.  Amino  Acid  Pools.  Distribution, 
Formation  and  Function  of  Free  Amino  Acids.  Elsevier,  Amsterdam. 


UPTAKE  OF  AMINO  ACIDS  BY  OPHIACTIS  185 

BELSER,  W.  L.,  1959.     Bioassay  of  organic  micronutrients  in  the  sea.     Proc.  Nat.  Acad.  Sci., 

45:  1533-1542. 
BELSER,  W.  L.,   1963.     Bioassay  of  trace  substances.     In:   The   Sea,   Ideas  and  Observations. 

Goldberg  et  al,  Ed.,  Wiley,  New  York,  vol.  2:  220-231. 
CLARK,     MARY,     1964.     Biochemical     studies    on     the    coelomic    fluid     of    Ncphtys    lunnhcrgi 

(Polychaeta:  Nephtyidae),  with  observations  on  changes  during  different  physiological 

states.     Biol.  Bull.,  127:  63-84. 
FLORKIN,    M.,    1962.     La    regulation    isosmotique    intracellulaire    chez    les    invertebres    marins 

euryhalins.     Bull.  I' Acad.  Royale  dc  Bclg.,  48:  687-694. 
KITTREDGE,  J.  S.,  D.  G.  SiMONSON,  E.  ROBERT  AND  B.  JELINEK,   1962.     Free  amino  acids   of 

marine    invertebrates.     In:    Holden,    J.    T.,    Ed.     Amino    Acid    Pools.     Distribution, 

Formation  and  Function  of  Free  Amino  Acids.     Elsevier,  Amsterdam. 
LANGE,  R.,   1963.     The  osmotic  function  of  amino  acids  and  taurine  in   the  mussel,  Mytihts 

cdulis.     Comp.  Biochem.  Physiol.,  10:  173-179. 
LANGE,  R.,  1964.     The  osmotic  adjustment  in  the  echinoderm,  Strongylocentrotus  drocbachicnsis. 

Comp.  Biochem.  Physiol.,  13:  205-216. 
POTTS,  W.  T.  W.,   1958.     The  inorganic  and  amino  acid  composition  of  some  lamellibranch 

muscles.    /.  Exp.  Biol.,  35:  749-764. 
SHAW,  J.,  1958.     Osmoregulation  in  the  muscle  fibers  of  Carcimis  nmcnas.    J.  Exp.  Biol.,  35: 

920-929. 
SMITH,   I.,   1960.     Chromatographic  and  Electrophoretic  Techniques.     Interscience   Publishers, 

New  York.     617  pp. 
STEPHENS,  G.  C,  1962.     Uptake  of  organic  material  by  aquatic  invertebrates.     I.     Uptake  of 

glucose  by  the  solitary  coral,  Fungia  scutaria.     Biol.  Bull.,  123:  648-659. 

STEPHENS,  G.  C.,  1963.     Uptake  of  organic  material  by  aquatic  invertebrates.     II.     Accumula- 
tion  of   amino   acids    by    the   bamboo   worm,    Clymenella    torquata.     Comp.    Biochem. 

Physiol.,  10:  191-202. 
STEPHENS,  G.  C.,  1964.     Uptake  of  organic  material  by  aquatic  invertebrates.     III.     Uptake  of 

glycine  by  brackish-water  annelids.     Biol.  Bull.,  126:  150-162. 
STEPHENS,  G.  C.,  AND  R.  A.  SCHINSKE,  1961.     Uptake  of  amino  acids  by  marine  invertebrates. 

Liiuiwl.  and  Occanog.,  6:  175-181. 
STEPHENS,  G.  C.,  AND  R.  A.  VIRKAR,  1965.     Accumulation  and  assimilation  of  amino  acids  by 

the  brittle  star,  Ophiactis  simplex.    Amcr.  Zool.,  5:  661. 
STEPHENS,  G.  C.,  J.  F.  VAN  PILSUM  AND  DORRIS  TAYLOR,  1965.     Phylogeny  and  the  distribution 

of  creatine  in  invertebrates.     Biol.  Bull..  129:  573-581. 
VIRKAR,  R.  A.,  1963.     Amino  acids  in  the  economy  of  the  sipunculid  worm,  Golfingia  gouldii. 

Biol.  Bull.,  125:  396-397. 
VIRKAR,  R.  A.,  1965.     The  role  of  free  amino  acids  in  the  intracellular  isosmotic  regulation  in 

the  sipunculid  Golfingia  gouldii.     Amcr.  Zool.,  5:  660-661. 


THE    EFFECT    OF    TEMPERATURE    UPON    THE    GROWTH    OF 
LABORATORY-HELD  POSTLARVAL  PENAEUS  AZTECUS  x 

ZOULA  P.  ZEIN-ELDIN  AND  GEORGE  W.  GRIFFITH 
Bureau  of  Commercial  Fisheries,  Galveston,  Texas 

The  general  life-history  of  North  American  shrimp  of  the  genus  Penaeus  has 
been  known  for  some  time  (Weymouth,  Lindner  and  Anderson,  1933;  Pearson, 
1939;  Burkenroad,  1934).  Adults  of  the  white  shrimp,  P.  sctifcrus,  the  most 
intensely  studied  species,  spawn  offshore.  The  young  move  to  the  estuaries  and, 
after  a  period  of  rapid  growth,  return  to  the  offshore  spawning  grounds.  During 
the  estuarine  phase  of  the  life  cycle,  the  postlarvae,  and  later  the  juveniles,  are 
exposed  to  wide  variations  of  temperature  and  salinity.  Although  it  has  been 
suggested  that  the  lower  salinities  of  the  estuaries  are  necessary  to  the  growth  and 
survival  of  these  postlarval  penaeids  (Pearse  and  Gunter,  1957;  Gunter,  Christmas 
and  Killebrew,  1964),  recent  laboratory  studies  indicate  that  salinity  per  se  has 
little  effect  on  growth  of  postlarval  P.  aztecus  (Zein-Eldin,  1963). 

Zein-Eldin  and  Aldrich  (1965)  suggested  that  temperature  was  of  greater 
significance  than  salinity  for  growth  and  survival  of  P.  aztecus.  Their  experiments 
were,  however,  conducted  at  only  four  temperatures:  11°,  18°,  25°,  and  32°  C. 
The  resulting  data  indicated  greater  differences  in  growth  rate  between  groups 
held  one  month  at  11°  and  18°  C.  or  those  at  18°  and  25°  C,  than  between  groups 
held  at  25°  and  32°  C.  The  greatest  growth-differential  per  7  degrees  was  between 
18°  and  25°  C. 

As  a  result  of  these  experiments,  we  decided  to  make  a  more  exhaustive  study 
of  the  effects  of  temperature  in  the  range  (15°-35°  C.)  commonly  encountered  by 
the  postlarvae. 

METHODS 

Postlarval  P.  astecus  were  obtained  from  the  surf  zone  of  the  Gulf  of  Mexico 
at  the  entrance  to  Galveston  Bay  and  kept  in  the  laboratory  for  24  hours  before 
introduction  into  experimental  aquaria.  Postlarvae  were  tested  at  five  constant 
temperatures  in  each  of  two  series.  In  Series  1,  we  tested  at  temperatures  of  15° 
through  25°  C.  at  intervals  of  2.5°,  and  in  Series  2,  temperatures  of  25°  through 
35°  C.  at  the  same  intervals.  Animals  were  obtained  for  Series  1  in  April,  1964, 
from  water  of  about  23°  C.,  and  for  Series  2  in  August,  1964,  from  water  of 
about  29°  C. 

Groups  of  20  postlarvae  were  placed  in  glass  aquaria  containing  4  liters  of 
continuously  aerated  and  filtered  bay  water  (salinity  approximately  25/£c).  Five 
such  aquaria,  prepared  as  described  by  Zein-Eldin  (1963),  containing  a  total  of 
100  animals,  were  held  in  darkness  at  each  constant  temperature  in  B.O.D.-type 

1  Contribution  No.  216,  Bureau  of  Commercial  Fisheries  Biological  Laboratory,  Galveston, 
Texas. 

186 


EFFECTS  OF  TEMPERATURE  ON  P.  AZTECUS 


187 


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A 22.5  (98) 


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30          35 


FIGURE  1.     Mean  increase  in  weight  of  P.  o5^n/j  postlarvae  exposed  to  different  temperatures 
(°C.)  for  one  month.     Figures  in  parentheses  indicate  per  cent  survival. 


188 


EFFECTS  OF  TEMPERATURE  ON  P.  AZTECUS 


189 


34-, 


29- 


24- 


19- 


I 
l- 
o 


14- 


10- 


SERIES     2 


25 


35 


35, 


< 

UJ 


30- 


25- 


20- 


15- 


10- 


SERIES     i 


.  25   C 


10  20 

TIME      (DAYS) 


30 


40 


FIGURE  2.     Mean  increase  in  length  of  P.  astecus  postlarvae  exposed  to 
different  temperatures  for  one  month. 


190 


ZOULA  P.  ZEIN-ELDIN  AND  GEORGE  W.  GRIFFITH 


INITIAL 


SERIES 


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H  17.5s 


INITIAL 


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25° 


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LENGTH     (MM.) 


35 


40 


50 


FIGURE  3.     Length  range  (mm.)  of  postlarval  P.  azteciis  surviving  exposure  to 
various  temperatures  for  one  month. 

incubators.     The  incubators  were  adjusted  over  a  period  of  36  hours  from  room 
temperature  (approximately  23°  C.)  to  the  final  experimental  temperature. 

Experimental  animals  were  fed  sufficient  live  nauplii  of  brine  shrimp  (Artemia) 
to  keep  food  constantly  available  during  the  month  of  the  study.     At  each  sampling 


l.Or 


0.8 


0.6 


LU 

I- 
< 

cr 


0.4 


O 
cr 


0.2 


15 


20  25 

TEMPERATURE     (°C.) 


30 


35 


FIGURE  4.       Growth  rate    (mm. /day)    of  P.  aztccus  postlarvae  held  one  month  at  different 
temperatures.     Rate  determined  by  inspection  from  straight-line  portions  of  curves  in  Figure  2. 


EFFECTS  OF  TEMPERATURE  ON  P.  AZTECUS 


191 


period  (about  5-day  intervals),  two  postlarvae  were  taken  from  each  aquarium. 
The  10  animals  from  a  given  temperature  constituted  a  sample.  Postlarvae  were 
weighed  and  measured  individually  as  described  by  Zein-Eldin  (1963).  At  the 
completion  of  the  study,  all  remaining  shrimp  were  weighed  and  measured  individ- 
ually to  check  the  reliability  of  the  sampling  procedure  (Table  I)  and  to  determine 
the  percentage  survival  at  each  temperature. 


RESULTS 


Grozvth 


The  final  mean  size  of  the  postlarvae,  whether  derived  from  length  or  weight 
(Figs.  1  and  2,  and  Table  I),  increased  with  temperature  between  15°  and  32.5°  C. 
Growth  rate,  however,  decreased  markedly  at  35°  C.,  an  effect  evident  in  the 
samples  taken  on  the  llth  day.  The  greatest  difference  in  growth  between  adjacent 
temperatures  occurred  between  17.5°  and  20°  C.  and  between  22.5°  and  25°  C. 
(Fig.  3).  As  a  result,  the  increase  in  growth  rate  per  unit  of  temperature  was 
greatest  in  the  temperature  range  17.5°  to  25°  C.  This  differential  effect  of 
temperature  is  illustrated  in  Figure  4,  an  S-shaped  curve  with  a  maximum  (at  30° 


ICOi- 


80 


60 


cc 

(T> 

\- 
H 
LU 


40 


LJ 
CL 


20 


0 


15 


20  25 

TEMPERATURE      (°C.) 


30 


FIGURE  5.     Per  cent  survival  of  P.  aztecus  postlarvae  held  at  different  temperatures 

for  one  month, 


192 


ZOULA  P.  ZEIN-ELDIN  AND  GEORGE  W.  GRIFFITH 


to  32.5°  C.)  typical  of  invertebrate  growth  responses  to  temperature  (Needham, 
1964,  p.  416).  In  Series  2,  final  size  did  not  differ  significantly  between  groups 
exposed  to  temperatures  of  27.5°  to  32.5°  C.  (Fig.  3). 

Survival 

The  relation  of  survival  to  temperature  was  somewhat  different.  The  per- 
centage survival  increased  with  temperature  between  15°  and  20°  C.,  remained 
above  90%  at  22.5°  and  25°  C.,  but  dropped  at  temperatures  above  25°  C.  (Fig.  5). 
At  35°  C.,  no  animals  remained  after  the  15th  day.  A  similar  decrease  in  survival 
at  32°  C.  as  compared  with  25°  C.  was  noted  in  earlier  experiments  in  our 
laboratory  (Table  II). 

Gross  production 

Gross  production,  as  estimated  by  comparing  the  total  weight  gains  of  the  post- 
larvae  surviving  exposure  to  each  temperature,  was  used  to  assess  the  combined 

TABLE  II 
Comparison  of  growth  of  P.  aztecus  postlarvae  from  various  experiments 


Experiment 
no. 

Date 

Volume 
of 
experi- 
mental 
unit 
(liters) 

No.  of 
animals 
per 
experi- 
mental 
unit 

Illumi- 
nation* 

Temper- 
ature 
(°C.) 

Mean 
increase 
in  length 
(mm.) 

Dura- 
tion of 
experi- 
ment 
(days) 

Mean 
growth 
rate 
(mm./ 
day) 

A'/A"** 

Survival 
(%) 

GS-lf 

4/62 

45 

100 

+ 

24.0 

19.0 

28 

0.68 

1.00 

36 

GS-2f 

8/62 

45 

100 

+ 

26.0 

17.4 

29 

0.60 

1.00 

100 

GTS-  Iff 

3-4/63 

45 

100 

+ 

25.0 

22.3 

28 

0.80 

1.00 

100 

GT-F1 

4/64 

45 

100 

+ 

25.0 

25.2 

30 

0.84 

1.00 

100 

Series  1# 

4/64 

4 

20 

— 

25.0 

21.0 

31 

0.68 

1.00 

91 

GTS-3 

8/64 

45 

90 

+ 

25.0 

20.3 

28 

0.72 

1.00 

100 

Series  2## 

8/64 

4 

20 

— 

25.0 

17.0 

29 

0.59 

1.00 

82 

GTS-lft 

3-4/63 

45 

100 

+ 

32.0 

32.0 

28 

1.14 

1.38 

58 

GTS-2 

8/63 

45 

40 

+ 

32.0 

30.4 

29 

1.05 

— 

34 

GTS-3 

8/64 

45 

90 

+ 

32.0 

27.2 

28 

0.97 

1.35 

100 

Series  2## 

8/64 

4 

20 

— 

32.5 

24.6 

29 

0.85 

1.46 

32 

GTS-lft 

3-4/63 

45 

100 

+ 

18.0 

7.4 

28 

0.26 

0.32 

100 

GTS-2 

8/63 

45 

40 

+ 

18.0 

5.2 

29 

0.18 

— 

95 

GT-F1 

4/64 

45 

100 

+ 

18.0 

5.9 

30 

0.20 

0.24 

100 

Series  1# 

4/64 

4 

20 

— 

17.5 

2.5 

31 

0.08 

0.12 

78 

GTS-3 

8/64 

45 

90 

+ 

18.0 

2.0 

28 

0.07 

0.10 

100 

GTS-lft 

3-4/63 

45 

100 

+ 

11.0 

0.5 

28 

0.02 

0.025 

92 

GT-F1 

4/64 

45 

100 

+ 

11.0 

0.0 

30 

0.00 

0.00 

26 

GTS-3 

8/64 

45 

90 

+ 

11.0 

1.1 

28 

0.04 

0.05 

5 

;  +  indicates  continuous  fluorescent  illumination;  —  continuous  darkness. 

*  A*  =  mean  growth  rate  at  temperature  t;  A25  =  mean  growth  rate  at  25°. 

t  Data  from  Zein-Eldin,  1963. 
ft  Data  from  Zein-Eldin  and  Aldrich,  1965. 

/Animals  from  same  population  as  those  in  GT-F1. 
##  Animals  from  same  population  as  those  in  GTS-3. 


EFFECTS  OF  TEMPERATURE  ON  P.  AZTECUS 


193 


6  - 


O  4 


0 


20  25 

TEMPERATURE     (°C.) 


30 


35 


FIGURE  6. 


Increase  in  total  weight  of  postlarval  P.  astecus  surviving  different 
temperatures  for  one  month. 


effects  of  growth  and  mortality  (Fig.  6).  The  total  weight  gain  was  maximal  at 
temperatures  of  25°  and  27.5°  C. ;  under  laboratory  conditions,  the  increased 
mortality  at  higher  temperatures  apparently  has  a  greater  effect  on  production 
than  has  the  accelerated  growth  of  survivors. 

Food  conversion 

The  efficiency  of  food  conversion  in  these  experiments  was  10%  to  15%  lower 
than  that  determined  in  previous  work  in  which  large  tanks  and  continuous  light 
were  used  (Zein-Eldin  and  Aldrich,  1965).  Efficiencies  were  approximately  30% 
at  all  temperatures  except  15°,  17.5°,  and  35°  C.  The  efficiency  declined  to  15% 
at  17.5°  C.  and  to  9%  at  15°  C. 

Temperature  relations 

Growth  rates  at  25°  C.  were  greater  in  Series  1  than  in  Series  2  (Figs.  1  and  2). 
The  previous  temperature  history  of  the  animals  in  the  two  series  did  not  appear 
to  explain  the  differences  in  either  growth  rate  or  survival.  If  past  temperature 
history  were  a  major  factor  in  determining  growth  rate  and  survival,  animals 
obtained  in  August  would  be  expected  to  grow  faster  and  survive  better  at  high 


194  ZOULA  P.  ZEIN-ELDIN  AND  GEORGE  W.  GRIFFITH 

temperatures  (30°  C.  and  greater)  than  those  obtained  in  April  when  temperatures 
were  considerably  lower.  Conversely,  spring  animals  might  be  expected  to  grow 
and  survive  somewhat  better  at  temperatures  below  20°  C.  than  animals  obtained  at 
higher  August  temperatures.  To  compare  various  wild  populations  exposed  to  the 
same  conditions,  we  examined  the  growth  data  from  several  experiments  (identified 
only  by  code  numbers  in  Table  II)  performed  in  our  laboratory  during  the  past 
few  years.  Spring  postlarvae  did  grow  slightly  more  rapidly  at  18°  C.,  but  animals 
collected  in  August  neither  grew  more  rapidly  nor  survived  better  at  32°  to  32.5°  C. 
than  those  collected  in  the  spring  (Table  II). 

To  obtain  further  information  concerning  the  effects  of  temperature  on  a  given 
population,  the  mean  increase  in  length  of  animals  held  at  25°  C.  in  a  given  experi- 
ment was  chosen  as  a  standard,  and  the  growth  at  other  temperatures  was  compared 
with  that  at  25°  C.  (mean  growth  at  temperature  T  divided  by  mean  growth  at 
25°  C. ;  Table  II).  The  ratio  of  growth  to  that  of  the  standard  was  nearly  constant 
at  11°  or  32°  C.,  but  was  variable  between  17.5°  and  18°  C.  The  variability 
at  17.5°  to  18°  C.  may  arise  from  the  relatively  great  effect  of  temperature  on  the 
growth  rate  at  this  temperature  range. 

The  somewhat  reduced  growth  rates  in  these  series  were  apparently  caused 
by  crowding.  Higher  growth  rates  were  attained  by  animals  from  the  same 
postlarval  populations  when  reared  in  larger  illuminated  tanks  (Experiments 
GT-F1  and  GTS-3  of  Table  II).  If  the  growth  rates  of  animals  from  Experiment 
GT-F1  are  compared  with  those  of  Series  1,  or  rates  from  GTS-3  with  those  of 
Series  2,  rates  in  the  two  series  are  0.8  of  those  in  the  larger  tanks  and,  thus, 
correspondingly  larger  volumes  of  water.  This  reduced  growth  may  have  been 
the  result  of  the  smaller  water  volume  per  animal  (crowding)  rather  than  the  lack 
of  light,  since  subsequent  experiments  have  indicated  that  effects  of  light  are 
negligible. 

It  seems  apparent  that  in  laboratory  studies,  actual  growth  rates  of  the  animals 
depend  upon  the  particular  natural  population  of  postlarvae  used.  Comparisons 
of  the  relative  effects  of  temperature  are,  however,  valid  within  a  given  group 
of  animals. 

ECOLOGICAL  IMPLICATIONS 

A  relation  between  temperature  and  growth  rate  of  P.  astecus  has  also  been 
suggested  in  reports  on  field  studies  by  St.  Amant,  Corkum  and  Broom  (1963), 
and  Ringo  (1965),  who  noted  an  apparent  spurt  of  growth  when  the  water  tempera- 
ture exceeds  20°  C.  Our  results  indicate  that  this  pattern  is  a  direct  effect  of 
temperature  on  the  growth  rate.  The  laboratory  studies  reported  here  also  confirm 
the  suggestion  of  Zein-Eldin  and  Aldrich  (1965)  that  the  influence  of  temperature 
on  growth  of  postlarval  brown  shrimp  is  most  marked  in  the  18°  to  25°  C.  range. 

Within  the  range  of  15°  to  20°  C.,  small  differences  in  temperature  have  a  pro- 
nounced effect  on  the  time  needed  for  the  completion  of  postlarval  development 
in  the  laboratory  (Fig.  7).  The  calculated  time  required  for  an  average  laboratory- 
held  postlarva  to  increase  from  12  to  25  mm.  decreases  from  260  clays  at  15°  to 
108  days  at  17.5°,  and  to  36  days  at  20°  C.  Temperatures  greater  than  20°  C. 
bring  about  relatively  minor  decreases  in  the  time  required  to  complete  postlarval 
development.  That  more  rapid  growth  may  occur  in  nature,  where  fluctuations 


EFFECTS  OF  TEMPERATURE  ON  P.  AZTECUS 


195 


250 


200 


150 


CO 
Q 

LU 


100 


50 


25 


•  -SERIES    I 
O  -SERIES  2 


15' 


20°  25° 

TEMPERATURE     (°C.) 


30C 


FIGURE  7.     Number  of  days  required  for  a  12-mm.  postlarva  of  P.  astccus  to  grow  to  25  mm. 
at  different  temperatures  (based  on  slopes  in  Figure  4). 

in  temperature  are  the  rule,  must  be  re-emphasized.  It  is  probable,  however,  that 
the  growth  rate  below  20°  C.  is  too  slow  to  be  readily  observable  in  successive 
field  samples. 

Further  observations  are  recjuired  to  determine  the  degree  to  which  other  natural 
factors,  such  as  food  and  light,  influence  the  growth  of  postlarval  P.  astecus,  as  well 
as  that  of  P.  setifcrns. 

SUMMARY 

1.  The  growth  of  postlarval  brown  shrimp,  Penaeits  aztecus,  was  studied  in  the 
laboratory  at  constant  temperatures  of  15°  through  35°  C. 


196  ZOULA  P.  ZEIN-ELDIN  AND  GEORGE  W.  GRIFFITH 

2.  Growth  increased  with  temperature  up  to  32.5°  C.     Maximal  increases  of 
growth  rate  per  unit  of  temperature  were  observed  in  the  temperature  range  of 
17.5°  to  25°  C. 

3.  Survival  for  one  month  was  markedly  decreased  at  32.5°  C.,  and  no  animals 
survived  at  35°. 

4.  The  results  suggest  that  in  the  laboratory  gross  production  is  optimal  at 
temperatures  of  22.5°  to  30°  C. 

5.  Non-lethal  temperatures  can  have  a  strong  effect  on  the  time  required  to 
complete  postlarval  development. 

LITERATURE  CITED 

BURKENROAD,  M.  D.,  1934.  The  Penaeidea  of  Louisiana  with  a  discussion  of  their  world 
relationships.  Bull.  Aincr.  Mus.  Nat.  Hist.,  68:  61-143. 

GUNTER,  G.,  J.  Y.  CHRISTMAS  AND  R.  KILLEBREW,  1964.  Some  relations  of  salinity  to  popula- 
tion distributions  of  motile  estuarine  organisms,  with  special  reference  to  penaeid 
shrimp.  Ecology,  45:  181-185. 

NEEDHAM,  A.  E.,  1964.  The  Growth  Process  in  Animals.  D.  Van  Nostrand  Co.,  Inc.,  Prince- 
ton, N.  J.,  522  p. 

PEARSE,  A.  S.,  AND  G.  GUNTER,  1957.  Salinity.  In:  Treatise  on  Marine  Ecology  and 
Paleontology.  Vol.  1,  J.  W.  Hedgpeth,  ed.  Geological  Society  of  America,  Memoir 
67,  129-158,  N.  Y. 

PEARSON,  J.  C.,  1939.  The  early  life  histories  of  some  American  Penaeidae,  chiefly  the  commer- 
cial shrimp  Pcnacus  sctiferus  (Linn.).  Bull.  U.  S.  Bur.  Fisheries,  49(30)  :  1-73. 

RINGO,  R.  D.,  1965.  Dispersion  and  growth  of  young  brown  shrimp.  U.  S.  Fish  IVildl.  Serv., 
Cir.  230,  68-70. 

ST.  AMANT,  L.  S.,  K.  C.  CORKUM  AND  J.  G.  BROOM,  1963.  Studies  on  growth  dynamics  of  the 
brown  shrimp,  Pcnacus  aztcciis,  in  Louisiana  waters.  Proc.  Gulf.  Caribb.  Fish.  Inst., 
15:  14-26. 

WEYMOUTH,  F.  W.,  M.  J.  LINDNER  AND  W.  W.  ANDERSON,  1933.  Preliminary  report  on  the 
life  history  of  the  common  shrimp  Pcnacus  sctiferus  (Linn.).  Bull.  U.  S.  Bur. 
Fisheries,  4B(14)  :  1-26. 

ZEIN-ELDIN,  Z.  P.,  1963.  Effect  of  salinity  on  growth  of  postlarval  penaeid  shrimp.  Biol. 
Bull,  125:  188-196. 

ZEIN-ELDIN,  Z.  P.,  AND  D.  V.  ALDRICH,  1965.  Growth  and  survival  of  postlarval  Pcnacus 
aztccus  under  controlled  conditions  of  temperature  and  salinity.  Biol.  Bull.,  129: 
199-216. 


MECHANICAL  FORCES  AS  A  CAUSE  OF  CELLULAR 
DAMAGE  BY  FREEZING  AND  THAWING 

OLA  BODVAR  REITE 

Institute  for  Experimental  Medical  Research,  Ulleraal  Hospital,  Oslo,  Nonvay 

During  microscopic  examination  of  mammalian  red  blood  cells  exposed  to 
freezing  and  thawing  Smith,  Polge  and  Smiles  (1951)  did  not  observe  any  intra- 
cellular  ice  crystals  between  the  time  the  ice  formed  in  the  surrounding  Ringer's 
solution  and  the  time  the  cells  became  hemolyzed.  When  amoebae  in  pond  water 
were  subjected  to  a  similar  procedure,  intracellular  ice  formation  occurred.  The 
presence  of  internal  ice  crystals  in  the  amoebae  was  always  associated  with  rupture 
of  cell  membranes  and  the  amoebae  in  question  never  revived.  Their  experiments 
reveal  that  there  may  be  differences  among  animal  cells  in  their  responses  to  freezing 
at  a  fixed  temperature.  The  role  played  by  ice  formation  per  sc  is  uncertain. 

There  are  several  potential  factors  involved  with  freezing  injury  to  animal  cells. 
On  the  one  side  we  have  the  mechanical  forces  attendant  upon  ice  crystal  formation 
and  on  the  other  the  physical  and  chemical  changes  such  as  hypertonicity  or  shift 
in  pH  associated  with  withdrawal  of  water  from  solution.  The  mechanical 
factors  involved  must  be  expected  to  be  less  dependent  on  the  length  of  time  the  cells 
are  exposed  to  freezing  than  are  the  physical  and  chemical  ones.  Intracellular  ice 
formation  is  most  likely  to  occur  in  supercooled  cells  (Mazur,  1963).  Cells  will 
become  supercooled  at  freezing  rates  which  are  so  high  that  intracellular  water  can 
not  pass  through  the  cell  membrane  rapidly  enough  to  keep  the  concentration  of 
solutes  inside  the  cell  in  equilibrium  with  that  of  its  surroundings.  Assuming 
similar  qualities  for  their  cell  membranes  a  suspension  of  small  cells  would  therefore 
have  to  be  exposed  to  more  rapid  freezing  to  show  intracellular  ice  formation  than 
would  a  suspension  of  large  cells.  The  red  blood  cells  of  the  congo  eel  have  a 
diameter  which  is  10  times  the  diameter  of  the  mammalian  red  blood  cells  studied 
by  Smith,  Polge  and  Smiles  (1951).  Visual  observation  during  freezing  at  the 
freezing  rate  required  to  produce  intracellular  crystallization  in  mammalian  red 
blood  cells  may  be  impossible,  while  the  red  blood  cells  of  the  congo  eel  are  likely 
to  become  frozen  internally  at  freezing  velocities  which  permit  observations  of  the 
freezing  process.  For  this  reason  red  blood  cells  from  congo  eels  were  utilized 
in  an  attempt  to  disclose  whether  freezing  injury  due  to  mechanical  forces  could  be 
observed  separate  from  eventual  damage  resulting  from  chemical  changes.  The 
cells  could  be  studied  during  freezing  and  thawing  over  periods  short  enough  to 
avoid  hemolysis  produced  by  changes  other  than  the  mechanical  ones  associated  with 
ice  crystal  formation. 

METHODS 

Fresh  samples  of  venous  blood  from  the  congo  eel  (Amphiuma  tridactylum) 
with  nucleated  red  blood  cells  of  a  diameter  in  the  range  of  70-90  //.  were  diluted 

197 


198 


OLA  BODVAR  REITE 


with  Ringer's  solution  and  a  drop  spread  on  a  1 -mm. -thick  slide  of  the  acrylic  resin 
"Perspex"  and  covered  with  a  0.2-mm.  coverslip.  A  small  piece  of  solid  carbon 
dioxide  in  tinfoil  wras  then  placed  at  the  edge  of  the  coverglass,  and  the  freezing  of 
the  diluted  blood  was  watched  through  a  Leitz  Ortholux  microscope  with  Leica 
camera  for  photomicrography.  Magnifications  between  40  X  and  200  X  were  used. 
A  thin  layer  of  glycerol  on  the  coverglass  facilitated  observation  by  avoiding 
condensation  of  water. 

RESULTS 

With  the  piece  of  solid  carbon  dioxide  in  place,  ice  formation  in  the  diluted 
blood  began  immediately.     The  ice  crystal  front  grew  into  the  preparation  and 


FIGURE  1.  The  ice  front  is  advancing  through  diluted  blood  from  the  congo  eel.  Shrinkage 
of  the  red  blood  cells  starts  as  soon  as  they  are  reached  by  the  spear-shaped  ice  crystals.  The 
volume  occupied  by  ice  compared  to  the  volume  of  the  fluid  space  among  the  ice  crystals  indicates 
the  portion  of  water  withdrawn  from  solution.  Magnification  100  X. 

water  was  withdrawn  from  solution.  Shrinkage  of  the  red  blood  cells  started  when 
they  were  reached  by  the  ice  front  (Fig.  1)  and  continued  concurrent  with  the 
decrease  of  fluid  space  among  the  ice  crystals.  At  first  ice  crystals  appeared 
exclusively  in  the  suspending  medium.  Behind  the  ice  front,  however,  ice  crystals 
formed  suddenly  within  one  after  the  other  of  the  shrunken  cells.  The  crystals  were 
so  small  that  the  light  was  scattered  and  the  cell  interior  became  opaque  and 
appeared  quite  black  (Fig.  2,  A).  When  a  definite  extent  of  extracellular  ice 
formation  and  thereby  a  certain  degree  of  cooling  was  reached,  the  phenomenon 
would  occur  at  various  distances  behind  the  ice  front.  The  spontaneous  intracellu- 
lar  freezing  took  place  only  as  long  as  the  ice  front  was  advancing.  The  opaque 


CELLULAR  DAMAGE  FROM  FREEZING 


199 


area  sometimes  appeared  to  occupy  the  whole  cell  interior,  but  in  most  experiments 
only  the  nuclear  area  and  its  immediate  surroundings  became  opaque.  If  the  piece 
of  solid  carbon  dioxide  at  the  edge  of  the  coverglass  was  removed  when  a  few  of 
the  red  blood  cells  were  frozen  internally,  the  ice  front  receded  (Fig.  2,  B).  With 
the  exception  of  the  cells  with  intracellular  ice  crystals  the  shrunken  cells  regained 
their  shape,  but  some  of  them  showed  a  ragged  cell  surface  (Fig.  2,  C).  When 


> 


'/if 


V 


A  '> 


V 


•"f; 


FIGURE  2.  Diluted  blood  from  the  congo  eel  observed  in  three  successive  situations,  A,  B, 
and  C,  all  observations  made  on  the  same  field.  A  :  The  whole  field  is  frozen  and  in  a  few 
of  the  cells  intracellular  ice  formation  is  visibly  manifest  from  the  black  appearance  of  their 
nuclear  area.  B  :  The  ice  front  is  receding.  Dehydrated  cells  without  intracellular  ice  crystals 
start  regaining  their  normal  shape.  The  opacity  of  the  nuclear  area  of  cells  with  intracellular 
ice  crystals  disappears.  C :  Thawing  is  complete.  Some  cells  which  have  been  dehydrated 
show  a  ragged  cell  surface,  but  only  the  cells  with  intracellular  ice  crystal  formation  are 
hemolyzed.  The  nuclei  from  the  hemolyzed  cells  are  left  intact.  Due  to  movements  of  fluid 
during  thawing  many  of  the  cells  have  changed  position,  but  five  cells  with  intracellular  ice 
crystals  can  be  observed  (as  groups  of  three  and  two  cells,  respectively)  in  the  lower  part  of 
the  field  (A).  These  cells  are  easy  to  trace  through  B  and  C.  Magnification  100  X. 

the  ice  crystals  of  the  internally  frozen  cells  melted,  the  cells  did  not  imbibe  water 
to  return  to  normal  size.  Their  hemoglobin  simply  spread  out  into  the  suspending 
medium  and  apparently  intact  nuclei  were  left  at  the  former  sites  of  the  whole 
cells  (Fig.  2,  C). 

The  ice  front  advanced  more  rapidly  the  nearer  to  the  piece  of  solid  carbon 
dioxide  the  field  under  observation  was  chosen.  This  was  reflected  in  the  sequence 


200  OLA  BODVAR  REITE 

of  events  as  observed  through  the  microscope.  The  description  above  and  both 
figures  refer  to  a  field  some  distance  away  from  the  piece  of  solid  carbon  dioxide. 
Here  the  individual  cells  were  easy  to  follow  both  during  freezing  and  thawing. 
Further  away  the  ice  front  advanced  very  slowly,  and  the  cells  seemed  to  become 
completely  dehydrated  without  any  signs  of  intracellular  ice  formation.  Close  to 
the  edge  where  the  freezing  was  initiated  intracellular  ice  formation  occurred  almost 
immediately  in  all  cells  and  dehydration  could  hardly  be  observed.  Estimation  of 
freezing  temperatures  from  the  amount  of  fluid  space  between  extracellular  ice 
crystals  indicated  that  the  slowly  frozen  cells  would  tolerate  short-time  freezing  to 
lower  temperature  than  the  temperature  at  which  all  cells  were  destroyed  at  high 
freezing  velocities. 

By  adjusting  the  size  of  the  piece  of  solid  carbon  dioxide  a  frozen  area  which 
never  reached  the  opposite  edge  of  the  coverglass  was  obtained.  Different  fields 
within  this  frozen  area  were  kept  under  observation  for  2-3  minutes  with  the  ice 
front  stagnant.  The  cells  nearest  to  the  edge  where  freezing  started  were  all  frozen 
internally.  Somewhat  further  away  some  cells  were  frozen  internally  and  some 
were  dehydrated  but  unfrozen.  Towards  the  ice  front  only  dehydrated  cells  were 
found.  Upon  thawing  all  cells  in  the  previously  frozen  area  were  hemolyzed 
except  for  a  few  ones  close  to  the  ice  front. 

DISCUSSION 

The  observations  made  during  the  present  investigation  show  that  two  potential 
factors  in  cellular  injury  from  freezing  and  thawing  can  be  studied  as  separate 
processes  by  choosing  appropriate  experimental  material  and  freezing  rates.  As 
an  advocate  for  the  great  role  of  physical  and  chemical  factors  in  freezing  injury  to 
animal  cells.  Lovelock  (1953^.1957)  presented  experimental  support  for  a  theory 
involving  a  mechanism  for  cellular  damage  by  freezing  which  would  act  without 
intracellular  formation  of  ice.  He  showed  that  phospholipids  and  cholesterol  were 
lost  from  the  membranes  of  red  blood  cells  suspended  in  solutions  of  sodium 
chloride,  and  that  this  loss  was  augmented  with  increasing  concentration.  The 
results  were  identical  whether  this  increased  concentration  was  brought  about  by 
freezing  or  by  the  addition  of  sodium  chloride  to  the  initial  solution.  The  red  blood 
cells  in  this  way  became  more  permeable  to  cations,  and  with  an  excess  of  cations 
the  cells  would  slowly  swell  and  hemolyze.  Eventually  they  would  be  rapidly 
entered  by  water  molecules  and  immediately  hemolyzed  when  returned  to  physio- 
logical saline  during  thawing.  The  slowly  frozen  red  blood  cells  from  the  congo  eel 
which  were  dehydrated  without  any  signs  of  intracellular  ice  formation  and  yet 
became  hemolyzed  if  kept  in  this  state  for  a  few  minutes  before  thawing  apparently 
were  destroyed  according  to  this  theory. 

Sloviter  (1962)  emphasizes  the  possible  damaging  effect  of  mechanical  forces 
attendant  upon  the  formation  of  ice.  He  found  that  the  extent  of  hemolysis  after 
freezing  and  thawing  of  mammalian  red  blood  cells  suspended  in  non-electrolytes 
in  the  presence  of  different  concentrations  of  sodium  chloride  did  not  increase 
with  an  increase  in  ionic  strength  of  the  surrounding  medium  of  the  cells.  The 
destruction  of  red  blood  cells  by  sudden  intracellular  ice  formation,  as  shown  in 
Figure  2  in  the  present  paper,  demonstrates  that  such  a  mechanism  for  cellular 


CELLULAR  DAMAGE  FROM  FREEZING  201 

injury  may  occur.  Although  this  process  as  studied  in  the  present  investigation 
seemed  to  be  closely  associated  with  the  formation  of  ice  crystals  inside  the  cells, 
a  weakening  of  the  cell  membranes,  due  to  increased  concentration  of  salts,  may  of 
course  precede  and  promote  the  intracellular  freezing.  External  ice  crystals  may 
also  tear  or  penetrate  the  cell  membrane  and  induce  ice  formation  in  supercooled 
cells. 

The  ice  formed  inside  the  red  blood  cells  of  the  congo  eel  was  not  observed  as 
individual  ice  crystals,  but  as  a  result  of  a  scattering  of  the  light  when  the  crystals 
occurred  in  great  number.  The  opaque  area  in  most  experiments  covered  only  a 
field  somewhat  greater  than  the  cell's  nucleus,  corresponding  to  the  thickest  part  of 
the  cell.  Supercooling  followed  by  rapid  intracellular  crystallization  would  be 
expected  to  promote  the  formation  of  ice  throughout  the  cell.  The  failure  to 
observe  individual  ice  crystals  in  the  thin  portions  of  the  cell  may  perhaps  be 
ascribed  to  the  low  magnification  used  in  this  study.  That  supercooling  is  necessary 
for  intracellular  ice  formation  is  indicated  by  the  fact  that  the  process  was  always 
associated  with  an  advancing  ice  front.  \Yhen  the  ice  front  is  stagnant  the  indi- 
vidual cells  are  exposed  to  a  constant  temperature  and  apart  from  an  eventual 
recrystallization  with  changes  in  ice  crystal  size,  no  alterations  in  the  suspending 
medium  will  take  place.  The  concentrations  of  solutes  inside  the  cells  will  be  in 
equilibrium  with  those  of  the  outside.  Very  slow  freezing  will  also  allow  equi- 
librium to  be  maintained  and  supercooling  \vill  be  minimal. 

The  process  of  sudden  intracellular  ice  formation  with  the  appearance  of  cell 
opacity  during  freezing  was  described  by  Smith,  Polge  and  Smiles  (1951)  to  occur 
at  about  —8°  C.  for  the  amoeba.  After  thawing  of  the  amoeba,  its  cell  membrane 
was  ruptured  and  cytoplasmic  granules  drifted  out  into  the  medium.  Smith  and 
Smiles  (1953)  found  the  same  phenomenon  to  take  place  between  —6°  and  --12°  C. 
in  preparations  of  tissues  from  guinea  pig  testis.  The  internally  frozen  cells  from 
guinea  pig  testis  were  also  disintegrated  after  thawing.  This  shows  that  the 
response  of  the  red  blood  cells  of  the  congo  eel  to  freezing  and  thawing  is  related 
to  the  response  of  other  cell  types  investigated,  but  intracellular  ice  crystal  forma- 
tion seems  not  to  be  inevitably  lethal.  Salt  (1959)  has  demonstrated  that  the  large 
cells  in  the  fat  body  of  the  goldenrod  gall  fly,  Eurosta  solidaginis,  can  survive  freez- 
ing even  if  intracellular  ice  crystals  have  been  present. 

Considering  the  frozen  area  where  the  ice  front  was  kept  stagnant  for  some 
minutes  as  described  in  the  present  paper,  it  appears  that  several  ways  of  damage 
are  demonstrated.  Near  the  piece  of  solid  carbon  dioxide  rapid  freezing  with 
intracellular  ice  formation,  which  destroyed  all  cells,  occurred.  A  short  distance 
behind  the  ice  front  the  exposure  to  increased  salt  concentration  during  freezing 
probably  was  the  only  cause  of  injury.  At  an  intermediate  distance  some  cells 
seemed  to  be  destroyed  by  one  of  these  processes  and  some  by  the  other  one.  The 
few  cells  quite  close  to  the  ice  front  which  were  found  to  recover  after  thawing 
show  that  freezing  at  low  velocity  to  temperatures  just  below  zero  is  not  fatal 
provided  the  exposure  is  of  short  duration.  This  may  correspond  to  the  finding  of 
Lovelock  (1953)  that  the  critical  temperature  range  for  red  blood  cells  starts  at 
—  3°  C.  Once  ice  is  formed  in  the  preparation,  the  temperature  at  the  ice  front 
will  be  equal  to  the  freezing  point  of  the  solution,  and  a  temperature  of  —3°  C. 
or  lower  must  therefore  be  sought  a  short  distance  behind  the  ice  front  and 


202  OLA  BODVAR  REITE 

towards  the  piece  of  solid  carbon  dioxide.  Judged  from  the  present  investigation 
it  appears  that,  dependent  on  cell  type  and  cooling  rate,  mechanical  forces  may  be 
the  predominant  factor  in  cellular  damage  from  freezing  and  thawing  under  some 
circumstances  as  may  physical  and  chemical  changes  under  others. 

To  avoid  intracellular  ice  formation  it  is  necessary  to  cool  the  cells  at  a  slow 
rate.  That  rapid  cooling  promotes  intracellular  freezing  is  also  shown  for  sea 
urchin  eggs  (Asahina,  1961)  and  for  yeast  cells  (Nei,  1960;  Mazur,  1961).  How- 
ever, during  slow  cooling  the  time  of  exposure  of  the  cells  to  increased  salt  concen- 
trations is  prolonged  as  long  as  the  temperature  is  above  the  eutectic  points  of  these 
solutes.  To  obtain  minimum  damage  two  requirements  therefore  are  to  be  met 
with.  The  cells  must  be  cooled  slowly  enough  to  prevent  intracellular  ice  formation 
and  rapidly  enough  to  minimize  the  damaging  effects  of  exposure  to  increased  salt 
concentrations.  It  is  interesting,  then,  to  note  that  for  mammalian  red  blood  cells 
such  an  optimum  cooling  velocity  for  minimum  hemolysis  has  been  demonstrated 
(Gehenio  and  Luyet,  1958). 


I  am  grateful  to  Dr.  Joseph  Engelberg  for  careful  reading  of  the  manuscript. 
The  investigation  was  supported  by  a  research  training  award  from  the  Norwegian 
Research  Council  for  Science  and  the  Humanities. 

SUMMARY 

1.  The  present   investigation   was   initiated    in   an   attempt   to    dissociate   two 
potential  factors  in  cellular   injury  from  freezing  and   thawing:   damage   due   to 
mechanical  forces  attendant  upon  ice  crystal  formation  and  damage  due  to  physical 
and  chemical  changes  associated  with  withdrawal  of  water. 

2.  Red  blood  cells  in  diluted  blood  from  the  congo  eel  were  covered  and  sub- 
jected to  microscopic  examination  during  freezing  induced  by  a  piece  of  solid  carbon 
dioxide  placed  at  the  edge  of  the  coverglass.     Ice  crystals  grew  into  the  preparation, 
first  rapidly  and  then  more  slowly  the  further  from  the  piece  of  solid  carbon  dioxide 
they  advanced.     The  sequence  of  events  as  observed  through  the  microscope  was 
different  for  different  freezing  velocities. 

3.  Rapid  freezing  caused  intracellular  ice  formation  and  this  internal  freezing 
was  always  associated  with  hemolysis  even  if  followed  by  immediate  thawing.     At 
slow  freezing  the  cells  became  dehydrated  without  any  signs  of  intracellular  ice 
formation.     Such  cells  would  recover  if  thawing  occurred  within  a  few  seconds, 
but  they  were  all  hemolyzed  after  prolonged  exposure. 

4.  It  is  concluded  that,  dependent  on  the  freezing  rate,  either  mechanical  forces 
or  physical  and  chemical  "factors  may  be  the  main  cause  of  cellular  damage  from 
freezing,  the  mechanical  forces  being  predominant  at  rapid  freezing. 

LITERATURE  CITED 

ASAHINA,  E.,  1961.     Intracelluiar  freezing  and  frost  resistance  in  egg-cells  of  the  sea  urchin. 

Nature,  191:1263-1265. 

GEHENIO,  P.  M.,  AND  B.  J.  LUYET,  1958.     Hemolysis  and  freezing  velocity.     Fed.  Proc.,  17:  52. 
LOVELOCK,  J.   E.,   1953.     The  haemolysis  of  human   red   blood   cells   by   freezing  and   thawing. 

Biochem.  Biophys.  Ada,  10:  414-426. 


CELLULAR  DAMAGE  FROM  FREEZING  203 

LOVELOCK,   J.   E.,   1957.     Denaturation  of  lipid   protein  complexes   as   a   cause   of   damage   by 

freezing.     Proc.  Roy.  Soc.  London,  Scr.  B,  147:  427-433. 
MAZUR,  P.,  1961.     Physical  and  temporal  factors  involved  in  the  death   of  yeast  at  subzero 

temperatures.     Biophys.  J.,  1:  247-264. 
MAZUR,  P.,  1963.     Kinetics  of  water  loss  from  cells  at  subzero  temperatures  and  the  likelihood 

of  intracellular  freezing.    /.  Gen.  Physiol.,  47:  347-369. 
NEI,  T.,  1960.     Effects  of  freezing  and  freeze-drying  on  microorganisms.     In :  Recent  Research 

in  Freezing  and  Drying.     Ed.  by  Parkes,  A.  S.  &  Smith,  A.  U.     Blackwell  Scientific 

Publications,  Oxford,  pp.  78-86. 

SALT,  R.  W.,  1959.     Survival  of  frozen  fat  body  cells  in  an  insect.     Nature,  184:  1426. 
SLOVITER,    H.    A.,    1962.     Mechanism    of   haemolysis    caused   by   freezing    and    its    prevention. 

Nature,  193:884-885. 
SMITH,  A.  U.,  C.  POLGE  AND  J.  SMILES,  1951.     Microscopic  observation  of  living  cells  during 

freezing  and  thawing.     /.  Roy.  Micr.  Soc.,  71 :  186-195. 
SMITH,  A.  U.,  AND  J.  SMILES,  1953.     Microscopic  studies  of  mammalian  tissues  during  cooling 

to  and  rewarming  from  —79°  C.    /.  Roy.  Micr.  Soc.,  73:  134-139. 


INFLUENCE  OF  INDIVIDUAL  AMINO  ACIDS  ON  UPTAKE  AND 

INCORPORATION  OF  VALINE,  GLUTAMIC  ACID  AND 

ARGININE  BY  UNFERTILIZED  AND  FERTILIZED 

SEA  URCHIN  EGGS  x 

ALBERT  TYLER,  JORAM  PIATIGORSKY  AND  HIRONOBU  OZAKI  2 

Division  of  Biology,  California  Institute  of  Technology,  Pasadena,  California  91109 

In  the  course  of  investigations  (cf.  Tyler,  1965a)  into  the  mechanism  of  the 
initiation  of  protein  synthesis  hy  sea  urchin  eggs,  some  variable  results  were 
obtained  in  tests  with  dactinomycin  (actinomycin  D).  This  inhibitor  of  DNA- 
primed  RNA  synthesis  stimulated  incorporation  of  labeled  valine  into  protein  in 
four  experiments  with  suspensions  of  eggs  that  contained  many  oocytes  but 
failed  to  do  so  in  several  subsequent  tests.  The  nutritional  status  of  the  animals, 
and  consequently  of  the  eggs,  was  considered  as  one  possible  source  of  this 
variation.  Tests  were  therefore  made  of  the  effects  of  glucose,  which  experiments 
by  Honig  and  Rabinovitz  (1965)  had  shown  could  prevent  or  relieve  dactinomycin- 
induced  inhibition  of  protein  synthesis  in  sarcoma-37  cells.  However,  glucose  did 
not  enable  dactinomycin  to  enhance  incorporation  of  amino  acid  into  protein  by  sea 
urchin  eggs.  Tests  were  then  made  with  mixtures  of  amino  acids.  Again  no 
stimulation  was  obtained  with  dactinomycin  on  the  incorporation  of  a  labeled  amino 
acid.  In  these  tests  another  phenomenon  appeared,  namely,  a  marked  inhibition 
by  the  amino  acid  mixture  on  the  incorporation  of  the  labeled  amino  acid.  The 
experiments  on  the  oocytes,  including  the  erratic  dactinomycin  effect,  will  be 
reported  elsewhere  (Piatigorsky,  Ozaki  and  Tyler,  1966),  while  the  present  account 
will  deal  mainly  with  exploration  of  the  competition  among  amino  acids. 

That  the  rate  of  uptake  of  one  amino  acid  may  be  inhibited  by  the  presence 
of  others  has  been  shown  in  many  experiments  with  intact  cells  of  various  organisms 
(see  Wilbrandt  and  Rosenberg,  1961;  Christensen,  1962,  1964;  Johnstone  and 
Scholefeld,  1965,  for  review).  In  general,  the  inhibition  is  found  to  occur  between 
members  of  the  same  general  class  of  amino  acid  and  is  interpreted  as  being  due  to 
a  competition  for  transport  across  the  cell  surface. 

Since  sea  urchin  eggs  are  the  subject  of  increasing  numbers  of  investigations  of 
amino  acid  incorporation  into  protein  by  the  intact  cells,  it  seemed  to  us  desirable  to 
determine  whether  or  not  such  competition  at  the  cell  surface  occurs  with  this 
material,  too,  and  if  so,  to  examine  the  interrelationships  among  the  amino  acids. 
While  this  work  was  in  progress  a  preprint  was  received  of  an  article  by  Mitchison 
and  Cummins  (1966)  concerning  the  uptake  of  labeled  valine  and  cytidine  by  sea 

1  Supported  by  grants   from   the   National    Institutes   of   Health    (GA1    12777   and   2G-86) 
and  the  National  Science  Foundation  (GB-28). 

2  Damon  Runyon  Cancer  Research  Fellow. 

3  We  wish  to  acknowledge  the  effective  technical  assistance  of  Peter  N.  Redington,  Edgar 
E.  Vivanco  and  Jeffrey  W.  Greene. 

204 


AMINO  ACID  UPTAKE  BY  ECHINOID  EGGS  205 

urchin  eggs  at  various  stages  of  development.  In  tests  with  eggs  at  one  hour  after 
fertilization  they  report  a  marked  inhibition  of  the  uptake  of  C14-valine  by  each  of 
five  neutral  amino  acids  (L-leucine,  DL-isoleucine,  DL-alanine,  DL-phenylalanine, 
DL-threonine)  and  slight  inhibition  by  one  basic  amino  acid  (DL-lysine). 

Our  experiments  show  that  these  findings  hold  also  for  unfertilized  eggs  and 
provide  further  evidence,  from  measurements  of  both  accumulation  and  subsequent 
incorporation  of  amino  acid  into  protein,  supporting  that  of  Mitchison  and  Cummins 
that  the  inhibition  operates  as  a  competition  for  entrance  into  the  cell.  We  have 
extended  the  measurements  to  include  all  twenty  of  the  "coded"  amino  acids  tested, 
with  both  fertilized  and  unfertilized  eggs,  for  ability  to  inhibit  both  uptake  and 
incorporation  into  protein  of  a  neutral  (valine),  acidic  (glutamic  acid)  and  basic 
(arginine)  amino  acid.  In  the  present  article  the  amino  acids  that  are  termed 
basic  are  histidine,  arginine  and  lysine.  The  acidic  group  .includes  aspartic  acid, 
glutamic  acid  and  their  derivatives  asparagine  and  glutamine.  The  remaining 
thirteen  of  the  "coded"  amino  acids  are  placed  in  the  neutral  group. 

MATERIALS  AND  METHODS 

Eggs  were  obtained  from  the  sea  urchin  Lytechinus  plctus  by  the  method  of 
KCl-injection,  the  suspension  temporarily  acidified  to  pH  5  to  remove  the  gelatinous 
coat,  and  an  aliquot  removed  for  counting  (Tyler  and  Tyler,  1966).  For  the 
tests  of  uptake  and  incorporation  the  eggs  were  incubated  with  the  C14-labeled  amino 
acid  and  the  C12-amino  acid  being  explored,  in  a  total  volume  of  0.25  ml.  of  artificial 
sea  water,  at  pH  8.0,  in  polystyrene  test  tubes,  for  the  specified  time  and  at  20°  C. 
At  the  end  of  the  incubation  period  a  large  excess  (1  ml.  of  an  ice-cold  0.1  M 
solution)  of  the  C12-amino  acid,  corresponding  to  the  C14-amino  acid,  was  added 
as  quencher.  For  the  measurements  of  uptake  the  eggs  were  thoroughly  washed 
with  ice-cold  artificial  sea  water  and  transferred  with  distilled  water  to  filter  papers 
which  were  rapidly  dried  and  placed  directly  in  the  scintillation  fluid  4  in  which 
radioactivity  was  determined  (Tri-Carb  spectrometer)  with  about  50%  efficiency. 
For  the  measurements  of  incorporation  the  same  filter  papers  were  rehydrated  by 
transfer  through  absolute  alcohol,  95%  alcohol  and  5%  trichloroacetic  acid  (TCA). 
They  were  then  processed,  as  usual  (Tyler,  1966),  with  hot  TCA,  the  alcohols,  and 
ether,  and  transferred  to  the  vials  of  scintillation  fluid  for  determination  of  in- 
corporation of  the  labeled  amino  acid  into  protein. 

RESULTS 
1.  Inhibition  of  uptake  of  Cl*-valinc  by  an  amino  acid  mixture 

As  indicated  above  the  initial  experiments  on  the  effect  of  additional  amino  acids 
on  the  incorporation  of  labeled  valine  were  done  in  connection  with  tests  of  the 
action  of  dactinomycin.  Table  I  gives  the  results  of  two  experiments  in  which  the 
incorporation  of  C14-valine  into  protein  was  measured  in  the  presence  and  absence  of 
a  mixture  of  amino  acids  (Borsook  et  al.,  1957)  with  and  without  dactinomycin. 
The  inhibiting  effect  of  the  amino  acid  mixture  is  marked,  regardless  of  whether  or 
not  dactinomycin  is  present.  The  latter  had  no  significant  effect  on  C14-valine 

*2.88  g.  PPO  (2,5-diphenyloxazole)  and  0.34  g.  dimethyl  POPOP  (l,4-bis-2-(4-methyl-5- 
phenyloxazolyl) -benzene)  per  liter  of  toluene. 


206 


A.  TYLER,  J.  PIATIGORSKY  AND  H.  OZAKI 


TABLE  I 

Action  of  an  L-amino  acid  mixture*  and  of  dactinomycin**  c.n  incorporation  of  Cu-valine  into 
protein  by  unfertilized  eggs  of  L.  pictus,  incubated  for  30  minutes  at  20°  C. 


Counts  per  minute  per  104  eggs 

Experiment 

C14-valine 
(sp.  act. 
185  C./.U) 

Without  dactinomycin 

With  0.015  mg./ml.  dactinomycin 

/ic./ml. 

Without  amino 

With  amino 

Without  amino 

With  amino 

acid  mixture 

acid  mixture 

acid  mixture 

acid  mixture 

1 

0.50 

8327 

216 

10519 

178 

9226 

284 

12112 

173 

2 

0.42 

1050 

27 

1078 

14 

1701 

24 

1313 

22 

*  Composition  and  final  concentrations  in  mmoles/1. :  Alanine,  0.33 ;  arginine,  0.08 ;  aspartic 
acid,  0.48;  cysteine,  0.06;  glutamine,  1.33;  glycine,  0.89;  histidine,  0.40;  isoleucine,  0.05;  leucine, 
0.67;  lysine,  0.30;  methionine,  0.06;  phenyl  alanine,  0.26;  proline,  0.23;  serine,  0.28;  threonine, 
0.28;  tryptophan,  0.05;  tyrosine,  0.14. 

**  Gift  of  Merck,  Sharp  and  Dohme,  Rahway,  N.  J.;  courtesy  of  Dr.  H.  B.  Woodruff. 

incorporation  in  these  experiments.  Differences  between  the  two  experiments  in 
the  absolute  values  for  incorporation  of  C14-valine  may  reflect  differences  in  size 
of  the  endogenous  free  valine  pool  in  the  eggs. 

The  presence  of  the  added  amino  acids  did  not,  then,  enable  the  eggs  to  show  a 
stimulated  incorporation  of  C14-valine  in  response  to  dactinomycin,  that  had  been 
previously  noted  with  some  batches  of  eggs  of  Lyt  echinus  (see  introduction). 

2.  Pretreatment  ivith  amino  acids 

In  order  to  determine  whether  the  inhibiting  effect  of  the  additional  amino 
acids  is  on  the  accumulation  of  valine  by  the  eggs  or  on  its  subsequent  incorporation 

TABLE  II 

Effect  of  pretreatment  with  an  amino  acid  mixture  (a.a.  mix.}  on  the  uptake  of  Cu-valine* 
by  unfertilized  eggs  of  L.  pictus,  incubated  for  1  hour  at  20°  C. 


Pretreatment  for 
1  hour  in  : 

Counts  per  minute  per  104  eggs 

Total  uptake 

Incorporation  into  material  precipitable  by 
5%  trichloro-acetic  acid 

In  presence  of 
a.a.  mix. 

In  absence  of 
a.a.  mix. 

In  presence  of 
a.a.  mix. 

In  absence  of 
a.a.  mix. 

S.W. 

80 

23470 

4 

580 

100 

27714 

10 

638 

a.a.  mix. 

94 

26442 

8 

1332 

132 

27774 

6 

1338 

0.53  /ic./ml. ;  sp.  act.  185  c./M. 


AMINO  ACID  UPTAKE  BY  ECHINOID  EGGS  207 

into  protein,  tests  were  made  on  eggs  that  had  been  pretreated  with  the  amino  acid 
mixture  and  washed  just  before  addition  of  the  C14-valine.  Table  II  gives  the 
results  of  one  such  experiment.  The  total  uptake  of  C14-valine,  as  well  as  the 
incorporation  into  protein,  were  determined.  As  the  data  show,  preliminary 
exposure  of  the  eggs  to  the  mixture  of  amino  acids  has  no  effect  on  the  subsequent 
uptake  of  the  C14-valine,  either  in  the  presence  or  in  the  absence  of  the  C12-amino 
acid  mixture.  But,  uptake  is  almost  completely  suppressed  by  the  amino  acid 
mixture  present  during  the  period  of  incubation  with  the  C14-valine.  The  effect 
on  uptake  can,  then,  account  for  the  inhibition  of  incorporation  in  the  experiments 
shown  in  Table  I. 

The  data  of  Table  II  also  show  inhibition  of  incorporation  into  protein  in  those 
eggs  concurrently  exposed  to  the  amino  acid  mixture,  regardless  of  prior  exposure 
to  the  amino  acid  mixture.  Furthermore  there  is  an  apparent  increase  in  incorpora- 
tion by  those  eggs  exposed  to  the  amino  acid  mixture  before  incubation  with  the  C14- 
valine  alone.  In  three  additional  experiments  an  increase  was  obtained  in  one, 
while  no  appreciable  difference  was  observed  in  the  other  two.  At  present,  then, 
there  is  no  consistent  evidence  that  preincubation  with  other  amino  acids  results 
in  an  increased  incorporation  of  C14-valine  into  protein. 

Despite  the  washing  following  the  pretreatment  period  the  eggs  probably  retain 
most  of  the  accumulated  amino  acids.  This  seems  clear  from  experiments  of  other 
investigators  (Nakano  and  Monroy,  1958;  Mitchison  and  Cummins,  1966)  and  is 
indicated  here  by  the  large  quantity  of  acid-soluble  radioactivity  remaining  in  the 
washed  eggs.  One  may  conclude,  then,  that  retained  amino  acids  do  not  influence 
the  uptake  of  another  amino  acid,  namely,  valine.  One  or  more  of  the  amino  acids 
in  the  added  mixture  evidently  inhibit  the  uptake  of  valine  when  concurrently 
present  in  the  medium.  This  was  explored  further  with  the  individual  amino 
acids  and  with  fertilized  as  well  as  with  unfertilized  eggs. 

3.  Effect  of  one  amino  acid  on  the.  uptake  and  incorporation  of  another 
(a)   C^-valine 

Uptake,  and  incorporation  into  protein,  of  C14-valine,  C14-glutamic  acid  and  C14- 
arginine  by  unfertilized  and  fertilized  eggs  were  measured  individually  in  the 
presence  of  an  excess  (ca.  3000  X )  of  each  of  the  other  19  "coded"  amino  acids. 
In  some  experiments  the  labeled  amino  acid  was  tested  against  the  other  19  amino 
acids  at  the  same  time.  In  others  about  half  of  the  amino  acids  were  tested  at  one 
time,  as  noted  in  the  legends  for  the  figures.  The  results  are  represented 
graphically  in  Figures  1,  2  and  3.  Tables  III  and  IV  present  ratios  of  the  average 
uptake  of  the  labeled  amino  acid  in  the  presence  of  the  added  C12-amino  acid  to  that 
in  its  absence.  Ratios  for  incorporation  are  similarly  presented.  Table  III 
includes  results  of  an  additional  series  of  tests  of  incorporation  (see  footnote  to 
table).  In  Figures  1,  2  and  3,  for  each  experiment,  the  control  values  (indicated 
by  NONE)  are  given  at  the  top.  These  are  followed  by  the  values  obtained  for 
each  of  the  added  amino  acids  arranged  in  a  decreasing  (using  the  larger  of  each 
of  the  duplicate  values)  order  of  uptake. 

For  the  unfertilized  eggs  the  two  experiments  of  Figure  1  with  C14-valine  show 
marked  (greater  than  50%)  inhibition  of  uptake  by  SER,  ARC,  ASN,  GLN,  ALA, 


208 


A.  TYLER,  J.  PIATIGORSKY  AND  H.  OZAKI 


UNFERT.  EGGS 
C14  -  VALINE 
Uptake/IOeggs/hr 
I  ncorp./50eggs/hr 
EXPER.  -  I 

EXPER.  -  II-i 


25  50 

— I 1 1 1 1 


NONE 


PERT.  EGGS 
C14- Valine 
Uptake  /   egg   /hr 
Incorp./    egg  /hr 


10 


15 


20 


25 


CPM 


FIGURE  1.  Cu-L-valine  uptake,  and  incorporation  into  protein,  by  unfertilized  and  fertilized 
(one  hour  after  fert.)  eggs  of  L.  pictus  in  presence  of  various  individual  C^-L-amino  acids. 
Incubation  was  for  one  hour  in  a  total  volume  of  0.25  ml.  of  artificial  sea  water  containing, 
per  tube,  940  eggs  (unfert.,  expt.  I),  2860  eggs  (unfert,  expt.  II)  or  4314  eggs  (fert.),  and 
0.83  juc./ml.  of  the  C14-valine  (sp.  act.  208.5  c./M).  The  added  amino  acids  were  each  at  0.012 
M  except  TYR  which  was  at  0.0004  M.  The  tests  were  all  run  in  duplicate  and  the  individual 
results  are  represented  by  each  member  of  the  pairs  of  bars.  For  the  unfertilized  eggs  the  tests 
were  done  with  9  of  the  C12-amino  acids  (expt.  I)  on  one  day  and  with  the  remaining  10  (expt. 
II)  on  another  occasion,  using  eggs  from  a  different  female.  In  the  experiment  with  the 
fertilized  eggs  all  19  of  the  C^-amino  acids  were  tested  at  one  time.  For  the  entries  to  the  left 
of  the  figure:  None  =  no  added  C^-amino  acid;  ALA  =  alanine ;  ARG  =  arginine ;  ASN  =  as- 
paragine ;  ASP  =  aspartic  acid;  CYS  =  cysteine ;  GLU  =  glutamic  acid;  GLN  =  glutamine; 
GLY  =  glycine ;  HIS  =  histidine ;  ILU  =  isoleucine;  LEU  =  leucine;  LYS  =  lysine;  MET 
=  methionine ;  PHE  =  phenylalanine ;  PRO  =  proline ;  SER  =  serine ;  THR  =  threonine ; 
TRY  =  tryptophan ;  TYR  =  tyrosine ;  VAL  =  valine. 


AMINO  ACID  UPTAKE  BY  ECHINOID  EGGS 


209 


CYS,  THR,  TYR,  HIS,  ILU,  LEU,  MET,  PHE,  TRY,  listed  in  decreasing 
order  of  the  average  values,  as  given  in  Table  III.  For  all  except  the  first  three  of 
these  the  inhibition  of  uptake  is  greater  than  75%,  and  for  all  except  the  first  four 
the  inhibition  of  uptake  is  greater  than  90%.  The  values  for  degree  of  inhibition 
of  incorporation  are  similar  to  those  for  inhibition  of  uptake  for  each  of  the  tested 
amino  acids  except  for  GLN  where  incorporation  is  much  less  inhibited  (24  to 
30%)  than  is  uptake  (79%). 


UNFERT.   EGGS 

Cl4-Glutamic  acid 

EXPER.  E  EXPER.I 

Uptake/ 10  eggs/hr 

lncorp./200  eggs/hr 


PERT.  EGGS 
Cl4-Glutamic  acid 
Uptake /IO  eggs/hr 
Incorp./  lOeggs/hr 


8 


16 

CPM 


24 


32 


FIGURE  2.  C"-L-glutamic  acid  uptake  and  incorporation  into  protein ;  same  description  as 
for  Figure  1,  except  that  egg  numbers  were  3650  (unfert.,  expt.  I),  1570  (unfert,  expt.  II) 
and  1190  (fert.). 


210 


A.  TYLER,  J.  PIATIGORSKY  AND  H.  OZAKI 


For  the  fertilized  eggs  the  amino  acids  that  effect  greater  than  50%  inhibition 
of  both  uptake  and  incorporation  are  the  same  as  for  the  unfertilized  eggs,  except 
that  the  following  are  now  brought  just  within  this  group:  GLN  (incorp.),  GLY 
(uptake)  and  SER  (incorp.).  At  the  75%  level  of  inhibition  the  same  amino  acids 


t L {. L £ 1 £ L L {. 


UNFERT.    EGGS 

C14  -  Arginine 
Uptake  /I0eggs/hr 
lncorp./500eggs/hr 


LYS 


NONE 
ASP 


12 


16 


PERT.  EGGS 

Cl4-Arginine 

Uptake /I0eggs/hr 

Incorp./  lOOeggs/hr 


16 


24 


32 


40 


CPM 


FIGURE  3.  C14-L-arginiue  uptake  and  incorporation  into  protein ;  same  description  as  for 
Figure  1,  except  that  egg  numbers  were  4170  (unfert.)  and  4140  (fert),  that  L.  anamesus 
instead  of  L.  pictus  was  used  in  the  experiment  with  the  unfertilized  eggs  and  that  the  sp.  act. 
of  the  Cu-arginine  was  222  c./M. 


are  effective  except  for  TYR  (uptake  and  incorp.)  and  TRY  (incorp.).  Even  at 
the  90%  level  of  inhibition  most  of  the  inhibiting  amino  acids  are  the  same  as 
for  the  unfertilized  eggs  with  respect  both  to  uptake  and  incorporation,  as  com- 
parisons of  the  values  in  Tables  III  and  IV  show. 


AMINO  ACID  UPTAKE  BY  ECHINOID  EGGS 


211 


The  ainino  acids  that  effect  the  high  (90%  or  hetter)  degree  of  inhibition  of 
uptake  of  C14-valine,  both  for  unfertilized  (ALA,  CYS,  THR,  TYR,  HIS,  ILU, 
LEU,  MET,  PHE  and  TRY)  and  for  fertilized  (CYS,  HIS,  ILU,  LEU,  MET 
and  PHE)  eggs  all  belong  to  the  neutral  group,  with  the  exception  of  HIS  which 
is  only  weakly  basic.  This  holds  also  for  the  inhibition  of  incorporation  into 
protein. 

TABLE  III 

Influence  of  individual  amino  acids  on  the  uptake  and  incorporation  into  protein  of  a  neutral,  an 
acidic  and  a  basic  amino  acid  by  unfertilized  eggs  of  Lytechinus  pictus,*  incubated  for 

1  hour  at  20°  C. 


Ratios  of  cpm's  for  mixture  of  C12-  and  C14-amino  acid  to  cpm's  for  Cl4-amino  acid  alone 

"Competing" 
C^-amino 

CI4-L-Valine 

C14-L-Glutamic  acid 

C14-L-Arginine 

acid  at 

(3.9  X  10-6  M) 

(3.9  X  10~6  M) 

(3.7  X  10-6  M) 

0.012  M 

Total 
uptake 

Incorp. 

Incorp.** 

Total 
uptake 

Incorp. 

Incorp.** 

Total 
uptake 

Incorp. 

Incorp.** 

Alanine 

0.07 

0.17 

0.08 

0.49 

0.34 

0.42 

0.45 

0.22 

0.35 

Arginine 

0.34 

0.44 

0.54 

1.07 

2.36 

1.31 

— 

— 

— 

Asparagine 

0.33 

0.64 

0.83 

0.04 

0.20 

0.09 

0.93 

1.07 

0.70 

Aspartic  acid 

1.09 

1.35 

0.92 

0.01 

0.29 

0.12 

1.38 

1.65 

1.94 

Cysteine 

0.05 

0.15 

0.01 

0.18 

0.07 

0.69 

0.81 

0.47 

0.83 

Glutamic  acid 

0.80 

0.71 

0.81 

— 

— 

-  — 

0.83 

0.43 

0.95 

Glutamine 

0.21 

0.76 

0.70 

0.17 

0.00 

0.20 

0.51 

1.42 

0.90 

Glycine 

0.62 

0.64 

0.80 

0.65 

0.55 

0.49 

1.11 

1.32 

0.68 

Histidine 

0.00 

0.01 

0.46 

0.09 

0.85 

0.26 

0.23 

0.92 

0.39 

Isoleucine 

0.00 

0.01 

0.02 

0.65 

0.70 

0.47 

0.36 

0.19 

0.14 

Leucine 

0.00 

0.01 

0.01 

0.72 

0.44 

0.55 

0.06 

0.07 

0.09 

Lysine 

0.51 

0.58 

0.60 

1.12 

0.79 

0.51 

0.01 

0.35 

0.06 

Methionine 

0.00 

0.02 

0.01 

0.46 

0.52 

0.26 

0.22 

0.07 

0.01 

Phenylalanine 

0.00 

0.01 

0.01 

0.65 

0.46 

1.15 

0.35 

0.15 

0.34 

Proline 

0.95 

0.89 

0.89 

0.31 

0.33 

0.52 

0.93 

1.15 

0.87 

Serine 

0.48 

0.63 

0.71 

0.37 

0.27 

1.13 

0.33 

0.08 

0.23 

Threonine 

0.05 

0.17 

0.13 

0.21 

0.57 

0.72 

0.66 

1.00 

0.46 

Tryptophan 

0.00 

0.03 

0.04 

0.64 

0.49 

0.52 

0.24 

0.00 

0.20 

Tyrosine** 

0.05 

0.15 

0.12 

1.08 

0.56 

0.66 

1.12 

0.65 

0.68 

Valine 



•  — 

— 

0.40 

0.35 

0.53 

0.62 

0.10 

0.39 

*  Lytechinus  anamesus  used  in  experiments  with  C14-L-arginine,  columns  1  and  2.     Eggs  of 
this  species  resemble  closely  those  of  L.  pictus. 

**  Separate  experiment  in  which  only  incorporation  into  protein  was  measured. 
***  At  0.0004  M. 

(b)   C14-Glutamic  acid 

For  the  unfertilized  eggs  all  but  five  (ARC,  GLY,  ILU,  LYS  and  TYR)  of  the 
19  C12-amino  acids  cause  greater  than  50%  inhibition  of  uptake  or  incorporation,  or 
both,  of  the  C14-glutamic  acid.  With  the  fertilized  eggs  all  but  one  (ARG)  do  so. 
The  75%  (or  more)  inhibition  level  with  unfertilized  eggs  is  attained  by  ASN, 
ASP  (uptake),  CYS,  GLN,  HIS  (uptake),  and  THR  (uptake).  At  this  level,  for 
the  fertilized  eggs,  these  same  amino  acids,  except  for  ASP,  are  effective  as  are 
also  HIS  (incorp.),  PRO,  SER,  THR  (uptake)  and  VAL  (uptake).  At  the  90% 


212 


A.  TYLER,  J.  PIATIGORSKY  AND  H.  OZAKI 


level  with  the  unfertilized  eggs  there  are  ASN  (uptake),  ASP  (uptake),  CYS 
(incorp.)  and  GLN  (incorp.).  With  the  fertilized  eggs  90%  inhibition  is  given 
only  by  CYS  and  GLN. 

For  the  inhibition  of  uptake  and  incorporation  of  C14-glutamic  acid  there  again 
appears  to  be  a  relationship  to  type  of  amino  acid.  Thus  strong  inhibition  is  given 
by  ASP,  ASN  and  GLN  which  are  all  grouped  in  the  acidic  category.  Only  CYS 
and  THR,  of  the  neutrals,  and  HIS,  of  the  basics,  strongly  inhibit  uptake  by  the 

TABLE  IV 

Influence  of  individual  amino  acids  on  the  uptake  and  incorporation  into  protein  of  a  neutral,  an  acidic 
and  a  basic  amino  acid  by  fertilized  eggs  of  Lytechinus  pictus,  1  }iour  after  fertilization. 

Incubation  -was  for  1  hour  at  20°  C. 


"Competing" 
C12-amino  acid 
at  0.012  M 

Ratios  of  cpm's  for  mixture  of  C12-  and  C14-amino  acid  to  cpm's  for  C14-amino  acid  alone 

O-L-Valine 
(3.9  X  10-6  j|,/) 

Cu-L-Glutamic  acid 
(3.9  X  10-6  ji/) 

C14-L-Arginine 
(3.7  X  10-6  M) 

Total 
uptake 

Incorporation 

Total 
uptake 

Incorporation 

Total 
uptake 

Incorporation 

Alanine 

0.11 

0.18 

0.32 

0.29 

0.55 

0.48 

Arginine 
Asparagine 
Aspartic  acid 
Cysteine 
Glutamic  acid 

0.68 
0.40 
0.78 
0.09 
0.85 

0.67 
0.52 
0.74 
0.14 
0.81 

0.85 
0.11 
0.31 
0.03 

0.68 
0.11 
0.27 
0.04 

0.78 
1.08 
0.53 
1.11 

0.65 
0.92 
0.52 
0.97 

Glutamine 

0.21 

0.29 

0.08 

0.01 

0.53 

0.38 

Glycine 
Histidine 

0.46 
0.04 

0.54 
0.05 

0.39 
0.15 

0.38 
0.17 

0.94 
0.24 

0.85 
0.21 

Isoleucine 

0.02 

0.02 

0.37 

0.33 

0.38 

0.36 

Leucine 

0.01 

0.01 

0.32 

0.37 

0.08 

0.08 

Lysine 
Methionine 

0.67 
0.01 

0.75 
0.02 

0.48 
0.27 

0.48 
0.30 

0.02 
0.25 

0.03 
0.23 

Phenylalanine 

0.01 

0.01 

0.32 

0.38 

0.45 

0.40 

Proline 

0.64 

0.71 

0.16 

0.21 

1.06 

0.91 

Serine 

0.42 

0.43 

0.18 

0.20 

0.58 

0.50 

Threonine 

0.13 

0.17 

0.16 

0.38 

0.75 

0.61 

Tryptophan 
Tyrosine* 
Valine 

0.24 
0.36 

0.27 
0.43 

0.29 
0.46 
0.21 

0.33 
0.46 
0.30 

0.26 
1.05 
0.75 

0.21 
0.83 
0.62 

*  At  0.0004  M. 

unfertilized  eggs,  and  these  same  amino  acids  plus  PRO,  SER  and  VAL  are  simi- 
larly effective  with  the  fertilized  eggs. 

(c)   C14- Arginine 

Inhibition  greater  than  50 %,  for  the  uptake  and  incorporation,  or  both,  of  C14- 
arginine,  was  obtained  with  all  of  the  added  C12-amino  acids  with  the  exception  of 
ASN,  ASP,  GLU,  GLY,  PRO,  THR  and  TYR  for  the  unfertilized  eggs  and,  in 
addition,  CYS  and  VAL  for  the  fertilized  eggs.  At  the  75%  level  of  inhibition, 
only  ALA,  HIS,  ILU,  LEU,  LYS,  MET,  PHE,  SER,  TRY  and  VAL  for  the 


AMINO  ACID  UPTAKE  BY  ECHINOID  EGGS 


213 


unfertilized  eggs  and  I  ITS,  LEU,  LYS,  MET  and  TRY  for  the  fertilized  remain 
inhibitory.  At  the  90%  level  of  inhibition  of  uptake  and/or  incorporation  LEU, 
LYS,  MET,  SER,  TRY  and  VAL  remain  for  the  unfertilized  eggs  while  only 
LEU  and  LYS  are  effective  in  the  fertilized  eggs. 

It  is  evident  that  amino  acids  categorized  as  acidic  did  not  significantly  inhibit 
the  uptake  of  C14-arginine.  In  fact,  ASP  showed  a  slight  enhancement  of  uptake 
and  incorporation  for  the  unfertilized  eggs  but  this  effect  was  not  repeated  with 
the  fertilized  eggs.  Only  a  few  neutral  amino  acids  appreciably  inhibited  C14- 
arginine  uptake.  On  the  other  hand,  both  basic  amino  acids,  LYS  and  HIS, 
showed  strong  inhibition  for  both  unfertilized  and  fertilized  eggs. 

4.  Effect  of  fertilisation  on  uptake  of  amino  acids  and  on  incorporation  into  protein 

Apart  from  the  inhibitory  effects  of  added  amino  acids,  the  data  of  Figures  1, 
2  and  3  also  permit  incorporation  to  be  compared  with  uptake  with  regard  to  the 
changes  they  undergo  upon  fertilization  for  C14-valine,  C14-glutamic  acid  and  C14- 
arginine.  This  information  is  summarized  in  Table  V.  It  is  clear  that  uptake  of 

TABLE  V 

Effect  of  fertilization  on  uptake  of  amino  acids  and  on  incorporation  into  protein  by  eggs  of  Lytechimis 
(from  data  of  Figures  1,  2  and  3;  average  values  of  cpm's  per  103  eggs  for  1  hour  incubation) 


Cu-Valine 

CI4-Glutamic  acid 

C14-Arginine 

Uptake 
(U) 

Incorp. 
(I) 

i/u 

Uptake 
(U) 

Incorp. 
(I) 

i/u 

Uptake 
(U) 

Incorp. 
(I) 

i/u 

Unfertilized 
Fertilized 

11361 
21738 

576 
13398 

0.05 
0.62 

3172 
3116 

91.7 

901 

0.03 
0.29 

1398 

3029 

7.2 
173 

0.005 
0.057 

Fert./Unfert. 

1.91 

23 

12.4 

0.98 

9.8 

9.67 

2.17 

24 

11.4 

all  three  of  these  amino  acids  is  high  in  the  unfertilized  egg.  Upon  fertilization 
there  is  an  approximately  two-fold  increase  in  uptake  of  valine  and  of  arginine,  and 
no  appreciable  change  in  uptake  of  glutamic  acid,  at  the  stated  external  concentra- 
tions. The  data  for  incorporation,  however,  show  the  usual  great  stimulation  that 
occurs  upon  fertilization.  In  the  present  experiments  these  amount  to  23-  to  24-fold 
for  valine  and  arginine,  and  10-fold  for  glutamic  acid.  If  incorporation  is  expressed 
in  terms  of  uptake  (columns  4,  7  and  10  of  Table  V)  then  the  increase  upon 
fertilization  is  of  the  order  of  10-fold  for  all  three  amino  acids,  at  the  indicated 
concentrations  and  incubation  time. 

These  comparisons  are  made  apart  from  considerations  of  possible  feedback 
inhibition  of  uptake,  particularly  in  the  fertilized  eggs,  and  of  possible  effect  of 
depletion  of  labeled  amino  acid  from  the  medium.  The  data  of  Mitchison  and 
Cummins  (1966)  show  that  with  C14-valine  at  a  concentration  of  0.14  mM  there  is 
no  appreciable  feedback  inhibition  of  uptake  by  fertilized  sea  urchin  eggs  during 
a  period  of  one  hour.  The  concentration  of  valine  in  the  present  tests  (0.0039  mM) 
is  very  much  less  than  this.  Therefore,  feedback  inhibition  is  unlikely.  While 
similar  information  is  not  available  for  glutamic  acid  and  for  arginine  the  present 


214  A.  TYLER,  J.  PIATIGORSKY  AND  H.  OZAKI 

data  would  indicate  that  feedback  inhibition  is  not  likely  to  have  occurred  to  any 
very  appreciable  extent  in  these  experiments. 

With  regard  to  depletion  of  the  labeled  amino  acid  from  the  medium,  calculations 
from  the  data  presented  in  Figures  1,  2  and  3  show  that  the  average  concentrations 
in  the  medium  at  the  end  of  the  incubation  period  are  reduced  by  approximately  2% 
for  glutamic  acid,  5%  for  arginine  and  40%  for  valine.  It  is  only  for  valine,  then, 
that  the  value  for  uptake  by  the  fertilized  eggs  may  be  appreciably  affected  by 
depletion  of  the  label.  The  40%  reduction  by  the  end  of  the  incubation  period 
would  mean  an  approximately  20%  average  decrease  in  uptake,  assuming  linearity 
between  uptake  and  concentration.  This  does  not  require  altering  the  above 
statement  of  an  approximately  two-fold  increase  upon  fertilization. 

The  external  concentration  employed  in  tests  of  valine-uptake  is  about  one- 
fifth  that  found  by  Mitchison  and  Cummins  (1966)  to  give  maximum  rate  of  uptake 
with  fertilized  eggs  of  Paracentrotus  lividus.  These  workers,  using  concentrations 
well  above  that  giving  maximum  rate  of  uptake  for  fertilized  eggs,  report  a  consider- 
able increase  in  uptake  upon  fertilization.  This  may  be  estimated  from  their 
Figure  1  to  amount  to  15-  to  30-fold.  It  would  appear,  then,  that  the  amino  acid 
concentrations  at  which  the  present  measurements  were  made  were  in  a  range  at 
which  the  uptake  rate  relative  to  the  maximum  attainable  for  the  unfertilized  egg 
was  higher  than  that  for  the  fertilized  egg.  This  may  also  mean  that  the  maximum 
rate  is  reached  at  lower  concentrations  for  unfertilized  than  for  fertilized  eggs. 

DISCUSSION 

The  present  results  provide  information  of  use  in  studies  of  changes  in  protein 
synthesis  upon  fertilization  and  early  development  of  sea  urchin  eggs.  The  demon- 
stration by  Mitchison  and  Cummins  (1966),  with  fertilized  sea  urchin  eggs,  of  the 
ability  of  one  amino  acid  to  inhibit  the  accumulation  of  another,  has  been  confirmed, 
and  the  tests  have  been  extended  to  include  all  twenty  of  the  "coded"  amino  acids 
in  the  presence  of  a  characteristic  neutral,  acidic  and  basic  amino  acid  in  both 
unfertilized  and  fertilized  eggs.  The  analysis  has  shown  that  competition  occurs 
primarily  between  amino  acids  that  belong  to  the  same  group.  However,  these 
interrelationships  are  not  exclusive  and  there  is  a  certain  degree  of  overlap. 

As  noted  in  the  introduction  there  have  been  many  studies  (e.g.,  Wilbrandt  and 
Rosenberg,  1961;  Scholefeld,  1961;  Jacquez,  1961a,  1961b;  Christensen,  1962, 
1964 ;  Christensen  ct  al.,  1962 ;  Oxender  and  Christensen,  1963 ;  Johnstone  and 
Scholefeld,  1965;  Guroff  et  al.,  1964;  Larsen  et  al.,  1964;  Spencer  and  Brody, 
1964;  Adamson  et  al.,  1966;  Alvarado,  1966)  with  cells  of  various  other  kinds  of 
organisms,  in  which  the  influence  of  one  amino  acid  on  the  uptake  of  another  has 
been  examined.  Competition  is  found  to  occur  largely  within  the  separate  groups 
but  there  are  many  exceptions.  The  same  general  conclusions  apply  to  the  results 
of  our  experiments. 

The  concentration  of  the  competing  amino  acid  in  each  of  our  tests  with  valine 
is  many  thousands  of  times  higher  than  that  at  which,  according  to  Mitchison 
and  Cummins  (1966),  the  maximum  rate  of  uptake  is  attained.  This  is  probably 
true  also  for  glutamic  acid  and  arginine  although  the  plateau  levels  for  these  have 
not  been  determined.  We  may  infer,  then,  that  the  experiments  reveal  all  instances 
in  which  a  particular  amino  acid  has  some  appreciable  ability  to  compete  for  entrance 


AMINO  ACID  UPTAKE  BY  ECHINOID  EGGS  215 

into  the  cell  with  the  three  amino  acids  tested.  The  correlations  between  the 
uptake  of  an  amino  acid  and  the  incorporation  into  protein  are  very  good  for  the 
unfertilized  eggs  and  even  better  for  the  fertilized  eggs  where  the  values  are  higher 
and  variation  is  correspondingly  lower.  Thus,  the  inhibition  that  one  amino  acid 
effects  on  the  incorporation  of  another  evidently  takes  place  at  the  uptake  site. 
That  this  site  operates  independently  of  the  sites  of  protein  synthesis  is  suggested 
by  the  wide  divergences  between  uptake  and  incorporation  with  respect  to  the 
changes  in  these  properties  that  are  observed  upon  fertilization. 

As  noted  above,  and  as  is  summarized  in  Table  V,  the  unfertilized  eggs  exhibit 
a  relatively  high  capacity  for  uptake  of  the  three  test  amino  acids,  and  the  increase 
upon  fertilization  is  evidently  rather  small.  The  high  amino  acid  uptake  rate  of 
the  unfertilized  egg  contrasts  with  other  uptake  systems  studied  in  sea  urchin  eggs. 
For  example,  phosphate  uptake  (Whiteley,  1949;  Whiteley  and  Chambers,  1961) 
and  nucleoside  uptake  (Nemer,  1962;  Piatigorsky  and  Whiteley,  1965;  Mitchison 
and  Cummins,  1966)  are  very  strongly  suppressed,  as  is  the  transport  of  many 
other  substances  in  the  unfertilized  sea  urchin  egg  (cf.  Monroy,  1965 ;  Rothschild, 
1956). 

Apart  from  the  theoretical  considerations  that  are  of  interest  in  the  transport  of 
amino  acids  into  cells,  one  may  utilize  the  data  presented  here,  in  combination  with 
measurements  of  the  maximum  rates  at  which  the  various  labeled  amino  acids  are 
incorporated  into  protein,  to  specify  the  more  effective  mixtures  of  amino  acids 
for  labeling  nascent  protein  in  sea  urchin  eggs.  Measurements  of  rates  of  in- 
corporation of  individual  labeled  amino  acids  as  a  function  of  concentration  have 
been  made  on  eggs  of  Lytechinus  at  one  hour  after  fertilization  (Tyler,  1965b  and 
unpublished).  From  these  measurements  the  presently  available  values  for  the 
(approximately)  maximum  incorporation  into  protein,  in  m/xmoles  incorporated 
in  one  hour  by  10*  eggs  (and  the  values  for  the  external  concentrations,  in 
junioles/ml.,  at  which  maximum  or  near  maximum  incorporation  is  first  attained 
given  in  parentheses)  are  as  follows:  ALA-2.3(30),  ARG-2(>60),  ASN-0.5(2.8), 
ASP-1.7(>1.9),  CYS-CYS-0.3(sat.  in  s.w.),  GLU-4.5(40),  GLN-2.6(0.1),  GLY- 
1(3.8),  HIS-O.S(O.l),  ILU-3.S(0.3),  LEU- 1.6 (0.03),  LYS-0.5(120),  MET- 
0.2(0.03),  PHE-0.4(0.1),  PRO-1(>0.24),  SER-2(8),  THR-2.5(1.0),  TRY- 
2(sat.  in  s.w.),  TYR-0.2(0.4),  and  VAL-3.3(0.1). 

Depending  upon  how  the  various  parameters  are  evaluated  and  combined  a 
number  of  highly  effective  mixtures  may  be  formulated.  The  general  procedure 
is  to  maximize  incorporation  into  protein  while  minimizing  effects  of  competition 
among  the  amino  acids.  It  is  assumed  that  the  labeled  amino  acids  would  be 
available  at  about  the  same  specific  activity.  One  example  of  a  group  of  amino  acids 
that  would  yield  high  radioactivity  of  nascent  protein  is :  ILU,  ARG,  GLU  and 
PRO.  The  addition  of  other  amino  acids  would  tend  to  reduce  incorporation  by 
vdrtue  of  competition  of  uptake.  However,  one  may  substitute  for  each  of  these 
certain  other  "competing"  amino  acids  that  have  reasonably  high  values  of  in- 
corporation when  tested  individually.  For  example,  if  VAL  were  substituted  for 
ILU  there  would  not  be  a  very  great  over-all  change  in  the  values  for  incorporation 
given  by  the  mixture.  Similarly  ASP  could  be  substituted  for  GLU  without  large 
effect,  as  could  LYS  for  ARG.  Obviously,  there  are  many  more  complex  mixtures 
and  substitutions  that  might  be  formulated,  but  since  the  present  tests  were  made 


216  A.  TYLER,  J.  PiATIGORSKY  AND  H.  OZAKI 

with  only  57  of  the  380  possible  combinations,  further  assessment  of  the  most 
effective  mixtures  does  not  seem  warranted  at  this  time. 

SUMMARY 

1.  Tests  were  made  of  the  uptake  and  incorporation  into  protein  of  a  neutral 
(C14-valine),  an  acidic  (C14-glutamic  acid)  and  a  basic  (C14-arginine)  amino  acid 
in  the  presence  of  a  mixture  of  other  amino  acids  and  in  the  presence  of  a  great 
excess  (3000-fold)  of  each  of  the  other  "coded"  amino  acids  by  unfertilized  and 
fertilized  eggs  of  Lytech'mus  pictus. 

2.  The  results  showed  competition  occurring  principally  among  amino  acids 
belonging  to  the  same  group.     For  C14-valine  the  amino  acids  that  effected  strong 
inhibition   (90%  or  greater)   of  uptake  with  unfertilized  eggs  were  ALA,  CYS, 
THR,  TYR,  HIS,  ILU,  LEU,  MET,  PHE  and  TRY,  and  with  fertilized  eggs 
were   CYS,   HIS,   ILU,   LEU,   MET  and   PHE.     For   C14-glutamic   acid   90% 
inhibition  of  uptake  was  given  by  ASN  and  ASP  with  unfertilized  eggs  and  by  CYS 
and  GLN  with  fertilized  eggs.     Finally,  strong  inhibition  of  C14-arginine  uptake 
was  demonstrated  by  LYS  and  LEU  with  both  unfertilized  and  fertilized  eggs. 
Similar   results  were   obtained   in   the   corresponding   tests   of   incorporation   into 
protein.       The    inhibitory    effects    on    incorporation    are,    then,    attributable    to 
competition  for  uptake. 

3.  In  contrast  to  the  relatively  low  capability  of  the  unfertilized   egg  to  in- 
corporate amino  acid  into  protein  it  possesses  a  relatively  high  ability  to  accumulate 
amino  acids  from  the  surroundings.     For  C14-valine  and  C14-arginine,  the  uptake 
rate  by  the  unfertilized  egg  was  approximately  half  of  that  of  the  fertilized  egg, 
while  for  C14-glutamic  acid  the  pre-  and  post-fertilization  rates  of  uptake  were 
approximately  the  same. 

4.  The  percentage  of  accumulated  Cl4-amino  acid  that  was  incorporated  in  one 
hour  into  protein  in  these  experiments  with  valine,  glutamic  acid  and  arginine  was 
5,  3  and  0.5,  respectively,  in  the  unfertilized  eggs  and  60,  30  and  6,  respectively,  in 
the  fertilized  eggs.     When  expressed  in  terms  of  uptake,  and  assuming  no  large 
change  in  the  pool  of  free  amino  acid  in  the  egg,  there  is  an  approximately  10-fold 
increase  in  incorporation  into  protein  upon  fertilization  for  each  of  these  three 
amino  acids. 

5.  The  results,  also,  enable  formulations  to  be  made  of  the  kinds  of  combinations 
of  labeled  amino  acids  that  would  be  the  more  highly  effective  in  labeling  nascent 
proteins  of  sea  urchin  eggs.     One  such  combination  would  be  ILU,  ARG,  GLU 
and  PRO  with  each  of  these  being  replaceable  by  certain  alternative  "competing" 
amino  acids  as  indicated  in  the  text. 

LITERATURE  CITED 

ADAMSON,  L.  F.,  S.  G.  LANGELUTTIG  AND  C.  S.  ANAST,  1966.  Amino  acid  transport  in 
embryonic  chick  bone  and  rat  costal  cartilage.  Biochnn.  Biophys.  Acta,  115:  345-354. 

ALVARADO,  F.,  1966.  Transport  of  sugars  and  amino  acids  in  the  intestine :  Evidence  for  a 
common  carrier.  Science,  151:  1010-1013. 

BORSOOK,  H.,  E.  H.  FISCHER  AND  G.  KEIGHLEY,  1957.  Factors  affecting  protein  synthesis  in 
vitro  in  rabbit  reticulocytes.  /.  Biol.  Chcm.,  229:  1059-1070. 

CHRISTENSEN,  H.  N.,  1962.     Biological  Transport.     W.  A.  Benjamin,  Inc.,  New  York. 


AMINO  ACID  UPTAKE  BY  ECHINOID  EGGS  217 

CHRISTENSEN,  H.  N.,  1964.     Free  amino  acids  and  peptides  in  tissues.     In:  Mammalian  Protein 

Metabolism.     H.  N.  Munro  and  J.  B.  Allison,  Editors.     Academic  Press,  New  York, 

pp.  105-124. 
CHRISTENSEN,  H.  N.,  H.  AKEDO,  D.  L.  OXENDER  AND  C.  G.  WINTER,  1962.     On  the  mechanism 

of  amino  acid  transport   into  cells.     In :    Amino   Acid   Pools.     J.   T.    Holden,   Editor. 

Elsevier  Publishing  Co.,  Amsterdam,  pp.  527-538. 
GUROFF,  G.,  G.  R.  FANNING  AND  M.  A.  CHIRIGOS,  1964.     Stimulation  of  aromatic  amino  acid 

transport  by   p-fluorophenylalanine   in   the   sarcoma-37   cell.     /.    Cell.   Comp.   Physiol., 

63:323-331. 
HONIG,   G.   R.,   AND  M.   RABINOVITZ,    1965.     Actinomycin   D :    Inhibition   of   protein   synthesis 

unrelated  to  effect  on  template  RNA  synthesis.     Science,  149:  1504-1506. 
JACQUEZ,  J.  A.,   1961a.     Transport  and  exchange  diffusion  of  L-tryptophan   in  Ehrlich  cells. 

Amcr.  J.  Physio!.,  200:  1063-1068. 
JACQUEZ,   J.    A.,    1961b.     The   kinetics    of   carrier-mediated    active    transport    of   amino    acids. 

Proc.  Natl,  Acad.  Sci.,  47:  153-162. 
JOHNSTONE,  R.  M.,  AND  P.  G.  ScHOLEFELD,  1965.     Amino  acid  transport  in  tumor  cells.     In: 

Advances  in  Cancer  Research,  Vol.  9.     A.  Haddow  and  S.  Weinhouse,  Editors.     Aca- 
demic Press,  New  York,  pp.  144-227. 
LARSEN,  P.  R.,  J.  E.  Ross  AND  D.  F.  TAPLEY,  1964.     Transport  of  neutral,  dibasic  and  N-methyl- 

substituted  amino  acids  by  rat  intestine.     Biochim.  Biophys.  Ada,  88:  570-577. 
MITCHISON,  J.   M.,  AND  J.   E.   CUMMINS,    1966.     The   uptake   of  valine  and  cytidine   by   sea 

urchin  embryos  and  its  relation  to  the  cell  surface.     /.  Cell.  Sci.,  1 :  35-47. 
MONROY,  A.,  1965.     Chemistry  and  Physiology  of  Fertilization.     Holt,  Rinehart  and  Winston, 

New  York. 
NAKANO,  E.,  AND  A.  MONROY,  1958.     Incorporation  of  S35-methionine  in  the  cell  fractions  of  sea 

urchin  eggs  and  embryos.     Ex  p.  Cell  Res.,  14:  236-244. 
NEMER,  M.,  1962.     Characteristics  of  the  utilization  of  nucleosides  by  embryos  of  Paracentrotus 

lividus.    J.  Biol.  Chem,,  237:  143-149. 
OXENDER,  D.  L.,  AND  H.  N.  CHRISTENSEN,  1963.     Distinct  mediating  systems  for  the  transport 

of  neutral  amino  acids  by  the  Ehrlich  cell.    /.  Biol.  Chcm.,  238:  3686-3699. 
PIATIGORSKY,  J.,  H.  OZAKI  AND  A.  TYLER,   1966.     RNA-  and  protein-synthesizing  capacity  of 

isolated  oocytes  of  the  sea  urchin  Lytechinus  pictus.     Dcvcl.  Biol.  (in  press). 
PIATIGORSKY,    J.,    AND   A.    H.    WHITELEY,    1965.     A    change    in    permeability    and    uptake    of 

["CJuridine     in     response    to    fertilization     in    Strongylocentrotus    purpuratns    eggs. 

Biochim.  Biophys.  Ada,  108:  404-418. 

ROTHSCHILD,  LORD,  1956.     Fertilization.     John  Wiley  and  Sons,  Inc.,  New  York. 
TYLER,  A.,  1965a.     The  biology  and  chemistry  of  fertilization.     Amcr.  Nat.,  99:  309-334. 
TYLER,  A.,  1965b.     Incorporation  of  amino  acids  into  protein  by  non-nucleate,  nucleate  and  poly 

u-treated  sea  urchin  eggs.     Amcr.  Zool.,  5:  635-636. 
TYLER,  A.,  1966.     Incorporation  of  amino  acids  into  protein  by  artificially  activated  non-nucleate 

fragments  of  sea  urchin  eggs.     Biol.  Bull.,  130:  450-461. 
TYLER,    A.,   AND    B.    S.    TYLER,    1966.     The   gametes;    some    procedures    and    properties.     In: 

Physiology  of  Echinodermata,  Ch.  27.     R.  A.  Boolotian,  Editor.     John  Wiley  and  Sons, 

Inc.,  New  York  (in  press). 
SCHOLEFELD,  P.  G.,  1961.     Competition  between  amino  acids  for  transport  into  Ehrlich  ascites 

carcinoma  cells.     Canad.  J.  Biochem.  Physiol.,  39:  1717-1735. 
SPENCER,  R.  P.,  AND  K.  R.  BRODY,   1964.     Intestinal  transport  of  cyclic  and  noncyclic  imino 

acids.     Biochim.  Biophys.  Acta,  88:  400-406. 
WHITELEY,  A.  H.,  1949.     The  phosphorus  compounds  of  sea  urchin  eggs  and  the  uptake  of 

radiophosphate  upon  fertilization.     Amcr.  Nat.,  83:  249-267. 
WHITELEY,  A.  H.,  AND  E.  L.  CHAMBERS,  1961.     The  differentiation  of  a  phosphate  transport 

mechanism  in  the  fertilized  egg  of  the  sea  urchin.     Symp.  on  Germ  Cells  and  Develop- 
ment (Institut.  Intern.  d'Embryologie  and  Fondazione  A.  Baselli),  387-401. 
WILBRANDT,  W.,  AND  T.  ROSENBERG,  1961.     The  concept  of  carrier  transport  and  its  corollaries 

in  pharmacology.     Pharmacol.  Revs.,  13:  109-183. 


ERRATA 

In  the  paper  by  Hayward  and  Ball  in  volume  131,  number  1,  of  THE  BIOLOGICAL 
BULLETIN,  the  first  lines  on  page  100  should  read  as  follows  :  "of  any  added  stimula- 
tion, the  mean  value  for  the  rate  of  oxygen  consumption  of  brown  adipose  tissue 
was  2.33  and  3.29  times  that  of  liver  and  heart,  respectively.  The  addition  of 
epinephrine  caused  an  average  increase  of  350%  in  the  respiratory  rate  of  brown 
adipose  tissue,  and  was  without  effect  upon  liver  or  heart  slices.  In  .  .  ." 

On  page  102,  line  7  of  the  Summary,  the  value  should  read  "1374  /il.  CX/100  mg. 
fresh  tissue/hr." 


Vol.  131,  No.  2  October,  1966 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED   BY   THE    MARINE   BIOLOGICAL  LABORATORY 


DISTRIBUTION    OF    URCEOLARIA    SPINICOLA    (CILIATA, 

PERITRICHIDA)  ON  THE  SPINES  OF  THE  SEA  URCHIN 

STRONGYLOCENTROTUS  DROEBACHIENSIS 

C.  DALE  BEERS 

Department  of  Zoology,  University  of  North  Carolina,  Chapel  Hill,  North  Carolina  27514, 
and  Mount  Desert  Island  Biological  Laboratory,  Salisbury  Coi'c,  Maine 

Most  of  the  described  species  of  Urceolaria  occur  epizoically  on  various  fresh- 
water and  marine  invertebrates  (Hirshfield,  1949).  Probably  U.  patellae,  from 
the  ctenidia  of  the  European  limpet  Patella  I'ulgata,  and  U.  niitra,  from  the  external 
surface  of  fresh-water  triclads,  are  the  best  known,  owing  to  the  ecological  studies 
of  Brouardel  (1941,  1947)  and  Reynoldson  (1950,  1955).  The  only  species  known 
at  present  from  echinoids  is  U.  spinicola  Beers,  1964,  which  occurs  in  abundance 
on  the  spines  and  pedicellariae  of  Strongylocentrotus  droebacJiicnsis,  at  least  in  the 
waters  adjoining  Mount  Desert  Island,  Maine.  Since  the  ciliate  appears  to  be 
obligately  epizoic  on  the  urchin,  its  geographic  range  is  probably  coextensive  with 
that  of  the  host.  In  general,  U.  spinicola  has  the  form  of  a  short  cylinder,  which 
measures  about  60  //,  in  diameter  and  25  ju.  in  height.  By  means  of  its  specialized 
basal  disc,  it  adheres  firmly  to  the  spines  and  pedicellariae,  although  it  is  capable 
of  limited  locomotion,  either  by  sliding  along  the  substratum  or,  less  commonly,  by 
swimming  freely  in  the  medium. 

The  preceding  study  (Beers,  1964)  was  concerned  chiefly  with  the  structure  and 
identification  of  the  ciliate  and  with  its  actual  occurrence  on  the  urchins  of  Mount 
Desert  Island.  Its  distribution  on  the  spines  received  only  incidental  mention, 
although  it  presented  some  remarkable  features.  The  evidence  indicated,  for 
example,  that  short  spines  had  many  more  urceolarias  per  spine  than  long  ones,  and 
that  the  ciliates  attached  to  long  spines  were  concentrated  on  the  proximal  halves 
of  the  spines.  The  reference  to  long  and  short  spines  does  not  mean  primary  and 
secondary  ones.  The  spines  of  any  specimen  of  3\  droebachiensis  differ  greatly  in 
length,  but  the  intergrades  between  the  extremes  are  practically  countless.  Thus, 
the  spines  cannot  be  separated  into  two  categories  (Hyman,  1955,  p.  424).  In 
view  of  the  fundamental  similarity  of  the  spines,  any  differential  distribution  of  I', 
spinicola  on  their  surfaces  assumes  added  interest.  Therefore,  a  more  thorough 
study  of  the  distribution  was  undertaken  in  the  summer  of  1965,  again  on  the 

219 

Copyright  ©  1966,  by  the  Marine  Biological  Laboratory 
Library  of  Congress  Card  No.  A38-518 


220  C.  DALE  BEERS 

urchins  of  Mount  Desert  Island.     The  results  are  presented  in  the  present  paper, 
which   deals  mainly   with   the  following  aspects   of  the   urchin-ciliate   association : 

(1)  the  occurrence  of  the  ciliate  on  spines  from  different  regions  of  the  urchin  test; 

(2)  the  density  of  the  ciliate  population  (intensity  of  epifaunation)  on  urchins  of 
different  sizes;    (3)   the  occurrence  of  the  ciliate  on  spines  of  different  lengths; 
and  (4)  its  distribution  on  individual  spines.     Once  the  distribution  on  the  spines 
is  definitely  established,  an  analysis  of  the  factors  responsible  for  such  distribution 
can  be  attempted,  but  this  aspect  of  the  study  is  deferred  for  the  present.     Any 
consideration  of  the  distribution  of  the  urceolarias  on  the  pedicellariae  is  likewise 
deferred. 

MATERIAL  AND  METHODS 

From  June  15  to  August  25,  1965,  specimens  of  6".  droebachiensis  were  collected 
as  needed  from  the  inshore  waters  of  Mount  Desert  Island.  They  were  taken 
from  three  localities :  Laboratory  Point,  Bartlett  Narrows  and  Long  Ledge. 
Laboratory  Point  means  the  waters  of  Frenchman  Bay  adjacent  to  the  Laboratory 
area.  In  the  summer  the  Bay  is  relatively  calm  and  littoral  urchins  are  subjected 
to  the  minimum  of  wave  action.  Thus,  their  spines  show  very  little  weathering 
at  the  tips.  Although  the  mean  tidal  range  of  the  Bay  amounts  to  3.25  m.,  the 
amount  of  organic  matter  in  the  water  and  the  bacterial  count  were  evidently  high 
in  1965,  since  much  of  the  Bay  was  closed  to  the  taking  of  mussels  and  clams  for 
table  use.  In  general,  the  waters  of  Bartlett  Narrows,  a  strait  in  Blue  Hill  Bay, 
are  likewise  free  of  turbulence  and  in  1965  they  were  relatively  uncontaminated. 
Long  Ledge,  well  removed  from  Laboratory  Point  and  Bartlett  Narrows,  presents 
a  somewhat  different  habitat.  The  waters  are  quite  uncontaminated,  the  Ledge 
is  exposed  to  the  winds,  and  a  surf  is  constantly  present.  Thus,  the  long  spines  of 
inshore  urchins  are  much  eroded  distally. 

Counts  of  the  urceolarias  were  made  on  detached  fresh  spines.  A  small  piece 
of  the  test  was  excised  from  a  recently  collected  urchin  and  removed  to  a  watch 
glass  of  sea  water  under  the  dissecting  binocular,  with  the  spines  uppermost.  The 
piece  was  held  down  by  a  blunt  needle,  the  tip  of  a  small  scalpel  was  brought  against 
the  base  of  a  spine,  and  the  spine  was  detached  by  a  quick  movement  of  the  scalpel. 
When  a  sample  of  several  contiguous  spines  was  desired,  the  spines  were  detached 
in  turn,  beginning  at  the  margin  of  the  piece.  The  number  of  urceolarias  dislodged 
by  the  procedure  was  negligible. 

It  is  practically  impossible  to  count  the  urceolarias  In  situ  on  a  spine,  largely 
because  of  its  opacity.  In  order  to  count  them,  the  detached  spines  were  transferred 
in  groups  of  five  or  ten  to  a  watch  glass  of  distilled  water.  When  a  fresh  spine 
is  immersed  in  distilled  water,  any  urceolarias  on  it  are  immediately  immobilized 
and  after  3-5  min.  they  become  detached.  If  the  spine  is  shaken  gently  with 
forceps,  they  drop  to  the  bottom  of  the  watch  glass,  where  they  can  be  counted 
accurately. 

With  reference  to  the  distribution  of  U.  spinicola  on  the  urchin,  the  following 
three  regions  of  the  test  were  distinguished :  a  circumoral  region,  meaning  the  some- 
what flattened  surface  which  is  normally  in  contact  with  the  substratum ;  an  ambital 
or  circumferential  region  ;  and  an  aboral  region,  meaning  the  expanse  between  the 
ambitus  and  the  periproct. 


DISTRIBUTION  OF  URCEOLARIA 


221 


The  ages  of  certain  of  the  urchins  were  estimated  from  the  diameter  of  the 
test,  following  the  data  summarized  by  Swan  (  lc)6l.  Table  IV).  In  the  presenta- 
tion of  the  results,  comparisons  will  he  made  occasionally  between  the  numbers  of 
urceolarias  in  two  groups.  If  the  larger  number  exceeds  the  smaller  by  one-third 
or  more,  the  difference  is  judged  to  be  significant.  Minor  comments  on  methods 
will  be  supplied  as  needed. 

RESULTS 

1.   Occurrence  of  I',  spinicola  on  spines  from  different  regions  of  the  urchin  test; 
intensity  of  epifaunation  on  urchins  of  different  sizes 

Urchins  of  various  sizes  (measured  by  the  diameter  of  the  test)  were  examined 
from  each  of  the  three  localities.  Their  respective  sizes  are  listed  in  column  1  of 
Table  I  and  their  corresponding  ages  in  column  2,  in  so  far  as  estimates  of  age 
are  available.  Most  of  the  sizes  represent  recognized  year-classes,  but  some  urchins 
of  undetermined  age  are  also  included.  Five  urchins  of  each  of  nine  sizes  were 
examined  from  Laboratory  Point.  Unfortunately,  urchins  9-18  mm.  in  diameter 
were  unavailable  at  Bartlett  Narrows  and  Long  Ledge,  but  five  of  each  of  the 


TABLE  I 

Occurrence  of  U.  spinicola  on  urchins  (S.  droebachiensis)  of  different  sizes  from 

three  localities  on  Mount  Desert  Island,  Maine  (Laboratory  Point, 

Bartlett  Narrows  and  Long  Ledge).  Summer  1965 


Diameter  of 
test  in  mm. 

Age  in  years 
from  time  of 
settling 

Locality 

Average  number  of  urceolarias  per  spine 

Circu  moral 
region 

Ambital 
region 

Aboral 
region 

Entire 
urchin 

9-10 

1 

Lab.  Point 

1.3 

1.0 

0.5 

0.9 

12-14 

? 

Lab.  Point 

2.3 

1.7 

0.7 

1.6 

16-18 

? 

Lab.  Point 

18.4 

19.1 

10.1 

15.9 

24-26 

2 

Lab.  Point 

20.9 

23.7 

12.6 

19.1 

30-38 

? 

Lab.  Point 

28.2 

32.4 

13.9 

24.8 

40-42 

3 

Lab.  Point 

37.8 

35.1 

26.4 

33.1 

46-54 

4 

Lab.  Point 

43.1 

41.6 

24.3 

36.3 

55-60 

5 

Lab.  Point 

22.9 

26.8 

16.2 

22.0 

62-74 

6  + 

Lab.  Point 

2.7 

3.4 

2.8 

3.0 

24-26 

2 

Bart.  Xar. 

9.8 

8.2 

4.5 

7.5 

30-38 

? 

Bart.  Xar. 

22.1 

25.8 

9.6 

19.2 

40-42 

3 

Bart.  Xar. 

11.0 

9.2 

3.6 

7.9 

46-54 

4 

Bart.  Xar. 

10.5 

8.2 

4.1 

7.0 

55-60 

5 

Bart.  Xar. 

1.8 

1.2 

0.6 

1.2 

62-70 

6  + 

Bart.  Xar. 

0.8 

0.6 

0.6 

0.7 

24-26 

2 

Long  Ledge 

4.7 

S  ^ 

4.6 

4.8 

30-38 

? 

Long  Ledge 

4.0 

2.9 

2.2 

3.0 

40-42 

3 

Long  Ledge 

2.1 

1.9 

0.7 

1.6 

46-54 

4 

Long  Ledge 

2.3 

2.3 

1.4 

2.0 

55-60 

5 

Long  Ledge 

2.6 

2.4 

1.0 

2.0 

62-72 

6  + 

Long  Ledge 

0.8 

0.5 

0.4 

0.6 

C.  DALE  BEERS 

remaining  sizes  were  examined  from  these  areas.  The  average  number  of 
urceolarias  per  spine  was  determined  from  a  spine-sample  taken  from  each  of  the 
three  regions  of  each  urchin.  Such  a  sample  consisted  of  any  ten  contiguous  spines 
from  an  excised  piece  of  test.  Thus,  each  entry  in  columns  4,  5  and  6  of  Table  I 
represents  an  average  based  on  50  spines.  It  is  understood  that  the  spines  of  any 
sample  varied  considerably  in  length  (usual  range,  1.0-15.0  mm.,  but  reduced  to 
0.5-5.0  mm.  in  samples  from  small  urchins) . 

Turning  to  Table  I,  consider  the  average  number  of  urceolarias  per  spine  on 
different  regions  of  the  test,  beginning  with  the  urchins  from  Laboratory  Point. 
In  any  of  the  nine  size-classes,  the  average  number  of  ciliates  per  spine  was 
approximately  the  same  on  the  circumoral  and  ambital  regions  (columns  4  and  5). 
For  example,  in  size-class  24—26  mm.  the  average  numbers  were  20.9  and  23.7, 
respectively  (no  significant  difference).  On  the  other  hand,  the  average  number 
on  the  aboral  spines  (column  6)  was  decidedly  smaller  in  all  the  size-classes,  with 
one  exception — the  class  consisting  of  the  largest  urchins  (62-74  mm.),  which  had 
very  few  ciliates  per  spine,  regardless  of  the  region.  In  general,  the  foregoing 
comments  also  apply  to  the  urchins  from  Bartlett  Narrows,  although  the  average 
number  of  ciliates  per  spine  was  smaller  without  exception.  With  reference  to  the 
Long  Ledge  urchins,  the  ciliate  populations  were  extremely  sparse  and  the  average 
number  of  urceolarias  per  spine  was  therefore  much  reduced.  Indeed,  the  ciliate 
counts  were  so  small  that  comparisons  between  the  respective  regions  of  the  test 
are  scarcely  practicable.  Nevertheless,  the  general  features  of  the  distribution  were 
in  agreement  with  those  already  described. 

Referring  again  to  Table  I,  consider  the  average  number  of  urceolarias  per  spine 
on  urchins  of  different  sizes ;  that  is,  the  intensity  of  epifaunation  of  the  entire  urchin 
(column  7,  each  entry  of  which  is  based  on  a  total  of  150  spines).  With  reference 
to  the  urchins  from  Laboratory  Point,  the  average  number  of  ciliates  per  spine 
increased  with  the  size  of  the  urchin,  until  a  diameter  of  46-54  mm.  (or  an  age  of 
about  4  years)  was  attained.  On  urchins  larger  than  these,  the  number  decreased 
abruptly.  The  scarcity  of  urceolarias  on  urchins  62  mm.  or  larger  in  diameter 
(presumed  to  be  at  least  6  years  of  age)  was  remarkable.  Indeed,  on  many 
urchins  of  this  size  it  was  impossible  to  find  any  urceolarias,  either  on  the  spines  or 
pedicellariae.  In  general,  the  foregoing  remarks  also  apply  to  the  urchins  from 
Bartlett  Narrows,  although  the  average  number  of  ciliates  per  spine  was  consistently 
smaller  and  the  maximal  number  occurred  on  urchins  30-38  mm.  in  diameter,  some 
of  which  were  probably  3  years  of  age.  On  the  Long  Ledge  urchins  the  average 
number  of  urceolarias  per  spine  was  small,  and  comparisons  between  successive 
sizes  are  therefore  less  meaningful.  Nevertheless,  the  trend  in  the  intensity  of 
epifaunation  agreed  with  that  already  mentioned. 

Spine-samples  from  the  ambulacral  and  interambulacral  areas  of  certain  urchins 
were  also  examined  comparatively,  although  the  results  are  not  presented  in  tabular 
form.  Without  exception,  the  average  number  of  urceolarias  per  spine  was  essen- 
tially the  same  on  the  two  areas.  For  example,  on  a  41 -mm.  urchin  from  Labora- 
tory Point,  the  average  number  per  spine  was  27.3  on  an  ambulacral  area  and  26.7 
on  an  adjacent  interambulacral  area,  based  on  a  sample  of  50  spines  removed  at 
random  from  each  area.  Evidently  the  presence  of  the  tube  feet  does  not  affect  the 
occurrence  of  the  ciliate. 


DISTRIBUTION  OF  URCEOLARIA 


223 


In  summary,  the  results  show  (1)  that  U.  spinicola  is  more  abundant  on  the 
circumoral  and  ambital  spines  than  on  the  aboral  ones;  (2)  that  it  occurs  in 
equivalent  numbers  on  the  ambulacral  and  interambulacral  areas;  (3)  that  the 
density  of  the  ciliate  population  increases  gradually  as  the  urchin  grows  and  attains 
its  maximum  on  urchins  40-54  mm.  in  diameter;  and  (4)  that  the  density  decreases 
markedly  on  urchins  62-74  mm.  in  diameter,  many  of  which  bear  no  urceolarias 
whatsoever. 

2.  Occurrence  of  U.  spinicola  on  spines  of  different  lengths  from  three  regions  of 
the  urchin  test 

Considerable  numbers  of  spines  were  detached  from  each  of  the  three  regions 
of  five  urchins  (diameter,  40-42  mm.)  from  Laboratory  Point,  and  the  number  of 
urceolarias  per  spine  was  recorded.  These  records  supplied  numerous  counts  for 

TABLE  II 

Occurrence  of  U.  spinicola  on  spines  of  different  lengths  from  three  regions  of  the  urchin 

test.  Number  of  urchins,  5.  Diameter  of  test,  40-42  mm.  Spine-sample,  10; 

namely,  2  spines  of  each  length  from  each  region  of  each  urchin 


Average  number  of  urceolarias  per  spine 

Spine  length 

in  mm. 

Circumoral  region 

Ambital  region 

Aboral  region 

Entire  urchin 

0.6-0.9 

8.8 

7.4 

10.2 

8.8 

1.0-1.9 

29.5 

31.3 

21.0 

27.3 

2.0-2.9 

28.9 

32.4 

18.4 

26.6 

3.0-3.9 

42.9 

30.3 

14.9 

29.4 

4.0-4.9 

28.8 

21.5 

10.1 

21.5 

5.0-5.9 

14.6 

13.4 

7.1 

11.7 

6.0-6.9 

8.0 

6.4 

3.8 

6.1 

7.0-7.9 

6.3 

4.6 

4.5 

5.1 

8.0-8.9 

5.8 

3.9 

1.6 

3.8 

9.0-9.9 

3.1 

2.5 

0.6 

2.1 

10.0-10.9 

* 

2.7 

1.8 

2.3 

11.0-16.0 

* 

2.8 

1.2 

2.0 

*  None  of  this  length  present. 

spines  of  many  different  lengths.  Representative  data  on  the  relation  of  the  number 
of  urceolarias  to  the  length  of  the  spine  are  presented  in  Table  II.  For  descriptive 
purposes,  most  of  the  spines  will  be  treated  as  "short"  and  "long"  ones.  Although 
these  terms  are  relative,  they  are  nonetheless  useful.  Spines  1.0-4.9  mm.  in  length, 
which  comprise  about  80%  of  the  spines  on  urchins  24  mm.  or  more  in  diameter, 
will  be  called  short  spines,  whereas  spines  5.0  mm.  or  more  in  length,  which 
comprise  about  15%,  will  be  called  long  spines.  Spines  shorter  than  1.0  mm., 
which  make  up  the  remainder,  are  therefore  uncommon  on  such  urchins. 

Reference  to  Table  II  shows  that  the  smallest  spines  (length,  0.6-0.9  mm.)  of  all 
three  regions  had  relatively  few  ciliates  per  spine.  The  average  number  varied  from 
7.4  to  10.2,  and  the  average  for  the  entire  urchin  (based  on  30  spines)  was  8.8. 
Presumably  most  of  these  spines  were  immature  ones  which  had  not  acquired 
their  full  complement  of  urceolarias.  Spines  1.0-4.9  mm.  in  length,  on  the  other 


224  C.  DALE  BEERS 

hand,  had  the  largest  numbers  of  ciliates  per  spine;  for  example,  the  average 
number  on  the  circumoral  spines  varied  from  28.8  to  42.9.  Then,  spines  5.0  mm. 
or  more  in  length  had  decreasing  numbers  of  ciliates,  and  in  general  the  average 
number  per  spine  varied  inversely  with  the  length  of  the  spine.  In  agreement 
with  the  data  of  Table  I,  spines  from  the  circumoral  and  ambital  regions  had 
approximately  equal  (and  maximal)  numbers  of  ciliates,  whereas  those  from  the 
aboral  region  had  fewer,  although  certain  exceptions  appear  in  Table  II. 

In  general,  the  distribution  of  urceolarias  shown  in  Table  II  was  typical  of 
urchins  24—60  mm.  in  diameter  from  Laboratory  Point.  For  example,  50  short 
spines  detached  at  random  from  five  25-mm.  urchins  had  an  average  of  17.2 
urceolarias  per  spine  whereas  50  long  ones  had  only  4.4  per  spine.  Similarly,  50 
short  spines  from  five  urchins  55-58  mm.  in  diameter  had  29.4  ciliates  per  spine, 
and  50  long  ones  had  only  4.8.  Thus,  the  results  show  conclusively  that  the 
short  spines  of  urchins  24-60  mm.  in  diameter  bear  many  more  urceolarias  per 
spine  than  the  long  ones. 

Some  further  aspects  of  the  urchin-ciliate  association  can  be  mentioned  at  this 
point.  With  respect  to  any  individual  urchin,  the  number  of  urceolarias  on  the 
spines  of  a  particular  length  is  extremely  variable.  For  example,  on  ten  ambital 
spines  of  length  2.0-2.9  mm.  from  a  50-mm.  urchin,  the  number  varied  from  12  to 
57;  on  ten  spines  of  length  6.0-6.9  mm.,  from  1  to  15  ;  and  on  ten  of  length  10.0- 
16.0  mm.,  from  0  to  9.  It  is  evident,  furthermore,  that  the  number  will  vary  with 
the  intensity  of  epifaunation  of  the  host.  The  largest  number  of  urceolarias  found 
on  any  spine  in  the  entire  study  was  157  on  an  ambital  spine  3.2  mm.  long  from 
a  31-mm.  urchin.  If  an  urchin  bears  a  somewhat  dense  urceolaria  population  (of 
the  degree  indicated  in  Table  II),  ciliates  will  be  found  on  practically  every  short 
spine,  including  those  of  the  periproct,  but  their  occurrence  on  long  spines  is 
unpredictable.  It  is  a  remarkable  fact,  which  is  at  present  unexplained,  that 
urceolarias  are  absent  on  many  of  the  longest  spines  (length,  10.0-16.0  mm.), 
even  though  the  urchin  as  a  whole  harbors  a  dense  population. 

3.  Distribution  of  U.  spinicola  on  individual  spines  of  different  lengths 

It  has  been  shown  that  short  spines  bear  significantly  more  urceolarias  per 
spine  than  long  ones,  but  there  is  a  further  peculiarity  in  the  distribution.  Briefly, 
the  ciliates  are  not  always  distributed  uniformly  along  the  spine  ;  on  long  spines 
they  are  concentrated  on  the  basal  (proximal)  half.  The  regional  distribution  on 
individual  spines  was  studied  by  cutting  detached  spines  in  half  transversely  and 
counting  the  urceolarias  on  the  respective  halves.  A  45-mm.  urchin  from  Labora- 
tory Point  was  selected  for  special  examination,  since  such  urchins  usually  had 
undamaged  spines  and  substantial  epifaunations. 

The  counts  compiled  from  various  spine-samples  from  this  urchin  are  sum- 
marized in  Table  III.  From  this  Table  it  is  seen  that  spines  0.6-0.9  mm.  in  length 
from  any  of  the  three  regions  had  approximately  equal  numbers  of  urceolarias  on 
the  basal  and  distal  halves.  Likewise,  spines  1.0-1.9  and  2.0-2.9  mm.  in  length 
had  equivalent  numbers  on  their  respective  halves.  Spines  3.0-3.9  mm.  in  length, 
on  the  contrary,  had  approximately  three  times  as  many  on  the  basal  half  as  on  the 
distal,  and  spines  4.0-4.9  mm.  in  length  showed  a  still  greater  difference  in  numbers 
between  the  halves.  Finally,  spines  5.0  mm.  or  more  in  length  had  on  the  basal  half 


DISTRIBUTION  OF  URCEOLARIA 


225 


many  times  the  number  on  the  distal  half.  The  spines  of  two  additional  urchins,  a 
34-mm.  specimen  from  Bartlett  Narrows  and  a  31 -mm.  one  from  a  lobster  trap  in 
11  m.  of  water  in  Frenchman  Bay,  were  subjected  to  a  similar  analysis  with  results 
in  full  agreement  with  those  of  Table  III. 

\Vith  reference  to  the  long  spines,  the  data  as  presented  in  Table  III  are 
inadequate  to  show  the  true  distribution  on  them.  For  example,  on  spines  5.0—6.9 
mm.  in  length,  most  of  the  urceolarias  of  the  basal  half  were  actually  restricted 
to  the  basal  third,  and  on  spines  7.0  mm.  or  greater  in  length,  to  the  basal  fourth  or 
even  the  fifth.  Unfortunately,  lack  of  time  prevented  me  from  cutting  such  spines 
into  four  parts  and  counting  the  ciliates  on  the  respective  quarters.  Thus,  the 

TABLE  III 

Distribution  of  U.  spinicola  on  individual  spines  (basal  and  distal  halves,  respectively} 
from  a  45-mm.  urchin.  Spine-sample:  5  of  each  length  from  each  region 


Spine  length 
in  mm. 

Average  number  of 
urceolarias  on 
circumoral  spines 

Average  number  of 
urceolarias  on 
ambital  spines 

Average  number  of 
urceolarias  on 
aboral  spines 

Basal  half 

Distal  half 

Basal  half 

Distal  half 

Basal  half 

Distal  half 

0.6-0.9 

8.2 

6.8 

8.8 

10.2 

9.0 

7.6 

1.0-1.9 

11.4 

12.2 

16.8 

18.8 

12.6 

11.4 

2.0-2.9 

21.2 

20.4 

22.4 

18.0 

12.4 

11.2 

3.0-3.9 

34.0 

11.2 

35.8 

9.6 

7.6 

2.6 

4.0-4.9 

27.2 

7.8 

21.4 

6.2 

4.6 

0.0 

5.0-5.9 

10.8 

0.2 

9.0 

0.0 

5.8 

0.4 

6.0-6.9 

5.0 

0.2 

6.6 

0.2 

3.0 

0.2 

7.0-7.9 

4.4 

0.4 

2.2 

0.0 

1.4 

0.0 

10.0-15.0 

* 

* 

0.8 

0.0 

0.4 

0.0 

*  None  of  this  length  present. 

statement  is  based  merely  on  an  inspection  of  the  spines  and  not  on  actual  counts, 
but  I  believe  that  it  is  nonetheless  correct. 

In  summary,  the  data  of  Table  III  permit  the  following  generalization:  on 
spines  3.0  mm.  or  less  in  length,  the  urceolarias  are  distributed  uniformly  along 
the  length  of  the  spine ;  on  spines  longer  than  3.0  mm.,  they  are  largely  restricted  to 
the  basal  half  of  the  spine. 

4.  Consideration    of  sonic   factors   affecting    flic    distribution    of    fr.    spinicola   on 
individual  spines 

\Yith  reference  to  such  factors,  various  possibilities  suggest  themselves.  For 
example,  does  the  distribution  coincide  with  the  ciliation  of  the  spines?  Hyman 
(1955,  p.  438)  points  out  that  in  echinoids  the  epidermis  of  the  spines  is  "more  or 
less  ciliated"  and  that  "the  ciliation  tends  to  disappear  with  age  except  around  the 
spine  base.  .  .  ."  In  my  experience  with  the  spines  of  5".  droebachiensis,  however, 
carmine  particles  are  swept  energetically  toward  the  distal  ends  of  all  the  spines 
regardless  of  their  length,  indicating  that  much,  if  not  all,  of  the  spine  surface  is 
ciliated.  Thus,  the  evidence  indicates  that  the  absence  of  urceolarias  on  the  distal 
portions  of  long  spines  does  not  result  from  the  absence  of  cilia.  Furthermore, 


226  C.  DALE  BEERS 

specimens  of  U.  spinicola  which  have  been  gently  brushed  oft"  the  spines  are 
capable  of  adhering  firmly  to  various  non-ciliated  surfaces,  such  as  glass,  metal 
and  granite.  Although  these  observations  are  not  extensive,  they  show  at  least 
that  a  ciliated  surface  is  not  necessary  for  the  firm  attachment  of  U.  spinicola. 

A  second  possibility  affecting  distribution  relates  to  the  constant  movements 
of  the  pedicellariae  and  spines ;  that  is,  does  contact  of  the  pedicellariae  with  the 
long  spines  or  contact  of  such  spines  with  one  another  limit  the  distribution  of 
urceolarias  to  the  basal  portions  ?  The  movements  of  the  pedicellariae,  spines  and 
attached  ciliates  can  be  readily  observed  on  an  excised  piece  of  test.  The  stalks 
of  the  pedicellariae,  especially  those  of  the  triphyllous  and  tridentate  ones,  vary 
considerably  in  length,  and  in  their  movements  the  outer  surfaces  of  the  jaws 
commonly  rub  against  spines  of  various  lengths.  Indeed,  the  jaws  of  the  shorter 
pedicellariae  frequently  come  in  contact  with  the  ciliates  on  spines  2.0-3.0  mm. 
long.  When  touched,  the  urceolarias  move  away  from  the  area  of  contact,  but 
they  quickly  resume  their  former  distribution.  In  view  of  their  abundance  on  such 
spines,  it  is  evident  that  their  distribution  is  not  adversely  affected  by  contact  with 
the  pedicellariae.  Spines  may  likewise  touch  the  ciliates  on  other  spines,  but  with 
little  more  than  a  temporary  disturbance  of  the  distribution.  It  is  unusual  for  the 
jaws  of  a  pedicellaria  actually  to  seize  a  spine  and  thereby  injure  the  ciliates. 

If  it  is  assumed,  nevertheless,  that  mechanical  contact  affects  the  distribution 
unfavorably,  one  might  expect  the  ciliates  on  the  bases  of  long  spines  to  distribute 
themselves  uniformly  when  the  spines  are  detached  and  thereby  isolated  from  one 
another.  To  ascertain  whether  the  distribution  changes  under  such  conditions,  12 
spines  3.6-4.8  mm.  in  length,  which  had  urceolarias  on  their  basal  halves  only,  were 
detached  from  a  35-mm.  urchin  and  tranferred  to  two  Syracuse  watch  glasses  of 
filtered  sea  water  (six  spines  in  8  ml.  in  each  watch  glass;  water  changed  daily; 
normal  temperature  of  14°  C.  maintained).  The  average  number  of  ciliates  per 
spine  (counted  at  the  end  of  the  experiment)  was  32.  The  general  distribution  of 
the  ciliates  on  the  respective  quarters  of  each  spine  was  recorded  daily.  In  such 
an  experiment  it  is  difficult  to  compile  quantitative  data,  since  it  is  impossible  to 
count  accurately  the  number  of  urceolarias  on  any  part  of  a  relatively  opaque  spine. 
Fortunately,  such  data  were  not  needed,  for  the  changes  in  the  original  distribution 
were  almost  negligible.  For  example,  after  3  days  conditions  in  the  watch  glasses 
were  as  follows:  cilia  still  active  on  the  spines  (epidermis  living);  urceolarias 
firmly  attached  (none  swimming  freely),  moving  slightly  on  the  spine  surface 
(normal  behavior)  ;  two  urceolarias  on  the  penultimate  quarter  of  each  of  two 
spines ;  none  on  the  distal  quarters ;  the  remainder  on  the  basal  halves  as  originally. 
The  experiment  was  discontinued  2  days  later,  when  conditions  were  as  follows : 
29  ciliates  detached  and  motionless  near  their  respective  spines ;  one  swimming 
freely ;  14  on  the  penultimate  and  distal  quarters  of  certain  spines ;  the  remainder, 
totaling  340,  still  attached  to  the  basal  halves. 

The  experiment  was  repeated,  using  12  spines  4.6-6.4  mm.  in  length  from  a 
42-mm.  urchin  which  carried  an  especially  heavy  epifaunation.  The  average  num- 
ber of  urceolarias  per  spine  was  31.  On  eight  of  the  spines,  the  ciliates  were 
restricted  to  the  basal  quarter ;  on  the  remaining  four,  to  the  basal  half.  After  4 
days  the  original  distribution  was  unchanged,  except  for  four  ciliates  on  the  pen- 
ultimate quarter  of  one  spine.  Two  days  later,  when  the  ciliates  were  beginning 


DISTRIBUTION  OF  UUCKOI.AKIA 

to  die  and  detach,  this  distribution  still  prevailed.  It  is  evident,  therefore,  that 
when  spines  are  detached  and  isolated  from  contact  with  other  spines  or  pedi- 
cellariae,  the  distribution  of  the  urceolarias  undergoes  no  significant  change. 

DISCUSSION 

Since  the  presence  of  U.  spinicola  on  its  host  was  demonstrated  somewhat 
recently,  there  has  been  little  opportunity  for  an  intensive  study  of  the  host-ciliate 
relationship.  Nevertheless,  certain  features  can  be  discussed  briefly. 

Transmission  fro;;/  host  to  host.  In  U.  patellae,  Brouardel  (1947)  observed 
that  a  very  small  percentage  of  the  urceolarias  left  the  limpet  spontaneously  from 
time  to  time  and  swam  freely.  Somewhat  larger  numbers  detached  when  the  host 
was  in  an  unhealthy  or  moribund  condition,  and  agitation  of  the  medium  facilitated 
detachment.  Some  of  the  free-swimming  urceolarias  survived  for  6-8  hr.  in  sea 
water,  and  urceolaria-free  limpets  acquired  ciliates  when  immersed  in  the  water. 
Reynoldson  (1950)  concluded  that  [/T.  initra  was  dispersed  when  small  populations 
occasionally  assumed  a  free-swimming  habit. 

My  efforts  to  induce  U.  spinicola  to  leave  its  host  and  disperse  in  the  medium 
were  notably  unsuccessful.  Its  persistent  adhesion  to  detached  spines  has  been 
mentioned.  Its  behavior  was  also  studied  from  day  to  day  on  excised  pieces  of  test 
and  on  eviscerated  whole  tests.  A  few  of  the  ciliates  detached  and  swam  briefly, 
but  the  number  was  insignificant,  and  the  remainder  perished  in  situ.  Agitation 
of  the  medium,  whether  by  vigorous  stirring  or  by  directing  a  stream  of  sea  water 
on  the  urchin,  was  also  ineffective.  When  a  strong  stream  of  water  from  a  small 
glass  nozzle  was  directed  on  a  spine,  the  urceolarias  merely  retreated  to  the 
opposite  side  of  the  spine. 

Probably  the  natural  method  of  dispersal  can  be  determined  only  by  studying 
the  association  throughout  the  entire  year.  In  U.  patellae,  Brouardel  (1941) 
observed  well-defined  seasonal  variations  in  the  density  of  population,  which  was 
minimal  in  April  and  maximal  in  September  and  October.  He  found  that  dividing 
individuals  were  relatively  numerous  in  May,  but  very  scarce  in  January.  In  U. 
spinicola,  the  population  appears  to  lie  relatively  stable  in  the  summer  months. 
Dividing  individuals  are  scarce — a  fact  reported  earlier  (Beers,  1964)  and  con- 
firmed in  the  present  study- — and  the  population  density,  judged  by  counts  per 
spine,  seems  to  be  as  high  in  mid-June  as  in  late  August.  Evidently  U.  spinicola 
in  summer  is  physiologically  specialized  for  continued  adhesion  to  the  host  and  not 
for  dispersal.  Presumably  dispersal  to  new  hosts  occurs  at  other  times  of  the  year. 

Population  dcnslt\  in  relation  to  habitat  oj  the  Jwst.  In  U.  initra,  Reynoldson 
(1955)  found  that  fluctuations  in  the  ciliate  population  were  directly  correlated 
with  changes  in  the  bacterial  population  of  the  water.  Since  U.  spinicola  feeds 
primarily  on  bacteria,  its  high  incidence  on  the  urchins  of  Frenchman  Bay  is  at- 
tributed to  an  abundance  of  bacterial  food.  Similarly,  its  low  incidence  on  the 
littoral  urchins  of  Long  Ledge  is  attributed  largely  to  a  scarcity  of  food,  although 
the  abrasive  action  of  the  surf,  which  erodes  the  spines,  probably  reduces  the 
ciliate  population  through  mechanical  injury.  Presumably  the  waters  of  Bartlett 
Narrows  are  intermediate  with  respect  to  the  availability  of  food. 

For  the  present  I  am  unable  to  explain  why  U.  spinicola  is  less  abundant  on  the 
aboral  surface  of  the  host  than  elsewhere.  My  earlier  statement  (1964)  to  the 


C.  DALE  BEERS 

effect  that  "it  is  found  very  sparingly  on  the  spines  and  pedicellariae  of  the  equator" 
is  incorrect;  evidently  it  resulted  from  the  examination  of  inadequate  samples. 

Distribution  on  individual  spines.  Probahly  the  most  remarkable  feature  of  the 
distribution  of  U.  spinicola  concerns  its  abundance  on  short  spines,  its  scarcity  or 
absence  on  long  spines,  and  its  concentration  on  the  basal  portions  of  such  spines, 
when  it  is  present.  Attempts  to  correlate  the  distribution  with  the  ciliation  of  the 
spines  or  with  certain  mechanical  factors,  such  as  contact  with  other  spines,  were 
unsuccessful,  as  has  been  said.  It  may  be  argued  that  the  distribution  results  from 
an  avoidance  of  strong  water  currents  which  sweep  across  the  surface  of  the 
urchin  in  its  natural  habitat.  Actually,  such  currents  are  absent  at  Laboratory 
Point  and  elsewhere  in  Frenchman  Bay,  except  in  restricted  channels  of  strong  tidal 
flow.  Furthermore,  urchins  may  be  kept  in  good  health  for  many  days  in  an 
aquarium  containing  gently  running  sea  water,  provided  they  are  supplied  with 
suitable  food,  such  as  Lamlnaria.  In  the  absence  of  strong  water  currents,  these 
urchins  retain  their  urceolarias  in  abundant  numbers  for  at  least  10  days,  and  the 
distribution  on  the  spines  undergoes  no  observable  change.  Finally,  large  urchins 
(diameter,  62-74  mm. )  occupy  the  same  natural  habitat  as  smaller  ones.  Yet  U. 
spinicola  is  very  scarce  or  even  absent  on  the  spines  and  pedicellariae  of  large 
urchins.  It  is  evident,  therefore,  that  its  distribution  cannot  be  related  to  water 
currents. 

The  availability  of  bacterial  food  on  the  surface  of  the  urchin  remains  to  be 
considered.  It  may  be  argued  that  suitable  food  is  more  plentiful  near  the  surface 
of  the  urchin  than  at  the  free  extremities  of  the  long  spines.  If  the  correctness 
of  this  proposition  is  conceded,  it  still  does  not  explain  the  distribution  on  the 
spines.  For  example,  a  long  spine  is  usually  surrounded  by  a  group  of  short 
spines.  Yet  U.  spinicola  is  abundant  on  the  short  spines,  but  scarce  or  absent  on 
the  base  of  the  adjacent  long  spine.  In  this  connection  the  scarcity  or  absence  of 
the  ciliate  on  large  urchins  must  be  mentioned  again.  Presumably  bacterial  food 
is  quite  as  abundant  on  the  surface  of  these  urchins  as  on  smaller  ones. 

It  is  evident  that  an  explanation  of  the  distribution  must  be  sought  in  factors 
other  than  those  already  mentioned.  For  the  present  I  am  disposed  to  conclude 
that  the  distribution  is  related  to  certain  intrinsic  properties  of  the  spines  themselves, 
perhaps  to  the  histological  structure  of  the  spine  epidermis.  The  conclusion  implies 
that  the  spine  surface  is  not  a  uniform  substratum.  Although  ciliated  columnar 
cells  predominate  in  the  epidermis  of  echinoids,  various  types  of  gland  cells  are 
also  present,  as  Hyman  (1955,  p.  438)  indicates.  It  is  possible  that  the  distribution 
of  U.  spinicola  is  correlated  with  the  presence  of  certain  gland  cells,  and  it  is  hoped 
that  this  point  can  be  investigated. 


I  am  indebted  to  my  colleague,  Dr.  Alan  E.  Stiven,  for  useful  suggestions  and 
advice  relative  to  the  plan  of  the  investigation.  The  study  was  further  aided  by  a 
grant  from  the  Research  Council  of  the  University  of  North  Carolina. 

SUMMARY 

1.  At  Mount  Desert  Island,  Maine,  Urceolaria  spinicola  is  of  general  occurrence 
on  the  spines  of  Strongylocentrotus  drocbacliicnsis.  Two  aspects  of  the  urchin- 


DISTRIBUTION  OF  URCEOLARIA  229 

ciliate  relationship  were  studied,  largely  on  urchins  from  Frenchman  Bay :  the 
occurrence  of  the  ciliate  on  urchins  of  different  sizes  and  its  distrihution  on  spines 
of  different  lengths. 

2.  The  density  of  the  urceolaria  population  was  highest  on  urchins  measuring 
2-1—60  mm.  in  diameter  (test  only),  assumed  to  be  2-5  years  of  age  (average  num- 
ber of  ciliates  per  spine,  27).     Smaller  and  therefore  younger  urchins   (diameter, 
9-18  mm.)   had  fewer  per  spine   (average  number,  9).     On  the  largest  urchins 
(62-74  mm.),  assumed  to  be  at  least  6  years  of  age,  urceolarias  were  extremely 
scarce   (average  number  per  spine,   3).     Indeed,   many  urchins  of  this  size  had 
no  ciliates  whatsoever. 

3.  The  distribution  on  spines  of  different  lengths  was  studied  with  special  care 
on  41-mm.   urchins.     The  smallest  spines    (length,   0.6-0.9  mm.)    had   relatively 
few  urceolarias  per  spine  (average  number,  9),  whereas  spines  measuring  1.0-4.9 
mm.  in  length  had  the  largest  number  per  spine   (average,  36).     The  remaining 
spines  (length,  5.0-16.0  mm.)  were  seriated  according  to  length.     On  all  the  sizes, 
the  average  number  of  urceolarias  per  spine  was  well  below  the  maximum  of  36 
and  the  number  decreased  as  the  length  of  the  spine  increased.     Thus,  many  of  the 
longest  spines  lacked  ciliates.     On  spines  measuring  0.6  to  about  3.0  mm.  in  length, 
the  urceolarias  were  distributed  uniformly  along  the  length  of  the  spine ;  on  spines 
longer  than  3.0  mm.,  they  were  concentrated  on  the  basal  half  of  the  spine. 

4.  The  distribution  of  U.  splnicola  on  the  spines  could  not  be  related  convinc- 
ingly to  any  of  the  following  factors :  degree  of  ciliation  of  the  spines,  contact  of  the 
spines  with  one  another,  presence  of  water  currents  in  the  environment  or  avail- 
ability of  bacterial  food  on  the  surface  of  the  urchin.     Therefore,  it  is  concluded 
tentatively  that  the  distribution  is  related  to  the  intrinsic  properties  of  the  spine 
epidermis,  perhaps  to  the  distribution  of  gland  cells  in  it. 

LITERATURE  CITED 

BEERS,  C.  D.,   1964.     Urceolaria  spinicola  n.  sp.,  an  epizoic  ciliate    (Peritrichida,   Mobilina)    of 

sea-urchin  spines  and  pedicellariae.     /.  Proiozool.,  11:  430-435. 
BROUARDEL,  J.,  1941.     Variation  saisonniere  de  la  densite  de  population  et  du  nombre  de  divisions 

de  I'Urceolaria  patellae  (Cuenot)    (Infusoires).     Bull.  Mus.  Hist.  Nat.,  Paris,  Ser.  2, 

13:  314-317. 
BROUARDEL,    J.,    1947.     fitude    de    mode    d'infestation    des    patelles    par    1'  Urceolaria    patellae 

(Cuenot).     Bull.  hist.  Oceanogr.,  Monaco.  No.  911,  pp.  1-7. 
HIRSHFIELD,  H.,  1949.     The  morphology  of  Urceolaria  karyohbia,  sp.  nov.,   Trichodina  tegula, 

sp.  nov.,  and  Scyphidia  ubiquita,  sp.  nov.,  three  new  ciliates  from  southern  California 

limpets  and  turbans.     /.  Morph.,  85:  1-33. 

HYMAN,  L.  H.,  1955.     The  Invertebrates :  Echinodermata.     McGraw-Hill,  New  York. 
REYNOLDSON,    T.   B.,    1950.     Natural    population    fluctuations    of    Urceolaria    initra    (Protozoa, 

Peritricha)  epizoic  on  flatworms.     /.  Animal  Ecol.,  19:  106-118. 
REYNOLDSOX,    T.    B.,    1955.     Factors    influencing   population    fluctuations    of    Urceolaria    initra 

(Peritricha)  epizoic  on  freshwater  triclads.     /.  Animal  Ecol.,  24:  57-83. 
SWAN",    E.    F.,    1961.     Some   observations    on    the    growth    rate    of    sea    urchins    in    the    genus 

Strongylocentrotns.     Biol.  Bull.,  120:  420-427, 


THE  GENETICS  OF  ARTEMIA  SALINA. 
VI.    SUMMARY  OF  MUTATIONS  x 

SARANE  T.  BOWEN,  JEAN  HANSON,  PHILIP  BOWLING  AND  MAN-CHIU  POON 

Academy  of  Sciences,  Golden  Gate  Park,  San  Francisco  94138  and  Department  of  Biology, 
San  Francisco  State  College,  San  Francisco,  California  94132 

Artemia  salina  is  of  interest  to  geneticists  because  some  populations  are  diploid, 
triploid,  tetraploid,  or  pentaploid  (see  reviews  by  Goldschmidt,  1952;  Barigozzi, 
1957;  and  Stefani,  1964).  Although  the  cytology  of  the  brine  shrimp  has  been 
studied  for  many  years,  it  is  only  recently  that  attempts  have  been  made  to  analyze 
mutant  traits  governed  by  one  locus.  Cervini  (1965)  has  described  the  spontaneous 
recessive  autosomal  mutation  "curly"  (rr)  which  causes  ventral  curling  of  the 
abdomen.  In  our  study  of  gonochoristic  diploid  populations,  we  have  found  seven 
mutations  and  many  sex  mosaics  and  eye  color  mosaics.  The  purpose  of  this 
paper  is  to  describe  these  morphological  variations. 

MATERIALS  AND  METHODS 

Genetic  techniques,  glassware,  and  feeding  schedule  were  described  in  detail 
earlier  (Bowen,  1962).  In  brief,  two  or  three  nauplii  were  placed  in  each  vial  of 
culture  medium  (50  g.  NaCl  per  liter  of  sea  water).  Once  a  week,  0.05  or  0.10  cc. 
of  yeast  suspension  (1  cc.  dry  brewers'  yeast  mixed  with  9  cc.  of  medium)  was 
added  to  each  vial.  Inbred  stocks  are  maintained  at  21-24°  C. ;  shrimps  reach 
sexual  maturity  at  two  to  three  weeks  of  age.  Origins  of  the  inbred  stocks  and  of 
the  wild  populations  have  been  given  earlier  (Bowen,  1964,  1965). 

Macrophotographs  of  living  Artemia  were  taken  with  a  Brinkmann  camera 
(30"  bellows)  and  collimated  transmitted  light.  For  histological  preparations, 
shrimps  were  anesthetized  in  ether  and  a  few  legs  were  removed  to  allow  entry  of 
fixative ;  they  were  placed  in  Bouin's  for  24—48  hours,  stored  in  70%  ethanol, 
embedded  in  paraffin,  sectioned  at  10  /x  and  stained  in  haematoxylin  and  eosin. 

India  ink  was  injected  into  the  thorax  through  micropipettes  with  tips  of  2-A  p. 
(O.D.)  by  means  of  a  de  Fonbrune  micromanipulator.  Best  results  were  obtained 
when  shrimps  were  first  anesthetized  with  ether  and  then  placed  within  an  enclosure 
improvised  from  a  plastic  slide  (Bowling,  1963).  At  the  time  of  injection,  the 
culture  medium  was  drawn  off  to  prevent  loss  of  ink  from  the  micropipette. 
Artemia  is  able  to  survive"  for  a  few  minutes  outside  the  liquid  environment. 

MORPHOLOGY  OF  WILD-TYPE  ARTEMIA 

Fransemeier  (1939),  Weisz  (1947)  and  Dutrieu  (1960)  have  described  embry- 
onic development.  Heath  (1924)  and  Weisz  (1946,  1947)  have  outlined  the 

1  Supported  by  grants  from  the  National  Science  Foundation  (NSF  G-13219  and  GB-3836). 
We  would  like  to  thank  Mrs.  Jean  Cons  who  made  the  histological  preparations  and  Mrs. 
Jean  Chapman  who  discovered  the  mosaic  shown  in  Figure  15. 

230 


MUTATIONS  IN  ARTEMIA  231 

changes  during  larval  development.  We  have  used  Heath's  diagrams  to  determine 
the  instar  of  immature  shrimps.  The  morphology  of  the  adult  has  been  described 
by  Weisz  (1947)  and  Lochhead  (1950).  The  adult  body  consists  of  head,  11 
thoracic  segments,  two  genital  segments,  and  6  abdominal  segments  (Weisz,  1947, 
p.  81).  Each  of  the  11  thoracic  somites  bears  a  pair  of  phyllopodia  (Figs.  2  and  5  ). 
The  last  abdominal  somite  is  fused  to  the  telson  which  bears  the  caudal  furca 
(Fig.  5). 

The  head  of  Artemia  bears  a  pair  of  slender  antennules  and  a  larger  pair  of 
antennae.  The  antennae  show  sexual  dimorphism,  being  larger  and  modified  for 
clasping  in  the  male  (Figs.  1,  2  and  5). 

The  median  eye  consists  of  three  cups,  or  ocelli.  It  is  red  in  the  first  instar  and 
does  not  gain  black  pigment  until  the  second  (Vaissiere,  1961,  p.  29).  By  the 
third  instar,  black  pigment  is  usually  present  in  the  rudiments  of  the  lateral  com- 
pound eyes  also.  The  normal  compound  eye  is  seen  in  Figures  7  and  12.  The 
cuticle,  which  is  secreted  by  the  epidermal  cells,  is  not  thickened  to  form  a  lens. 
Each  ommatidium  consists  of  a  cone  surrounded  by  the  four  crystalline  cells  which 


FIGURE  1.     Ventral  view  of  head  of  normal  female  (left),  normal  male  (center)  and 
Ctt/+  female  with  curved  antennae  (right). 

secreted  it  and  a  proximal  rhabdome  surrounded  by  a  retinula.  The  rhabdome 
lacks  the  alternating  layers  of  microtubules  found  in  other  crustaceans  (Eguchi  and 
Waterman,  1965).  Each  retinula  usually  contains  5  principal  cells  and  a  sixth 
accessory  cell  (Debaisieux,  1944,  p.  13).  The  retinular  cells  contain  the  photo- 
stable  black-brown  pigment  which  gives  the  wild-type  eye  its  black  color.  Each 
retinular  cell  is  a  primary  neuron  which  penetrates  the  basement  membrane  and 
continues  as  an  axon  in  the  fascicular  zone  of  the  eyestalk.  There  are  two  optic 
ganglia:  the  distal  lamina  ganglionaris  and  a  proximal  medulla  (Fig.  7).  Nerves 
from  the  ganglia  enter  the  supra-esophageal  ganglion. 

The  gonads  of  both  sexes  are  straight  cylinders  lying  above  and  lateral  to  the 
gut  in  the  two  genital  segments  and  first  few  abdominal  segments.  Gametes  leave 
the  anterior  ends  of  the  gonads  by  means  of  ducts.  On  each  side  of  the  body,  the 
male  has  a  U-shaped  seminal  vesicle,  vas  deferens,  and  penis.  The  female  has  two 
oviducts  (lateral  pouches)  which  convey  the  eggs  into  a  single  median  uterus 
wherein  they  undergo  segmentation.  Four  grape-like  clusters  of  shell  glands 
empty  their  secretions  into  the  uterus.  The  oviducts  and  uterus  lie  within  a 
ventral  median  swelling,  the  ovisac  (Fig.  2). 


232 


BOWEN,  HANSON,  BOWLING  AND  POON 


FIGURES  2-4. 


MUTATIONS  IX  AIMT.M  \  \ 


233 

^UP 


FIGURE  5.     Dorsal  view  of  living  normal  male  Artcmia. 

FIGURE  6.  Dorsal  view  of  cyclops  male.  The  distance  between  the  lines  on  the  right  is 
0.5  mm. 

MORPHOLOGICAL  VARIATIONS 
A.   Variations  in  morphology  <>j  "a'ilil  populations 

Wild-type  Artcmia  look  very  much  alike.  This  is  surprising  when  one  con- 
siders their  geographical  isolation :  they  are  found  in  salt  lakes  and  salterns  on 

FIGURE  2.  Lateral  view  of  living  normal  female  brine  shrimp,  showing  two  genital  and  6 
abdominal  segments.  The  arrow  indicates  the  spine  on  the  ovisac. 

FIGURE  3.  Lateral  view  of  s/s  female  which  has  extreme  stump  expression.  Only  two 
genital  segments  are  present. 

FIGURE  4.     Lateral  view  <if  living  s/s  female  with  moderate  stump  phenotyp',-. 


234 


BOWEN,  HANSON,  DOWLING  AND  POON 


FIGURE  7.  Dorsal  view  of  normal  compound  eye  of  living  brine  shrimp.  The  medulla 
(ME),  lamina  ganglionaris  (LA)  and  the  cones  of  the  ommatidia  (CO)  are  clearly  seen. 
A,  anterior  border ;  P,  posterior  border  of  eye.  The  other  photographs  on  this  page  are 
oriented  in  a  similar  manner. 

FIGURES  8,  9,  AND  10.  Dorsal  views  of  living  shrimps  of  c/c  genotype,  showing  variation 
of  expression  of  crinkle  phenotype. 

FIGURE  11.  Dorsal  view  of  eye  of  living  garnet  shrimp  (g/g  genotype).  In  areas  where 
the  retinular  cells  have  degenerated,  the  eye  is  transparent.  Few  axons  remain  in  the  fascicular 
zone  (between  lamina  and  basement  membrane). 


MUTATIONS   IX   AKTHMIA  235 

six  continents.  Furthermore,  certain  populations  are  known  to  be  reproductively 
isolated.  Whereas  most  American  populations  are  gonochoristic,  many  European 
populations  are  parthenogenetic.  American  diploid  gonochoristic  populations  are 
also  reproductively  isolated  from  the  diploid  gonochoristic  population  from  San 
Bartolomeo  near  Cagliari,  Sardinia  (Kuenen,  1939,  p.  387;  Bowen,  1965).  The 
gonochoristic  Mono  Lake,  California,  population  is  a  sibling  species  which  cannot 
survive  in  sea  water  or  concentrated  brines  in  which  all  the  other  populations 
thrive  (Bowen,  1964). 

When  reared  under  identical  environmental  conditions,  some  wild-type  popula- 
tions can  be  distinguished  by  quantitative  differences  such  as  ratio  of  lengths  of 
abdomen  and  trunk  (see  data  and  review  by  Gilchrist,  1960).  We  have  examined 
two  parthenogenetic  populations  (from  Sete,  France,  and  from  Rottnest  Island,  near 
Perth,  Australia)  and  six  gonochoristic  populations  (from  Europe,  North  America 
and  South  America)  and  have  detected  only  a  few  differences  of  a  qualitative  nature. 
For  example,  females  of  the  Quemado,  New  Mexico,  population  have  a  small 
projection  on  their  antennae  which  is  absent  in  other  females  (Bowen,  1964).  Both 
males  and  females  from  the  San  Bartolomeo  population  lack  the  spikes  on  the 
genital  segments  (seen  in  Figures  2  and  3)  which  are  present  in  other  populations 
(Bowen,  1965). 

B.  Description  of  seven  mutant  genes 

Six  of  the  seven  mutations  listed  below  appeared  when  non-irradiated  stocks 
were  inbred  by  sibling  matings ;  one  (garnet)  appeared  in  progeny  of  x-irradiated 
shrimps.  Two  mutations  (white  and  curved)  were  found  by  S.  T.  B. ;  the  other 
five  were  discovered  by  J.  H.  Five  are  completely  recessive;  two  (crinkle  and 
curved)  have  expression  in  a  fraction  of  the  heterozygotes.  Six  of  the  seven 
mutations  are  carried  in  our  laboratory  in  pure-breeding  cultures ;  the  exception  is 
cyclops  which  occurs  in  high  frequency  in  stock  #1.  Five  are  autosomal,  one 
(white)  is  partially  sex-linked,  and  the  mode  of  inheritance  of  one  (cyclops)  is  not 
completely  known. 

1.  Curved  (Cit).  Males  homozygous  for  the  mutant  gene  are  normal;  ex- 
pression is  therefore  said  to  be  sex-limited.  Expression  in  the  females  is  variable, 
ranging  from  enlarged,  sharply  bent  antennae  (easily  seen  without  a  microscope) 
to  antennae  which  are  normal  in  size  but  which  have  a  small  projection  on  the 
posterior  surface  (visible  only  when  the  female  is  anesthetized  and  examined  under 
30  X  magnification).  Females  with  extreme  curved  expression  have  antennae 
similar  to  those  of  normal  males  (Fig.  1 ) . 

The  first  females  with  curved  antennae  were  discovered  in  1965  among  the 
progeny  of  a  cross  between  inbred  stocks  #5  and  #12.  These  females  were  mated 
to  males  from  an  inbred  wild-type  stock  derived  from  salterns  on  Pichilingue  Island, 
Mexico.  Of  the  313  hybrid  female  progeny,  56  showed  strong  expression  of 
curved.  In  retrospect,  it  seems  probable  that  the  first  females  were  Cu/+  geno- 
type. The  hybrid  progeny  were  inbred  for  four  generations  with  constant  selection 
of  females  for  strong  expression  of  curved.  The  result  was  stock  #49  which  has 
high  incidence  of  curved  (Table  I). 

The  degree  of  bending  of  female  antennae  increases  if  animals  are  reared  at 


236 


BOWEN,  HANSON,  BOWLING  AND  POON 


12 


NRC 


BM 


13 


FIGURES  12-13. 


MUTATIONS  IN  ARTEMIA 


237 


27°  C.  instead  of  22°  C.  At  both  temperatures,  frequency  of  females  with  strong 
expression  (detected  without  use  of  a  compound  microscope)  increases  as  the 
population  ages.  The  Artcinia  described  in  Table  I  were  reared  at  27°  C.  and 
classified  at  an  age  of  five  weeks. 

Females  from  the  wild-type  Pichilingue  inbred  stock  were  mated  to  stock  #49 
males.  Data  in  the  second  line  of  Table  I  show  that  31/52,  or  60%  of  the  Fj 
females  had  curved  antennae,  indicating  that  this  trait  is  determined  by  a  gene 
with  incomplete  dominance  which  can  be  transmitted  through  the  male.  This 
excludes  the  possibility  that  the  curved  trait  is  governed  by  a  gene  on  the  Y 
chromosome;  the  female  is  the  heterogametic  sex  (XY)  in  Artcinia  (Bowen,  1963a, 
1965;  Stefani,  1963).  The  F1  females  were  backcrossed  to  +/+  Pichilingue 
males.  The  data  in  the  last  line  of  Table  I  show  that  their  daughters  had  curved 

TABLE  I 

•Segregation  of  gene  for  curved  antennae  which  has  expression  only  in  fannies 
(classification  at  5  weeks  of  age) 


Parental  cross 


Number  with 

Number  with 

Description 

Presumed 

strong**  ex- 

some*** ex- 

genotype 

pression/total 

pression/total 

curved  stock  #49  9  9   X  stock  #49  <?  & 

Cu/Cu  X  Cu/Cu 

43/46 

46/46 

non-curved  Pich.*   9  9   X  stock  #49  c?  cf 

+  /+  X  Cu/Cu 

31/52 

41/52 

non-curved  Pich.   99  X  c?  o71  FI  (Pich.   9  9 

X  #49  d1) 

+  /+  X  Cu/  + 

6/22 

8/22 

curved  9  9  FI  (Pich.  9  9  X  #49  rf)  X  Pich.  d"  cf 

CK/  +  X  +/  + 

12/44 

16/44 

Female  progeny  classified 
as  curved 


*  Pichilingue  inbred  wild-type  stock. 

**  Strong  expression  indicates  that  unanesthetized  females  were  classified  as  curved  after 
observation  under  a  dissecting  microscope  (7  X). 

***  Some  curved  expression  includes  those  with  strong  expression  and  those  with  mild  ex- 
pression (seen  only  on  anesthetized  females  under  30  X). 

antennae.  This  demonstrates  that  curved  is  not  located  on  the  X  chromosome. 
(If  curved  were  on  the  X,  the  Fx  females  would  be  XCuY+  and  would  be  unable  to 
transmit  the  mutant  gene  to  their  female  offspring.) 

We  conclude  that  curved  is  a  dominant  autosomal  sex-limited  gene  with  incom- 
plete penetrance  and  variable  expression.  Females  with  mild  expression  have  a 
projection  of  the  posterior  surface  of  their  antennae,  as  do  wild-type  Quemado 
females. 

2.  Stump  (Y).  This  autosomal  recessive  mutation  was  discovered  in  1960 
during  inbreeding  of  wild-type  shrimps  from  salterns  on  San  Francisco  Bay.  In 
some  s/s  shrimps,  the  abdomen  is  normal.  In  others,  it  is  twisted  dorsally  (Fig. 
4),  or  it  lacks  from  one  to  six  segments.  The  female  shown  in  Figure  3  lacked  all 

FIGURE  12.  Longitudinal  section  of  normal  compound  eye.  Black  pigment  is  within  the 
retinular  cells. 

FIGURE  13.  Longitudinal  section  of  eye  of  shrimp  with  ^v/^v  (white)  genotype.  The  basal 
membrane  (BM)  and  the  nuclei  of  the  retinular  cells  (NRC)  are  seen.  The  retinular  cells 
contain  opaque  white  pigment.  All  histological  preparations  (Figs.  12-17)  were  prepared  in 
the  same  manner  (haematoxylin  and  eosin). 


238 


BOWEN,  HANSON,  BOWLING  AND  POON 


MUTATIONS  IN  ARTKMIA  239 

six   abdominal   segments.     Although    her   second   genital    segment   was   attached 
directly  to  the  telson,  she  had  normal  fertility. 

Matings  of  stump  males  to  stump  females  gave  rise  to  a  stock  of  s/s  shrimps  in 
which  only  37%  of  the  shrimps  showed  sufficient  expression  to  be  classified  as 
stump  (when  viewed  under  a  7  X  dissecting  microscope).  From  matings  within 
this  s/s  stock,  the  ratio  of  stump  to  non-stump  progeny  was  the  same  when 
extreme  stump  parents  were  selected  as  when  non-stump  parents  were  selected. 
This  suggests  that  the  .y  gene  has  low  penetrance  in  homozygotes. 

3.  Red   (r}.     This  autosomal  recessive  mutation  appeared  during  inbreeding 
of  a  stock  from  Great  Salt  Lake,  Utah,  in  1960.     Segregation  data  and  descriptions 
of  r/r,  r/+   and    +/+    shrimps  appeared   earlier    (Bowen,    1962).     Briefly,   r/r 
shrimps  have  colorless  compound  eyes  from  the  third  to  the  fifth  instar.     Then 
deposition  of  red  pigment  begins  in  the  posterior  border  and  rapidly  progresses 
anteriorly   (Fig.   18).      Median  eye  and  compound  eyes  are  bright  red  from  the 
seventh  through  the  thirteenth  instars  (about  two  to  three  weeks  of  age).     Shortly 
after  sexual  maturity,  brown-black  pigment  appears  in  the  caudal  retinular  cells 
of  the  compound  eyes.     Deposition  of  black  pigment  also  progresses  anteriorly, 
masking  the  red  pigment  within  48  hours  (Fig.  14).     The  median  eye  may  also 
darken,   but  more  often  remains  red.     A  similar  mutation  governs   rate  of   eye 
pigment  production  in  the  amphipod,  Gamniarus  chevreuxi.     The  gene  d  (delayed 
melanin)  delayed  deposition  of  pigment  until  the  amphipods  were  sexually  mature 
(Ford  and  Huxley,  1929). 

4.  Cyclops   (cy).     During  development  of  cyclopean  metanauplii,   the  lateral 
eyes  give  the  illusion  of  moving  forward  and  fusing  together  in  the  midline  as  a 
single  large  compound  eye  (Figs.  6  and  20).     The  eyes  are  in  the  normal  location  at 
the  fourth  instar ;  fusion  is  complete  by  the  ninth  instar.     Histological  preparations 
indicate  that  ganglia  and  nerves  of  the  two  optic  stalks  fuse.     The  eye  of  the 
cyclopean  Artemia  is  similar  to  the  normal  eye  of  the  cladoceran,  Leptodora. 

Cyclopean  Artemia  occur  sporadically  in  stock  #1  (r/r  genotype)  which  is 
descended  from  the  Great  Salt  Lake  population.  Nine  cyclops  were  observed  in 
this  stock  in  1961.  Two  died  before  sexual  maturity.  Of  those  that  matured, 
four  were  male  and  three  were  female.  Only  two  produced  offspring.  In  the 
first  successful  mating,  a  cyclopean  male  (r/r}  was  outcrossed  to  a  wild-type 
female.  Of  the  27  progeny,  only  9  lived  to  maturity ;  all  were  non-cyclopean  r/+ 
shrimps.  These  were  bred  inter  se  but  none  of  the  255  F2  progeny  was  cyclopean. 
In  the  second  successful  mating,  a  cyclopean  female  was  mated  to  her  brother 
and  produced  42  nauplii,  of  which  11  reached  maturity.  One  was  a  male  cyclops 
which  failed  to  produce  progeny.  His  sibs  were  mated  inter  se;  of  140  offspring, 
75  reached  maturity  and  all  had  r/r,  non-cyclopean  eyes.  This  finding  is  quite 
different  from  the  results  obtained  when  the  sibs  of  another  cyclops  were  mated : 
three  of  the  51  progeny  were  cyclopean. 

FIGURE  14.  Longitudinal  section  through  eye  of  a  sexually  mature  shrimp  of  r/r  genotype. 
A,  anterior ;  P,  posterior.  Deposition  of  black  pigment  has  begun  in  retinular  cells  in  the 
posterior  part  of  the  eye.  The  anterior  portion  is  still  bright  red. 

FIGURE  15.  Section  through  eye  of  sex  mosaic  #14  which  was  also  mosaic  for  eye  color. 
There  is  a  central  patch  of  white  tissue  surrounded  by  pigmented  tissue  of  r/r  genotype  (black 
pigment  in  this  mature  shrimp).  Note  that  pigmented  and  white  retinular  cells  lie  side  by  side 
with  no  areas  of  intermediate  color. 


240 


BOWEN,  HANSON,  BOWLING  AND  POON 


The  study  of  cyclops  was  abandoned  because  the  cyclopean  shrimps  had  low 
viability  and  fertility.  The  trait  may  be  governed  by  a  recessive  gene  which  has 
low  viability  or  low  penetrance,  or  it  may  be  affected  by  more  than  one  locus.  A 
future  study  might  be  made  of  stock  #1  in  varying  ionic  environments  or  at  different 
temperatures,  in  an  attempt  to  increase  the  frequency  of  cyclopean  shrimps. 

5.  Crinkle  (c).  In  immature  shrimps  with  c/c  genotype,  the  compound  eyes 
are  normal.  After  sexual  maturity,  some  retinular  cells  detach  from  the  ommatidia 
with  the  result  that  the  eye  becomes  mottled  in  appearance.  In  c/c  shrimps  six 
weeks  old  or  older,  the  distal  ends  of  some  retinular  cells  lie  in  the  eye  stalk  rather 
than  in  the  normal  eye  field.  A  characteristic  "crinkle  patch"  (containing  retinular 
cells  but  lacking  cones)  appears  in  the  anterior  dorsal  region  of  the  stalk,  medial 
to  the  basement  membrane  (Figs.  8,  9  and  10) . 

The  first  crinkle-eyed  shrimp  appeared  in  1960  in  an  inbred  stock  derived 
from  salterns  on  San  Francisco  Bay  (Bowen,  1963a).  From  a  backcross  of  c/  + 
to  c/c  shrimps,  116/274,  or  42  %  of  the  progeny  had  crinkle  phenotype  at  the  age 
of  five  weeks.  The  frequency  had  increased  to  114/224,  or  $\c/r,,  when  these  back- 
cross  progeny  reached  7  weeks  of  age,  because  expression  of  c/c  genotype  becomes 

TABLE  II 

Segregation  of  the  gene  g 


Classification  of  progeny 

(at  4  weeks  of  age) 

Type  of  mating 

Total 

Wild 

Garnet 

gig  X  gig 

0 

301 

301 

&  +  /  +  X   9  gig 

105 

0 

105 

<?  g/g  X   9  +/  + 

2<>7 

0 

297 

rf1  +/g  X   9  g/g 

158 

133 

291 

<?  g/g  X   9  +/R 

261 

240 

501 

more  pronounced  with  age.  Unfortunately,  the  crinkle  gene  has  some  expression 
in  a  small  fraction  of  heterozygotes  and  this  frequency  is  also  increased  with  age. 
Evidence  for  this  is  seen  in  data  from  a  heterozygous  ¥l  population.  At  four  weeks 
of  age,  3/90  c/+  shrimps  had  crinkle  eyes;  at  10  weeks  of  age,  8/53  were 
crinkle-eyed.  The  frequency  of  expression  in  c/+  heterozygotes  may  vary  with 
environment  and  genetic  background  as  well  as  with  age. 

6.  Garnet  (g).  This  mutant  eye  color  first  appeared  in  1961  in  the  F2  of  two 
shrimps  from  San  Francisco  cysts  which  had  received  10  kr  of  x-irradiation 
(Bowen,  1963b).  After  a  pure-breeding  stock  was  established,  reciprocal  crosses 
were  made  between  garnet  and  wild-type  and  testcrosses  were  made  of  the  Flt 
The  data  in  Table  II  indicate  that  garnet  eye  color  is  due  to  an  autosomal  recessive 
gene  which  has  complete  penetrance  in  the  homozygote  and  no  expression  in  the 
heterozygote. 

Eye  color  becomes  progressively  lighter  as  the  g/g  shrimp  ages.  In  the  first 
three  instars,  the  eyes  cannot  be  distinguished  from  wild-type.  However,  at  one 
week  of  age  (fourth  to  seventh  instar),  the  eyes  become  dark  brown  or  red-brown 
(garnet).  This  mutation  affects  both  eye  color  and  structure.  At  sexual  maturity 


MUTATIONS   IX  ARTEMIA 


241 


•  l«  ••" 


FIGURE  16.  Longitudinal  section  through  tip  of  the  eye  of  a  garnet  (//A/)  shrimp. 
Whereas  the  rhabdome  remains  intact,  the  retinular  cells  have  degenerated,  leaving  cell 
fragments  filled  with  garnet  pigment  above  and  below  the  basement  membrane. 

FIGURE  17.  Longitudinal  section  through  eyestalk  of  a  garnet-eyed  shrimp.  The  retinular 
cell  axons,  which  normally  lie  between  the  basement  membrane  and  the  distal  optic  ganglion, 
have  degenerated. 


242 


BOWEN,  HANSON,  BOWLING  AND  POON 


FIGURES  18-21. 


MUTATIONS  IN  ARTEMIA  243 

(three  weeks  of  age),  the  garnet  eyes  often  have  an  irregular  proximal  border. 
Many  retinular  cells  degenerate,  causing  the  eye  to  be  flecked  with  clear  areas.  By 
6  to  8  weeks  of  age,  the  compound  eyes  of  g/g  shrimps  have  irregular  patches  of 
garnet  pigment  only  at  the  periphery  of  the  eye ;  some  eyes  are  almost  colorless 
(Fig.  11).  The  median  eye  is  often  unpigmented  also.  In  histological  prepara- 
tions, it  is  seen  that  retinular  cells  detach  from  the  rhabdome  and  basement  mem- 
brane. Many  disintegrate.  Those  that  remain  are  small  spherical  cells  (or 
cell  remnants)  which  lie  medial  to  the  basement  membrane  or  in  the  periphery  of 
the  eye  field  (Fig.  16).  Note  that  the  rhabdome  remains  structurally  intact  after 
degeneration  of  the  retinular  cells.  At  the  age  of  two  months,  no  axons  can  be 
seen  in  the  zone  between  basement  membrane  and  distal  ganglion  (Fig.  17).  By 
this  time,  garnet-eyed  shrimps  have  lost  the  tendency  to  orient  with  their  ventral 
surface  toward  a  light  source. 

Correlated  with  a  decreasing  pigmentation  of  the  retinular  cells  is  an  increasing 
deposition  of  garnet  pigment  in  other  specialized  cells.  By  the  sixth  instar, 
garnet  pigment  is  seen  in  the  antennal  glands.  By  the  eighth  instar,  pigment  is 
present  in  the  phagocytic  storage  cells  and  in  the  maxillary  glands.  When  g/g 
shrimps  reach  four  weeks  of  age  or  more,  they  have  conspicuous  pigmented  areas 
around  the  caudal  walls  of  the  lobes  of  the  stomach,  on  the  lateral  surface  of  the 
anterior  part  of  the  digestive  tract,  and  on  the  outside  of  the  anterior  portion  of 
the  heart.  These  are  sites  where  phagocytic  storage  cells  are  concentrated.  To 
demonstrate  this,  wild-type  shrimps  were  injected  with  India  ink.  Ink  was  found 
in  cells  in  the  phyllopodia,  in  the  maxillary  glands,  and  scattered  along  the  outer 
walls  of  the  heart.  These  phagocytic  cells  were  particularly  dense  on  the  outside 
of  the  lateral  walls  of  the  gut  in  the  anterior  thorax.  In  Figure  19,  the  character- 
istic "inverted  U"  distribution  of  ink-filled  phagocytic  cells  across  the  dorsal  surface 
of  the  stomach  walls  is  seen.  The  garnet  pigment  in  mature  g/g  shrimps  has 
an  identical  distribution. 

In  order  to  determine  the  mechanisms  of  cellular  degeneration  and  of  pigment 
dissolution,  a  study  should  be  made  of  the  ultrastructure  of  the  eye  in  g/g  shrimps, 
with  particular  attention  to  changes  in  the  lysosomes  with  age.  It  is  interesting  to 
note  that  a  recessive  mutation  (albino)  which  brought  about  complete  degeneration 
in  retinular  cells  appeared  in  the  amphipod  Gammarus  chevreuxi  (reviewed  by 
Sexton  and  Clark,  1936,  p.  365). 

7.  White  (w).  The  gene  for  white  eyes  is  recessive  and  partially  sex-linked; 
that  is,  the  white  locus  is  on  the  homologous  segment  of  the  sex  chromosomes 

FIGURE  18.  Ventral  view  of  living  sixth  instar  metanauplius  of  r/r  genotype.  The  median 
eye  is  red.  The  anterior  portions  of  the  two  compound  eyes  lack  pigment.  Red  pigment  is 
being  laid  down  in  posterior  ommatidia ;  this  process  will  proceed  anteriorly  until  the  entire 
eye  becomes  red. 

FIGURE  19.  Dorsal  view  of  head  and  upper  thorax  of  living  wild-type  female  which  has 
been  injected  with  India  ink.  Note  ink-filled  phagocytic  cells  concentrated  in  a  U-shaped 
area  above  the  posterior  walls  of  the  stomach  lobes  (SL). 

FIGURE  20.     Dorsal  view  of  eye  of  living  cyclops  male. 

FIGURE  21.  Lateral  view  of  genital  segment  of  living  mosaic  male  #3  which  has  one 
"penis"  which  is  a  mixture  of  ovisac  and  penis  structures  proximally  and  phyllopodium  struc- 
tures distally.  The  arrow  indicates  the  spine  of  the  "ovisac" ;  SV,  seminal  vesicle  filled 
with  sperm. 


244  BOWEN,  HANSON,  DOWLING  AND  POON 

(Bowen,  1963a).  In  matings  of  XWXW  males  to  XWY+  females  or  to  X+YW  females, 
crossing  over  can  lie  detected.  The  amount  of  recombination  between  the  white 
locus  and  the  sex  locus  varies  from  0.05 %  to  20%,  depending  upon  which  female 
line  is  tested.  The  characteristic  crossover  frequency  is  transmitted  matroclinously 
(Bowen,  1965). 

The  gene  it'  has  no  expression  in  the  heterozygote  and  has  complete  penetrance 
in  the  homozygote.  The  lateral  and  median  eyes  in  u'/iv  shrimps  are  white 
throughout  the  lifespan  with  one  complication :  some  zv/u'  shrimps  develop  a  pink 
or  bright  orange  cast  to  their  eyes.  Orange  pigment  may  be  in  the  retinular 
cells  and/or  in  the  nerve  and  ganglia  in  the  eye  stalk.  Attempts  to  select  for 
orange  color  in  breeding  experiments  have  failed.  Further  evidence  that  this 
trait  is  not  heritable  is  the  fact  that  if  w/iv  shrimps  with  orange  eye  color  are 
transferred  to  fresh  culture  medium,  the  color  will  fade  within  a  few  weeks,  which 
suggests  that  the  orange  tinge  must  be  due  to  the  storage  of  some  material  obtained 
from  the  food. 

If  one  compares  white  eyes  and  garnet  eyes  (Figs.  13  and  16),  one  sees  that 
aging  garnet  eyes  become  transparent  due  to  degeneration  of  retinular  cells,  whereas 
white  eyes  contain  opaque  white  pigment  in  their  retinular  cells. 

C.  Gene  interactions  (r.  g  and  c}  and  linkage 

Shrimps  with  c/c ;  y/y  genotype  have  reddish-brown  eyes  as  they  approach 
sexual  maturity.  Three  weeks  later,  crinkle  patches  on  the  eye  stalk  appear  but 
they  are  difficult  to  see  because  at  this  time  the  retinular  cells  degenerate  under 
control  of  the  garnet  gene.  Shrimps  with  c/c ;  r/r  genotype  have  dark  red  eyes 
as  they  approach  sexual  maturity.  Three  weeks  later,  the  main  eye  fields  turn 
black,  but  the  crinkle  patches  remain  red. 

The  most  useful  genetic  marker  is  the  mutation  for  white  eyes.  It  has  complete 
penetrance  in  the  homozygote,  no  expression  in  the  heterozygote,  and  is  easily 
classified  at  all  ages  and  in  all  environments.  The  other  mutations  fail  to  meet 
one  or  more  of  these  criteria.  For  this  reason  and  also  because  of  complex  inter- 
actions between  the  mutations  affecting  the  eyes,  linkage  tests  are  tedious  to  carry 
out.  At  the  present  time,  the  only  linkage  relationship  which  has  been  established 
is  that  between  the  white  locus  and  the  sex  locus  (Bowen,  1965). 

D.  Epistasis  and  tests  for  allelisui  (eye  color  genes:  r,  g  and  w) 

In  order  to  determine  if  the  three  eye  color  mutations  were  allelic,  the  following 
stocks  were  crossed :  garnet  X  red,  garnet  X  white,  and  red  : :  white.  Because  the 
Fx  progeny  from  the  three  crosses  were  wild-type,  we  conclude  that  the  three 
mutations  are  not  alleles. 

The  gene  w,  when  homozygous,  is  epistatic  to  the  gene  r.  Stock  #10  breeds 
true  for  white-eyed  males  (XWXW;  r/r)  and  red-eyed  females  (XWY+;  r/r)  because 
crossing  over  between  the  X  and  Y  is  suppressed  in  this  stock. 

White  is  also  epistatic  to  garnet.  Evidence  for  this  is  seen  in  the  results  of  a 
cross  of  garnet-eyed  shrimps :  g/g ;  X+YW  females  to  g/g  \  X+XW  males.  Twenty- 
seven  per  cent  (57/211)  of  the  progeny  were  white-eyed.  Of  the  57  white-eyed 
shrimps,  53  were  females  and  4  were  males  resulting  from  crossing  over. 


MUTATIONS  IN  AKTEMIA  245 

E.  Discussion  oj  the  mechanism  oj  gcuc  action  (r,  g  and  w) 

The  black  eye  pigment  of  wild-type  Artcuiia  is  an  ommochrome  (Becker,  1942 j. 
Probably  the  gene  for  garnet  eyes  affects  ommochrome  degradation  rather  than 
ommochrome  synthesis  for  two  reasons :  ( 1 )  retinular  cells  of  young  g/g  shrimps 
contain  normal  black  pigment,  and  (2)  retinular  cells  of  sexually  mature  g/g 
shrimps  degenerate  as  a  result  of  the  action  of  the  garnet  gene. 

We  wrill  discuss  three  alternative  hypotheses  for  the  mode  of  action  of  the 
gene  for  white  eve  color : 

j 

1.  The  gene  zv  may  act  on  the  stroma  of  the  eye  pigment  granule,  either  by 
causing  a  complete  absence  of  the  stroma  or  by  producing  a  defect  in  its  structure. 
This  hypothesis  would  account  for  the  fact  that  z^/zv ;  r/r  and  zv/zv ;  g/g  shrimps 
have  white  eye  color.     The  white  eyes  should  be  examined  with  the  electron  micro- 
scope to  determine  if  pigment  granules  are  absent  or  changed  in  structure.     Nolte 
(1961)  has  reported  a  great  reduction  in  the  number  of  granules  in  the  retinulae 
of  st/st  and  v/v  Drosophila  which  lack  ommochrome  pigment. 

2.  The  gene  zc  may  alter  retinular  cell  membrane  permeability  in  such  a  way 
as    to    prevent    the    entrance    of    ommochrome    precursors    into    the    cell.     This 
hypothesis  would  also  account  for  the  fact  that  the  gene  for  white  is  epistatic  to 
the  genes  for  red  and  garnet. 

3.  (a)   The  genes  for  white  and  for  red  eyes  may  be  changes  in  structural 
genes  which  code  enzymes  in  the  biosynthetic  pathway  of  ommochrome  eye  pigment. 
Because  white  is  epistatic  to  red,  the  enzyme  controlled  by  the  white  locus  would 
act  earlier  in  the  ommochrome  synthetic  pathway  than  the  enzyme  controlled  by 
the  red  locus,      (b)  Along  the  same  line  of  reasoning,  it  is  possible  that  w  and  r 
may  be  changes  at  operator  or  represser  loci  which  indirectly  control  enzymes  in 
ommochrome  synthesis.     Some  doubts  about  hypothesis  #3  have  been  raised  by 
the  discovery  of  mosaic  shrimps  with  compound  eyes  which  contain  black  and  white 
retinular  cells  lying  next  to  one  another  (described  under  section  G  of  this  paper). 
If  the  white  cells  lacked  an  ommochrome  precursor,  one  might  expect  it  to  enter 
the  white  cells  by  diffusion  from  adjacent  normally  pigmented  cells,  resulting  in 
a  gradient   of  white,   intermediate,   and   normal   cells.     Because    no    intermediate 
cells  were  seen,  we  conclude  that  if  hypothesis  #3  is  valid,  the  gene  zv+  must  govern 
an   enzyme  which   catalyzes   the   synthesis   of   a   non-diffusable   precursor   of   the 
ommochrome  eye  pigment. 

E.  Modifications  of  nnknozoi  origin 

In  a  study  of  the  effects  of  x-irradiation  upon  encysted  blastulae  (Bowen. 
1963b)  variants  were  discovered  among  the  inbred  descendants  of  shrimps  in  the 
2-kr  and  10-kr  x-irradiation  treatments.  There  were  five  independent  occurrences 
of  absence  of  setae  on  the  exopodites  and  three  occurrences  of  swollen  abdomen. 
Other  variants  were :  bent  abdomen,  kidney-shaped  eyes,  swollen  branchiae,  and  a 
ventral  median  projection  on  the  fifth  segment  of  the  abdomen.  The  only  viable 
pure-breeding  stock  that  could  lie  developed  from  the  progeny  of  x-irradiated 
shrimps  was  the  garnet-eye  stock. 

Many  variants  occurred  in  non-irradiated  stocks.     One  male  had  no  antennae 


246  BOWEN,  HANSON,  BOWLING  AND  POON 

whatsoever.  Several  animals  in  one  r/r  line  showed  deposition  of  the  red  pigment 
delayed  until  sexual  maturity.  Many  shrimps  in  a  wild-type  stock  lacked  a  median 
eye.  There  were  five  independent  occurrences  of  shortened,  twisted  abdomens  (in 
addition  to  stump,  described  above).  These  morphological  variants  could  not  be 
developed  into  mutant  stocks  for  one  of  these  reasons :  the  shrimps  died  without 
progeny,  sib  matings  gave  all  wild-type  F2  progeny,  the  traits  had  low  viability  or 
penetrance,  or  the  stock  lost  vigor  with  inbreeding. 

F.  Mosaics 

Eighteen  mosaics  are  described  in  Tables  III  and  IV.  Eleven  were  sex  mosaics, 
three  were  mosaics  combining  genitalia  and  phyllopodia  tissue,  one  was  a  female 
with  abnormal  legs  on  one  side,  one  was  a  male  lacking  an  antenna,  and  two  were 
metanauplii  which  had  eyes  of  unequal  sizes.  The  last  two  hatched  from  cysts 
given  a  lethal  dose  of  50,000  r  x-irradiation  and  died  before  maturity  (Bowen, 
1963b). 

The  three  phyllopodia-genitalia  mosaics  (mosaics  1,  2  and  3  in  Table  III)  sug- 
gested that  structures  within  the  phyllopodia  are  homologous  with  those  in  the 
genital  segments.  Mosaic  #3  is  a  white-eyed  male  descended  from  a  cross  between 
a  black-eyed  mother  (XWY+)  and  a  white-eyed  father  (XWXW)  from  stock  #9.  On 
each  side  of  the  body,  there  is  a  normal  testis  filled  with  sperm  and  a  male  antenna. 
On  the  right  side,  the  last  four  thoracic  appendages  are  shortened  and  deformed. 
On  the  left  side  of  the  genital  segments,  there  is  a  normal  penis  containing  a  vas 
and  seminal  vesicle.  On  the  right,  the  external  genitalia  are  a  mixture  of  ovisac 
and  penis  structures  proximally ;  distally,  the  structure  becomes  a  phyllopodium 
(Fig.  21). 

Of  the  11  sex  mosaics  (numbers  8  to  18,  in  Tables  III  and  IV),  three  were 
perfect  bilateral  gynandromorphs.  Each  had  a  testis  containing  sperm  on  one 
side  and  an  ovary  producing  yolky  eggs  on  the  other.  One  has  been  described  in 
detail  earlier  (Bowen  and  Hanson,  1962).  Another  sex  mosaic  (number  11  in 
Table  III)  consisted  of  male  tissue  except  for  the  presence  of  shell  glands  filled 
with  brown  secretion.  Mosaic  No.  18  (Table  IV)  consisted  of  female  tissue 
except  for  one  perfect  male  antenna.  The  presence  of  a  small  amount  of  tissue 
characteristic  of  one  sex  in  an  animal  composed  for  the  most  part  of  cells  of  the 
other  sex  is  usually  interpreted  as  evidence  that  sex  is  determined  autonomously. 
We  can  be  certain  that  if  a  sex  hormone  is  present  in  Artemia,  it  does  not  suppress 
the  differentiation  of  cells  with  the  chromosome  constitution  of  the  opposite  sex. 
In  each  of  the  11  sex  mosaics,  the  internal  organs  were  male  or  female  rather 
than  intersexual  in  character.  The  external  shape  of  the  antennae  and  genitalia 
were  sometimes  intermediate,  but  this  could  be  attributed  to  mixtures  of  cells  with 
male  or  female  genotypes  in  the  epidermis.  Therefore,  all  the  mosaics  were  in 
accord  with  the  hypothesis  that  each  cell  is  either  male  or  female  in  phenotype. 

Although  we  know  that  female  Artemia  are  XY  and  males  are  XX,  we  do  not 
know  whether  the  female  phenotype  is  due  to  (1)  female-determining  gene(s)  on 
the  Y  or  to  (2)  the  balance  of  the  two  sets  of  autosomes  (bearing  genes  for 
femaleness)  to  the  single  X  chromosome  (bearing  genes  for  maleness).  Each  of 
the  five  sex  mosaics  in  Table  IV  resulted  from  the  cross  of  a  white  male  from 
stock  #9  to  a  w/+  female.  It  was  hoped  that  some  insight  into  sex  determination 


MUTATIONS  IN  ARTEMIA 


247 


TABLE  III 

Descriptions  of  thirteen  mosaic  shrimps 


Mosaic 
no. 


6,7 

8,  9,  10 
11 


12 


13 


Code* 


px 

X 

px 
px 


px 


Description 


Normal  male  antennae  and  male  reproductive  organs  in  genital  segments.  Five 
appendages  on  left  side  of  thorax  are  mixtures  of  male  and  female  genitalia. 

Both  testes  filled  with  sperm,  normal  male  antennae.  External  genitalia  on 
both  sides  are  a  mixture  of  penis,  ovisac,  and  phyllopod  structures.  Vas  is  con- 
tinuous with  internal  structures  of  phyllopod. 

On  both  sides,  there  is  a  normal  male  antenna  and  a  testis  filled  with  sperm. 
Left  side:  normal  penis  containing  vas  and  seminal  vesicle. 

Right  side:  the  genitalia  are  a  mixture  of  ovisac  and  penis  structures  proximally; 
distally  there  is  a  phyllopodium  (Fig.  21). 

Female  with  normal  antennae,  ovaries,  and  genitalia.  On  two  thoracic  seg- 
ments, one  leg  is  shortened,  another  slants  dorsally. 

Normal  male  genitalia  and  testis  on  both  sides.  One  antenna  has  normal  male 
shape ;  other  is  a  broad  stump. 

Two  metanauplii ;  each  has  eyes  of  disparate  size. 

Three  perfect  bilateral  gynandromorphs. 

Right  side:  male  antenna,  testis  filled  with  sperm,  male  genitalia  (although 
penis  lacks  external  spine).  Left  side:  antenna  is  intermediate  in  shape  and  lacks 
frontal  knob.  Testis  is  filled  with  sperm.  External  genitalia  are  mixture  of  ovisac 
and  penis  structures;  within  lie  seminal  vesicle,  vas,  and  shell  glands  (attached 
to  vas  deferens). 

Right  side:  small  lump  in  place  of  antenna,  testis  filled  with  sperm.  Although 
penis  is  normal,  seminal  vesicle  is  straight  and  vas  is  missing.  Left  side:  normal 
male  antenna,  testis  filled  with  sperm ;  external  genitalia  are  a  mixture  of  ovisac 
and  penis  structures.  A  short  spherical  seminal  vesicle  is  filled  with  sperm. 

Perfect  female  antenna  on  left,  intermediate  antenna  on  right  lacks  frontal 
knob.  Normal  ovisac.  In  this  immature  specimen,  the  sex  of  the  gonad  and  acces- 
sory organs  could  not  be  determined. 


*  x,  mosaic  hatched  from  x-irradiated  cyst  (50,000  r) ; 
px,  mosaic  found  in  progeny  of  x-irradiated  shrimps  (2,000  or  10,000  r); 
s,  spontaneous  occurence  in  non-irradiated  stocks. 

could  be  gained  from  study  of  these  mosaics  whose  sex  chromosomes  carry  eye 
color  markers.  However,  no  conclusions  can  be  drawn  because  each  of  the 
mosaics  in  Table  IV  could  have  resulted  from  (1)  a  normal  zygote  with  somatic 
non-disjunction  occurring  in  an  early  cleavage  division,  (2)  a  binuclear  oocyte  with 
both  nuclei  fertilized,  or  (3)  a  binuclear  oocyte  with  only  one  nucleus  fertilized. 
Goldschmidt  (1952,  p.  123)  reported  finding  binuclear  oocytes  in  California 
Artemia.  This  problem  is  further  complicated  by  crossing  over  between  the  X 
and  Y ;  an  estimate  of  crossover  frequency  in  the  five  mothers  can  be  obtained  from 
the  third  vertical  column  in  Table  IV. 

Histological  examination  of  mottled  eyes  (in  mosaics  #17  and  18)  revealed 
that  retinular  cells  containing  white  pigment  were  adjacent  to  retinular  cells  with 
wild-type  black  pigment.  In  mosaic  #14,  white  ommatidia  were  adjacent  to  red 
ommatidia  which  turned  black  as  the  animal  aged.  There  were  no  intermediate 
cells  bearing  a  reduced  amount  of  dark  pigment  (see  Fig.  15).  This  indicates 
that  there  is  no  diffusable  substance  produced  by  wild-type  or  by  r/r  retinulae 
which  is  lacking  in  white  retinulae;  that  is,  the  type  of  pigment  is  determined 


248 


BOWEN,  HANSON,  BOWLING  AND  POON 


TABLE  IV 

Five  mosaic  progeny  of  non-irradiated  parents 
(w/w  fathers  and  w/+  mothers) 


Mosaic 
no. 

Parents* 

Siblings  and 
progeny  of 
similar  matings 

Description  of  mosaic 

Mother 

Father 

Left  side 

Right  side 

14 

Xw»  Y+* 

X  9  X  9 

^Vw     -A.w 

1  pigm.  d" 

2  white  9  9 
513  pigm.   9  9 
379  white  cf  o" 

Red  median  eye 

Some  white  retinular 
cells  surrounded  by 
r/r  cells  (red, 
changing  to  black 
at  maturity). 
Normal  male  antenna. 

White  compound  eye. 
Proximal  segment  of 
antenna  male; 
distal  part  female. 

Male  gonad,  genitalia,  and  sperm 
present  on  both  sides. 

15 

XW9  Y+<> 

X  9  X  9 

-t*-w     -<*--w 

623  white  o*  o" 
877  pigm.   9  9 

Red  median  eye 

Red  compound  eyes 

Normal  male  antenna. 
Shell  glands  present. 
Mixed  male  and 
female  genitalia. 

Normal  female  an- 
tenna. 
Female  ovisac  and 
shell  glands. 

16 

Xw"  Y+Q 

Xw9  Xw« 

69  pigm.   9  9 
59  white  cf  c?1 
8  white  9  9 
15  pigm.  cfcf 

White  compound  eye. 
Larger  antenna,  mixed 
male  and  female 
characteristics. 
Female  gonad,  yolky 
eggs. 
Genitalia  mixed. 

Black  compound  eye. 
Smaller  antenna, 
mixed  male  and  fe- 
male characteristics. 
Female  gonad,  yolky 
eggs. 
Female  genitalia;  ovi- 
duct, 2  shell  glands, 
half-uterus. 

17 

X+SF  Yw" 

xw«  xw« 

264  white  9  9 
294  pigm.  cfc? 

31  white  c?1  cf 
36  white  9  9 

Black  and  white  retin- 
ular cells  in  eye. 

White  compound  eye. 

Normal  female  antennae,  gonads  and 
genitalia  on  both  sides  of  body. 

18 

Xw9  Y+SF 

Xw«  Xw* 

149  white  tf  <? 

149  pigm.    9  9 
8  white  9  9 

6  pigm.  cf  o" 

White  and  black  (mottled)  median  eye 

Black  compound  eye. 
Female  antenna. 

White  compound  eye. 
Male  antenna. 

Male  gonad,  genitalia  and  sperm  present 
on  both  sides. 

*  Iii  each  genotype,  subscripts  indicate  the  allele  on  the  sex  chromosome;  superscripts  desig- 
nate origin  of  the  differential  segment  of  the  sex  chromosome  (inbred  stocks  5,  9,  11;  and  wild 
populations:  SF,  San  Francisco  Bay;  Q,  Quemado,  New  Mexico). 


MUTATIONS  IN  ARTEMIA  249 

autonomously  by  the  genes  rather  than  through  the  mediation  of  hormones  or 
other  diffusable  substances. 

SUMMARY 

1.  Seven  mutant  genes  of  the  brine  shrimp  have  been  studied.     The  mutation 
s,  stump,  shortens  the  abdomen ;  in  extreme  cases,  all  six  abdominal  segments  are 
missing.     An  autosomal  sex-limited  mutation,  Cu,  curved,  determines  that  females 
will  have  small  curved  antennae  similar  to  those  of  the  male.     Two  mutations  (w, 
white  and  r,  red)  affect  color  of  the  eye  and  two  mutations  alter  eye  structure  (cy, 
cyclops  and  c,  crinkle).     The  garnet  mutation,  g,  affects  both  color  and  structure 
of  the  eye. 

2.  Five  of  the  mutant  genes  are  autosomal,  one  (white)  is  partially  sex-linked, 
and  the  mode  of  inheritance  of  one  (cyclops)  is  not  completely  known. 

3.  Injections  of  India  ink  were  used  to  demonstrate  the  distribution  of  phago- 
cytic  cells.     These  cells  also  take  up  pigment  released  by  degenerating  retinular 
cells  in  garnet-eyed  shrimps. 

4.  The  1 1  sex  mosaics  are  consistent  with  the  hypothesis  that  each  cell  is  male  or 
female  rather  than  intersexual  in  character. 

5.  Four  shrimps  had  eyes  which  were  mosaic  for  red  and  white  or  for  black 
and  white  retinular  cells.     This  suggests  that  eye  pigment  is  determined  auton- 
omously ;  that  is,  there  is  no  diffusable  factor  produced  by  red  or  wild-type  retinular 
cells  which  is  lacking  in  white  cells. 

6.  The  gene  for  white  eyes,  when  homozygous,  is  epistatic  to  the  genes  for 
garnet  and  for  red  eyes.     Three  possible  modes  of  action  of  the  gene  w  are 
discussed. 

LITERATURE  CITED 

BARIGOZZI,    C.,    1957.     Differentiation    des    genotypes    et    distribution    geographique    d'Artemia 

salina  Leach:  donnees  et  problems.    Annee  Biol.,  33:  241-250. 
BECKER,  E.,  1942.     t)ber  Eigenschaften,  Verbreitung  und  die  genetisch-entwicklungsphysiolo- 

gische  Bedeutung  der  Ommatin-  und  Ommingruppe   (Ommochrome)   bei  den  Arthro- 

poden.    Zeitschr.  Verebungslehre,  80:  157-204. 
BOWEN,  S.  T,,  1962.     The  genetics  of  Artemia  salina.     I.    The  reproductive  cycle.     Biol.  Bull., 

122:25-32. 
BOWEN,  S.  T.,  1963a.     The  genetics  of  Artemia  salina.     II.    White  eye,  a  sex-linked  mutation. 

Biol.  Bull,  124:  17-23. 
BOWEN,  S.  T.,  1963b.     The  genetics  of  Artemia  salina.     III.    Effects  of  x-irradiation  and  of 

freezing  upon  cysts.    Biol.  Bull.,  125:  431-440. 
BOWEN,  S.  T.,  1964.     The  genetics  of  Artemia  salina.     IV.    Hybridization  of  wild  populations 

with  mutant  stocks.     Biol.  Bull,  126:  333-344. 
BOWEN,  S.  T.,  1965.     The  genetics  of  Artemia  salina.    V.   Crossing  over  between  the  X  and  Y 

chromosomes.     Genetics,  52:  695-710. 
BOWEN,  S.  T.,  AND  J.  HANSON,  1962.     A  gynandromorph  of  the  brine  shrimp,  Artemia  salina. 

Genetics,  47 :  277-280. 
CERVINI,  A.  M.,  1965.     II  mutante  spontaneo  "curly"  (cr)  del  ceppo  diploide  anfigonico  Hildago 

di  Artemia  salina.    Atti  Ass.  Genet.  It.,  10:  343-345. 
DEBAISIEUX,  P.,  1944.     Les  yeux  de  Crustaces.     Structure,  developpement,  reactions  a  1'eclaire- 

ment.     La  Cellule,  50:  5-122. 
DOWLING,  P.  M.,  1963.     A  new  microinjection  technique  for  Artemia  salina  L.     Unpublished 

M.A.  thesis,  San  Francisco  State  College  Library,  San  Francisco,  California. 
DUTRIEU,  J.,  1960.     Observations  biochimiques  et  physiologiques  sur  le  developpement  d' 'Artemia 

salina  Leach.    Arch.  Zool.  Exp.  Gen.,  99:  1-133. 


250  BOWEN,  HANSON,  BOWLING  AND  POON 

EGUCHI,  E.,  AND  T.   H.   WATERMAN,   1965.     Rhabdom   fine   structure  and  visual   function   in 

crustacean  compound  eyes.     Fed.  Proc.,  24:  275. 
FORD,  E.  B.,  AND  J.  S.  HUXLEY,  1929.     Genetic  growth  rate  factors  in  Gammarus.    Arch.  f. 

Entw.,  117:  67-79. 
FRANSEMEIER,    L.,    1939.     Zur    frage    der    Herkunft    des    metanauplialen    Mesoderms    und    die 

Segmentbildung  bei  Artemia  salina  Leach.    Zeitschr.  f.   Wiss.  Zool.    (Leipzig),   152: 

439-472. 
GILCHRIST,  B.  M.,  1960.     Growth  and  form  of  the  brine  shrimp  Artemia  salina   (L.).     Proc. 

Zool.  Soc.  London,  134:  221-235. 
GOLDSCHMIDT,  E.,  1952.     Fluctuation  in  chromosome  number  in  Artemia  salina.    J.  Morph.,  91: 

111-133. 

HEATH,  H.,  1924.     The  external  development  of  certain  phyllopods.    /.  Morph.,  38:  453-475. 
KUENEN,   D.  J.,    1939.     Systematical   and  physiological   notes   on   the   brine   shrimp,   Artemia. 

Arch.  Neerl.  Zool.,  (Leiden),  3:  365-449. 
LOCHHEAD,  J.  H.,  1950.     Artemia.    In:  Selected  Invertebrate  Types  (pp.  394-399).     Edited  by 

F.  A.  Brown,  Jr.     John  Wiley  and  Sons,  Inc.,  New  York. 
NOLTE,  D.  J.,  1961.     The  pigment  granules  in  the  compound  eyes  of  Drosophila.    Heredity,  16: 

25-38. 
SEXTON,  E.  W.,  AND  A.  R.  CLARK,  1936.     A  summary  of  the  work  on  the  amphipod  Gammarus 

chevreuxi  Sexton  carried  out  at  the  Plymouth  Laboratory  (1912-1936).    /.  Mar.  Biol. 

Assoc.,  21 :  357-414. 
STEFANI,   R.,   1963.     La  digametia  femminile   in  Artemia  salina   Leach   e   la  costituzione   del 

corredo    chromosomico    nei    biotipi    diploide    anfigonico    e    diploide    partenogenetico. 

Caryologia,  16:  625-636. 
STEFANI,   R.,   1964.     The  origin  of  males   in  parthenogenetic   populations   of  Artemia   salina. 

(Bilingual  edition  in  Italian  and  English.)     Riv.  Biol.,  57:  147-162. 
VAISSIERE,  R.,  1961.     Morphologic  et  histologie  comparees  des  yeux  des  Crustaces  Copepodes. 

Arch.  Zool.  Exp.  Gen.,  100:  1-125. 
WEISZ,  P.  B.,  1946.     The  space-time  pattern  of  segment  formation  in  Artemia  salina.    Biol. 

Bull. ,91:  119-140. 
WEISZ,   P.  B.,   1947.     The  histological  pattern  of  metameric  development  in  Artemia  salina. 

J.  Morph.,  81 :  45-95. 


MOTILITY  AND  AGING  OF  ARBACIA  SPERM  x 

JOSEPH  M.  BRANHAM  2 
Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts  02543 

It  is  well  known  that  diluted  sea  urchin  sperm  have  a  relatively  short  effective 
life  span.  In  a  few  hours  or  days  they  lose  the  ability  to  activate  eggs,  become 
immotile  and  their  respiration  ceases  (Gemmill,  1900;  Cohn,  1918;  Gray,  1928, 
1931;  Rothschild,  1951;  Tyler,  1953;  Rothschild  and  Tyler,  1954;  Bishop,  1962; 
Mann,  1964). 

The  loss  of  vitality  of  sperm  is  reported  to  be  associated  with  the  exhaustion 
of  energy  reserves  (Gemmill,  1900;  Cohn,  1918;  Tyler,  1953).  Gemmill  recog- 
nized that  sperm  were  more  active,  and  also  pointed  out  (p.  171)  that  "on  com- 
paring the  movement  of  spermatozoa  in  different  mixtures  [dilutions],  one  finds 
that  the  difference  in  activity  is  not  sufficiently  marked  to  account  for  the  very 
early  loss  of  vitality  of  spermatozoa  in  the  weaker  mixtures  simply  in  terms  of 
exhaustion  of  energy."  The  relationship  between  the  motility  of  sea  urchin  sperm 
and  the  rate  at  which  they  lose  the  ability  to  activate  eggs  remains  somewhat  un- 
certain, primarily  because  it  is  difficult  to  evaluate  quantitatively  the  motility  of 
spermatozoa. 

The  problems  of  finding  the  motility  status  of  semen  samples  are  manifold. 
Microscopic  examination  to  determine  sperm  activity  involves  many  variables  that 
are  difficult  to  control  (Bishop,  1962;  Rikmenspoel,  1962;  Rothschild,  1953;  van 
Duijn,  1963,  1964).  The  impedance  change  frequency  method  for  rating  motility 
(Rothschild  1948a)  allows  better  control  of  these  variables  but  is  unsuitable  with 
diluted  semen.  A  simple  method  of  rating  sperm  motility  which  avoids  some  of 
these  problems  was  devised  for  this  study. 

The  first  part  of  this  report  deals  with  an  analysis  of  the  method  for  rating 
motility.  The  second  part  is  an  investigation  of  senescence  of  sperm,  utilizing  the 
method  for  rating  motility.  The  following  results  show  that  under  the  conditions 
of  these  experiments  the  concentration,  motility  and  fertility  of  sperm  suspensions 
diminished  most  rapidly  soon  after  dilution  and  more  slowly  later  on.  The  rapid 
initial  decline  in  fertility  and  concentration  was  prevented  by  experimentally  im- 
mobilizing the  spermatozoa,  but  immotile  semen  ultimately  lost  fertility  at  about 
the  same  time  as  motile  semen. 

MATERIAL  AND  METHODS 

Arbacia  punctulata  was  furnished  by  the  Supply  Department  of  the  Marine 
Biological  Laboratory  at  Woods  Hole,  Mass.  Gametes  were  obtained  by  electrical 
stimulation  of  the  intact  animals  (cf.  Costello  et  al.,  1957).  Sperm  were  collected 

1  Research  supported  by  the  Lalor  Foundation  through  a  faculty  summer  research  grant. 

2  Present  address  :  Institute  of  Animal  Genetics,  Edinburgh. 

251 


252  JOSEPH  M.  BRANHAM 

in  100-  or  250-ml.  beakers  of  sea  water  by  immersing  the  aboral  surface  of  the 
urchin,  along  with  one  electrode  of  the  stimulator,  and  applying  the  other  electrode 
to  the  oral  surface.  Sperm  accumulated  in  piles  on  the  bottom  of  the  beaker  in  a 
relatively  undiluted  state.  The  time  of  the  first  stirring  of  the  sperm  into  suspen- 
sion was  considered  the  beginning  of  the  experiment.  Eggs  were  shed  in  a  similar 
fashion  into  100  ml.  of  sea  water,  but  only  a  few  thousand  at  a  time  so  that  the 
same  female  could  be  used  repeatedly  to  produce  fresh  eggs. 

Sperm  concentration  was  determined  by  counting  in  a  Neubauer  hemocytometer 
and/or  by  measuring  light  absorption  with  a  Klett-Summerson  colorimeter  (green 
filter,  cf.  Iverson,  1964). 

Fertilizing  capacity  was  determined  by  diluting  sperm  in  two-fold  steps,  then 
adding  about  500  eggs  to  each  dilution  (total  volume  5  ml.).  Fertilizing  capacity 
was  rated  numerically  by  taking  the  reciprocal  of  the  sperm  concentration  at  which 
less  than  100%  cleavage  was  attained  and  above  which  all  eggs  were  fertilized. 

Motility  was  rated  by  comparing  the  sedimentation  of  living  and  formalin-killed 
portions  of  sperm  suspensions.  Sedimentation  was  enhanced  by  motility.  In  order 
to  rate  motility  experimentally,  two  15-ml.  portions  of  diluted  sperm  suspensions 
(usually  about  20  million  cells/ml.)  were  withdrawn  and  one  portion  killed  with 
0.02%  formalin.  Sperm  concentration  of  the  two  portions  was  determined  by 
optical  density  (O.D.)  measurements.  Both  samples  were  then  centrifuged  at 
200  g,  20°  C.  for  20  minutes  in  conical  tubes  in  swinging  buckets.  After  centrifuga- 
tion  5-ml.  portions  of  the  supernatant  were  withdrawn  from  one  centimeter  above 
the  bottom  of  the  tubes  and  their  O.D.  determined.  The  difference  between  the 
O.D.  before  and  after  centrifugation  was  proportionate  to  the  number  of  sperm 
sedimented.  This  difference  was  always  greater  for  suspensions  of  motile  sperm. 
The  difference  between  the  decrease  in  O.D.  of  live  and  formalin-killed  suspensions 
was  assumed  to  result  from  the  "downward"  migration  of  sperm  in  the  motile 
sample,  and  was  considered  to  be  the  motility  score  of  the  sample  (cf.  van  Duijn, 
1963,  for  discussion  of  sperm  migration  rate).  This  score  was  determined  by 
the  equation 

M  =  dL  -  dK, 

where  M  is  the  motility  score,  dL  is  the  change  in  O.D.  ( X  100)  of  the  living 
sample,  presumably  resulting  from  sedimentation  plus  "downwards"  swimming  of 
sperm,  and  dK  is  the  change  in  O.D.  (X  100)  of  the  formalin-killed  sample  repre- 
senting sedimentation  unaltered  by  motility.  Under  these  conditions  the  value  of 
M  for  50  freshly  diluted  sperm  varied  from  3  to  41  with  a  mean  value  of  14,  with  15 
recurring  most  frequently. 

EXPERIMENTS  AND  RESULTS 
Motility  by  centrifugation 

Motility  can  be  rated  quantitatively  by  finding  measurable  differences  between 
motile  and  immobile  samples  of  sperm  that  are  otherwise  equivalent.  It  was  found 
that  such  differences  resulted  when  live  and  dead  sperm  suspensions  were  centri- 
fuged. Living,  motile  sperm  sedimented  faster  than  formalin-killed  ones,  and  the 
difference  could  be  measured  as  described  above.  A  possible  explanation  of  the 


ARBACIA  SPERM  AGING 


253 


difference  in  sedimentation  rates  is  that  sperm  oriented  head  "downwards"  in  the 
centrifugal  field  (cf.  Rothschild,  1962)  because  their  tails  are  more  buoyant  than 
their  heads  (Kihlstrom,  1958;  Beatty,  1964),  and,  therefore,  motile  sperm  swam 
"downwards"  faster  than  dead  ones  sank. 

The  hypothesis  that  sperm  were  oriented  by  centrifugal  force  was  tested  in 
the  centrifuge  microscope.  Formalin-killed  sperm  were  seen  to  be  oriented  head 
"downwards."  Living  sperm  moved  very  rapidly  "downwards,"  but  it  was  impos- 
sible to  see  any  orientation  because  of  the  rapid  movement  of  the  sperm  super- 
imposed on  the  flashing  field  of  the  microscope,  and  so  it  remained  uncertain 
whether  or  not  living  sperm  were  oriented  in  the  centrifugal  field. 

The  following  experiments  tend  to  support  the  assumption  that  living  sperm 
sedimented  faster  than  dead  ones  because  they  swam  "downwards."  Sperm  from 
the  pellet  that  formed  when  living  sperm  were  centrifuged  were  highly  active 

TABLE  I 

Motility  and  fertilizing  capacity  of  various  fractions  of  centrifuged  sperm* 


Experiment 
number 

Motility** 

Fertilizing  capacity*** 

Pellet 

Control 

Pellet 

Supernatant 

Control 

1 

9 

3 

0.22 

0.03 

0.21 

2 

9 

4 

5.0 

0.13 

2.5 

3 

15 

4 

2.5 

1.4 

— 

*  Sperm  were  centrifuged  20  minutes  at  200  g.  The  upper  5  ml.  of  the  supernatant  and  the 
sperm  in  the  pellet  were  then  withdrawn  by  pipette.  Pellet  sperm  were  resuspended  in  sea  water 
to  the  original  concentration.  These  fractions  were  then  tested  for  motility  or  fertilizing  capacity. 
The  original  uncentrifuged  suspension  served  as  a  control  for  aging. 
**  Motility  score  (M)  is  in  Klett  units. 

***  Fertilizing  capacity  is  the  reciprocal  of  sperm  concentration  wherein  just  less  than  100% 
fertilization  was  attained  (  X  10~6). 

when  viewed  under  a  microscope.  Sperm  remaining  in  the  supernatant  seemed  less 
active.  When  sperm  from  the  pellet  were  resuspended  in  sea  water  and  tested 
for  motility  by  centrifugation,  they  had  higher  motility  scores  than  control  sperm 
of  the  same  age,  and  were  more  effective  at  fertilizing  eggs  than  either  sperm  from 
the  supernatant  or  uncentrifuged  control  sperm  (Table  I).  This  could  mean  that 
sperm  were  improved  by  being  packed  into  a  pellet,  or,  as  seems  more  likely,  that 
the  most  active  and  effective  ones  were  concentrated  by  centrifugation. 

The  experiments  on  aging  reported  below  also  tend  to  confirm  that  the  difference 
in  sedimentation  rates  resulted  from  motility.  The  proportion  of  live  sperm 
sedimented  (dL)  was  greatest  at  first  when  samples  were  visibly  most  active  and 
gradually  decreased  until  it  equalled  the  proportion  of  dead  sperm  sedimented  (dK) 
as  the  samples  aged  and  became  immotile  (Fig.  1).  Similarly,  sperm  immobilized 
at  low  pH  or  by  narcosis  with  carbon  dioxide  (cf.  Mohri  and  Yasumasu,  1963) 
sedimented  at  the  same  rate  as  formalin-killed  sperm :  that  is  to  say,  hardly  at  all 
(Fig.  2).  It  therefore  seems  likely  that  the  formalin-killed  sperm  sedimented  more 


254 


JOSEPH  M.  BRANHAM 


slowly  than  live  ones  because  their  motility  was  inhibited  rather  than  because  of 
some  extraneous  effect  of  the  formalin. 

Motility  rating  by  centrifugation  could  be  influenced  by  the  sperm  concentration 
(Rothschild,  1956a;  Tampion  and  Gibbon,  1963).  Table  II  compares  the  values 
obtained  when  the  motility  of  the  same  sperm  sample  was  determined  at  different 
concentrations.  Sometimes,  but  not  always,  the  more  dilute  sperm  gave  lower 
motility  values.  This  may  have  resulted  from  more  rapid  aging  by  more  dilute 
sperm  (Rothschild,  1948b).  Dilute  sperm  tended  to  lose  motility  very  rapidly  at 
first  (Fig.  1),  but  as  Gray  (1928)  observed,  sea  urchin  semen  samples  vary.  He 
reported  that  the  respiration  of  some  samples  declined  rapidly  after  dilution,  while 
others  showed  some  lag  before  beginning  to  decline.  The  concentration  effect  on 


Hours 

FIGURE  1.  Change  in  sperm  concentration,  fertilizing  capacity  and  motility  with  time. 
Initially  Arbacia  semen  was  diluted  to  18  X  10"  cells/ml,  in  500  ml.  of  sea  water.  Periodically 
the  suspension  was  stirred  and  portions  tested  for  motility  and  fertilizing  capacity.  Motility  is 
in  Klett  units.  Fertilizing  capacity  is  expressed  as  the  reciprocal  of  the  concentration  wherein 
just  less  than  100%  fertilization  was  attained  (units  X  10~6).  Sperm  concentration  is  in  millions 
of  sperm/ml,  (units  X  10"). 

motility  rating  that  was  sometimes  observed  may  also  have  reflected  physical 
interaction  between  sperm  (Taylor,  1952;  Rikmenspoel,  1962;  Tampion  and  Gib- 
bon, 1963;  van  Duijn,  1963).  The  motility  score  used  in  the  experiments  reported 
below  is  probably  valid  only  if  sperm  concentration  and  time  after  dilution  are  taken 
into  account. 

Sperm  senescence 

This  somewhat  quantitative  method  for  rating  motility  was  used  to  investigate 
the  relationship  between  motility  and  the  loss  of  fertilizing  capacity  by  aging  sea 
urchin  spermatozoa. 

In  the  initial  experiments  semen  was  diluted  about  2000-fold  in  filtered  sea  water 


ARBACIA  SPERM  AGING 


255 


and  allowed  to  stand  in  100-  or  250-ml.  beakers  at  room  temperatures.  Periodically 
the  suspensions  were  thoroughly  stirred  and  portions  tested  by  centrifugation  for 
motility,  and  in  serial  dilution  for  fertilizing  capacity.  Sperm  concentration  was 
determined  by  absorptiometry  at  each  interval  and  confirmed  occasionally  by  direct 
counts. 

Under  the  conditions  of  these  experiments  (25  in  all)  sperm  concentration  as 
well  as  motility  and  fertilizing  capacity  declined  with  time  (Fig.  1).     In  different 


100 


90 


so 


7O 


60 


o     so 


4O 


30 


20 


10 


8-O 


,HCL 


7-5 


7O 


65 


6  O 


PH 


FIGURE  2.  Effects  of  acid  and  carbon  dioxide  on  Arbacia  sperm  motility.  Sperm  were 
shed  into  250  ml.  of  sea  water  to  18  X  10"  sperm/ml.,  then  divided  into  three  equal  portions. 
One  portion  served  as  control,  the  second  was  acidified  by  bubbling  with  6.8%  CO3  in  air,  and 
the  third  was  acidified  with  HC1.  The  motility  of  the  three  portions  was  determined  simul- 
taneously by  centrifugation.  The  process  was  repeated  at  different  pH  values  with  fresh  sperm 
shed  from  the  same  male.  The  data  are  presented  as  percentage  of  the  control  sperm  motility. 

samples  the  average  rate  of  decrease  in  concentration  varied  from  0.25  X  106 
sperm/ml,  lost  each  hour  to  5  X  106  sperm/ml,  lost  each  hour.  There  was  no 
evidence  that  the  loss  of  sperm  resulted  from  sedimentation  or  the  adherence  of 
sperm  to  the  walls  of  the  container.  The  decrease  in  sperm  concentration  had  to 
be  considered  in  motility  and  fertilization  capacity  determinations,  so  concentration 
was  determined  and  new  portions  killed  for  motility  determination  at  each  time 
interval. 


256 


JOSEPH  M.  BRANHAM 


Motility  and  fertilizing  capacity  of  sperm  declined  most  rapidly  soon  after 
dilution  and  more  slowly  later  on  (Fig.  1).  Motility  became  imperceptible,  as 
rated  by  the  centrifuge  method,  after  about  six  hours.  Such  "immobile"  sperm 
were  seen  under  the  microscope  to  be  twitching  slightly  but  not  progressing. 
Fertilizing  capacity,  on  the  other  hand,  persisted  for  30  to  40  hours  after  dilution 
(as  determined  directly  in  three  experiments  and  estimated  from  semilog  plots  of 
the  data  from  the  other  experiments).  Actively  moving  sperm  were  observed 
trapped  in  the  jelly  coat  of  eggs  inseminated  with  dilutions  of  aged,  apparently 
immotile  sperm.  This  suggested  that  sperm  were  stimulated  to  renewed  activity 
under  the  conditions  of  fertilization.  The  possibilities  were  considered  that  such 
rejuvenation  could  have  resulted  from  (1)  further  dilution  (Rothschild,  1956a)  or 
(2)  stimulation  by  substances  exuding  from  eggs  (Hathaway,  1963). 

TABLE  II 
The  effect  of  sperm  concentration  on  motility  determination  by  centrifugation* 


Sperm  sample 

Dilution 

1 

2 

3 

4 

5 

6 

7 

8 

9 

1 

C** 

22 

33.5 

61 

36 

47 

19 

31 

17.5 

19 

M*** 

15 

17 

7 

6 

25 

19 

7 

15 

15 

2 

C 

11 

20.5 

29 

18 

18 

14 

16.5 

16 

17 

M 

15 

6 

6 

6 

6 

15 

7 

9 

15 

3 

C 



9.5 

13 

9 

8 

11 

7.5 

14 

16 

M 

— 

6 

3 

6 

4 

8 

7 

10 

17 

*  Semen  was  serially  diluted  and  tested  for  motility. 
**  Concentration  is  in  millions  of  sperm  per  ml. 
***  M  =  Motility  score  in  Klett  units. 

Sea  urchin  sperm  are  known  to  be  stimulated  to  a  burst  of  activity  by  dilution 
in  sea  water  (cj.  Mann,  1964,  p.  343).  The  rejuvenation  of  sperm  suggested 
above  could  have  resulted  from  such  a  "dilution  effect."  Sperm  activity  in  the  aged 
samples  could  have  been  suppressed  by  exhaustion  of  oxygen  or  accumulation  of 
respiratory  CO2  with  a  concomitant  fall  in  pH  (Rothschild,  1956a;  Mohri  and 
Yasumasu,  1963),  and  then  restored  by  further  dilution.  In  order  to  test  this 
hypothesis,  air  was  bubbled  through  sperm  suspensions  as  they  aged  (two  experi- 
ments). Aerated  sperm  lost  motility  at  a  uniform  rate  while  the  control  sperm  lost 
motility  more  rapidly  at  first  and  more  slowly  later  on.  Some  motility  persisted  in 
the  control  samples  for  several  hours  after  the  aerated  ones  lost  perceptible  motility. 
Concentration  and  fertilizing  capacity  declined  more  rapidly  in  aerated  than  in 
control  suspensions.  The  pH  was  constant  (8.0)  in  the  aerated  sample  but  fell  to 
pH  7.6  in  the  controls  during  the  first  hour,  and  then  remained  constant.  Roths- 
child (1956b)  reported  that  a  pH  increase  from  7.84  to  8.00  resulted  in  a  400% 
increase  of  the  respiratory  rate  of  Echinus  esculentus  sperm.  Mohri  and  Horiuchi 
(1961),  on  the  other  hand,  reported  that  Japanese  sea  urchin  sperm  (Hemicentrotus 
piilcherrimus,  Pseudoccntrotus  depresses  and  Anthocidaris  crassispina)  were  little 


ARBACIA  SPERM  AGING  257 

affected  by  varying  the  pH  from  7.0  to  8.5.  In  order  to  evaluate  the  significance  of 
the  observed  drop  in  pH  on  Arbacia  sperm  suspensions  the  effects  of  acid  and 
carbon  dioxide  on  motility  were  compared  with  the  centrifuge  method  for  rating 
motility.  In  three  experiments  the  hydrogen  ion  concentration  of  freshly  shed 
sperm  suspensions  was  adjusted  to  a  given  pH  in  the  range  of  8.0  to  6.0,  either 
with  HC1  or  by  bubbling  6.8%  CO2  in  air  through  the  suspension.  The  motility 
of  acidified  sperm  was  then  compared  with  control  sperm  and  results  expressed  as 
the  percentage  of  control  motility  remaining  (Fig.  2).  The  results  clearly  indicated 
that  CO,  inhibited  motility  at  hydrogen  ion  concentrations  which  had  little  or  no 
effect  on  Arbacia  sperm  motility.  The  fall  in  pH  from  8.0  to  7.6  observed  in 
the  preceding  experiments  on  aging  could  have  appreciably  suppressed  motility  if 
it  resulted  from  accumulating  CO2. 

It  was  of  interest  to  see  if  the  fertile  life  of  diluted  sea  urchin  sperm  could  be 
prolonged  by  depressing  their  motility  (cf.  VanDemark,  Koyama  and  Lode,  1965). 
It  has  long  been  known  that  sperm  immobilized  with  acid  or  CO2  can  fertilize  eggs 
after  dilution  in  sea  water  (Cohn,  1918).  In  four  experiments  sperm  were  diluted 
and  allowed  to  age  in  sea  water  adjusted  to  pH  6.5  or  7.0  with  HC1  or  CO2.  The 
pH  was  adjusted  occasionally  so  that  it  did  not  fluctuate  more  than  0.1  unit  from 
the  desired  value.  Periodically  samples  were  tested  for  sperm  concentration  and 
fertilizing  capacity.  In  these  experiments  the  fertilizing  capacity  of  acidified  sperm 
remained  at  the  high  initial  value  for  about  24  hours  and  then  declined  rapidly  so 
that  all  sperm  became  infertile  after  about  32  hours.  Control  sperm  (in  sea  water, 
pH  8.0-7.8)  lost  fertilizing  capacity  rapidly  in  the  first  few  hours  and  then  more 
slowly  until  all  fertilizing  capacity  was  lost  at  about  32  hours  (cf.  Fig.  1).  In  one 
experiment  sperm  were  completely  immotile  in  sea  water  adjusted  to  pH  6.5  with 
either  HC1  or  CO2  (cf.  Fig.  2).  The  fertilizing  capacity  of  both  acidified  samples 
was  initially  about  25%  less  than  the  control  value,  but  persisted  undiminished 
for  22  hours  and  then  declined  rapidly  in  both  samples.  At  pH  7.0  (three  experi- 
ments) sperm  in  sea  water  with  HC1  were  slightly  motile  while  those  in  sea  water 
with  CO,  were  immotile.  The  fertilizing  capacity  of  both  samples  remained  the 
same  as  the  initial  control  value  until  the  rapid  decline  began  after  about  24  hours. 
The  fertilizing  capacity  of  sperm  in  HCl-ad justed  sea  water  began  to  decline  about 
two  hours  before  the  ones  in  CO2-adjusted  sea  water  in  all  three  experiments.  In 
these  experiments  the  sperm  concentration  diminished  less  rapidly  in  the  acidified 
suspensions  than  in  the  control  suspensions.  In  one  experiment  the  control  sperm 
were  all  gone  after  14  hours,  while  the  concentration  of  sperm  held  at  pH  7.0  did 
not  diminish  in  32  hours.  In  a  fifth  experiment  50  units  of  penicillin  per  ml.  were 
used  to  control  bacteria  (Mohri,  1957).  In  this  six-hour  experiment,  cell  loss 
was  reduced  by  penicillin  to  the  level  observed  in  the  immobilized  suspensions,  while 
the  concentration  of  sea  water  control  sperm  diminished  0.26  million  cells  per  ml. 
hour.  The  penicillin  had  a  marked  detrimental  effect  on  motility  and  fertilizing 
capacity,  however,  so  the  experiment  was  discontinued. 

A  second  hypothesis  accounting  for  the  rejuvenation  of  aged  sperm  is  that  sub- 
stances exuding  from  eggs  could  reactivate  "immobile"  sperm  (Hathaway,  1963), 
just  as  the  surface  of  the  chorion  near  the  micropyle  of  fish  eggs  activated  motionless 
sperm  (Yanagimachi,  1957).  It  proved  unfeasible  to  test  this  hypothesis,  how- 
ever, because  the  water  in  which  eggs  had  stood  caused  irreversible  agglutination 


258  JOSEPH  M.  BRANHAM 

of  formalin-killed  sperm  (cf.  Tyler  and  Bishop,  1963)  and  thus  rendered  motility 
rating  by  the  centrifuge  method  unreliable. 

CONCLUSIONS 

Senescence  of  Arbacia  sperm  in  the  experiments  reported  above  occurred  in 
several  phases.  Soon  after  dilution,  sperm  motility,  fertilizing  capacity  and  con- 
centration declined  rapidly.  After  about  six  hours  motility  became  imperceptible  by 
the  rating  method  used  here.  Thereafter  the  concentration  and  fertility  of  the 
remaining  sperm  declined  much  more  slowly  (Fig.  1).  Ultimately  after  30  to  40 
hours,  all  fertility  was  lost. 

Fertility  and  sperm  concentration  both  declined  most  rapidly  soon  after  dilution 
when  motility  was  greatest  (Fig.  1).  This  initial  rapid  decline  was  prevented  by 
suppressing  motility  with  hydrogen  ions  and/or  carbon  dioxide.  It  therefore  seems 
likely  that  the  loss  of  sperm  from  suspension  and  the  initial  diminution  of  fertility 
were  both  associated  with  exhaustion  of  energy  supplies.  Afzelius  and  Mohri 
(1966)  demonstrated  that  reduction  in  mitochondrial  cristae  apparently  resulted 
from  the  catabolism  of  phospholipids  and  suggested  that  sea  urchin  sperm  might 
burn  up  structural  elements  for  energy.  It  is  conceivable  that  the  observed  decrease 
in  sperm  concentration  resulted  from  such  autolysis. 

Bacterial  contamination  could  have  been  involved  in  the  destruction  of  sperm 
and  the  loss  of  fertility  by  the  remaining  ones.  The  decline  in  sperm  fertility  and 
concentration,  however,  was  most  rapid  soon  after  dilution  when  bacterial  contami- 
nation should  have  been  minimal,  and  the  rate  of  decline  diminished  later  when 
bacterial  effects  should  have  been  more  pronounced.  It  seems  more  likely  that 
bacteria  had  a  role  in  the  final  phase  of  sperm  aging,  when  all  fertility  disappeared 
from  the  samples,  rather  than  in  the  striking  initial  changes.  This  final  loss  of 
fertility  was  probably  due  to  factors  other  than  exhaustion  of  energy  reserves,  since 
it  occurred  at  about  the  same  time  whether  motility  was  suppressed  or  not  (cf. 
Mann,  1964,  p.  349). 

Gray  (1931)  emphasized  that  sperm  suspensions  are  heterogeneous  populations 
of  cells.  He  recognized  this  heterogeneity  in  his  data  as  the  varying  rate  of  decline 
of  respiration  as  sperm  aged,  and  attributed  it  to  physiological  variability  in  the 
composition  of  individual  cells.  The  data  presented  above  can  also  be  interpreted 
in  this  way. 

Figure  1  suggests  that  the  semen  samples  consisted  of  a  population  of  short- 
lived sperm  that  disappeared  during  the  first  phase  of  aging  and  a  second  population 
that  persisted  through  the  second  phase.  Perhaps  the  short-lived  sperm  exhausted 
their  energy  supplies  rapidly  while  the  second  population  maintained  a  reserve  of 
energy.  Slow-speed  centrifugation  is  apparently  a  useful  method  for  separating 
sperm  according  to  motility.  It  therefore  presents  a  tool  to  investigate  further 
physiological  variability  within  semen  samples  and  the  significance  of  this  variability 
to  studies  of  gamete  physiology  and  development. 


I  wish  to  thank  the  Will  Corporation  and,  specifically,  Mr.  Willis  Noland  for 
the  use  of  a  microscope  for  the  summer.  I  am  indebted  to  Dr.  Yukio  Hiramoto  for 
his  help  in  the  examination  of  sperm  in  the  centrifuge  microscope,  and  to  Dr.  Ralph 


ARBACIA  SPERM  AGING  259 

Hathaway  for  a  pressure  siphon  used  for  withdrawing  samples  from  centrifuge 
tubes  while  leaving  the  sediments  undisturbed. 

SUMMARY 

1.  The  aging  of  semen  was  investigated  in  terms  of  sperm  motility.     A  method 
for  rating  motility  was  devised.     It  was  based  on   the   observation   that   motile 
sperm  sedimented  faster  than  formalin-killed  controls  when  subjected  to  low-speed 
centrifugation. 

2.  The  aging  of  semen  was  found  to  involve  the  loss  of  motility,  the  loss  of 
fertilizing  capacity  and  disappearance  of  sperm  from  suspension.     All  three  of  these 
factors  declined  most  rapidly  at  first  and  more  slowly  later  on.     The  rapid  loss 
of  fertilizing  capacity  and  the  disappearance  of  sperm  could  be  delayed  by  suppress- 
ing motility  with  hydrogen  ions  or  carbon  dioxide. 

LITERATURE  CITED 

AFZELIUS,  B.  A.,  AND  H.  MOHRI,  1966.     Mitochondria  respiring  without  exogenous  substrate. 

A  study  of  aged  sea  urchin  spermatozoa.     Exp.  Cell  Res.,  42:  10-17. 
BEATTY,  R.  A.,  1964.     Density  gradient  media  for  mammalian  spermatozoa.     Proc.  5th  Internal. 

Congress  for  Animal  Reproduction   and  Artificial  Insemination    (Trento,   1964),    3: 

276-281. 

BISHOP,  D.  W.,  1962.     Sperm  motility.     Physiol.  Rev.,  42:  1-59. 

COHN,  E.  J.,  1918.     Studies  in  the  physiology  of  spermatozoa.    Biol.  Bull,  34:  167-218. 
COSTELLO,  D.  P.,  M.  E.  DAVIDSON,  A.  EGGERS,  M.  H.  Fox  AND  C.  HENLEY,  1957.     Methods  for 

Obtaining  and  Handling  Marine  Eggs  and  Embryos.     Marine  Biological  Laboratory, 

Woods  Hole,  Mass. ;  pp.  184-190. 
VAN  DUIJN,   C.,  JR.,   1963.     Fertilizing  capacity  of  spermatozoa  in  relation  to  their  motility 

characteristics  and  duration  of  survival.     1.    Kinetic  theory  of  probability  of  fertiliza- 
tion.    Rapp.   Inst.   Veeteelk.   Onderz   "Schoonoord"   Ziest.     No.   648,   409   pp. 
VAN  DUIJN,  C.,  JR.,  1964.     A  rational  method  for  estimating  fertility  of  spermatozoa  in  vitro. 

Proc.   5th   Internal.    Congress  for  Animal   Reproduction    and   Artificial   Insemination 

(Trento,  1964),  4:  323-328. 
GEMMILL,  J.  F.,  1900.     On  the  vitality  of  ova  and  spermatozoa  of  certain  animals.    /.  Anat. 

Physiol.,  34:  163-181. 

GRAY,  J.,  1928.     The  senescence  of  spermatozoa.    /.  Exp.  Biol.,  5:  345-361. 
GRAY,  J.,  1931.     The  senescence  of  spermatozoa  II.    /.  Exp.  Biol.,  8:  202-210. 
HATHAWAY,  R.  R.,  1963.     Activation  of  respiration  in  sea  urchin  spermatozoa  by  egg  water. 

Biol.  Bull,  125:486-498. 
IVERSON,    S.,    1964.     Evaluation   of   the   number    of   spermatozoa    in   bull    semen.     Comparison 

between  electronic  counting,  light  scattering  and  absorptiometry.    /.  Agri.  Sci.,  62: 

219-223. 
KIHLSTROM,  J.  E.,  1958.     Specific  gravity  of  different  parts  of  bull  spermatozoa.     Ark.  Zool., 

11:  569-573. 
MANN,  T.,  1964.     Biochemistry  of  Semen  and  of  the  Male  Reproductive  Tract.     Methuen  and 

Co.,  Ltd.,  London,  493  pp. 
MOHRI,  H.,  1957.     Endogenous  substrates  of  respiration  in  sea  urchin  spermatozoa.    /.  Fac.  Sci., 

Univ.  Tokyo,  B:  51-63. 
MOHRI,  H.,  AND  K.  HORIUCHI,  1961.     Studies  on  the  respiration  of  sea  urchin  spermatozoa. 

III.     Respiratory  quotient.    /.  Exp.  Biol,  38:  249-257. 
MOHRI,  H.,  AND  I.  YASUMASU,  1963.     Studies  of  the  respiration  of  sea  urchin  spermatozoa.     V. 

The  effects  of  PCO2.    /.  Exp.  Biol,  40:  573-586. 
RIKMENSPOEL,  R.,  1962.     Biophysical  approaches  to  the  measurement  of  sperm  motility.    In: 

Spermatozoan  Motility.     Ed.  by  D.  W.  Bishop;  AAAS  Pub.  no.  72,  Washington,  D.  C. ; 

pp.  31-54. 


260  JOSEPH  M.  BRANHAM 

ROTHSCHILD,  LORD,  1948a.     The  activity  of  ram  spermatozoa.    /.  Exp.  Biol.,  25:  219-226. 
ROTHSCHILD,  LORD,   1948b.     The  physiology  of  sea  urchin  spermatozoa.     Senescence  and  the 

dilution  effect.    /.  Exp.  Biol.,  25:  353-378. 

ROTHSCHILD,  LORD,  1951.     Sea  urchin  spermatozoa.    Biol.  Rev.,  26:  1-27. 
ROTHSCHILD,  LORD,  1953.     A  new  method  of  measuring  the  activity  of  spermatozoa.    /.  Exp. 

Biol.,  30:  178-199. 
ROTHSCHILD,  LORD,  1956a.     The  physiology  of  sea  urchin  spermatozoa.     Action  of  pH,  dinitro- 

phenol,   dinitrophenol  +  Versene,   and   usnic   acid   on   O2  uptake.    /.   Exp.   Biol.,   33 : 

155-173. 
ROTHSCHILD,  LORD,  1956b.     The  respiratory  dilution  effect  in  sea  urchin  spermatozoa.     Vie  et 

Milieu,  7:  405-412. 
ROTHSCHILD,  LORD,  1962.     Sperm  movement.     Problems  and  observations.     In:  Sperm  Motility 

ed.  by  D.  W.  Bishop;  AAAS  Pub.  no.  72,  Washington,  D.  C.,  pp.  13-29. 
ROTHSCHILD,  LORD,  AND  A.  TYLER,  1954.     The  physiology  of  sea  urchin  spermatozoa.     Action 

of  Versene.    /.  Exp.  Biol,  31 :  252-259. 
TAMPION,  D.,  AND  T.  A.  GIBBONS,  1963.     Swimming  rate  of  bull  spermatozoa  in  various  media 

and  the  effect  of  dilution.     /.  Reprod.  &  Fertil,  5(2)  :  259-275. 
TAYLOR,  SIR  GEOFFREY,  1952.     The  action  of  waving  cylindrical  tails  in  propelling  microscopic 

organisms.     Proc.  Roy.  Soc.  London,  Sec.  A,  211:  225-239. 
TYLER,  A.,  1953.     Prolongation  of  life-span  of  sea  urchin  spermatozoa  and  eggs  with  metal 

chelating  agents    (amino  acids,  Versene,  DEDTC,  oxine,  cupron).     Biol.  Bull.,  104: 

224-239. 

TYLER,  A.,  AND  D.  W.  BISHOP,  1963.     Immunological  phenomena.    In:  Conference  on  Physio- 
logical   Mechanisms    Concerned    with    Conception.     Pergamon    Press,     New    York ; 

pp.  458-465. 
VANDEMARK,  N.  L.,  K.  KOYAMA  AND  J.  R.  LODE,  1965.     Factors  affecting  immobilization  of 

bovine  spermatozoa  with  CO2  and  their  subsequent  reactivation.    J.  Dairy  Sci.,  48(5)  : 

586-591. 
YANAGIMACHI,  R.,   1957.     Some  properties  of  the   sperm  activating  factors   in  the  micropyle 

area  of  the  herring  egg.     Anat.  Zool.  Japan,  30:  114-124. 


THE    REPRODUCTIVE    CAPACITY    OF   ARTEMIA    SUBJECTED   TO 
SUCCESSIVE  CONTAMINATIONS  WITH  RADIOPHOSPHORUS  *• 2 

DANIEL  S.  GROSCH 
Department  of  Genetics,  N.  C.  State  University,  Raleigh,  N.  C.  27607 

The  dangers  of  contaminating  the  environment  with  radioisotopes  or  other 
deleterious  substances  have  received  much  publicity.  Often  the  main  biological 
consideration  has  been  the  number  of  adult  individuals  of  susceptible  species  seen 
or  caught  during  a  particular  season.  These  numbers  are  then  compared  with 
records  of  other  years  in  an  attempt  to  infer  damage  or  to  claim  unimpairment. 
Unfortunately,  adult  abundance  equalling  that  of  an  area  before  exposure  does  not 
necessarily  indicate  recovery  from  genetic  damage  to  the  population.  The  repro- 
ductive capacity  of  an  organism  may  be  adequate  to  compensate  for  infecundity  and 
the  death  of  immature  stages.  Therefore,  to  reveal  the  consequences  of  contamina- 
tion, the  quantitative  aspects  of  natality  and  survival  to  maturity  must  be  studied. 

In  our  laboratory,  Artemia  populations  are  being  used  in  studies  of  induced 
changes  in  the  components  of  biological  fitness  after  subjection  to  a  variety  of 
agents.  Results  for  the  last  four  years  of  an  8-year  study  of  radiophosphorus  effects 
are  reported  below.  Most  notable  is  the  consistent  demonstration  that  the  number 
of  adults  can  be  identical  in  different  mass  cultures,  but  the  reproductive  potential 
of  populations  with  different  ancestral  histories  differs  considerably. 

The  fitness  components  obtained  from  Artemia  pair  mating  tests  over  a  four- 
year  period  following  radioisotope  exposure  of  ancestors  were  reported  earlier 
(Grosch,  1962) .  Subsequently  several  of  the  original  populations  have  been 
subjected  to  further  experimentation  and  analyses  which  reveal  persistent  inferiority 
of  reproductive  performance  in  pair  matings.  In  addition  we  have  determined 
whether  subpopulations  can  survive  repeated  contaminations,  an  important  problem 
in  radioecology.  Our  results  indicate  that  species  density  or  standing  crop  data 
can  be  misleading.  Although  adults  in  an  experimental  culture  may  be  abundant, 
their  reproductive  potential  may  be  inadequate  to  withstand  further  damage  by 
radiations. 

MATERIALS  AND  METHODS 

Two  isotopes,  Zn65  and  P32,  differing  in  rays,  half-life,  and  metabolic  fate,  were 
selected  for  Artemia  experiments  because  of  their  persistence  in  Columbia  River 
food  chains  (Foster  and  Davis,  1955).  All  Zn65  populations  are  now  extinct,  not 

1  These  experiments   were  begun   with   the  help   of   summer   assistants    supported   at   the 
Marine   Biological   Laboratory  by  U.    S.   Atomic   Energy   Commission   funds.     Currently   the 
author  receives  support  from  U.  S.  Public  Health  Service  research  grant  ES-00044,  Division  of 
Environmental  Engineering  and  Food  Protection. 

2  Contribution   from   the   Genetics   Department,    North    Carolina    Agricultural    Experiment 
Station,  Raleigh,  North  Carolina.     Published  with  the  approval  of  the  Director  of  Research  as 
Paper  No.  2137  of  the  Journal  Series. 

261 


262  DANIEL  S.  GROSCH 

only  the  30  /ic./3L  culture  of  the  1962  (Grosch)  report,  but  also  several  20  ^, 
cultures  followed  subsequently.  On  the  other  hand,  many  of  the  P32  cultures 
survived.  One  of  these  is  a  90  juc./3L  culture  which  is  providing  useful  data.  In 
nature,  although  it  comprises  less  than  \%  of  the  radioactivity  in  Hanford  wastes 
which  contain  more  than  a  dozen  different  nuclides,  P32  accounts  for  40%  to  95% 
of  the  radioactivity  of  most  Columbia  River  invertebrates  and  fish.  This  reflects 
the  biological  demand  for  an  element  incorporated  into  genetically  important  nucleic 
acids  and  energy  storage-transfer  systems. 

The  three-liter  mass  cultures  derived  from  the  diploid  amphigonic  strain  of 
Artemia  salina  have  been  maintained  in  cylindrical  gallon  jars  for  nearly  a  decade 
at  room  conditions  in  Woods  Hole.  During  the  summer  they  have  received  1 
ml.  of  yeast  suspension  daily.  During  the  winter  they  typically  evaporated  to  less 
than  one-third  of  the  summer  volume.  In  spring  the  cultures  were  reconstituted 
by  adding  distilled  water  to  dissolve  salt  encrustations  and  activate  cysts.  Putrefy- 
ing dead  algal  masses  were  removed  as  soon  as  Artemia  emergence  seemed  com- 
plete. With  such  attention  a  population  of  250  to  300  adults  has  quickly  developed 
in  every  three-liter  container  except  in  strains  nearing  extinction.  The  control 
cultures  maintained  simultaneously  with  experimental  cultures  under  identical  con- 
ditions, were  derived  from  ancestors  which  have  never  been  exposed  to  radioisotopes 
or  other  deleterious  agents  of  technological  origin. 

Artemia  culture  techniques  have  been  improved  during  the  years  in  which  jar 
populations  have  been  maintained  and  studied.  Although  Artemia  is  an  organism 
assumed  to  be  exceptionally  tolerant  because  the  adults  survive  for  days  in  a  wide 
range  of  salinities,  previous  work  (Grosch,  1962)  indicated  that  reproductive  per- 
formance was  improved  by  increasing  the  salinity  above  that  of  sea  water.  From 
their  own  experience  other  geneticists  (Goldschmidt,  1952;  Bowen,  1962)  decided 
to  culture  Artemia  in  water  saltier  than  sea  water.  Prior  to  1962  an  increase  in 
salinity  for  our  mass  cultures  resulted  only  from  the  slow  process  of  evaporation. 
Since  1962  NaCl  has  been  added  routinely  to  mass  cultures.  Present  practice  is 
to  bring  them  to  50  grams  of  added  NaCl  per  liter  before  maturation  of  the 
summer's  first  generation.  In  1964  and  1965  water  of  increased  salinity  was  used 
also  for  pair  mating  tests.  Bowen's  routine  medium  was  adopted,  50  grams  of  NaCl 
per  liter  of  filtered  sea  water. 

Each  subpopulation  to  be  subjected  to  an  addition  of  radioisotope  was  obtained 
by  transferring  20  adult  pairs  to  a  gallon  jar  containing  three  liters  of  brine. 
For  pair  mating  studies,  15  young  pairs  were  transferred  from  mass  culture  as 
soon  as  the  male  had  clasped  the  female.  Since  arbitrary  matching  was  not  prac- 
ticed, the  pairs  studied  are  representative  of  those  contributing  to  the  future  of 
the  population  from  which  they  were  withdrawn.  Each  pair  was  placed  in  its  own 
quart  jar.  The  average  number  of  days  between  the  transfer  to  quart  jars  and 
the  death  of  members  of  mated  pairs  is  taken  as  a  measure  of  adult  life  span. 

Jars  containing  parental  pairs  and  the  jars  to  which  their  broods  were  trans- 
ferred were  maintained  under  constant  illumination  from  a  bank  of  fluorescent 
tubes.  The  water  temperature  ranged  between  25°  and  28°  C.  All  jars  were 
examined  daily  at  the  time  of  feeding  with  yeast  suspension  (0.3  ml.).  When 
present,  cysts  were  filtered,  dried  and  resuspended  in  filtered  sea  water  for 
emergence  tests. 


SUCCESSIVE  P32  CONTAMINATIONS 


263 


RESULTS  AND  INTERPRETATIONS 

A  persistent  population  descended  from  ancestors  exposed  to  30  pC.  of  P32 
produced  hundreds  of  adults,  generation  after  generation  and  year  after  year  in 
mass  culture.  However,  subcultures  were  unable  to  survive  a  second  30-/uc.  dose 
until  four  years  or  a  minimum  of  12  generations  had  elapsed.  Furthermore,  as 
shown  in  Figure  1,  additional  years  and  generations  passed  before  subcultures 
managed  to  survive  a  third  30-/xc.  dose  of  P32.  Evidently  the  carrying  capacity 
(300  adults)  of  a  three-liter  culture  was  easily  achieved  by  Artemia  of  experimental 
lineage.  Differences  between  strains  were  revealed  only  by  investigating  repro- 
ductive performance.  For  this  purpose  we  employ  isolated  parental  pairs. 


30;uc 

ADDITIONS      1958 


1959 


1960 


1961 


1962 


1963 


-HO 


4™ 


FIGURE  1.  The  origin  and  subsequent  fate  of  three-liter  experimental  cultures  of  Artemia 
which  received  30-/uc.  doses  of  P32.  The  black  arrows  indicate  subcultures  given  radiophos- 
phorus.  Cultures  marked  with  an  X  did  not  survive.  After  three  months  no  adult  offspring- 
have  appeared  in  the  1965  "fourth  addition"  jar  and  no  horizontal  arrow  representing  continued 
survival  of  the  strain  is  shown.  The  subculture  of  control  origin  which  received  the  first 
addition  of  P32  was  discarded  in  1963. 


An  adult  female  can  exhibit  both  oviparity  and  viviparity  (Lochhead,  1961). 
In  pair  mating  tests,  control  females  were  not  strongly  inclined  toward  oviparity. 
They  gave  birth  to  relatively  more  live  young  and  deposited  fewer  cysts  than  did 
females  from  other  strains  tested.  Table  I  summarizes  records  for  the  last  four 
years  in  which  the  percentage  of  zygotes  encysted  has  been  lowest  for  control 
parents  year  after  year.  In  addition,  for  the  years  1962  and  1965,  the  lowest 
percentage  of  emergence  or  "hatchability"  of  the  cysts  was  found  in  the  controls. 
An  interesting  feature  of  the  experimental  results  has  been  the  steady  climb  in 
percentage  of  emergence.  Originally  in  1959,  one  year  after  the  population  had 
experienced  its  first  radiophosphorus  exposure,  emergence  was  less  than  25%.  In 


264 


DANIEL  S.  GROSCH 


TABLE  I 
Cyst  deposition  and  emergence  of  larvae  from  four  years'  records  of  pair  matings 


Strain  tested 

%of 
zygotes 
encysted 

1962 

% 
emerged 

%of 
zygotes 
encysted 

1963 

% 
emerged 

%of 
zygotes 
encysted 

1964 

%      H 

emerged 

%of 
zygotes 
encysted 

1965 

%     , 
emerged 

Control 

44.19 

30.66 

51.35 

49.26 

30.47 

65.93 

26.40 

27.60 

30-/iC.  Experiments 
First  P32  addition 

65.30 

36.17 

Second  P32  addition 

76.23 

40.32 

62.32 

42.82 

96.04 

66.94 

Third  P32  addition 

87.64 

63.32 

90-Atc.  P32 
Single  dose 

None 

None 

58.49 

26.82 

48.19 

58.09 

1965  emergence  has  reached  a  high  of  66.94%  for  a  strain  which  has  received 
two  doses  of  P32. 

The  results  reflect  the  performance  of  all  members  of  the  sample  rather  than 
that  of  only  a  few  females.  Every  female  which  produced  young  also  yielded  cysts, 
typically  as  her  first  brood  and  often  as  her  last.  This  contrasts  with  the 
records  in  our  1962  paper  in  which  only  controls  showed  a  majority  of  the  females 
depositing  cysts. 

Control  pairs  tended  to  produce  a  greater  number  of  broods.  In  addition  there 
were  more  zygotes  per  brood  and  of  these  more  survived  to  adulthood  than  survived 
in  tests  of  progeny  from  exposed  ancestors.  Table  II  summarizes  the  life  span 

TABLE  II 

The  life  span  and  fecundity  of  parents  from  pair  mating  tests  along  with 
survival  and  sex  ratio  of  offspring 


Adult  life 
span  in  days 

Broods 
per   9 

Zygotes  voided 

% 

surviving 
to  adult 

Mature 
adults 
per  9 

Sex  ratio 
oV9 

9 

c?1 

per  brood 

per   9 

1963 

Control 

16.3 

14.2 

1.2 

272.78 

327.34 

29.91 

97.91 

0.97 

30-juc.  second  addition 

16.6 

21.9 

1.4 

130.70 

182.98 

12.85 

23.51 

0.95 

90-Aic.  single  dose 

12.7 

12.8 

0.3 

74.33 

22.30 

0.58 

0.13 

1.33 

1964 

Control 

60.45 

66.65 

12.25 

187.35 

2295.04 

73.49 

1686.62 

0.80 

30-juc.  second  addition 

33.62 

44.38 

2.87 

133.98 

384.52 

49.93 

181.99 

0.88 

90-juc.  single  dose 

30.60 

31.60 

5.7 

177.84 

1013.68 

17.07 

173.35 

0.99 

1965 

Control 

55.45 

52.18 

10.18 

157.17 

1599.99 

59.45 

951.18 

0.85 

30-^c.  second  addition 

21.92 

31.17 

3.83 

106.26 

406.96 

47.70 

194.12 

0.91 

30-juc.  third  addition 

30.71 

36.71 

4.36 

109.59 

477.81 

56.71 

270.97 

0.81 

90-/zc.  single  dose 

32.08 

38.00 

3.63 

81.45 

295.66 

68.71 

203.15 

0.90 

SUCCESSIVE  P32  CONTAMINATIONS  265 

of  parents  and  their  reproductive  performance  for  1963  through  1965.  Included 
are  the  sex  ratios  for  adult  progeny.  Females  are  favored,  except  in  1963  for  the 
90-/i.c.  culture  which  was  then  not  doing  well.  These  results  differ  from  the  earlier 
sex  ratios  which  favored  males  in  many  irradiated  strains  (Grosch,  1962). 

Sex  ratio  can  be  discounted  but  the  problem  is  to  decide  if  improvement  in  any 
other  aspect  such  as  adaptive  value  is  involved  in  surviving  an  additional  radio- 
isotope  contamination.  Adaptive  value  can  be  defined  as  the  relative  capacity  of 
carriers  of  a  given  genotype  to  transmit  their  genes  to  the  gene  pool  of  the  following 
generation.  It  may  be  calculated  by  dividing  the  mature  adults  per  pair  of  treated 
ancestry  by  the  number  of  mature  adults  per  control  pair.  The  adaptive  values  for 
the  90-ju.c.  strain  have  shown  regular  improvement  from  nearly  zero  in  1962  through 
0.001,  0.10,  to  0.21  in  the  last  three  years.  On  the  other  hand  adaptive  values 
have  varied  for  the  30-ju.c.  doses,  due  partly  to  variability  in  control  performance 
which  is  taken  as  unity  in  deriving  the  value.  On  this  basis  an  average  of  191.99 
live  adults  in  1964  does  not  compare  so  well  with  194.1  in  1965  when  respective 
control  performance  is  used  as  the  basis  for  calculating  A.V.'s  of  0.11  and  0.20. 
Possibly  prolonged  patterns  of  weather  are  reflected  in  our  data ;  1964  and  1965 
were  different  types  of  summers. 

The  reproductive  advantage  of  controls,  expressed  as  mature  adults  produced 
per  parental  pair,  depends  in  part  upon  the  length  of  time  adults  survive  in  quart 
jars.  In  1963  before  the  Bowen  medium  was  adopted  for  pair  mating  tests,  the 
ratio  of  control  to  "second  addition"  progeny,  97.9:23.5,  was  about  4:1.  In  1964 
the  ratio  was  about  9:1  and  in  1965  the  ratio  was  about  5:1.  Part  of  the  difference 
comes  from  an  increase  in  survival  (mean  adult  life  span)  from  less  than  three 
weeks  to  approximately  two  months.  Although  the  poorest  experimental  groups 
are  now  attaining  life  spans  equivalent  to  or  exceeding  control  values  of  previous 
years,  the  difference  which  was  not  apparent  in  1963  has  become  pronounced. 
Controls  are  now  living  nearly  twice  as  long  as  adults  from  experimental 
populations. 

With  increased  life  span,  improvement  in  the  number  of  offspring  per  female 
was  inevitable  because  broods  have  been  deposited  with  regular  frequency.  The 
raw  data  are  too  extensive  for  tabulation  here.  Analysis  of  1965  brood  frequency 
showed  no  significant  difference  in  the  averages  of  the  interval  for  control  (3.00 
±0.91),  second  (3.20  ±0.27),  and  third  addition  (3.80  ±  0.94)  tests.  This 
pattern  in  which  females  void  broods  every  third  or  fourth  day  has  been  evident 
in  our  records  since  1959.  In  1963  when  pair  matings  were  still  maintained  in 
ordinary  sea  water  the  control  interval  was  3.86  ±  0.06  days  and  for  second  addition 
pairs  3.25  ±  0.30  days.  A  striking  and  unexplained  deviation  in  the  pattern  has 
appeared  for  the  90-/xc.  strain.  In  1964  the  interval  averaged  3.07  ±  0.02  days. 
In  1965  the  strain  showed  a  longer  period  between  broods;  the  interval  of  6.80 
±  0.98  days  differs  significantly  from  all  others  cited.  These  frequencies  which 
concern  only  the  pattern  between  the  first  and  last  brood  voided  cannot  be  inferred 
directly  from  the  number  of  broods  per  female  shown  on  Table  II.  The  time 
elapsing  between  isolation  of  pairs  and  deposit  of  first  brood  varies  as  does  the 
period  intervening  between  last  brood  and  death. 

Another  influence  of  life  span  appears  to  be  upon  brood  size  but  the  relationship 
is  not  simple.  In  young  adults  brood  size  is  correlated  with  the  increasing  size  of 


266 


DANIEL  S.  GROSCH 


250r 


200- 


BROOD       NUMBER 


10 


15 


FIGURE  2.  Brood  size  plotted  against  the  sequence  of  brood  deposit.  Each  curve  represents 
the  reproductive  record  of  the  individual  female  surviving  for  the  longest  period  of  time  in 
its  respective  group  of  1965  pair  mating  tests.  The  solid  circles  indicate  the  control  example. 
Triangles  represent  values  for  the  long-lived  individual  from  a  culture  which  received  a  single 
90-/u,c.  dose  of  P32  in  1959.  Squares  mark  values  for  the  examples  from  30-^c.  addition  experi- 
ments. The  number  of  successive  doses  in  the  history  of  the  culture  is  indicated  by  a  designa- 
tion at  the  end  of  the  curve,  2nd  =  2  and  3rd  =  3  doses. 


the  female.  Subsequently  brood  size  declines,  presumably  as  a  reflection  of  aging  or 
because  of  debility  associated  with  impending  death.  The  clearest  demonstration  of 
this  pattern  was  obtained  by  plotting  brood  size  against  brood  sequence  for  each 
individual  female.  A  peak  and  subsequent  decline  is  characteristic  of  records 
for  all  females  surviving  15  days,  the  time  usually  required  for  voiding  about  five 


SUCCESSIVE  PS2  CONTAMINATIONS  267 

broods.  Plotting  the  average  brood  size  is  an  unsatisfactory  procedure  in  this 
case.  Particularly  for  experimental  females  the  pattern  of  fecundity  tends  to  be 
obscured  in  pooled  data  by  the  broods  contributed  by  short-lived  females. 

In  order  to  reveal  potentialities  in  fecundity,  patterns  of  brood  size  for  females 
of  maximum  longevity  from  each  of  the  populations  sampled  by  pair  mating  tests 
in  1965  are  shown  in  Figure  2.  Peak  productivity  was  achieved  by  the  fifth 
or  sixth  brood.  The  subsequent  decline  was  pronounced  for  females  of  experi- 
mental ancestry  but  moderated  by  a  long  plateau  for  the  control  sample.  As 
evidence  that  the  example  is  representative  of  the  control  pattern  we  can  state  that 
7  of  the  first  10  points  are  nearly  identical  with  the  respective  average  values,  and 
the  remaining  three  points  lie  within  one  standard  error  of  the  pertinent  control 
values.  The  control  sample  which  averaged  10  broods  before  death  (Table  II) 
contained  many  individuals  which  survived  beyond  the  period  of  maximum 
fecundity.  On  the  other  hand,  experimental  females  which  averaged  only  3  or  4 
broods  before  death  probably  failed  to  reach  their  maximum  fecundity. 

Although  deficient,  the  fecundity  of  "second  addition"  pairs  has  not  varied  in 
a  manner  which  would  explain  the  response  of  the  mass  culture  to  additional  isotope 
exposures.  A  possible  key  may  be  the  survival  to  adulthood.  These  percentage 
values  fit  into  a  pattern : 

Year  1959  1960  1961  1962  1963  1964  1965  Addition 

Survival  24.4  27.6  57.6  61.0  First 

12.8  49.9  47.7  Second 

Not  until  1962  was  a  second  addition  tolerated.  Survival  to  adulthood  was  reduced 
severely  by  the  treatment  and  a  third  addition  was  not  tolerated  until  the  moderate 
recovery  demonstrated  in  1964  had  occurred. 

DISCUSSION 

A  twice-repeated  series  of  P32  doses  had  indicated  that  a  single  addition  of 
90  p.c.  approached  the  threshold  dose  for  population  extinction  (Grosch,  1962). 
From  the  simple  standpoint  of  dose  arithmetic  it  seemed  possible  that  subcultures 
from  a  strain  that  had  survived  30  ju,c.  should  be  able  to  withstand  additional  doses. 
This  was  not  the  case,  and  pair  mating  studies  revealed  severe  genetic  damage 
to  reproductive  ability  from  one  30-/x,c.  treatment.  Additional  equivalent  doses 
were  tolerated  only  after  a  period  during  which  the  germ  plasm  presumably  under- 
went a  partial  purge. 

The  pair  mating  tests  summarized  above  reveal  that  maintenance  of  populations 
numbering  300  adults  amounts  to  only  a  small  fraction  of  the  control  potential. 
For  example,  if  300  adults  comprise  150  pairs  each  capable  of  providing  951  mature 
offspring  (1965  average),  the  potential  number  of  offspring  amounts  to  the  product, 
142,650.  However,  when  the  carrying  capacity  determined  by  a  density-dependent 
process  is  300,  only  a  fraction  of  the  reproductive  potential  can  be  utilized.  This 
fraction  300/142,650  =  0.002  or  0.2%.  By  the  same  reasoning,  only  1%  of  the 
"second  addition"  culture's  potential  is  required  to  maintain  the  ceiling  level  of 
300  in  a  three-liter  culture.  In  the  past,  a  number  of  cases  was  observed  in  which 
to  30%  of  the  reproductive  potential  was  used  to  maintain  300  adults  in  a 


268  DANIEL  S.  GROSCH 

three-liter  culture.  This  type  of  situation  proved  precarious  and  many  such  cul- 
tures became  extinct.  Conceivably  even  more  extreme  situations  may  occur  in 
nature,  and  in  unfavorable  circumstances  80%  to  100%  of  the  reproductive  potential 
of  an  organism  may  be  required  to  maintain  the  frequencies  of  adults  found  in  census. 

As  pointed  out  by  Grant  (1963),  we  don't  really  have  enough  quantitative 
information  at  present  about  the  actual  number  of  genetic  deaths  a  population  can 
tolerate  and  still  survive  under  various  conditions.  Possibly  more  than  50%  of 
the  Artemia  reproductive  potential  must  be  held  in  reserve  to  buffer  populations 
against  extinction.  If  so,  in  natural  situations  impaired  by  human  activities, 
other  organisms  may  be  balanced  even  more  precariously  than  Artcinia.  Actually 
Artemia  may  have  an  advantage  over  most  animals.  Possibly  when  shrimp  popula- 
tions reach  the  ceiling  level  their  excess  productivity  can  be  switched  into  encysted 
zygotes  rather  than  expended  in  juvenile  mortality. 

Fertility  requirements  are  very  high,  usually  too  high,  for  a  species  acquiring 
many  deleterious  two-allele  loci  with  high  selective  differentials.  Several  geneticists 
(Lerner,  1958;  Wallace  and  Dobzhansky,  1959;  Grant,  1963;  Wallace,  1963)  have 
speculated  about  the  number  of  offspring  which  must  survive  if  a  population  is  to 
avoid  extinction  following  the  induction  of  simple  dominant  or  recessive  lethals. 
The  maximum  number  of  offspring  is  limited  by  the  number  of  functional  eggs 
produced  per  female.  In  this  attribute  Artemia  exceeds  domestic  animals  and  most 
insects.  On  the  other  hand,  Artemia  fecundity  is  not  particularly  exceptional  when 
compared  with  the  range  for  fish  and  aquatic  invertebrates  (Altman  and  Dittmer, 
1962).  The  cost  in  segregation  of  inferior  homozygotes  may  be  met  by  a  fecund 
organism,  or  the  price  may  be  reduced  by  series  of  multiple  alleles  or  by  numbers 
of  independently  assorting  interchangeable  genes  (Grant,  1963). 

An  alternate  approach  is  to  view  lethality  as  the  product  of  lethal  gene  com- 
binations rather  than  the  product  of  lethal  genes  (Mayr,  1963).  The  genes  which 
interact  harmoniusly  in  the  population's  gene  pool  were  brought  together  by 
natural  selection  acting  over  a  long  period  of  time.  Disharmonius  combinations 
can  follow  the  induction  of  genie  diversity.  Recently  bichromosomal  synthetic 
semilethals  have  been  demonstrated  in  Drosophila  pseudoobscura  (Dobzhansky 
et  al.,  1965).  Individuals  homozygous  for  specific  second  and  third  chromosomes 
showed  viability  down  in  the  semilethal  range.  Conceivably  disharmonius  inter- 
actions also  can  occur  in  heterozygous  genotypes.  So  many  beneficial  effects  of 
heterozygosity  have  been  described  that  we  are  too  inclined  to  regard  all  heterozy- 
gosity  as  good,  but  for  example,  a  loss  of  epistatic  balance  among  interacting  loci 
can  override  the  beneficial  effect  of  high  heterozygosity  (Mayr,  1963). 

To  date  we  have  been  unable  to  demonstrate  increased  genetic  fitness  in 
irradiated  Artemia  such  as  shown  by  Wallace  (1956)  in  Drosophila  and  by 
Crenshaw  (1965)  in  Tribolium,  but  admittedly  a  demonstration  of  the  phenomenon 
may  require  a  more  inbred  strain  of  shrimp  than  is  yet  available.  Also  our  testing 
has  been  limited  to  a  particular  season.  Possibly  in  certain  seasons  (or  years)  it 
might  be  possible  for  experimental  Artemia  to  equal  or  exceed  the  controls  in 
fitness.  On  the  other  hand,  Sokal  and  Huber  (1963)  reported  heterozygote 
intolerance  to  crowding  in  one  Tribolium  experiment,  and  Sankaranarayanan 
(1965)  found  Drosophila  subpopulations  plateauing  at  70  to  75%  viability  in  x-ray 
experiments.  This  level  achieved  in  five  generations  after  cessation  of  irradiation 


SUCCESSIVE  P32  CONTAMINATIONS  269 

indicated  more  rapid  recovery  than  we  have  observed  with  Artemia,  but  gives  no 
evidence  of  superiority  of  irradiated  strains. 

In  prolific  mass  cultures,  crowding  is  more  pronounced  than  that  experienced  by 
broods  in  quart  jars.  Until  this  year  survival  to  adulthood  in  experimental  broods 
in  quart  tests  has  differed  obviously  from  control  values,  and  any  effect  of  crowding 
has  been  obscured.  However,  even  the  90-yu.c.  strain  has  improved  to  the  degree 
that  its  fewer  zygotes  per  brood  (81.45)  enable  compensation  in  a  higher  percentage 
of  survival  to  adulthood  in  the  1965  results  of  Table  II.  When  68.71  %  survival 
was  compared  with  the  control's  59.45%,  the  contingency  chi  square  calculated  from 
the  raw  data  was  11.48  with  P  <  0.001.  On  the  other  hand,  106  to  109  shrimp  per 
quart  jar  do  not  provide  a  situation  significantly  different  from  the  control's  157 
per  quart.  In  these  cases  survival  to  adulthood  does  not  exceed  that  of  controls. 

Fly  crowding  experiments  furnish  some  interesting  parallels  and  differences. 
Survivorship  near  60%  can  be  demonstrated  through  a  wide  range  of  fly  densities 
for  a  variety  of  strains,  and  in  house  fly  experiments  Sullivan  and  Sokal  (1963) 
quote  67%  survivorship  for  a  density  of  160  per  bottle,  considered  "normal  condi- 
tions." Competition  became  impressive  only  at  1280  flies  per  bottle  and  extreme  at 
2560  per  bottle.  This  was  reflected  by  "negligible"  adult  emergence,  \%  or  less 
in  some  strains  (Bhalla  and  Sokal,  1964).  Thus  normal  density  in  fly  cultures 
gives  survival  similar  to  that  in  Artemia  brood  tests,  and  high  density  fly  experi- 
ments more  nearly  resemble  what  may  happen  in  prolific  mass  cultures  of  shrimp. 
Moderate  crowding  to  give  selection  pressure  of  intermediate  intensity  of  Tribolium 
provides  another  example  of  insect  survivorship  within  a  58  to  78%  range  (Sokal 
andHuber,  1963). 

Differences  from  insect  results  derive  from  differences  in  growth  pattern  and 
become  pronounced  as  development  nears  maturity.  Size  is  determinate  for 
holometabolous  insects  and  indeterminate  for  shrimp.  In  crowded  dipteran  popula- 
tions the  usual  response  is  a  maintenance  of  numbers  accompanied  by  a  reduction 
in  size  of  individuals  (Sullivan  and  Sokal,  1963).  Brine  shrimp  respond  by 
repressed  rate  of  growth  and  delayed  maturity  of  part  of  the  group  or  brood 
(Grosch,  1962),  suggesting  a  feedback  phenomenon  such  as  reported  by  Rose 
(I960)  for  fish  and  Amphibia.  Unfortunately,  for  purposes  of  comparison, 
cannibalism  occurs  among  crowded  fish. 

If  adults  function  as  growth  suppressors,  their  longevity  could  interfere  with  a 
turnover  of  generations.  No  data  are  available  on  the  effects  of  crowding  on  the 
longevity  of  Artemia  adults  but  with  moderate  fecundity  and  good  potential 
survival  to  adulthood,  parents  need  live  only  long  enough  to  produce  a  brood  or  two 
in  order  to  maintain  a  mass  population  in  three  liters.  Our  life  span  data  concern 
isolated  pairs.  Although  present  techniques  prolong  adult  survival,  individual 
examples  of  extreme  longevity,  such  as  reported  by  Lochhead  (1941)  and  Bowen 
(1962),  have  not  been  obtained.  However,  frequent  transfer  is  necessary  in  order 
to  assess  the  reproductive  performance  of  pairs  and  under  such  circumstances  even 
inanimate  objects  like  cafeteria  tumblers  have  a  predictably  limited  life  span 
(Brown  and  Flood,  1947). 

SUMMARY 

1.  This  paper  is  a  progress  report  on  four  additional  years  of  studying  Artemia 
in  and  from  mass  cultures  to  which  radioisotope  has  been  added. 


270  DANIEL  S.  GROSCH 

2.  Although  the  number  of  adults  seen  in  mass  cultures  may  be  equivalent, 
subcultures  of  control  and  experimental  strains  react  differently  to  radioisotope 
additions.     Strains  descended  from  ancestors  exposed  to   P32  do  not  necessarily 
survive  a  second  dose  even  though  total  dosage  does  not  exceed  the  extinction  dose 
given  as  a  single  addition.     A  period  of  recovery  involving  a  passing  of  generations 
must  intervene.     Depending  upon  culture  conditions,  this  may  involve  two  to  four 
years. 

3.  Pair  mating  tests  revealed  that  in  comparison  with  controls,  experimental 
strains : 

A.  Have  a  shorter  life  span. 

B.  Deposit  fewer  zygotes  per  brood. 

C.  Deposit  more  of  their  developed  zygotes  as  viable  cysts. 

D.  Show  poor  survival  to  adulthood  except  when  crowding  is  mitigated  by 
low  fecundity. 

4.  Because  the  frequency  of  brood  deposit  has  been  regular  in  both  control  and 
experimental  strains,  an  increase  in  total  progeny  has  accompanied  increased  life 
span.     The  one  exception  to  an  interval  of  three  to  four  days  between  deposits 
occurred  in  1965  records  of  a  strain  recovering  from  a  near  lethal  dose. 

5.  The  sex  ratios  among  adult  progeny  from  pair  matings  now  favor  females 
in  all  strains. 

6.  On  the  basis  of  pair  mating  tests,  maintenance  of  mass  cultures  at  an  observed 
level  of  300  adults  per  three  liters  requires  only  0.2%  of  the  reproductive  potential 
of  controls.     Cultures  of  experimental  origin  utilize  \%  or  more  of  their  potential 
to  maintain  the  same  total. 

7 ' .  The  proportional  number  of  larvae  surviving  to  adults  may  be  critical  in 
determining  whether  or  not  a  strain  can  tolerate  another  exposure  to  a  radioisotope. 

8.  Although  general  comparisons  may  be  drawn  to  crowding  experiments  in 
insect  populations,  a  complete  parallel  is  impossible  because  of  the  shrimp's  inde- 
terminate growth  pattern  (although  Artemia  populations  are  inversely  density- 
dependent). 

LITERATURE  CITED 

ALTMAN,   P.  L.,  AND  DOROTHY   S.   DITTMER    (Eds.),    1962.     Growth    Including   Reproduction 

and  Morphological  Development.     Federation  Amer.  Soc.  Exp.  Biol.,  Washington,  D.  C. 
BHALLA,  S.  C.,  AND  R.  R.  SOKAL,  1964.     Competition  among  genotypes  in  the  housefly  at  various 

densities  and  proportions  (The  Green  Strain).    Evolution,  18:  312-330. 
BOWEN,  SARANE  T.,  1962.     The  genetics  of  Artemia  salina.     I.    The  reproductive  cycle.     Biol. 

Bull,  122:25-32. 

BROWN,  G.  W.  AND  M.  M.  FLOOD,  1947.     Tumbler  mortality.     /.  Amer.  Statist.  Assoc.,  42:  562. 
CRENSHAW,  J.  W.,  JR.,  1965.     Radiation-induced  increases  in  fitness  in  the  flour  beetle  Tribolium 

confiisum.    Science,  149:  426-427. 
DOBZHANSKY,  T.,  B.  SPASSKY  AND  W.  ANDERSON,  1965.     Bichromosomal  synthetic  semilethals 

in  Drosophila  pseudoobscura.    Proc.  Nat.  Acad.  Sci.,  53:  482-486. 
FOSTER,  R.  F.,  AND  J.  J.  DAVIS,  1955.     The  accumulation  of  radioactive  substances  in  aquatic 

forms.     Int.  Conf.  Peaceful  Uses  Atomic  Energy,  13:  364-367. 
GOLDSCHMIDT,  E.,   1952.     Fluctuation  in  chromosome  number   in  Artemia  salina.    J.  Morph., 

91:  111-131. 

GRANT,  V.,  1963.    The  Origin  of  Adaptations.     Columbia  Univ.  Press,  N.  Y. 
GROSCH,  D.   S.,  1962.     The  survival  of  Artemia  populations  in  radioactive   sea  water.     Biol. 

Bull,  123:302-316. 


SUCCESSIVE  P32  CONTAMINATIONS  271 

LERNER,  I.  M.,  1958.    The  Genetic  Basis  of  Selection.    John  Wiley  &  Sons,  Inc.,  N.  Y. 

LOCHHEAD,  J.  H.,  1941.    Artcmia,  the  brine  "shrimp."     Turtox  News,  19:  41-45. 

MAYR,     E.,     1963.     Animal     Species     and     Evolution.      Harvard     Univ.     Press,     Cambridge, 

Massachusetts. 
ROSE,    S.    M.,    1960.     A   feedback   mechanism    of   growth    control    in    tadpoles.    Ecology,    41: 

188-189. 
SANKARANARAYANAN,  K.,  1965.     Further  data  on  the  genetic  loads  in  irradiated  experimental 

populations  of  Drosophila  melanogastcr.     Genetics,  51 :  153-164. 
SOKAL,  R.  R.,  AND  I.  HUBER,  1963.     Competition  among  genotypes  in  Tribolium  castaneum  at 

varying  densities  and  gene  frequencies  (the  sooty  locus).    Amer.  Nat.,  97:  169-184. 
SULLIVAN,  R.  L.,  AND  R.  R.  SOKAL,  1963.     The  effect  of  larval  density  on  several  strains  of 

the  house  fly.     Ecology,  44:  120-130. 
WALLACE,  B.,  1956.     Studies  on  irradiated  populations  of  Drosophila  melanog aster.    J.  Genetics, 

54:280-293. 
WALLACE,  B.,  1963.     Modes  of  reproduction  and  their  genetic  consequences.     Statistical  Genetics 

and  Plant  Breeding  NAS-NRC,  982:  3-17. 
WALLACE,  B.,  AND  T.  DOBZHANSKY,  1959.     Radiation,  Genes  and  Man.     Henry  Holt  and  Co., 

N.  Y. 


SOME  FUNCTIONS  OF  THE  URINARY  BLADDER  IN  A  CRAB 

WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 
Department  of  Life  Sciences,  University  of  California,  Riverside,  California  92502 

The  antennary  glands  of  crabs  generally  are  ineffective  as  organs  of  osmoregula- 
tion  inasmuch  as  the  urine  they  produce  remains  isosmotic  with  the  blood  under 
conditions  of  hypo-  or  hyperosmotic  stress.  On  the  other  hand,  as  indicated  in 
the  reviews  by  Lockwood  (1962)  and  Potts  and  Parry  (1964)  the  probable  primary 
function  of  these  renal  organs  is  ionic  regulation.  Of  particular  interest  is  the 
high  concentration  of  Mg++  attained  in  the  urine  of  crabs  during  immersion  in 
hypersaline  water. 

Prosser  et  al.  (1955)  demonstrated  in  the  shore  crab,  Pachygrapsus  crassipes, 
dramatic  increases  in  urine  Mg++  but  decreases  in  urine  Na+  as  the  environmental 
salinity  increased.  This,  they  attributed  to  competition  between  Na+  and  Mg++  for 
transport  across  the  membranes  of  the  antennary  gland,  Mg++  prevailing.  Green 
et  al.  (1959)  observing  a  similar  phenomenon  in  twro  species  of  Uca  also  suggested 
that  Mg++  and  Na+  compete  for  transport  and  that  active  movement  of  Na+  is 
reduced  by  such  competition  under  the  high  Mg++  load  found  in  hypersaline  water. 
These  authors  as  well  as  Riegel  and  Lockwood  (1961),  who  observed  the  phenome- 
non in  Carcinus,  considered  but  rejected  direct  Mg++-Na+  exchange  as  the 
mechanism  of  concentrating  Mg+*  at  the  expense  of  Na+.  Whatever  the  mechanism, 
the  phenomenon  seems  to  be  common  in  crabs  (Gross,  1964;  Gross  et  al.,  1966). 

Gross  (1964),  examining  a  series  of  crabs  from  aquatic,  amphibious  and 
terrestrial  modes  of  life,  revealed  that  animals  showing  high  degrees  of  terrestrial- 
ness  tended  to  concentrate  Mg++  more  highly  in  the  urine  than  the  more  aquatic 
crabs.  An  exception  was  the  terrestrial  Gecarcinus  lateralis  which  is  the  only 
brachyuran  crab  examined  to  date  incapable  of  concentrating  urine  Mg++  at  the 
expense  of  Na+.  Still,  it  was  shown  that  high  urine  Mg++  does  not  necessarily  re- 
flect strong  Mg++  regulation  in  the  blood.  For  example,  the  urine  Mg++  of  the 
amphibious  Uca  was  more  than  three-fold  that  of  the  aquatic  Cancer,  yet  the  blood 
Mg++  concentrations  of  these  two  species  were  about  the  same. 

Gross  and  Marshall  (1960)  demonstrated  that  the  concentration  of  Mg++  in  the 
urine  of  Pachygrapsus  is  independent  of  the  Mg++  influx  and  in  some  way  a  function 
of  the  osmotic  concentration  of  the  external  medium.  This  phenomenon  also  was 
demonstrated  in  Cardisoma  carnije.r,  Varuna  litterata  and  Sesarma  mcinerti  (Gross 
ctal,  1966). 

The  above  described  phenomena  lead  to  the  following  questions :  ( 1 )  What  is 
the  relationship  between  Mg++  concentration  in  the  urine  of  a  crab  and  the  amount 
of  Mg++  it  excretes?  (2)  By  what  means  does  a  crab  immersed  in  a  Mg++-free 
medium  of  high  salinity  concentrate  Mg++  in  its  urine?  (3)  By  what  means  does 
the  urine  Na+  concentration  become  reduced  as  the  urine  Mg++  concentration 
elevates  when  the  animal  is  transferred  from  dilute  to  concentrated  sea  water  ? 

272 


CRAB  BLADDER  FUNCTIONS  273 

The  present  investigation  produces  evidence  that  Mg++  concentration  in  the 
urine  depends  on  the  relative  length  of  time  the  latter  is  held  in  the  bladder.  Mg++ 
is  transported  across  the  walls  of  the  bladder  into  the  urine  at  different  rates  depend- 
ing on  the  blood  Mg++  concentration  and  a  direct  exchange  with  Na+  can  take  place 
which  effects  movement  of  water  between  blood  and  urine. 

MATERIALS  AND  METHODS 

The  shore  crab,  Pachygrapsus  crassipes  Randall,  which  is  a  known  hypo-  and 
hyperosmotic  regulator  (Jones,  1941;  Prosser  et  al.,  1955;  Gross,  1957)  was 
collected  at  Laguna,  California,  and  maintained  in  the  laboratory  at  15°  C.  in  100% 
artificial  sea  water  made  from  the  Utility  Chemical  Company  Seven-Seas  Marine 
Mix.  Only  intermolt  crabs  larger  than  15  grams  were  used  in  the  experiments. 
A  salinity  of  34.3%0  was  considered  to  be  100%  sea  water.  This  contained  the 
following  cation  concentrations :  Na+,  455  mM/1. ;  K+,  11.5  mM/1. ;  Ca++,  14.2  mM/1. 
and  Mg++,  55.5  mM/1.  Different  concentrations  of  sea  water  were  attained  by 
varying  the  amounts  of  water  added  to  these  salts.  MgCU  was  added  to  test  media 
where  the  Mg*4"  concentration  was  to  be  higher  than  normal.  Also,  artificial  sea 
water  for  experiments  concerned  with  Mg++  depletion  was  made  up  using  the 
proportions  of  Na+,  K+,  Ca++,  Mg++,  Cl~  and  SO4=  given  in  the  tables  of  Barnes 
(1954)  with  the  pH  adjusted  to  8.0.  Na+  was  substituted  for  Mg++  when  the  latter 
was  deleted. 

Perfusion  fluid  used  to  simulate  plasma  and/or  primary  urine  contained  the 
following  concentrations  of  ions:  Na+,  483  mM/1. ;  K+.  7  mM/1. ;  Mg++,  10  mM/1. ; 
Ca++,  15  mM/1.,  Cl~,  520  mM/1.  and  SO/  10  mM/1.  This  approximates  the  blood 
cation  and  osmotic  concentration  of  Pachygrapsus  when  immersed  in  normal  sea 
water  (Gross,  1959;  1964).  The  Cl~  concentration  approximates  the  mean  blood 
concentration  (517  mM/1.,  S.D.,  11.3)  of  6  crabs  taken  from  100%  sea  water. 
Concentrations  of  SO4=  were  estimated  by  difference  assuming  Cl~  and  SO4=  as  the 
only  anions  and  considering  electro-chemical  balance.  Hereinafter  isosmotic 
perfusion  fluid  will  mean  a  solution  made  up  of  the  above  proportions  but  adjusted 
by  water  content  to  be  approximately  isosmotic  with  the  blood  for  crabs  immersed 
in  a  particular  salinity.  Blood  osmotic  concentrations  for  crabs  immersed  in 
different  salinities  are  given  by  Gross  (1957 ;  1964). 

Immersion  experiments  were  conducted  using  approximately  400  ml.  of  medium 
which  was  sufficient  to  assure  complete  immersion. 

Osmotic  concentrations  of  media  and  body  fluids  were  determined  by  means  of 
a  Mechrolab,  vapor  pressure  osmometer.  Na+  and  K+  were  determined  by  flame 
photometry;  Ca++  and  Mg++  by  ethylene  diamine  tetra  acetic  acid  (EDTA)  titra- 
tion  as  previously  described  (Gross,  1959;  Gross  et  al.,  1966)  ;  Cl~  by  the  method 
of  Schales  and  Schales  (1941)  ;  inulin  was  determined  by  the  resorcinol  method 
of  Schreiner  (1950). 

In  the  range  of  normal  sea  water  osmotic  concentrations  could  be  measured 
within  a  \%  error,  Na+,  about  2%,  K+,  less  than  10%,  Ca++  and  Mg++  less  than 
6%  and  microsamples  of  Cl~  to  less  than  10%.  Inulin  could  be  measured  with 
less  than  a  7%  range  of  error. 

Blood  was  extracted  by  puncturing  the  arthrodial  membranes  at  the  bases  of  the 
walking  legs  with  a  glass  pipette.  Urine  was  removed  from  the  nephropore  by 


274 


WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 


means  of  a  fine  glass  cannula.     Since  urine  is  clear  and  blood  turbid,  any  contamina- 
tion of  urine  with  blood  could  easily  be  detected.     Doubtful  samples  were  discarded. 

RESULTS 

It  has  been  shown  that  when  Pachygrapsus  is  immersed  in  100%  and  150%  sea 
water  the  urine  Mg++  concentrations  averaged  118  mM/1.  and  204  mM/1.,  respec- 
tively, the  corresponding  urine  concentration/blood  concentration  (U/B)  values 
for  Mg++  being  13.6  and  15.4  (Gross,  1959).  The  following  experiment  therefore 
was  performed  to  show  the  role  of  water  withdrawal  in  achieving  the  above  urine 
Mg++  concentrations  and  U/B  values.  The  bladders  of  crabs  which  had  been 
immersed  in  100%  or  158%  sea  water  were  drained,  and  the  animals  were  injected 
with  about  0.1  ml.  of  an  isosmotic  perfusion  of  fluid  containing  approximately  6% 
inulin.  The  crabs  then  were  reimmersed  in  the  media  from  which  they  were  taken, 
and  after  6  or  48  hours  the  urine  and  blood  were  sampled  for  inulin  analysis. 
Another  group  taken  from  100%  sea  water  was  also  thus  treated  but  was  kept  out 
of  water  rather  than  reimmersed.  Thus,  it  can  be  seen  that  the  U/B  values 
(Table  I)  were  so  low  that  water  withdrawal  cannot  be  a  major  factor  in  effecting 

TABLE  I 

Inulin  U/B  values  of  Pachygrapsus 


6  hours  exposure 

48  hours  exposure 

No. 

Mean 

S.D. 

No. 

Mean 

S.D. 

100%  sea  water 

13 

1.11 

0.13 

10 

1.92 

1.29 

158%  sea  water 

10 

1.16 

0.24 

13 

1.52 

0.65 

Air 

7 

1.09 

0.17 

5 

1.44 

0.12 

high  Mg++  concentrations  or  U/B  values.  Of  the  mean  U/B  values  presented  in 
Table  I  only  those  for  crabs  immersed  in  158%  sea  water  or  kept  in  air  for  48 
hours  are  significantly  different  from  unity  (P  <  0.02).  The  means  for  the  three 
6-hour  experiments  are  not  significantly  different.  The  means  for  the  three  48- 
hour  experiments  are  not  significantly  different,  and  there  is  no  significant  differ- 
ence between  6-  and  48-hour  treatments  for  either  salinity.  The  difference  in 
urine  Mg++  concentration  in  animals  immersed  in  normal  sea  water  compared  to 
those  immersed  in  hypersaline  water  therefore  cannot  be  achieved  by  differences  in 
water  withdrawal  from  the  urine.  Further  reference  will  be  made  to  data  in 
Table  I  later. 

After  24  hours  immersion  in  158%  Mg++-free  sea  water,  the  urine  Mg++  of 
15  crabs  averaged  235  mM/1.  (S.D.,  117)  whereas  the  mean  urine  Mg++  of 
18  crabs  immersed  for  24  hours  in  50%  sea  water  containing  65  mM/1.  of  Mg++ 
was  only  20.5  mM/1.  (S.D.,  8.90).  These  data,  which  confirm  the  observations 
of  Gross  and  Marshall  (I960),  clearly  show  that  the  ability  of  Pachygrapsus  to 
concentrate  Mg++  in  the  urine  is  neither  a  function  of  Mg++  influx  nor  the  concen- 
tration of  Mg++  in  the  medium. 


CRAB  BLADDER  FUNCTIONS 


275 


Figure  1  illustrates  the  frequency  distribution  for  the  urine  Mg++  concentrations 
of  51  crabs  sampled  in  the  field  where  only  normal  sea  water  was  available. 
Figure  2  shows  urine  Mg++  +  Ca++  concentrations  of  crabs  totally  immersed  in  a 
running  sea  water  aquarium  containing  100%  sea  water.  Small  quantities  of  urine 
(-—20  /u.1.)  \vere  periodically  sampled  from  the  same  nephropore  of  individual  crabs, 
10  ftl  of  which  were  analyzed  for  Mg++  +  Ca++.  Ca++  was  not  determined  because 
of  the  small  sample  size.  There  it  can  be  seen  that  the  concentration  of  Mg++  +  Ca++ 
varies  tremendously  with  time,  and  since  urine  Ca++  is  relatively  constant  in  concen- 
tration (approximately  20  mM/1.)  with  little  variance  (Gross,  1959),  the  large 
fluctuations  in  Figure  2  can  be  attributed  to  Mg++.  This  might  suggest  that  the 


15  n 


•5    10- 


I     5 


Meon=  98.0  mM/l  (S.D.  =  42.8) 


26-50       51-75 


76-100    101-125    126-150    151-175 
Urine  Mg  (mM/l) 


251-275 


FIGURE  1.     Frequency  distribution  for  urine  Mg++  concentrations  of  crabs  sampled  in  the  field 

where  only  100%  sea  water  was  available. 

Mg++-transporting  mechanism  fluctuates  in  its  rate  of  activity.  However,  another 
possibility  is  that  the  urinary  bladder  itself  has  a  transporting  function  with  respect 
to  Mg++.  That  is,  urine  entering  the  bladder  from  the  labyrinth  is  relatively  low 
in  Mg++.  If  the  urine  were  held  in  the  bladder  for  a  prolonged  period,  sufficient 
time  would  permit  elevation  of  the  Mg++  concentration.  Following  bladder  evacua- 
tion, then,  the  urine  Mg++  should  be  low.  When  a  hypo-regulating  crab  is  immersed 
in  hypersaline  media,  the  water  influx  would  be  slow,  the  bladder  would  be  evacuated 
with  low  frequency  and  urine  would  be  held  in  the  bladder  sufficiently  long  to 
permit  accumulation  of  Mg++.  On  the  other  hand,  in  low  salinities,  water  influx 
would  be  rapid  in  a  hyper-regulating  crab,  evacuation  of  the  bladder  would  be 


276 


WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 


frequent  and  no  time  permitted  for  Mg++  accumulation.  Such  a  model  would 
explain  the  high  concentration  of  urine  Mg++  for  crabs  immersed  in  158%  Mg++-free 
sea  water  and  low  Mg++  concentration  in  urine  of  crabs  immersed  in  50%  sea  water 
containing  high  Mg++.  This  would  also  explain  the  fluctuations  in  urine  Mg*+ 
shown  in  Figure  2.  That  is,  low  Mg++  concentrations  would  follow  bladder  evacua- 
tion and  high  Mg++  concentrations  would  precede  evacuation. 


250 1 


225- 


200- 


175- 


o 

u 

+ 

o> 


0) 


125- 


100- 


75- 


50- 


'A 


0 


234 
Time    in    Days 


FIGURE  2.     Fluctuations  in  the  concentration  of  urine  Mg++  +  Ca++  of  individual  crabs  immersed 
in  100%  sea  water.     Each  symbol  connected  by  line  represents  history  of  individual  crab. 

In  order  to  test  this  model,  one  of  the  paired  bladders  of  a  crab  immersed  in 
100%  sea  water  was  evacuated ;  the  crab  was  then  reimmersed  in  100%  sea  water 
and  after  a  given  period,  urine  from  the  same  bladder  was  sampled  for  Mg++ 
analysis.  Thus,  for  17  crabs  reimmersed  2-24  hours,  the  mean  Mg++  concentration 
was  69  mM/l.  (S.D.,  55)  and  for  16  crabs  reimmersed  48-96  hours,  the  mean 
Mg++  concentration  was  165  mM/l.  (S.D.,  99),  the  two  means  being  significantly 


CRAB  BLADDER  FUNCTIONS 


277 


500-1 


450- 


400- 


350- 


300- 


250- 


200- 


150- 


100- 


50- 


0-1 


SODIUM 


LJ 


10 


12 


7i   8 


10 


15 


MAGNESIUM 


6  18  42  -  48  92  -  96 

Time    in    Hours 

FIGURE  3.  Decreases  in  urine  Na+  concentration  accompanying  increases  in  urine  Mg++ 
concentration  in  crabs  immersed  in  100%  sea  water  as  a  function  of  time  after  bladder 
evacuation.  Mean  is  represented  by  horizontal  line ;  range  by  vertical  line  and  twice  the 
standard  error  on  either  side  of  the  mean  by  the  rectangle.  Numerals  indicate  number  of  cases. 


278  WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 

different  (P<0.01).  If  it  is  assumed  (in  the  above  experiment)  that  crabs 
immersed  for  the  longer  periods  also  retain  (on  the  average)  urine  in  the  bladder 
for  longer  periods,  these  data  suggest  that  urine  first  entering  the  bladder  is  rela- 
tively low  in  Mg++  concentration,  and  as  it  is  held  in  the  bladder  Mg++  is  added  to  it. 

Figure  3  illustrates  an  experiment  which  lends  support  to  this  suggestion.  The 
bladder  of  a  crab  that  had  been  immersed  in  100%  sea  water  was  drained  and 
flushed  with  perfusion  fluid  in  order  to  remove  high  concentrations  of  residual  Mg++. 
After  flushing  the  bladder,  the  fluid  was  removed  and  the  crab  with  the  empty 
1  iladder  was  reimmersed  in  100%  sea  water.  Following  a  given  period  of  immer- 
sion, the  urine  was  removed  from  the  same  bladder  and  analyzed  for  Mg++  and  Na+. 
As  can  be  seen  in  Figure  3,  6  hours  after  reimmersion,  the  mean  urine  Mg++  con- 
centration was  low  and  the  mean  urine  Na*  concentration  was  high.  As  the 
immersion  period  increased  and  presumably  the  average  period  of  urine  retention 
increased,  the  mean  urine  concentration  of  Mg++  increased  and  the  mean  urine  con- 
centration of  Na+  decreased.  For  both  urine  Na+  and  Mg++,  the  92-96-hour  group 
(mean)  was  greatly  different  from  the  6-hour  group  (P  <  0.001).  It  should  be 
pointed  out  that  urine  samples  taken  6  hours  following  bladder  evacuation  are  also 
isosmotic  with  the  blood.  Thus,  15  crabs,  which  included  5  immersed  in  100% 
sea  water,  5  immersed  in  158%  sea  water  and  5  kept  out  of  the  water,  had  an 
osmotic  U/B  mean  of  1.007  (S.D.,  0.0176). 

The  possibility  was  considered  that  the  empty  bladder  encouraged  a  rapid  surge 
of  fluid  through  renal  organ  and  that  insufficient  time  was  allowed  for  Mg++  to 
concentrate  in  the  urine  before  entering  the  bladder.  As  the  bladder  filled,  the 
flow  of  urine  through  the  labyrinth,  for  example,  would  be  retarded  and  the  subse- 
quent urine  entering  the  bladder  would  be  relatively  high  in  Mg++.  An  experiment 
therefore  was  conducted  showing  that  increases  in  Mg++  occur  in  the  urine  with  time 
when  the  bladder  is  full. 

Gross  and  Marshall  (1960)  gave  evidence  that  Pachygrapsus  does  not  lose  urine 
when  kept  out  of  the  water.  The  following  preliminary  experiment  was  conducted 
to  demonstrate  that  fluid  introduced  into  the  bladder  after  artificial  evacuation  will 
be  held  in  the  bladder  while  the  animal  is  kept  out  of  the  water.  Urine  from  one 
bladder  of  the  crab  was  emptied,  flushed  with  an  isosmotic  solution  colored  with 
indigo  carmine,  emptied  again  and  refilled.  If  there  was  no  immediate  sign  of 
leakage  due  to  injury  of  the  nephropore,  the  dried  animal  was  placed  in  a  dry 
plastic  container,  the  floor  of  which  was  covered  with  several  layers  of  white 
absorbent  tissue  paper.  In  such  a  situation  any  loss  of  "urine"  would  stain  the 
white  paper.  Of  20  animals  thus  tested  using  the  following  isosmotic  solutions : 
(a)  perfusion  fluid  for  24  hours  (10  crabs)  ;  (b)  NaCl  for  3  hours  (7  crabs)  and 
(c)  MgClo  for  3  hours  (3  crabs)  only  one  (NaCl)  lost  "urine"  but  this  still  had 
dye  in  the  "urine"  remaining  in  the  bladder,  indicating  that  only  part  of  the 
introduced  fluid  leaked  out.  All  other  crabs  retained  sufficient  color  in  the  bladder 
fluid  until  the  end  of  the  experiment  to  have  stained  the  white  paper  had  fluid  been 
lost.  Still,  after  24  hours  the  bladder  fluid  had  lost  considerable  color,  indicating 
absorption  of  the  dye.  Thus,  such  an  experiment  would  be  of  little  value  if  con- 
tinued for  more  than  one  day.  Nevertheless,  the  probability  is  high  that  isosmotic 
fluids  introduced  into  an  empty  bladder  will  remain  there  for  at  least  24  hours  if  the 
crab  is  kept  out  of  the  water.  It  should  also  be  noted  that  when  dye  is  introduced 


CRAB  BLADDER  FUNCTIONS  279 

into  one  of  the  paired  bladders,  it  does  not  appear  in  the  other  side,  indicating  that 
the  bladders  are  isolated  from  each  other. 

Next,  bladders  of  crabs  removed  from  100%  sea  water  were  evacuated,  rinsed 
and  filled  with  the  above-described  perfusion  fluid  containing  10  mM/1.  of  Mg++. 
The  animals  were  placed  in  dry  containers  and  after  selected  periods  the  bladder 
fluid  was  sampled  and  analyzed  for  Mg++.  The  bladder  fluid  of  10  animals  so 
treated  averaged  33.2  mM/1.  (S.D.,  11.0)  for  Mg+*  1-3  hours  after  introduction  of 
the  fluid,  whereas  the  bladder  fluid  of  8  crabs  averaged  64.5  mM/1.  (S.D.,  27.5) 
after  28-48  hours.  These  two  groups  are  significantly  different  (P  <  0.01 )  and 
only  part  of  this  difference  could  be  caused  by  water  withdrawal  (Table  I).  Thus, 
urine  Mg++  concentrates  with  time  in  a  full  bladder.  This  is  interpreted  to  mean 
that  the  walls  of  the  bladder  transport  Mg*+  into  the  urine  and  prolonged  retention 
of  urine  in  the  bladder  results  in  the  attainment  of  high  Mg++  concentrations  in 
the  urine. 

Evidence  has  been  produced  that  Pachygrapsus  does  not  lose  urine  when  out 
of  the  water.  On  the  other  hand,  when  the  bladder  is  emptied,  it  will  readily  fill 
even  though  the  crab  is  not  immersed.  Substantial  urine  samples  can  be  extracted 
from  the  bladders  of  most  "dry"  crabs  6  hours  after  bladder  evacuation.  Twenty- 
four  hours  after  emptying,  the  bladders  of  crabs  kept  in  dry  situations  seem  as  full 
as  those  of  immersed  crabs. 

Figure  4  illustrates  how  crabs  placed  in  dry  containers  with  empty  bladders 
(previously  rinsed  with  isosmotic  perfusion  fluid)  concentrate  Mg++  in  the  urine  with 
time  at  the  expense  of  Na+.  As  shown  for  the  immersion  experiments,  urine  Mg++ 
increases  with  time  after  bladder  evacuation,  but  Na+  decreases  with  time. 

Now  if  the  period  of  time  urine  is  held  in  the  bladder  dictates  the  concentration 
of  urine  Mg++,  then  blocking  the  nephropore  to  prevent  urine  release  should  result 
in  an  increase  in  the  Mg++  concentration  of  the  urine.  Thus,  one  of  the  paired 
nephropores  of  Pachygrapsus  was  blocked  with  epoxy  cement  and  after  the  animal 
was  immersed  in  50%  sea  water  for  24  hours,  urine  from  both  blocked  and  un- 
blocked sides  was  extracted  and  analyzed  for  Mg++.  In  every  case  (12)  urine  from 
the  blocked  bladder  was  higher  in  Mg++  than  urine  from  the  unblocked  bladder,  the 
mean  ratio,  blocked/unblocked  being  2.63  (S.D.,  1.19)  which  is  significantly 
different  from  unity  (P  <  0.001). 

Four  lines  of  evidence  have  been  presented  indicating  that  the  bladder  of 
Pachygrapsus  transports  Mg++  from  the  blood  into  the  urine,  thus  increasing  the 
concentration  of  Mg++  in  the  urine  with  time  as  it  is  retained  in  the  bladder  far 
beyond  that  which  could  be  caused  by  water  withdrawal  (Table  I)  :  (1)  Crabs 
immersed  with  empty  bladders  show  increased  urine  Mg++  concentrations  with  time  ; 
(2)  when  perfusion  fluid  is  substituted  for  urine  in  the  bladder,  the  Mg++  concentra- 
tion of  the  bladder  fluid  increases  with  the  period  the  crabs  are  kept  out  of  the 
water ;  ( 3 )  when  crabs  with  emptied  bladders  are  kept  out  of  the  water,  fluid  low 
in  Mg++  fills  the  bladder,  but  with  time  the  concentration  of  urine  Mg++  increases ; 
(4)  when  urine  from  blocked  and  unblocked  bladders  of  the  same  immersed  crab  are 
compared,  urine  from  the  blocked  side  is  higher  in  Mg++  than  urine  from  the 
unblocked  side. 

Since  the  phenomenon  illustrated  in  Figures  3  and  4  suggests  a  direct  Mg+*- 
Na*  exchange,  isosmotic  solutions  of  NaCl  or  MgCl,  were  substituted  for  urine  in 


280 


WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 


bladders  of  crabs  kept  out  of  water.  One  bladder  of  each  crab  first  was  evacuated 
of  urine,  rinsed  twice  with  isosmotic  test  solution  and  then  filled  with  a  volume  of 
test  solution  which  approximated  the  volume  of  urine  removed.  After  the  crab  was 
kept  for  a  given  period  in  air,  the  test  solution  was  removed  from  the  bladder  and 
analyzed  for  Na+  and  Mg++.  In  this  way  Na+  and  Mg++  concentration  changes  could 


500 1 


450- 


400- 


350- 


300- 


250 


200 


150 


100- 


50 


A 
A 

1 


A 

A 

A 


SODIUM 


A 
A 


MAGNESIUM 


12 


24 


36 


48 


60 


Time    in    Hours 


FIGURE  4.  Decreases  in  urine  Na+  concentration  accompanying  increases  in  urine  Mg++ 
concentration  in  crabs  kept  out  of  the  water  as  a  function  of  time  after  bladder  evacuation. 
Triangles  represent  urine  Na+ ;  circles  urine  Mg++.  Each  point  represents  a  single  determination. 


CRAB  BLADDER  FUNCTIONS 


281 


be  measured  in  the  bladder  fluid  and  assuming  constancy  of  bladder  fluid  volume, 
this  information  could  give  the  relative  number  of  Na+  ions  exchanged  for  Mg++  ions. 
Table  II  includes  all  cases  of  this  experiment  where  there  was  no  immediate  indica- 
tion of  leakage  from  the  nephropore  due  to  injury  and  where  there  was  sufficient 
concentration  change  of  both  ions  to  be  measured  quantitatively.  Thus,  it  can  be 
seen  for  both  NaCl  and  MgCl2  that  whenever  there  was  a  gain  in  Mg++  concentration 
in  the  bladder  fluid  there  was  a  loss  in  Na+  concentration  and  vice  versa.  The 


TABLE  II 

Na+-Mg++  exchange  through  bladder  wall  of  Pachygrapsus 


Bladder  solution 

Spec.  no. 

Na+  change 
(mM/l.) 

Mg++  change 
(mM/l.) 

Na+  change 

Time 
hrs. 

Mg++  change 

560  mM/l. 



+ 

NaCl 

1 

116 

82 

1.42 

1.0 

2 

80 

44 

1.82 

1.5 

3 

100 

55 

1.82 

2.0 

4 

126 

87 

1.45 

3.0 

5 

80 

31 

2.58 

3.0 

6 

47 

24 

1.96 

3.0 

7 

64 

36 

1.78 

3.0 

8 

56 

31 

1.81 

3.0 

9 

81 

69 

1.17 

18.0 

10 

79 

48 

1.64 

18.0 

11 

96 

72 

1.33 

18.0 

12 

78 

69 

1.13 

18.0 

13 

48 

34 

1.41 

18.0 

14 

130 

74 

1.76 

19.0 

360  mM/l. 

+ 

— 

MgCl2 

15 

52 

23 

2.26 

1.0 

16 

265 

218 

1.22 

1.0 

17 

235 

154 

1.53 

1.0 

18 

218 

127 

1.72 

1.0 

19 

420 

264 

1.59 

1.0 

20 

362 

233 

1.55 

1.0 

21 

60 

35 

1.71 

1.5 

22 

322 

221 

1.46 

2.0 

23 

241 

155 

1.55 

19.0 

24 

358 

219 

1.63 

19.0 

Mean 
S.D. 


1.64 
0.33 


mean  ratio,  Na+  concentration  change/Mg++  concentration  change,  was  1.64.  Now, 
assuming  no  net  anion  movements,  for  every  divalent  Mg++  ion  transported,  two 
monovalent  Na+  ions  should  be  exchanged.  Chloride  constitutes  most  of  the  urine 
anions  because  the  urine  for  ten  crabs  removed  from  normal  sea  water  had  a  mean 
osmotic  concentration  of  1040  mOsm/1.  (S.D.,  12.6)  and  a  mean  urine  chloride  of 
516  mM/l  (S.D.,  27.2).  Green  et  al.  (1959)  stated  that  if  Na+-Mg++  exchange 
occurred,  the  Na+  change/Mg++  change  should  be  2.  However,  the  loss  of  two  Na+ 


282  WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 

ions  for  every  Mg++  ion  gained  would  reduce  the  osmotic  concentration  of  the 
bladder  fluid.  Yet  isosmotic  NaCl  solution  introduced  into  empty  bladders  of  8 
crabs  remained  essentially  isosmotic  for  three  hours  when  the  animals  were  kept 
out  of  the  water,  the  mean  osmotic  urine/blood  value  being  1.01  (S.D.,  0.018). 
Since  the  Na+-Mg++  exchange  would  reduce  the  urine  osmotic  concentration,  water 
must  move  to  effect  the  isosmotic  condition  between  blood  and  urine.  Therefore, 
the  observed  Na+  change/Mg++  change  should  be  related  to  the  isotonic  coefficients 
for  NaCl  and  MgCU  which  were  empirically  determined  to  be  1.8  and  2.8,  respec- 
tively, at  the  initial  test  concentration  (Table  II).  Therefore,  the  Mg++  concen- 
tration change  X  2.8  =  Na+  concentration  change  X  1.8,  or  Na+  concentration 
change/Mg++  concentration  change  =  2.8/1.8  =  1.56,  a  value  which  closely  approxi- 
mates the  observed  value,  1.64  (Table  II).  This  close  agreement  is  interpreted  as 
evidence  that  a  direct  Na+-Mg++  exchange  can  indeed  take  place.  It  also  seems 
that  such  an  exchange  can  take  place  in  either  direction  across  the  membranes  of 
the  bladder.  This  in  turn  suggests  relative  impermeability  of  those  membranes 
to  chloride. 

However,  when  isosmotic  perfusion  fluid  was  used  instead  of  NaCl  or  MgCL, 
the  mean  Na+  concentration  change/Mg++  concentration  change  for  6  crabs  after 
24  hours  was  only  1.15  (S.D.,  0.14)  which  is  significantly  less  than  the  mean  1.64 
given  in  Table  II  (P  <  0.001).  The  longer  test  period  for  perfusion  fluid  was 
necessary  to  permit  a  measurable  cation  change.  A  possible  reason  for  these  con- 
flicting results  will  be  given  below. 

The  question  now  may  be  raised  as  to  the  dependence  of  Mg++  transport  on  Na+ 
active  transport.  An  attempt  therefore  was  made  to  block  active  transport  of  Na* 
from  the  lumen  of  the  bladder  into  the  hemocoele  by  ouabain  which  is  known  to 
inhibit  Na+  transport  (Judah  and  Ahmed,  1964).  One  bladder  of  the  crab  was 
drained  of  urine,  rinsed  with  isosmotic  perfusion  fluid  containing  10  mM/1.  Mg++ 
and  refilled  with  the  same  perfusion  fluid  containing  5  X  10"*  or  10~3  M  ouabain. 
The  crab  then  was  placed  in  a  dry  container  for  24  hours  after  which  time  the 
bladder  was  drained  again  and  urine  analyzed  for  Mg++.  The  mean  urine  Mg++ 
concentration  for  14  crabs  thus  treated  was  120  mM/1.  (S.D.,  88.5).  Even 
though  low  activity  of  the  crabs  indicated  that  the  ouabain  had  diffused  into  the 
blood  and  was  present  on  both  sides  of  the  bladder  membrane,  it  obviously  did  not 
prevent  accumulation  of  Mg++  in  the  bladder  fluid. 

The  mean  urine  Na+  concentration  of  13  crabs  after  this  treatment  was  460 
mM/1.  (S.D.,  27.4)  which  was  not  significantly  different  from  the  initial  Na+  con- 
centration (483  mM/1.).  However,  the  highest  urine  Mg++  concentrations  were 
accompanied  by  the  lowest  Na+  concentrations,  so  it  is  believed  that  either  Na+ 
movement,  in  this  case,  is  a  passive  process  or  ouabain  was  ineffective  in  blocking 
the  Na+  transport  mechanism  in  all  cases.  Nevertheless,  there  is  no  evidence  that 
Mg++  secretion  is  coupled  to  the  Na+  transport  mechanism,  but  there  is  further 
evidence  that  Mg++  accumulates  in  a  full  bladder  with  time.  It  might  be  that  the 
Mg++  ion  can  exchange  for  any  other  cation,  but  since  Na+  is  the  dominant  one,  loss 
of  Ca*+  or  K+  from  the  urine  in  exchange  for  Mg++  could  not  be  detected  by  the 
methods  used  in  this  investigation.  Exploratory  experiments  where  the  bladder 
was  filled  with  a  perfusion  fluid  in  which  choline  was  substituted  for  Na+  showed 
that  Mg++  was  concentrated  in  the  bladder  fluid  after  24  hours.  However,  Na+  was 


CRAB  BLADDER  FUNCTIONS  283 

also  high  in  the  bladder  fluid  and  had  obviously  diffused  from  the  blood  down  the 
steep  gradient.  Thus,  it  was  not  determined  whether  or  not  Mg++  was  exchanged 
for  choline. 

In  view  of  the  above  findings,  there  can  be  little  doubt  that  Mg++  is  concentrated 
in  the  urine  by  the  bladder  and  that  the  Mg++  concentration  is  a  function  of  the  time 
urine  is  retained  in  the  bladder.  Ho\vever,  evidence  was  produced  in  the  following 
experiments  that  the  rate  of  Mg++  transport  into  the  bladder  is  higher  when  the 
crab  is  in  hypersaline  water  than  when  in  normal  sea  water.  That  is,  the  amount 
of  Mg++  entering  the  bladder  6  hours  following  evacuation  is  greater  in  crabs  im- 
mersed in  hypersaline  water  than  those  immersed  in  normal  sea  water. 

In  Group  One  (10  animals),  the  bladder  of  the  crab  was  drained  and  flushed 
with  isosmotic  perfusion  fluid,  then  drained  again.  The  crab  was  reimmersed  in 
100%  sea  water  and  after  6  hours,  urine  from  the  same  bladder  was  sampled  for 
Mg++  analysis. 

In  Group  Two  (17  animals),  the  crab  was  first  immersed  in  158%  sea  water 
for  18-24  hours ;  the  bladder  was  drained,  rinsed  with  isosmotic  perfusion  fluid 
containing  about  15  mM/1.  Mg++  and  reimmersed  in  158%  sea  water  for  an  addi- 
tional 6  hours.  After  this  period  the  urine  was  completely  drained  from  the  same 
bladder  for  Mg++  analysis. 

In  Group  Three  (20  animals),  the  crab  was  first  immersed  in  158%  sea  water 
for  18  hours  ;  the  bladder  was  drained,  rinsed  with  isosmotic  perfusion  fluid  contain- 
ing about  15  mM/1.  Mg++  and  reimmersed  in  158%  Mg++-free  sea  water  for  an 
additional  6  hours.  The  urine  was  then  extracted  for  Mg'1"1"  analysis. 

Thus,  the  6-hour  urine  sample  for  crabs  immersed  in  100%  sea  water  (Group 
One)  averaged  54.5  mM/1.  (S.D.,  20.1),  whereas  for  crabs  immersed  in  158%  sea 
water  (Group  Two)  the  mean  urine  Mg++  was  113  mM/1.  (S.D.,  70.0).  A  second 
6-hour  sample  was  taken  from  Group  Two  (i.e.,  12  hours  after  rinsing  of  the 
bladder  and  reimmersion  in  158%  sea  water)  and  the  mean  urine  Mg++  then  was 
100  mM/l.  (S.D.,  48.5),  indicating  that  the  difference  between  6-hour  urine  Mg++ 
in  100%  and  158%  sea  water  treatments  is  not  merely  a  matter  of  residual  Mg++  in 
the  bladder  of  crabs  immersed  in  158%  sea  water  for  18  hours.  The  mean  urine 
Mg++  for  Group  Three  which  had  been  immersed  for  6  hours  in  158%  Mg++-free 
sea  water  was  120  mM/1.  (S.D.,  63.0) .  This  mean  as  well  as  those  for  Group  Two 
are  significantly  larger  than  the  mean  for  Group  One  (P  <  0.01). 

Inulin  U/B  values  (Table  I)  indicate  that  water  withdrawal  from  urine  is 
no  greater  for  crabs  immersed  in  158%  sea  water  than  for  those  immersed  in  100% 
sea  water.  Therefore,  the  different  urine  Mg++  concentrations  produced  during  the 
6-hour  period  by  crabs  in  the  two  salinities  cannot  be  explained  on  the  basis  of  water 
withdrawal. 

Inasmuch  as  the  6-hour  urine  sample  for  crabs  immersed  in  158%  Mg++-free 
sea  water  (Group  Three)  was  equally  as  high  in  Mg++  as  that  of  crabs  immersed  in 
158%  sea  water  containing  high  Mg++,  there  is  evidence  that  Mg++  transport  from 
blood  to  urine  is  independent  of  the  Mg++  concentration  in  the  external  medium  and 
in  turn  independent  of  the  Mg++  influx  from  the  external  medium  to  crab. 

The  higher  concentrations  of  urine  Mg++  observed  above  in  crabs  immersed  for 
6  hours  in  hypersalinities  over  those  immersed  for  6  hours  in  100%  sea  water 
may  indicate  that :  ( 1 )  the  rate  of  Mg++  transport  from  blood  into  urine  is  higher 


284 


WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 


when  the  crab  is  immersed  in  hypersaline  water  than  when  it  is  immersed  in  normal 
sea  water;  (2)  the  rate  of  Mg++  transport  is  constant,  but  the  volume  of  primary 
urine  formed  is  smaller  after  6  hours  in  158%  sea  water  than  after  6  hours  in  100% 
sea  water,  thus  effecting  a  higher  concentration  of  Mg++  in  the  urine  while  accumu- 
lating the  same  amout  of  Mg++,  and  (3)  there  is  reduced  primary  urine  accompanied 
by  increased  Mg++  transport  for  crabs  immersed  in  hypersalinities  compared  to  those 
in  normal  salinity.  If  primary  urine  were  formed  by  nitration,  its  rate  of  forma- 
tion would  be  expected  to  be  slower  when  the  crab  was  in  hypersalinities  than 
when  in  normal  salinity.  Lockwood  (1962)  discusses  the  possibility  of  renal 
filtration  among  crustaceans  in  general.  Kirschner  and  Wagner  (1965)  produce 
evidence  of  filtration  in  a  fresh-water  crayfish.  To  date,  no  reliable  values  have 
been  obtained  on  the  rate  of  primary  urine  production  in  Pachygrapsus  for  any 
treatment.  However,  evidence  will  be  produced  below  that  there  is  actually  a 


TABLE  III 

Elements  influencing  the  concentration  of  Mg++  in  urine 


Group  A 

Group  B 

Group  C 

50%  sea  water  with 
65  mAf/1.  of  Mg++ 
for  18  hours 
to 
100%  sea  water 
(6  hours) 

158%  sea  water  with 
59  mAf/1.  of  Mg++ 
for  18  hours 
to 
100%  sea  water 
(6  hours) 

158%  sea  water  with 
82  mAf/1.  of  Mg++ 
for  24  hours 
to 
158%  Mg++-free  sea  water 
(6  hours) 

No. 

Mean 

S.D. 

No. 

Mean 

S.D. 

No. 

Mean 

S.D. 

Urine  Mg++  (mAf/1.) 

8 

29.9 

13.2 

14 

60.6 

39.7 

11 

123.8 

66.6 

Blood  Mg++  (mAf/1.) 

11 

12.4 

2.11 

15 

14.8 

5.68 

12 

14.5 

1.75 

Blood  osmotic  concen- 
tration (%  sea  water) 

12 

93.7 

4.60 

15 

115.5 

2.51 

12 

135.0 

6.97 

higher  rate  of  Mg++  transport  for  crabs  in  hypersaline  water  than  for  those  immersed 
in  100%  sea  water. 

Assuming  for  the  moment  such  an  increase  in  Mg++  transport  does  occur,  then 
any  of  the  following  or  combination  of  the  following  could  be  responsible  for 
triggering  the  accelerated  rate  of  such  transport  from  blood  into  the  bladder :  ( 1 ) 
direction  of  passive  water  flux  between  animal  and  medium  ;  (2)  osmotic  concentra- 
tion of  the  external  medium;  (3)  osmotic  concentration  of  the  blood,  and  (4)  Mg++ 
concentration  in  the  blood.  Mg++  concentration  in  the  medium  and  Mg++  influx 
already  have  been  ruled  out  as  triggering  stimuli. 

The  experiment  summarized  in  Table  III  was  designed  to  test  the  direction  of 
passive  water  flux  and  blood  osmotic  concentration  as  factors  for  controlling  the 
rate  of  Mg++  transport  when  the  blood  Mg++  and  osmotic  concentration  of  the 
medium  were  held  constant.  Thus,  Group  A  was  immersed  for  18  hours  in  a 
medium  equivalent  to  50%  sea  water  in  osmotic  concentration,  but  containing  65 
mM/1.  of  Mg++  which  is  about  twice  that  present  in  50%  natural  sea  water.  After 
18  hours  immersion  one  bladder  of  the  crab  was  emptied,  rinsed  with  isosmotic 


CRAB  BLADDER  FUNCTIONS  285 

perfusion  fluid  and  emptied  again.  The  crab  then  was  immersed  in  100%  sea 
water  for  a  period  of  6  hours,  after  which  time  urine  was  removed  from  the  same 
bladder  for  Mg++  analysis. 

Group  B  was  immersed  for  18  hours  in  a  medium  equivalent  to  158%  sea  water 
in  osmotic  concentration  but  containing  59  mM/1.  of  Mg++  which  is  about  that 
found  in  100%  natural  sea  water  and  comparable  to  the  concentration  of  Mg++  in 
the  medium  for  Group  A  (above).  After  18  hours,  one  bladder  of  the  crab  was 
emptied,  rinsed  with  isosmotic  perfusion  fluid,  emptied  again  and  reimmersed  in 
100%  sea  water  for  6  hours.  After  this  period,  the  same  bladder  was  drained 
and  the  urine  analyzed  for  Mg++. 

For  the  second  step  of  this  experiment,  that  is,  immersion  in  100%  sea  water, 
the  blood  of  Group  A  was  osmotically  less  concentrated  than  the  medium  and  the 
blood  of  Group  B  was  osmotically  more  concentrated  than  the  medium  (Table  III). 
Thus,  with  respect  to  the  direction  of  passive  water  flux,  Group  A  was  simulating 
hypo-regulation  (passive  water  efflux)  and  Group  B  hyper-regulation  (passive 
water  influx)  which  normally,  when  observed  in  crabs  in  high  and  low  salinities,  are 
accompanied  by  high  and  low  urine  Mg++  concentrations,  respectively.  If,  then, 
the  direction  of  passive  water  flux  were  a  major  factor  in  triggering  the  acceleration 
of  Mg++  transport,  Group  A  should  have  produced  a  more  concentrated  urine  Mg++ 
during  the  6-hour  period  than  Group  B.  As  can  be  seen  in  Table  III,  however, 
Group  B  produced  the  more  concentrated  urine  Mg++  (P  <  0.02).  Since  the 
external  medium  was  the  same  for  both  groups,  the  cue  for  Group  B  to  produce  high 
urine  Mg"1"1"  could  not  have  come  from  the  external  medium  during  the  6-hour 
period.  Furthermore,  because  the  passive  water  flux  in  Group  B  was  inward  and 
in  Group  A  was  outward,  the  volume  of  primary  urine  formed  should  be  higher  in 
Group  B  than  in  Group  A,  again,  assuming  a  filtration  process.  It  is  interpreted 
that  the  rate  of  Mg++  transport  was  indeed  responsible  for  the  difference  between 
Groups  A  and  B  with  respect  to  urine  Mg++  concentration,  a  condition  caused  by  the 
preliminary  treatment  in  the  dilute  and  concentrated  sea  water.  There  is  evidence, 
then,  that  the  rate  of  Mg++  transport  is  elevated  when  the  salinity  of  the  external 
medium  is  increased.  Although  the  experiment  was  designed  to  maintain 
constant  concentrations  of  blood  Mg++  for  both  groups,  it  can  be  seen  that  the  mean 
blood  Mg++  concentration  of  Group  B  was  higher  than  that  of  Group  A  (P  <  0.02). 
Also,  the  blood  osmotic  concentration  of  Group  B  was,  by  design,  higher  than  that 
of  Group  A  (P  <  0.001).  Therefore,  high  blood  Mg++  and/or  osmotic  concentra- 
tions possibly  triggered  the  acceleration  of  Mg+*  transport. 

Group  C  was  treated  as  follows  in  an  attempt  to  lower  the  blood  Mg++  concen- 
tration to  that  of  Group  B,  but  to  elevate  the  blood  osmotic  concentration  above 
that  of  Group  B.  First,  the  crab  was  immersed  in  158%  sea  water  (82  mM/1. 
of  Mg++)  for  18  hours;  (2)  then  the  bladder  was  drained  and  rinsed  with  isosmotic 
perfusion  fluid;  (3)  the  crab  was  reimmersed  in  158%  sea  water  for  an  additional 
6  hours  when  the  bladder  was  again  drained,  rinsed  as  before,  and  (4)  the  crab 
was  reimmersed  in  158%  Mg++-free  sea  water  for  6  hours  after  which  the  urine  was 
sampled  for  Mg++  analysis.  In  the  above  procedure  initial  exposure  to  158%  sea 
water  containing  natural  amounts  of  Mg++  was  for  the  purpose  of  elevating  the  blood 
osmotic  concentration  by  prolonged  exposure  to  hypersaline  water;  transfer  to 
158%  Mg++-free  sea  water  for  the  brief  period  was  to  maintain  high  blood  osmotic 


286  WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 

concentrations,  but  to  reduce  the  blood  Mg++  concentration  to  approximately  the 
level  achieved  in  Group  B. 

As  seen  in  Table  III  the  mean  urine  Mg++  concentration  of  Group  C  is  higher 
than  that  of  Group  B  (P  <  0.02)  ;  mean  blood  Mg++  concentrations  for  the  two 
groups  are  not  significantly  different,  but  the  mean  blood  osmotic  concentration  of 
Group  C  is  considerably  greater  than  that  of  Group  B  (P  <  0.001).  It  might  seem 
that  high  blood  osmotic  concentration  triggers  the  acceleration  of  Mg++  transport. 
However,  values  in  Table  III  are  terminal  and  while  the  mean  blood  Mg++  values 
for  Groups  B  and  C  were  essentially  the  same,  it  is  likely  that  they  changed  during 
the  6-hour  period  when  the  sampled  urine  was  being  formed.  On  the  other  hand, 
as  pointed  out  above,  6-hour  urine  samples  from  crabs  immersed  in  158%  sea  water 
have  the  same  concentrations  of  Mg++  whether  or  not  Mg**  is  present  in  the  medium. 
There  does  seem  to  be  some  evidence  that  the  osmotic  concentration  of  the  blood 
gives  the  cue  for  setting  the  rate  of  Mg++  transport  into  the  bladder. 

Attempts  were  made  to  lower  blood  Mg++  further  while  maintaining  high  blood 
osmotic  concentrations  by  prolonged  immersion  in  Mg++-free,  hypersaline  water. 
However,  individual  responses  to  such  treatment  were  too  variable  (probably  due  to 
different  rates  of  blood  Mg++  depletion)  to  permit  adequate  resolution.  On  the 
other  hand,  blood  Mg++  concentrations  could  be  elevated  while  maintaining  the 
blood  osmotic  concentration  constant.  Thus,  crabs  removed  from  100%  sea  water 
were  injected  with  0.5  ml.  of  isosmotic  MgCl2  (360  mM/1.)  after  the  bladder  was 
evacuated  and  rinsed  with  isosmotic  perfusion  fluid.  The  crabs  were  reimmersed 
in  100%  sea  water  for  6  hours  after  which  the  urine  and  blood  were  sampled  for 
Mg++  analysis.  Thus,  the  mean  blood  Mg++  (19  cases)  was  20.6  mM/1.  (S.D., 
6.55)  and  the  mean  urine  Mg++  (12  cases)  was  158  mM/1.  (S.D.,  31.4).  The 
mean  blood  Mg++  was  significantly  higher  (P  <  0.001)  than  the  mean  value  10.0 
mM/1.  reported  for  normal  crabs  immersed  in  100%  sea  water  by  Gross  (1959)  ; 
the  mean  (6-hour)  urine  Mg++  was  significantly  higher  than  the  6-hour  urine  Mg++ 
(54.5  mM/1.)  reported  above  for  crabs  with  empty  bladders  immersed  in  100% 
sea  water  (P  <  0.001).  Six  crabs  treated  in  the  same  manner  but  injected  with 
0.5  ml.  of  isosomotic  perfusion  fluid  rather  than  MgCl2  had  a  mean  urine  Mg++ 
of  52.8  mM/1.  (S.D.,  20.6)  which  was  also  significantly  less  than  the  value  for 
the  Mg++  treatment  (P  <  0.001).  Since  the  injected  MgCl2  was  isosmotic  with 
the  blood,  the  critical  factor  in  elevating  the  urine  Mg++  appears  to  be  the  concentra- 
tion of  blood  Mg++.  There  is  evidence,  therefore,  that  the  rate  of  Mg++  transport 
into  the  bladder  is  influenced  by  the  concentration  of  Mg++  in  the  blood. 

It  is  concluded  that  the  concentration  of  urine  Mg++  in  Pachygrapsus  is  deter- 
mined by:  (1)  the  length  of  time  urine  is  retained  in  the  bladder,  and  (2)  the  rate 
of  transport  for  Mg++  into  the  bladder.  Factors  which  influence  the  rate  of  Mg++ 
transport  are:  (a)  the  concentration  of  blood  Mg+%  and  (b)  possibly  the  osmotic 
concentration  of  the  blood. 

There  is  no  evidence  that  osmotic  or  Mg++  concentrations  of  the  medium  directly 
influence  the  rate  of  Mg++  transport.  Neither  is  there  evidence  that  the  Mg++  flux 
or  the  direction  of  passive  water  flux  directly  influences  the  rate  of  Mg++  transport. 

DISCUSSION 

There  is  now  convincing  evidence  that  urine  first  entering  the  bladder  of  Pachy- 
grapsus has  a  low  concentration  of  Mg++  but  a  high  concentration  of  Na+.  In  time 


CRAB  BLADDER  FUNCTIONS  287 

the  urine  Mg++  concentration  increases  and  the  urine  Na+  concentration  decreases 
(Figs.  2,  3  and  4).  This  probably  is  accomplished,  in  part,  by  a  direct  Na+-Mg++ 
exchange  across  the  bladder  membranes. 

The  mean  Na+  concentration  change/Mg++  concentration  change,  1.64,  observed 
in  solutions  of  NaCl  or  MgCL  introduced  into  bladders  of  crabs  kept  out  of  the 
water  (Table  II)  is  compatible  with  this  scheme.  Yet,  as  shown  above,  when 
isosmotic  perfusion  fluid  was  used  instead  of  NaCl  or  MgCU  the  ratio  was  only  1.15, 
a  value  that  approximates  the  ratio  derived  from  differences  in  means  for  Na+  and 
Mg++  that  occur  with  time  in  Figure  3.  This  conflict  may  be  related  to  the  large 
Na+  gradient  between  blood  and  urine  created  by  the  introduction  of  pure  solutions 
of  NaCl  or  MgCL  into  the  bladder.  If  the  membranes  were  permeable  to  Na+ 
and  Mg++  but  far  less  permeable  to  Cl~,  the  rapid  diffusion  of  Na+  down  the  gradient 
across  the  membranes  would  necessitate  a  rapid  Mg++  exchange  because  of  the  low 
Cl~  permeability.  On  the  other  hand,  with  the  slow  transport  of  Mg++  that 
normally  occurs  into  the  urine,  the  probability  would  be  higher  that  a  given  Mg++ 
ion  could  be  accompanied  by  Cl~  ions,  thus  reducing  the  necessity  of  Na+  exchange 
for  electro-chemical  balance  and  therefore  reducing  the  value  for  Na+  concentration 
change/ Mg"1"1"  concentration  change. 

Should  Cl~  move  with  Mg++,  then  an  osmotic  increase  would  be  caused  in  the 
urine,  and  this  would  result  in  an  influx  of  water.  Yet,  the  efflux  of  exchanged  Na+ 
would  reduce  the  osmotic  concentration  of  the  urine,  thus  effecting  an  efflux  of 
water.  Since  inulin  U/B  values  (Table  I)  are  not  less  than  unity,  it  is  unlikely 
that  net  increases  in  bladder  fluid  are  caused  by  the  inward  movement  of  Cl"  with  the 
transported  Mg++. 

These  data  then  suggest  that  during  the  normal  processing  of  urine  in  the 
bladder,  there  is  a  direct  exchange  of  Na+  for  the  Mg++  that  is  secreted  into  the 
bladder,  but  also,  there  is  some  movement  of  Cl"  with  the  Mg++,  but  not  in  sufficient 
amounts  to  cause  a  net  gain  of  water  in  the  bladder. 

Riegel  and  Lockwood  (1961)  observed  increases  in  the  urine  Mg++  concentration 
of  Carcinus  and  decreases  in  urine  Na+  concentration  with  time  as  the  crab  was 
kept  out  of  water.  The  increase  in  Mg++  concentration  was  attributed  to  Mg++ 
secretion  and  water  withdrawal.  However,  these  authors  discounted  a  direct  Na+- 
Mg++  exchange  mechanism  because  during  the  test  period  (e.g.,  96  hours)  the  fall 
in  urine  Na+  concentration  (90  mM/1.)  seemed  too  small  to  account  for  the  rise  in 
urine  Mg++  concentration  (103  mM/1.)  on  the  basis  of  electro-chemical  balance. 
Now,  this  might  suggest  that  a  direct  Na+-Mg++  exchange  was  not  the  only 
process  involved,  but  it  does  not  rule  out  such  a  mechanism,  for  as  pointed  out 
above,  electro-chemical  balance  could  be  achieved  both  by  Na+-Mg++  exchange 
and  Cl~  movement.  Besides,  Riegel  and  Lockwood  point  out  that  there  is  water 
withdrawal  from  the  urine  and  in  such  a  situation,  the  movement  of  Na+  from  urine 
to  blood  would  be  partially  obscured  by  water  withdrawal  which  would  increase 
the  concentration  in  the  urine.  On  the  other  hand,  the  apparent  movement  of  Mg++ 
from  the  blood  to  the  urine  would  be  exaggerated  by  water  withdrawal  increasing 
the  Mg++  concentration.  In  end  effect,  withdrawal  of  water  would  reduce  the 
ratio,  Na+  concentration  change/Mg++  concentration  change,  below  that  anticipated. 

The  wide  range  of  urine  Mg++  concentrations  observed  in  Pachygrapsus  (Fig.  1 ) 
can  be  explained  largely  by  the  fluctuations  of  concentration  occurring  in  individual 


288  WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 

crabs  (Fig.  2)  which  as  indicated  above  probably  reflect  the  periods  of  bladder 
evacuation. 

A  distinction  should  be  made  between  the  concentration  of  urine  Mg++  and  the 
actual  net  excretion  of  Mg++ ;  the  rate  of  Mg++  transport  into  the  bladder  which  as 
indicated  above  can  be  varied  to  meet  the  load,  would,  of  course,  influence  both  of 
these,  but  where  there  is  a  prolonged  retention  of  urine  in  the  bladder  (e.g.,  in  a 
crab  immersed  in  hypersaline  water),  resulting  in  high  urine  Mg++,  the  steep  Mg++ 
gradient  between  blood  and  urine  would  likely  counteract  the  effect  of  accelerated 
transport.  There  is  no  evidence  of  a  good  correlation  between  the  ability  of  a  crab 
to  concentrate  Mg++  in  its  urine  and  its  ability  to  regulate  Mg++  in  the  blood  (Gross, 
1964).  Gross  and  Marshall  (1960)  produced  evidence  that  Pachygrapsus  loses 
more  Mg++  when  immersed  in  50%  sea  water  than  when  immersed  in  150%  sea 
water  even  though  the  urine  Mg++  of  crabs  in  the  dilute  medium  was  only  one-sixth 
the  concentration  of  that  for  the  crabs  in  hypersaline  media.  This  is  interpreted 
to  mean  that  although  the  active  rate  of  transport  for  Mg++  into  the  bladder  may 
have  been  less  for  crabs  in  the  dilute  medium  than  in  a  hypersaline  medium,  rapid 
water  influxes  in  the  former  precluded  retention  of  urine  in  the  bladder,  permitting 
no  time  for  the  buildup  of  a  Mg++  gradient,  thus  resulting  in  less  diffusion  of  Mg4* 
from  the  urine  back  to  the  blood  and  consequently  a  greater  net  transport  of  Mg++ 
into  the  urine  and  to  the  outside. 

It  has  been  shown  for  Carcinus  (Webb,  1940)  and  for  Cancer  (Gross,  1964) 
that  increased  Mg++  in  the  medium  is  reflected  in  higher  urine  Mg++  concentrations. 
Such  was  not  shown  for  Pachygrapsus  by  Gross  and  Marshall  (1960)  even  though 
the  blood  Mg++  concentration  was  elevated  by  the  treatment.  It  is  apparent  now 
that  Pachygrapsus  retains  urine  in  its  bladder  for  a  period  during  which  time  the 
urine  Mg++  concentration  is  built  up  (Figs.  2  and  3).  Such  a  phenomenon  would 
shroud  the  effect  of  accelerated  transport  of  Mg++  if  the  experiment  were  initiated 
on  crabs  with  full  bladders.  Thus,  a  crab  immersed  in  hypersaline  Mg++-free  sea 
water  will  appear  to  concentrate  urine  Mg++  as  if  the  ion  were  present  in  high 
concentrations  in  the  external  medium.  In  this  situation  Mg+*  will  continue  to 
be  pumped  into  a  full  bladder  probably  already  containing  a  high  concentration  of 
Mg++.  If  urine  is  not  evacuated,  the  Mg++  concentration  will  elevate  to  a  maximum 
level  determined  by  the  osmotic  concentration  of  the  isosmotic  blood  and  urine  and 
probably  by  the  magnitude  of  the  Mg++  gradient  between  blood  and  urine,  which,  in 
turn,  will  depend  on  the  rate  of  Mg++  transport  into  the  urine.  Until  bladder 
evacuation  occurs  no  Mg++  will  be  lost  by  this  route  and  decreases  in  blood  Mg++ 
caused  by  transport  of  this  ion  into  the  urine  could  be  offset  by  diffusion  of  Mg++ 
from  the  urine  back  into  the  blood.  Data  in  Table  II  show  that  Mg++  can  move 
from  urine  to  blood.  Also,  if  the  transport  of  Mg++  involves  a  direct  exchange  with 
Na*  as  the  evidence  above  suggests,  the  Na+  concentration  gradient  may  also  limit 
the  concentration  of  Mg++  in  the  urine. 

It  was  only  by  measuring  the  Mg++  concentration  in  urine  first  entering  the 
bladder  that  the  influence  of  blood  Mg++  on  the  rate  of  Mg++  transport  could  be 
shown  in  the  present  investigation.  In  the  cases  of  Carcinus  and  Cancer  where 
high  Mg++  concentrations  in  the  medium  are  reflected  in  high  urine  Mg++  concen- 
trations when  crabs  with  full  bladders  are  used  (Webb,  1940;  Gross,  1964),  the 
urine  probably  is  held  only  briefly  in  the  bladder,  no  time  being  permitted  to 


CRAB  BLADDER  FUNCTIONS  289 

elevate  the  Mg++  concentration  and  consequently  not  obscuring  the  influence  of 
blood  Mg++  on  the  urine  concentration  of  this  ion.  Gross  (1957)  produced  evidence 
that  the  exoskeleton  of  Pachygrapsus  is  less  permeable  than  that  of  Cancer.  Greater 
water  fluxes  would  be  expected  in  highly  permeable  animals  which,  in  turn,  would 
not  hold  urine  in  the  bladder  for  long ;  the  concentrations  achieved  for  urine  Mg++ 
would  be  expected  to  be  low  compared  with  a  relatively  impermeable  animal.  This 
invites  measurement  of  water  fluxes  in  an  array  of  crabs  to  determine  if  the  rate  of 
water  turnover  is  related  to  the  maximum  concentrations  of  Mg++  achieved  in 
the  urine. 

Obviously,  precise  measurements  of  urine  flow  would  allow  quantitative  evalua- 
tion of  the  assertions  made  here.  However,  meaningful  values  for  urine  flow  and 
the  consequent  ion  losses  would  have  to  be  made  under  conditions  where  evacuation 
of  urine  from  the  nephropore  was  allowed  to  proceed  in  a  natural  manner.  Gross 
and  Marshall  (1960)  have  calculated  urine  flow  in  Pachygrapsus  in  various 
salinities  from  mean  urine  Mg++  concentrations  and  mean  Mg++  losses  to  the 
medium.  Since  average  values  were  used,  relationships  between  urine  Mg++ 
concentrations  that  fluctuate  in  individuals  (Fig.  2)  and  Mg++  losses  could  not  be 
resolved. 

In  view  of  the  evidence  produced  above,  direct  catheterization  would,  by  drain- 
ing the  bladder,  deprive  it  of  its  normal  renal  function  and  probably  give  spurious 
values,  for  urine  flow  and  ion  loss.  Experiments  designed  to  measure  the  natural 
flow  of  urine  in  Pachygrapsus  are  in  progress,  but  reliable  data  have  not  yet  been 
obtained. 

The  urinary  bladder  of  Pachygrapsus  clearly  is  more  than  an  organ  of  storage. 
Although  the  anatomical  details  of  the  bladder  are  not  described,  exploratory  studies 
reveal  it  to  be  a  highly  complex,  lobed  structure  similar  to  those  described  for 
other  brachyurans  in  the  review  by  Balss  (1944)  where  histological  evidence 
suggests  a  secretory  function  of  the  bladder  wall. 


These  studies  were  supported  by  National  Science  Foundation  Grants,  GB- 
1092  and  GB-3969.  We  wish  to  express  our  gratitude  to  Prof.  E.  B.  Edney  for  his 
critical  reading  of  the  manuscript  and  to  Messrs.  John  Armstrong  and  Steven 
Peterson  for  their  able  technical  assistance. 

SUMMARY 

1.  The  concentration  of  urine  Mg++  in  immersed  specimens  of  Pachygrapsus  is 
independent  of  the  Mg++  influx  as  well  as  the  concentration  of  Mg++  in  the  medium. 
It  is,  however,  a  function  of  the  salinity  of  the  medium. 

2.  Low  U/B  values  for  inulin  indicate  that  water  withdrawal  has  little  effect  in 
causing  the  high  urine  Mg++  concentrations  and   Mg++  U/B   values  observed  in 
Pachygrapsus. 

3.  Repetitive  samplings  of  urine  from  individual  crabs  immersed  in  100%  sea 
water  reveal  that  the  urine  Mg++  concentration  fluctuates  with  time,  varying  as 
much  as  three-fold  in  a  single  crab.     This  is  not  believed  to  be  due  to  fluctuations 
in  the  Mg++  transport  mechanism. 


290  WARREN  J.  GROSS  AND  RONALD  L.  CAPEN 

4.  The  wide  range  of  urine  Mg+*  concentrations  observed  in  the  field  can  be 
explained  chiefly  on  the  basis  of  fluctuating  urine  concentrations  in   individuals 
rather  than  on  large  variations  in  the  ability  to  concentrate  Mg++. 

5.  There  is  evidence  that  the  membranes  of  the  bladder  transport  Mg++  from 
blood  to  urine,  and  the  concentration  of  Mg++  attained  in  the  urine  of  Pachygrapsus 
depends  on  the  length  of  time  that  urine  is  held  in  the  bladder.     Thus,  hypo- 
regulating  crabs  immersed  in  hypersaline  water  having  a  small  water  influx  will 
hold  urine  in  the  bladder  sufficiently  long  to  build  up  the   Mg++  concentration. 
Hyper-regulating  crabs  in  dilute  sea  water  with  a  large  water  influx  release  urine 
too  frequently  to  permit  Mg++  buildup.     This  explains  how  the  urine  Mg++  concen- 
tration can  be  independent  of  the   Mg++  concentration  in  the  medium,  but  is  a 
function  of  the  salinity  of  the  external  medium. 

6.  Fluctuating  urine   Mg++   concentrations   in   crabs   are   believed   to   indicate 
periods   of   bladder   evacuation,    low    Mg++   following   evacuation   and   high    Mg++ 
preceding  evacuation. 

7.  There  is  evidence  that  when  Mg++  is  transported  into  the  urine  through  the 
bladder  wall,  electro-chemical  balance  is  achieved  by  direct  exchange  with  Na+,  but 
also  by  some  movement  of  Cl~  with  the  Mg^.     Such  a  mechanism  is  compatible  with 
the  observed  decreases  in  urine  Na+  concentration  accompanying  increases  in  urine 
Mg++  concentration. 

8.  Crabs  treated  with  the  Na+  transport  inhibitor  ouabain  can  concentrate  Mg++ 
in  the  urine.     Thus,  there  is  no  evidence  that  Mg++  transport  is  coupled  to  the  active 
transport  of  Na+*. 

9.  Mg++  transport  from  blood  to  urine  is  more  rapid  when  the  crab  is  immersed 
in  high  salinities  than  when  immersed  in  low  salinities.     The  mechanism  controlling 
the  rate  of  Mg++  transport  seems  to  be  triggered  directly  by  the  Mg++  concentrations 
in  the  blood  and  possibly  by  the  blood  osmotic  concentration. 

10.  The  concentration  of  Mg++  attained  in  the  urine  of  a  crab  does  not  neces- 
sarily indicate  the  relative  ability  to  excrete  Mg++.    It  is  suggested  that  permeability 
of  the  animal  to  water  determines  the  rate  of  water  turnover  and  therefore  the  rate  of 
bladder  evacuation.     This,  in  turn,  limits  the  period  during  which  Mg++  can  be 
accumulated  in  a  given  volume  of  urine. 

11.  Direct  catheterization  of  Pachygrapsus  would  be  expected  to  deprive  the 
bladder  of  its  normal  renal  function,  thus  giving  spurious  values  for  urine  flow 
and  ion  losses. 

LITERATURE  CITED 

BALSS,  H.,  1944.     Decapoda.     In:   "Bronn's  Klassen  und  Ordnungen  des  Tierreichs,"   Bd.   5, 

Abt.  1,  Bch.  7,  Lfg.  4:562-591. 
BARNES,  H.,   1954.     Some  tables  for  the  ionic  composition  of  sea  water.    /.  Exp.  Biol.,  31: 

582-588. 
GREEN-,  J.  W.,  M.  HARSCH,  L.  BARR  AND  C  L.  PROSSER,  1959.     The  regulation  of  water  and 

salt  by  the  fiddler  crabs,  Uca  pagnax  and  Uca  pugilator.    Biol.  Bull.,  116:  76-87. 
GROSS,  W.  J.,  1957.     An  analysis  of  response  to  osmotic  stress  in  selected  decapod  Crustacea. 

Biol.  Bull.,  112:43-62. 
GROSS,  W.  J.,  1959.     The  effect  of  osmotic  stress  on  the  ionic  exchange  of  a  shore  crab.     Biol. 

Bull.,  116:  248-257. 
GROSS,  W.  J.,  1964.     Trends  in  water  and  salt  regulation  among  aquatic  and  amphibious  crabs. 

Biol.  Bull..  127:447-466. 


CRAB  BLADDER  FUNCTIONS  291 

GROSS,  W.  J.,  AND  L.  A.  MARSHALL,  1960.     The  influence  of  salinity  on  the  magnesium  and 

water  fluxes  of  a  crab.     Biol.  Bull.,  119:  440-453. 
GROSS,  W.  J.,  R.  LASIEWSKI,  M.  DENNIS  AND  P.   RUDY,   1966.     Salt  and  water  balance  in 

selected  crabs  of  Madagascar.     Comp.  Biochem.  Physiol.,  17:  641-660. 
JONES,  L.  L.,  1941.     Osmotic  regulation  in  several  crabs  of  the  Pacific  Coast  of  North  America. 

/.  Cell.  Comp.  Physiol,,  18:  79-91. 
JUDAH,  J.  D.,  AND  K.  AHMED,  1964.     The  biochemistry  of  sodium  transport.    Biol.  Rev.,  39: 

160-193. 
KIRSCHNER,  L.,  AXD  S.  WAGNER,  1965.     The  site  and  permeability  of  the  filtration  locus  in  the 

crayfish  antennal  gland.    /.  Exp.  Biol,  43:  385-395. 

LOCKWOOD,  A.  P.  M.,  1962.     The  osmoregulation  of  Crustacea.     Biol.  Rev.,  37:  257-305. 
POTTS,  W.  T.  W.,  AND  G.   PARRY,   1964.     Osmotic  and   Ionic   Regulation   in   Animals.     The 

Macmillan  Company,  New  York. 
PROSSER,  C.  L.,  J.  W.  GREEN  AND  T.  CHOW,  1955.     Ionic  and  osmotic  concentrations  in  blood 

and  urine   of  Pachygrapsus   crassipes  acclimated   to   different   salinities.    Biol.   Bull., 

109:  99-107. 
RIEGEL,  J.  A.,  AND  A.  P.  M.  LOCKWOOD,  1961.     The  role  of  the  antennal  gland  in  the  osmotic 

and  ionic  regulation  of  Carcinus  inacnas.    J.  Exp.  Biol,,  38:  491-499. 
SCHALES,  O.,  AND  S.  SCHALES,  1941.     A  simple  and  accurate  method  for  the  determination  of 

chloride  in  biological  fluids.     /.  Biol.  Chem.,  140:  879-884. 
SCHREINER,  G.,  1950.     Determination  of  inulin  by  means  of  resorcinol.     Proc.  Soc.  Exp.  Biol. 

andMed.,74:  117-120. 
WEBB,  D.  A.,  1940.     Ionic  regulation  in  Carcinus  maenas.    Proc.  Roy.  Soc.  London,  Ser.  B, 

129:  107-136. 


ESTIMATES    OF  POPULATION    DENSITY    AND    DISPERSAL    IN 

THE  NATICID  GASTROPOD,  POLINICES  DUPLICATUS,  WITH 

A   DISCUSSION    OF    COMPUTATIONAL    METHODS1 

W.  RUSSELL  HUNTER 2  AND  DAVID  C.  GRANTS.4 

Department  of  Zoology,  Syracuse  University,  Syracuse,  New  York  13210  and 
Department  of  Biology,  Yale  University,  New  Haven,  Connecticut  06520 

This  paper  reports  an  attempt  to  use  marking  of  individual  snails  and  capture- 
recapture  methods  to  assess  population  density  and  rates  of  dispersal  in  a  littoral 
population  of  Polinices  duplicatus  at  Barnstable  Harbor,  Cape  Cod,  Massachusetts. 
Earlier  work  by  the  present  authors  on  the  ecology  of  the  infauna  of  the  sand-flats 
in  the  Barnstable  area  (D.  C.  G.),  and  on  the  general  biology  of  Polinices  spp. 
(W.  R.  H.),  had  indicated  both  the  importance  and  the  difficulties  of  density 
estimates  in  populations  of  P.  duplicatus. 

Standard  methods  such  as  direct  counting  of  quadrats  are  completely  unsuitable, 
as  preliminary  surveys  for  the  present  work  showed.  Some  of  the  difficulties  are 
those  which  would  arise  with  any  moderately  large-sized,  and  relatively  widely 
dispersed  animal  capable  of  burrowing — others  are  peculiar  to  Polinices,  and  result 
from  aspects  of  the  behavior  of  "moon-snails."  For  example,  they  can  burrow  deep 
into  the  substratum,  can  remain  immobile  for  considerable  periods,  and  may  show 
marked  tidal  periodicity.  Thus  the  present  study  showed  that  the  number  of 
animals  visible  at  or  near  the  surface  of  the  sand  at  any  one  time  is  but  a  fraction 
of  the  total  inhabiting  the  area. 

However,  it  is  important  to  attempt  an  accurate  assessment  of  the  population 
density  in  Polinices — and  in  other  "ecologically  difficult"  species  such  as  the  horse- 
shoe crab,  Linmlus.  In  many  areas  of  tidal  flats  around  Cape  Cod  and  elsewhere, 
such  animals  are  among  the  important  "terminal  consumers"  in  the  majority  of 
infaunal  associations  of  invertebrates.  Further,  the  commercial  importance  of 
naticid  species  as  pests  of  shell-fisheries  has  long  been  recognized. 

Techniques  of  capture,  marking,  release  and  recapture  have  been  used  exten- 
sively in  studies  of  birds  and  small  mammals,  and  of  insects  such  as  Lepidoptera 
and  tsetse-flies.  Such  methods  were  developed  independently  by  Jackson  (1933, 
1937,  1939,  1948)  working  on  tsetse-flies,  and  by  Lincoln  (1930)  working  on 
ducks.  The  former  author,  in  association  with  R.  A.  Fisher,  evolved  a  more 
sophisticated  arithmetical  treatment  which  allowed  estimates  to  be  made  on  popula- 
tions of  changing  density.  Subsequent  work  on  Lepidoptera  (Fisher  and  Ford, 
1947;  Dowdeswell,  Fisher  and  Ford,  1940,  1949)  utilized  "trellis"  arrays  and 
allowed  estimates  of  death-rates  and  emergence-rates.  Some  of  the  complications 

1  Supported  in  part  by  a  grant  from  the  U.  S.  National  Institutes  of  Health,  GM  11693. 

2  Instructor    and    3  Assistant    Instructor,    Department    of    Invertebrate    Zoology,    Marine 
Biological  Laboratory,  Woods  Hole,  Massachusetts. 

4  Present  address :  Systematics-Ecology  Program,  Marine  Biological  Laboratory,  Woods 
Hole,  Massachusetts. 

292 


DENSITY  AND  DISPERSAL  IN  POLINICES  293 

arising  from  differential  behavior  within  the  populations  studied  have  been  dis- 
cussed by  workers  on  small  mammals  (for  example,  Evans,  1949,  on  house  mice) 
and  on  insects  (for  example,  Ay  re,  1962,  on  ant  colonies).  The  significance  of  the 
different  methods  of  analysis  used  on  capture-recapture  data  has  been  considered  by 
Schumacher  and  Eschmeyer  (1943),  Leslie  (1952;  see  also  Leslie  and  Chitty, 
1951;  Leslie,  Chitty  and  Chitty,  1953),  DeLury  (1958),  Turner  (1960)  and 
Andrewartha  (1961).  Treatments  which  can  be  used  to  estimate  the  bias  and 
precision  of  the  results  obtained  have  also  been  developed  (Bailey,  1951,  1952),  and 
the  methods  applied  to  a  variety  of  terrestrial  vertebrates  and  insects.  However, 
capture-recapture  methods  have  not  previously  been  applied  to  any  extent  in  studies 
of  marine  benthic  animals. 

METHODS 

Use  of  capture-recapture  methods  to  assess  population  density  is  based  on  a 
number  of  assumptions,  and  this  population  density  experiment  on  Polinices 
duplicates  was  designed  to  satisfy  as  many  of  these  as  possible.  Preliminary 
surveys  and  assessments  of  population  density  and  dispersal  by  other  methods 
made  this  feasible.  The  most  important  condition  is  that  the  marked  animals 
after  their  release  become  homogeneously  dispersed  through  the  unmarked  popula- 
tion before  resampling.  On  the  other  hand,  the  simplest  methods  of  calculation 
involve  the  assumption  that  resampling  takes  place  immediately  after  release  of 
marked  animals  (thus  before  the  population  is  altered  by  births,  deaths,  immigra- 
tion or  emigration).  Sampling  only  adult  snails  and  using  a  time  interval  of  24 
hours  removes  the  complications  due  to  births  or  deaths  in  a  long-lived  (4-7  years, 
Hunter,  unpublished)  animal  like  Polinices.  The  accuracy  of  population  estimates 
is  most  greatly  increased  by  having  second  and  third  recaptures  at  exactly  equal 
intervals  of  time,  and  further  increases  in  accuracy  can  be  effected  with  further 
recaptures.  However,  the  increased  accuracy  of  ten  recaptures  over  nine  is  not 
great,  and  considerations  of  effort  to  be  expended  made  a  six-day  capture-recapture 
series  optimal  for  the  present  work.  A  final  assumption  in  this  work  is  that 
marked  individuals  are  identical  in  terms  of  life  expectancy  and  behavior  with  un- 
marked individuals,  and  that  the  actual  marks  are  permanent.  The  conditions  of 
survival  of  individual  snails  and  of  marks  are  certainly  satisfied  for  the  six-day 
period,  but  a  temporary  disturbance  of  behavior  resulting  from  the  handling  involved 
in  marking  was  detected.  This  is  discussed  more  fully  below,  and  arithmetical 
procedures  which  circumvent  the  effects  of  this  behavioral  change  on  population 
assessments  are  set  out. 

The  populations  of  Polinices  duplicates  in  Barnstable  Harbor  are  of  enormous 
extent,  and  a  limited  area  for  the  capture-recapture  experiment  had  to  be  set  out. 
Its  size  had  to  be  practical  for  collection  of  all  snails  sighted,  that  is  of  all  the 
proportion  of  the  population  visible  at  or  near  the  surface.  On  the  other  hand  it 
had  to  support  a  sufficient  number  of  snails  both  to  avoid  the  complications  of 
"patchy"  distribution  and  to  give  the  increased  accuracy  resulting  from  samples  of 
100  or  more.  Lastly,  if  the  rate  of  capture,  and  the  total  area  collected,  could  be 
adjusted  so  that  the  recaptured  (marked)  snails  numbered  between  5%  and  25% 
of  each  sample  subsequently  captured,  the  accuracy  of  the  population  assessment 
would  be  relatively  high. 


294  W.  RUSSELL  HUNTER  AND  DAVID  C  GRANT 

In  the  early  summer  of  1962,  direct  counting  of  qviadrats  was  carried  out  in 
several  parts  of  Barnstable  Harbor,  and  the  numbers  of  Polinices  were  counted 
along  tidal  contours  to  find  an  area  of  relatively  uniform  density.  The  locality 
chosen  was  on  the  southern  shore  of  Barnstable  Harbor,  about  400  meters  east  of 
the  dredged  channel  into  Maraspin  Creek,  on  a  relatively  uniform  substrate  of 
muddy  sand,  at  latitude  41°42.6'N,  and  longitude  70°  17.9'  W.  The  size  of  the 
area  chosen  to  fit  the  conditions  described  above  (i.e.,  with  sufficient  population 
numbers,  but  small  enough  to  make  recapture  practical)  was  1600  square  meters, 
being  a  square  40  m.  by  40  m.  The  tidal  range  at  this  locality  is  6.8  feet  at  neaps 
and  13.1  feet  at  springs.  The  experimental  area,  laid  out  normal  to  the  littoral 
contours,  was  just  covered  at  low  water  of  neaps,  and  was  totally  exposed  only 
during  spring  tides.  The  area  was  actually  marked  out  as  a  central  20-m.  square 
(400  sq.  m.)  within  the  main  40-m.  square  (1600  sq.  m.)  by  eight  permanent  steel 
pegs.  During  each  days'  work  a  six-foot  wooden  stake  was  placed  at  each  peg. 
The  dispersal  experiment  discussed  later  was  carried  out  around  a  single  peg  which 
was  placed  about  250  m.  west  of  the  main  experimental  area. 

Since  the  whole  process  of  capture,  marking  and  redispersal  took  about  four 
hours,  and  since  it  was  planned  to  sample  at  every  second  low  tide  for  six  days 
(to  maintain  a  constant  interval  of  24.8  hours  between  samples),  the  choice  of  dates 
was  limited.  The  main  series  was  run  on  August  8  through  13,  1962  (hereafter 
referred  to  as  days  I  through  VI),  actual  times  of  predicted  low  water  ranging 
from  1215  to  1640  hours  D.S.T.  The  tidal  cycle  in  Barnstable  Harbor  is  slightly 
modified  from  a  single  semi-diurnal  pattern,  and  the  afternoon  range  is  less  than 
the  overnight. 

On  each  day  the  eight  stakes  were  placed  about  two  hours  before  low  water,  and 
collecting  of  the  sample  of  Polinices  duplicatiis  was  begun  immediately.  All  col- 
lecting was  done  by  the  authors  alone,  in  less  than  two  feet  of  water  and  during 
the  ebbing  tide.  The  area  of  the  main  (larger)  square  was  repeatedly  traversed  in  a 
series  of  strips  (about  1.6  m.  wide),  with  a  regular  alternation  of  starting  point 
between  the  two  collectors.  In  fact,  no  bias  occurred  between  them  with  regard 
to  total  numbers  of  animals,  size  of  animals,  or  number  of  marks  recaptured. 
Every  snail  sighted  in  the  area  was  collected,  until  no  sightings  occurred  over  a 
10-15-minute  interval,  and  this  always  resulted  in  a  sample  of  over  150  snails.  The 
time  taken  for  this  collection  varied  somewhat  with  visibility  (i.e.,  weather  and 
extent  of  wave  action)  and,  under  better  conditions,  occupied  about  90  minutes. 
On  one  occasion  (day  IV,  see  below)  conditions  were  so  bad  that  collecting  had  to 
continue  for  about  three  hours  (i.e.,  including  a  period  of  the  rising  tide).  The 
snails  collected  were  stored  in  buckets  of  frequently  changed  shallow  water.  Sort- 
ing and  marking  were  carried  out  on  a  small  table  set  up  in  the  water  near  the 
sample  area.  Sorting  consisted  of  removal  of  undersized  ( <  2  cm. )  specimens  of 
P.  duplicatus  and  all  specimens  of  the  closely  related  Lunatia  heros  which  had  been 
picked  up  accidentally.  These  "rejected"  snails  were  dispersed  some  distance  away. 
At  this  stage  on  days  II  through  VI,  all  marks  recaptured  were  counted,  and  the 
snails  then  given  the  appropriate  mark  for  that  day.  The  actual  numbers  at  this 
stage  were:  1—142,  11—132,  III— 145,  IV— 157,  V— 161,  VI— 136.  Preliminary 
marking  experiments  had  been  carried  out  earlier  in  the  summer  on  over  100 
snails.  Test  marks  on  all  parts  of  the  shell  of  about  15  model  dopes,  nail  polishes 


DENSITY  AND  DISPERSAL  IN  POLINICES 


295 


and  marking  pens  showed  that  seven  types  would  survive  for  over  three  weeks  even 
on  the  apex  of  the  snail.  To  avoid  any  differential  predation  of  marked  snails,  the 
mark  in  the  density  experiment  was  placed  in  the  umbilicus  of  the  shell  which  is 
not  normally  exposed  in  life,  either  to  predators  or  to  abrasion  in  the  sand.  In  fact, 
of  the  specimens  marked  during  the  main  series,  at  least  three  had  clearly  dis- 
tinguishable marks  when  recovered  eleven  months  later,  and  one  after  three  years. 
More  confidently  than  is  usual  in  such  experiments,  it  can  be  assumed  that  no 


TABLE  I 
Data  from  density  square 


Day 

I 

II 

ill 

IV 

V 

VI 

VIF 

Number  captured 

(174) 
142 

(141) 
132 

(164) 
145 

(177) 
157 

(185) 
161 

(183) 
136 

(420+x) 
420 

Unmarked 

142 

129 

122 

99 

94 

89 

147 

1 

'  I       (Pink) 

3 

19 

15 

21 

8 

36 

& 

II     (Red) 

3 

19 

14 

11 

48 

V 

III  (Black) 

22 

24 

10 

45 

So 

c 

IV    (Green) 

3 

9 

46 

c/3 

.  V     (Orange) 

1 

35 

r      I  +  II 

1 

1 

0 

I  +  III 

3 

3 

11 

en 

II  +  III 

1 

1 

2 

rt 

I  +  IV 

1 

5 

§ 

II  +IV 

1 

10 

4)  " 

15 

III  +  IV 

2 

15 

3 
O 

I  +  V 

6 

Q 

II  +V 

3 

III  +  V 

1 

7 

.  IV  +  V 

1 

Triples 

1A 

3B 

Number  marked 

142 

132 

145 

157 

161 

Pink 

Red 

Black 

Green 

Orange 

Notes:  Numbers  in  brackets  above  second  row  are  actual  captures  including  L.  heros  and 
undersized  specimens  of  P.  duplicatus. 
A  One  triple:  I,  II  and  III. 
B  Two  triples:  I,  III  and  V;  One  triple:  II,  III  and  IV. 

marks   were    abraded   or   otherwise   lost    during    the    six    days    of    the   principal 
observations. 

The  marking  was  always  carried  out  in  the  same  way.  Lots  of  10  snails  each 
were  removed  from  water  in  the  storage  buckets  and  caused  to  withdraw  totally. 
Their  shells  were  then  dried  with  paper  towels  and  the  umbilical  area  cleaned  with 
a  little  alcohol,  care  being  taken  to  avoid  the  aperture  and  operculum  of  the  shell. 
The  alcohol-cleaned  spot  then  received  the  day's  mark,  and  was  allowed  to  dry  for  a 
maximum  of  four  minutes  before  the  snails  were  returned  to  the  storage  buckets. 
Periodically,  batches  of  30-40  marked  snails  were  randomly  distributed  through 
the  dispersal  area  (i.e.,  the  inner  20-m.  square).  The  rationale  for  this  dispersal 


296  W.  RUSSELL  HUNTER  AND  DAVID  C.  GRANT 

will  be  discussed  below.  Thus,  the  snails  were  out  of  their  habitat  for  1.5-2  hours, 
but  out  of  water  only  during  the  actual  marking,  for  a  maximum  of  eight  minutes. 
The  arithmetic  procedures  employed  in  assessing  population  density  from  the  data 
thus  collected  will  be  discussed  in  the  section  on  analysis  and  interpretation. 

An  experiment  on  rates  of  dispersal  in  P.  duplicatus  was  carried  out  in  an 
adjacent  area  during  the  last  three  days  of  the  main  capture-recapture  series.  A 
total  of  393  marked  snails  was  released  around  a  single  fixed  point  and  their 
dispersal  followed  visually  for  30  minutes.  On  two  subsequent  days  concentric 
circles  were  staked  out  at  1,2,  3,  4,  5,  7,  and  10  meters  from  the  fixed  point,  and 
the  numbers  of  marked  and  unmarked  specimens  of  Polinices  within  each  annulus 
recorded.  Two  types  of  marks  were  used.  Of  the  393  marked  snails,  105  were 
freshly  marked  in  the  field  with  a  single  black  line  on  the  apex,  while  the  remaining 
288  had  been  captured  on  the  previous  day,  taken  to  the  laboratory,  marked  with  a 
black  cross  and  released  after  24  hours. 

In  all,  1237  snails  were  marked  in  these  series  of  experiments.  On  the  last  day, 
after  the  regular  sampling,  as  many  snails  as  possible  were  captured  over  a  period 
of  hours  in  both  the  "density  square"  and  the  "dispersal  circle."  Of  737  snails 
marked  and  released  in  the  density  experiment,  273  snails  bearing  339  marks  were 
recaptured  and  taken  to  the  laboratory  where  maximum  shell  diameter  was  meas- 
ured. Of  the  393  snails  marked  and  released  in  the  dispersal  experiment,  156 
were  finally  captured  and  measured.  Almost  a  year  later,  three  collections  total- 
ling 402  snails  were  made  in  the  density  area  and  two  marked  snails  were  collected. 
At  the  same  time,  but  independently  by  Dr.  Ralph  I.  Smith,  a  further  marked  snail 
was  found  about  400  m.  away.  Three  years  later,  a  total  of  183  snails  collected  in 
the  general  area  included  one  marked  snail  (from  the  main  density  experiment), 
which  was  found  about  300  m.  from  the  density  square. 

RESULTS 

The  capture-recapture  data  from  the  density  experiment  are  set  out  in  Table  I 
above.  The  numbers  in  brackets  in  the  column  of  total  captures  represent  the 
actual  numbers  collected  including  undersized  specimens  of  Polinices  duplicatus, 
and  specimens  of  the  closely-related  Lunatia  heros  (see  Methods  above).  As  can 
be  seen,  the  experiment  was  relatively  successful  in  terms  of  satisfying  the  several 
conditions  for  greater  accuracy  outlined  above.  For  example,  the  average  number 
captured  and  marked  in  the  regular  samples  was  147  and,  with  certain  exceptions, 
the  numbers  of  each  mark  recaptured  made  up  7.7%  to  19.3%  of  the  samples.  As 
noted  above,  there  was  no  detectable  bias  between  the  two  collectors  in  any  respect. 
From  the  data,  there  is  no  indication  that  any  individual  animal  exhibits  an  in- 
creased or  decreased  likelihood  of  capture.  Certain  data  in  Table  I  are  significant 
in  this  respect.  An  excess  of  double  marks  in  any  group  captured  would  indicate 
that  there  were  individual  snails  more  prone  to  capture.  This  can  be  tested  in 
several  ways.  For  example,  if  we  consider  the  column  for  the  regular  captures 
of  day  VI,  total  captured  snails  were  136,  of  which  39  bore  a  single  mark  and  8 
double  marks.  On  that  day  the  cumulative  total  of  marked  individuals  at  risk  was 
586  (by  addition  of  the  appropriate  values  for  unmarked  snails  newly  marked  each 
day)  and  the  corresponding  number  for  double  (and  triple)  marks  at  risk  was 
143.  Thus  the  capture-ratio  for  all  marks  was  47/586  or  0.080  and  for  double 


DENSITY  AND  DISPERSAL  IN  POLINICES  297 

marks  8/143  or  0.056.  Applied  at  several  levels,  these  sorts  of  ratios  do  not  indi- 
cate any  group  of  snails  more  prone  to  capture.  On  the  other  hand,  estimates 
could  be  also  biased  by  some  individuals  being  less  readily  captured.  A  crude  test 
of  this,  although  it  involves  a  nearly  circular  argument,  can  be  justified  by  the  fact 
that  all  individuals  captured  on  days  I  through  V  were  marked.  Using  the  line  for 
the  total  captures  of  day  VI  (that  is,  VIF),  the  unmarked  captured  number  147, 
and  several  of  the  different  estimates  calculated  below  (and  based  on  the  entire 
day's  collection)  place  the  total  snail  population  in  the  square  at  about  990  individ- 
uals. Assuming  this  to  be  correct,  there  would  be  404  unmarked  individuals  at 
risk  on  day  VI ;  thus  the  estimated  capture-ratio  for  unmarked  individuals  was 
147/404  or  0.36,  while  the  ratio  for  all  marked  individuals  was  273/586  or  0.47. 
This  could  demonstrate  the  absence  of  excessive  bias  resulting  from  individuals  being 
less  readily  captured,  except  for  the  hypothetical  extreme  case  where  a  significant 
proportion  of  the  population  remained  undetected  for  more  than  six  days.  On  the 
basis  of  observed  behavior,  this  extreme  hypothesis  is  unlikely.  It  is  worth  empha- 
sizing again  that  it  was  totally  impossible  for  the  marks  in  the  umbilicus  to  influence 
the  collectors  during  the  capture  of  expanded  snails. 

One  source  of  bias  was  detected  subjectively  during  the  sampling,  and  is  revealed 
in  the  data  of  Table  I.  The  normal  behavior  of  moon-snails  is  temporarily  dis- 
turbed by  the  handling  involved  in  capture  and  marking.  After  release  in  the 
inner  dispersal  square,  such  snails  usually  re-expand  within  eight  minutes  (all  do 
within  twenty  minutes),  and  soon  burrow  deep  into  the  substratum  where  they 
remain  expanded  but  immobile  for  some  time.  Of  course,  in  this  expanded  but 
buried  state  they  are  not  liable  to  detection  and  capture.  At  first,  it  was  known 
only  that  this  period  of  immobility  extended  for  more  than  24  hours  but  less  than 
49.6  hours  (or  four  tidal  cycles).  Then  it  was  observed  that,  although  snails 
marked  on  the  previous  day  were  not  present  in  any  numbers  during  the  regular 
sampling,  that  is,  during  a  90-minute  period  before  the  time  of  low  water),  they 
became  obvious  among  the  many  active  snails  on  the  surface  during  the  two  hours 
after  the  tide  had  begun  to  rise.  In  other  words,  recovery  from  marking  trauma 
is  complete  in  just  over  two  tidal  cycles,  or  about  25.5  hours.  There  is  other  evi- 
dence that  a  proportion  of  the  population  will  always  become  more  active  immedi- 
ately after  inundation  by  a  rising  tide,  and  that  this  can  involve  a  condition  of 
temporary  hyperthermia  (Hunter  and  Apley,  1965).  The  effect  of  trauma  is  re- 
flected in  the  number  of  marks  recaptured  after  only  one  day  (Table  I).  For 
example,  the  number  of  mark  I  captured  on  day  II  is  relatively  low.  (For  con- 
venience, we  can  refer  to  Rxy,  being  the  number  of  recaptures  on  day  y,  bearing  a 
mark  from  day  x.)  Thus  R12,  R23,  R45  and  the  regular  Rn6  are  all  relatively  low. 
On  the  other  hand  two  collections  do  not  show  this  effect:  day  IV  and  the  final 
collection  of  day  VI  (VIF).  Collecting  on  both  these  occasions  extended  into 
the  first  hours  of  the  rising  tide :  on  day  IV  because  of  bad  weather  conditions  slow- 
ing the  collecting  rate,  and  later  on  day  VI  as  a  result  of  the  deliberate  collection 
of  the  large  final  sample  over  a  period  of  hours.  In  several  of  the  calculations 
below,  the  data  of  R12,  R23,  R45  and  the  regular  R56  are  rejected  as  biased,  while  R34 
and  the  final  R56  can  be  utilized,  along  with  the  other  data  where  more  than  one  day 
has  elapsed  between  marking  and  recapture,  such  as  R15,  R25,  R35  etc.  Finally,  the 
marks  borne  by  the  four  snails  recaptured  with  triple  marks  are  detailed  below 


298 


W.  RUSSELL  HUNTER  AND  DAVID  C.  GRANT 


Table  I,  and  have  to  be  incorporated  into  the  number  of  single  and  double  marks 
recaptured  in  certain  of  the  calculations  below. 

The  data  from  the  dispersal  experiment  are  summarized  in  Table  II.  Although 
less  successful  than  the  density  experiment,  and  hardly  a  complete  measure  of  the 
possible  rates  of  dispersal,  a  few  significant  facts  emerge.  In  the  first  30  minutes, 
376  of  the  393  individuals  had  expanded  and  righted,  and  >  200  were  out  of  sight, 
having  burrowed  into  the  sand.  The  three  specimens  in  the  2.0-4.0-m.  annuli  were 
known  to  have  expanded  and  "sailed"  with  the  longshore  tidal  current,  but  several 
had  crawled  >  1  m.  and  at  least  two  had  covered  1.75  m.  Comparison  of  the  sight- 
ing records  (after  two  tides)  and  the  final  collections  (after  four)  shows  that  line- 
marked  specimens  recovered  more  rapidly  than  the  X-marked  ones  did  from  their 

TABLE  II 
Data  from  dispersal  experiment 


Annuli  in  meters 

0.-0.5 

O.S-l.O 

1.0-2.0 

2.0-3.0 

3.0-4.0 

4.0-5.0 

5.0-7.0 

7.0-10.0 

Totals 

{Cross 

288 

0 

0 

0 

0 

0 

0 

0 

288 

Line 

105 

0 

0 

0 

0 

0 

0 

0 

105 

Unmarked 

? 

? 

? 

? 

? 

? 

? 

? 

p 

After  30  min.  f  Cross 

\  (16*)approx. 

Sight            \  Line 

j         100-150f 

18f 

2t 

It 

n.d. 

n.d. 

n.d. 

137-187f 

records         I  Unmarked 

p 

? 

p 

? 

p 

n.d. 

n.d. 

n.d. 

? 

After  2  tides   f  Cross 

\.                                               j 

2 

0 

0 

0 

0 

0 

129 

127 

Sight            \  Line 

4 

5 

8 

1 

2 

0 

0 

20 

records         I  Unmarked 

2 

3 

1 

9 

7 

17 

24 

63 

A 

/"                                                         "\ 

f  Cross 
After  4  tides      Une 

8 
1 

38 
0 

71 

2 

19 
4 

7 
3 

4 
3 

1 
1 

n.d. 
n.d. 

148 
14 

Collects    [Unmarked 

0 

2 

2 

5 

7 

13 

16 

n.d. 

45 

*  These  16  snails  were  the  only  ones  not  yet  moving. 

f  Combined  marks. 

n.d.  =  No  data  available. 

prolonged  and  more  drastic  handling.  The  circumstances  affecting  the  density 
experiment  discussed  above  are  not  valid  here  since  both  the  sighting  records  and 
the  final  collection  were  made  during  the  first  hours  of  the  rising  tide.  These  data 
estimate  dispersal  rather  than  absolute  rates  of  movement,  and  some  subjective 
assessments  made  during  the  density  experiment  are  relevant.  Rates  of  movement 
may  be  high  (as  much  as  3-4  m.  in  15  minutes).  In  the  course  of  one  tidal  cycle, 
snails  were  observed  to  have  left  tracks  equalling  7-8  m.  However,  tracks  are  rarely 
straight,  and  often  elaborately  looped,  so  that  each  individual  snail  tends  to  remain 
in  the  same  general  area.  Observations  such  as  these  led  to  the  proportions  ar- 
ranged, in  the  density  experiment,  for  the  larger  (sampling)  area  and  the  inner 
(dispersal)  area  (40  m.  by  40  m.  enclosing  20  m.  by  20  m.).  The  minimum  width 
of  the  outer  zone  was  thus  10  m.  All  marks  appeared  in  the  outer  zone  within  two 
days,  and  by  the  sixth  day  considerable  numbers  of  first-day  marks  \vere  found 


DENSITY  AND  DISPERSAL  IN  POLINICES 


299 


just  within  the  outer  edge  of  the  larger  square.  Actually  at  least  one  specimen  of 
every  mark  was  found  just  within  the  edge  of  the  outer  square  during  the  final 
collection  of  day  VI  (VIF). 

ANALYSIS  AND  INTERPRETATION 

The  simplest  possible  estimate  from  capture-recapture  data,  involving  a  single 
recapture  of  a  single  previous  mark,  is  the  "maximum-likelihood"  estimate  given 
1  >y  the  formula : 

J^sy 

where  Psy  is  an  estimate  of  the  population  based  on  the  recaptures  on  day  y  of 
individuals  marked  on  day  x,  Nr  is  the  total  captured  on  day  y,  Mx  is  the  number 
of  individuals  bearing  a  mark  from  day  x  which  are  "at  risk"  on  day  y,  and  Rxv 
is  the  number  of  recaptured  marks  as  defined  earlier.  Both  for  this,  and  for 
other  more  complex  estimates  below,  it  is  worth  setting  out  the  data  in  a  trellis 

TABLE  III 

Trellis:  raw  data  transformed  to  single  marks 


Capture  days: 

I 

II 

III 

IV 

V 

VI 

VIF 

Number  of  marked  indiv.  captured 

0 

3 

23 

58 

67 

47 

273 

Number  of  marks  captured 

0 

3 

24 

60 

73 

55 

339 

-      I 

142 

— 

3 

20 

16 

26 

11 

60 

u  _ 

*  £ 

II 

132 

— 

•  — 

4 

21 

15 

13 

64 

•Q   ^ 

—     I-  S 

III 

145 

— 

— 

— 

23 

28 

17 

83 

=   ci 

~z.  5 

IV 

157 

— 

— 

— 

— 

4 

12 

78 

f—  H 

.  V 

161 

—  • 

— 

— 

— 

— 

2 

54 

Unmarked  indiv.  captured 

142 

129 

122 

99 

94 

89 

147 

Total  indiv.  captured 

142 

132 

145 

157 

161 

136 

420 

array  (as  used  by  Dowdeswell,  Fisher  and  Ford,  1940.  1949,  but  in  the  reorientated 
and  typographically  simpler  form  used  by  Andrewartha,  1961,  and  others).  In  the 
trellis  (Table  III),  the  raw  data  are  transformed  to  single  marks,  and  there  is 
consequently  a  slight  loss  of  information.  In  the  trellis  each  horizontal  row  repre- 
sents the  recaptures  of  one  day's  marking  on  successive  days,  and  each  vertical 
column  the  recaptures  of  the  available  marks  on  one  day.  Diagonals  across  the 
central  part  represent  the  recaptures  after  an  interval  of  one  day,  of  two  days  and 
so  on.  The  column  headed  VIF  gives  the  final  total  number  of  snails  captured  on 
day  VI  and  includes  the  regular  sampling  (column  VI).  On  the  diagonal  of  recap- 
tures after  a  lapsed  time  of  one  day  appear  the  recapture  figures  (R12,  Ro3,  R45 
and  Regular  R56)  which  are  rejected  on  behavioral  grounds.  All  the  other  RXJ- 
data  are  available  for  calculation  by  this  simple  maximum-likelihood  formula,  and 
there  are  thus  16  possible  estimates  of  Pxy.  The  estimates  of  P16,  P26J  P3e  etc- 
derived  from  the  VIF  totals  are  to  be  preferred  (since  based  on  larger  samples)  to 
the  regular  Vlth  day  series.  This  leaves  the  12  values  of  P^y  which  are  calculated 
and  form  the  seventh  column  of  Table  IV.  These  population  estimates  range  from 
733.7  (P36)  to  1416.8  (P25),  with  a  mean  of  1,018.4,  and  a  standard  deviation  (s.d.) 


300 


W.  RUSSELL  HUNTER  AND  DAVID  C.  GRANT 


of  222.4.  It  should  be  noted  that  this  s.d.  is  a  measure  of  the  variance  of  this 
group  of  12  results,  not  a  measure  of  variance  of  the  population  estimates  as  such. 
Bailey  (1951,  1952)  has  examined  the  precision  of  such  estimates  of  Pxy  and  shows 
a  measure  of  the  variance  to  be : 

.  (Mx)2Ny(Ny-  Rxy) 
xy  ~  (Rxy)3 

expressed  in  the  terms  used  in  the  present  paper.  Values  can  be  calculated  for 
the  data  in  Table  IV:  for  example,  for  the  estimates  over  two  days  (P24)  and  over 
five  days  (Pi6)  the  variance  values  are  40,172.5  and  14,114.8,  respectively.  The 

TABLE  IV 

Maximum  likelihood  estimate  of  population,  simple  computation 


Estimate 

On  mark 

Capture  day 

Ny 

M, 

Rxy 

NyMx 

Riy 

Pw 

I 

III 

145 

142 

20 

1029.5 

Pu 

I 

IV 

157 

142 

16 

1393.4 

P24 

II 

IV 

157 

132 

21 

986.9 

P»4 

III 

IV 

157 

145 

23 

989.8 

Pll 

I 

V 

161 

142 

26 

879.3 

P26 

II 

V 

161 

132 

15 

1416.8 

P35 

III 

V 

161 

145 

28 

833.8 

Pl6 

I 

VIF* 

420 

142 

60 

994.0 

P26 

II 

VIF* 

420 

132 

64 

866.3 

P36 

III 

VIF* 

420 

145 

83 

733.7 

P46 

IV 

VIF* 

420 

157 

78 

845.4 

?56 

V 

VIF* 

420 

161 

54 

1252.2 

Mean     1018.4 


Final  totals  for  Vlth  day  used  here. 


root  values,  which  can  be  considered  as  "standard  errors"  of  the  estimates,  are 
200.4  (for  P24)  and  118.8  (P16). 

It  is  noted  above  that  transforming  the  data  to  single  marks  results  in  a  loss  of 
information  on  the  occurrence  of  multiple  recaptures  or  double  marks,  which 
could  yield  an  estimate  of  population  size.  The  simple  formula  for  a  maximum- 
likelihood  estimate  can  be  adapted  for  double  marks : 


(wx)y 


Ny     M(WX) 

R 


(wx)y 


where  P(wx)y  is  an  estimate  of  the  population  based  on  the  recaptures  on  day  y  of 
individuals  with  marks  for  both  days  w  and  x,  and  the  other  terms  correspond. 
The  numbers  of  double  marks  "at  risk"  on  any  day  subsequent  to  the  second  mark- 
ing can  be  derived  from  Table  I,  and  are  the  terms  for  M(wx).  The  following  values 
are  available:  M(12)  =  3,  M(13)  =  20,  M(23)  =  4,  M(14)  =  16,  M(24)  =  21,  M(34) 
=  23,  M(15)  =  26,  M(25)  =  15,  M(35)  =  28,  and  M(45)  =  4.  Rejecting  the  values 
forR(12)3,  R(14)5andR(35)6  on  the  usual  behavioral  grounds,  and  omitting  all  calcula- 
tions based  on  R(wx)y  =  1,  then  we  have  the  11  estimates  of  P(wx)y  calculated  and 


DENSITY  AND  DISPERSAL  IN  POLINICES 


301 


given  in  Table  V.  These  range  from  560  to  2100,  with  a  mean  of  1091.2,  and  an 
s.d.  of  484.1.  Most  individual  variances  of  these  estimates  would  be  very  large 
because  of  the  low  double  recapture  rates  (low  values  of  R(wx)y),  and  they  are  not 
calculated  here.  The  fact  that  these  estimates  of  P(Wx)y  based  on  double  marks  are 
closely  comparable  to  the  values  of  Pxy  calculated  earlier  is  a  further  confirmation  of 
a  lack  of  "capture-prone"  bias  in  individual  snails. 

A  modification  of  the  simple  proportional  formula  was  proposed  by  Bailey  (1951, 
1952)  to  reduce  positive  bias.  He  demonstrates  (Bailey,  1951)  that  this  modified 
estimate  has  an  average  relative  bias  that  is  more  than  order  of  magnitude  lower 

TABLE  V 

Maximum  likelihood  estimates  of  population,  based  only  on  "double" -marked  individuals 


Capture  day 

On  mark 

Ny 

M(wi) 

R(wx) 

NyM(wi) 

R(wx) 

V 

I  +  III 

161 

20 

4 

805.0 

VI 

I  +  III 

136 

20 

3 

906.7 

VI 

III  +  IV 

136 

23 

2 

1564.0 

VIF 

I  +  III 

420 

20 

13 

646.2 

VIF 

ir+  in 

420 

4 

3 

560.0 

VIF 

r+iv 

420 

16 

5 

1344.0 

VIF 

II  +  IV 

420 

21 

11 

801.8 

VIF 

III  +  IV 

420 

23 

16 

603.8 

VIF 

I  +V 

420 

26 

8 

1365.0 

VIF 

II  +V 

420 

15 

3 

2100.0 

VIF 

III  +V 

420 

28 

9 

1306.7 

Mean  =  1091.2 


at  certain  levels  of  sample  and  population  size  and,  expressed  in  our  terms,  has 
the  form : 


P      = 

A  xy 


Mx  (N 


+    1) 


Bailey  (1951)  also  proposed  a  satisfactory  approximation  for  the  variance  of  this 
estimate  of  Pxy.  The  present  data  are  unlikely  to  require  such  correction  for  posi- 
tive bias.  However,  using  the  above  formula  on  the  data  for  single  marks  in 
Table  III,  yields  estimates  (not  set  out  in  the  tables)  with  a  mean  of  986.6,  an  s.d. 
of  202.9,  and  'standard  errors"  (using  Bailey's  approximation)  of  the  order  of 
183.4  and  115.1. 

A  great  deal  of  previous  capture-recapture  work,  particularly  with  fish  stocks, 
has  utilized  methods  involving  series  of  census  carried  out  on  stocks  in  which  the 
marks  were  not  distinguished  as  to  date  of  origin.  One  of  the  best  of  such 
methods  is  that  originated  by  Schumacher  and  Eschmeyer  (1943)  involving  census 
weighted  for  sample  size.  The  Schumacher-Eschmeyer  method  is  discussed  by 
DeLury  (1958)  and  Turner  (1960),  and  theoretical  arguments  given  by  DeLury, 
and  practical  reasons  by  Turner,  for  preferring  it  to  a  maximum-likelihood  method, 
in  certain  cases.  In  general  terms,  it  appears  that  a  maximum-likelihood  estimate  is 


302 


W.  RUSSELL  HUNTER  AND  DAVID  C.  GRANT 


still  most  efficient  if  sampling  is  truly  random,  and  if  random  dispersal  of  marks  is 
complete  by  the  time  of  re-sampling,  but  that  if  sampling  is  biased  for  any  reason  a 
method  involving  weighting  for  sample  size  is  to  be  preferred.  The  data  on 
Polinices  duplicatus  can  be  used  in  a  Schumacher-Eschmeyer  computation.  The 
general  formula  used  is : 

£  Nd  Md2 

P  (Schumacher-Eschmeyer  estimate)  =  _   „    .. 

2_  Kd  Md 

when  Md  is  the  total  number  of  marked  individuals  at  risk  on  day  d,  Nd  is  the 
number  captured  on  day  d,  Rd  is  the  number  of  recaptured  marked  individuals  in 
Nd.  We  are  concerned  here  with  numbers  of  individuals  recaptured  rather  than 
number  of  marks,  and  it  is  necessary  to  use  for  Md  the  cumulative  totals  for  each 
day  of  all  the  marked  individuals  at  risk.  These  can  be  derived  from  the  sums 
of  previously  unmarked  snails  collected  for  marking  (listed  in  column  2  of  Table  I). 
Thus  we  have  as  values  for  Md  on  day  11—142,  III— 271,  IV— 393,  V-^92  and 
VI — 586.  These  values  are  used  along  with  those  for  Nd  and  Rd  in  Table  VI. 

TABLE  VI 
Serial  census  method  for  Schumacher-Eschmeyer  estimate  of  population 


Day 

Md 

Nd 

Rd 

(RdMd) 

(NdMd2) 

dll 

142 

132 

3 

426 

2,661,648 

dill 

271 

145 

23 

6,233 

10,648,945 

dIV 

393 

157 

58 

22,794 

24,248,493 

dV 

492 

161 

67 

32,964 

38,972,304 

dVIF* 

586 

420 

273 

159,978 

144,226,320 

ERdMd 

E  NdMd* 

=  222,395 

=  220,757,710 

/.  P  =  992.6 
*  Final  totals  for  Vlth  day  only  used  here. 

The  estimate  of  P  derived  by  this  method  is  992.6.  In  spite  of  the  fact  that  con- 
siderable information  is  lost  by  treating  all  marks  as  one  type  of  mark,  this  Schu- 
macher-Eschmeyer estimate  is  remarkably  close  to  the  mean  of  the  maximum- 
likelihood  estimates  produced  by  Bailey's  modified  formula. 

It  is  probable  that  the  most  complete  utilization  of  capture-recapture  data  of  the 
present  sort  is  provided  by  a  series  of  computations  such  as  were  developed  by 
C.  H.  N.  Jackson  in  his  work  on  tsetse-fly  populations  in  association  with  R.  A. 
Fisher  (Jackson,  1933,  1937,  1939,  1948;  see  also  Fisher  and  Ford,  1947;  Dowdes- 
well,  Fisher  and  Ford,  1940,  1949).  The  theoretical  bases  of  these  methods  are 
discussed  by  Bailey  (1951)  and  by  Leslie  (1952),  and  simplified  examples  of  the 
computations  set  out  by  Dowdeswell,  Fisher  and  Ford  (1940)  and  by  Andrewartha 
(1961).  The  present  data  on  Polinices  duplicatus  can  be  used  both  in  Jackson's 
positive  method,  involving  recapture  on  a  number  of  occasions  after  one  marking, 
and  in  Jackson's  negative  method,  involving  a  single  recapture  of  marks  made  on  a 
series  of  occasions.  Table  VII  is  a  trellis  array  of  the  values  of  Rxv  to  be  used, 


DENSITY  AND  DISPERSAL  IN  POLINICES 


303 


TABLE  VII 

Trellis :  selected  recapture  data  as  used  in  Jackson  computations, 
with  calculated  relative  frequencies 


Capture  days: 

in 

IV 

V 

VIF 

(total) 

Number  marked 

A 

'      I 

142 

20 
(9.7) 

16 
(7.2) 

26 
(11.4) 

60 
(10.1) 

II 

132 

21 
(10.1) 

15 

(7.1) 

64 
(11.5) 

III 

145 

23 
(10.1) 

28 
(12.0) 

83 
(13.6) 

IV 

157 

78 
(11.8) 

V 

161 

54 
(8.0) 

Total  individuals  captured  : 

145 

157 

161 

420 

Note:  the  numbers  in  brackets  are  relative  recapture  frequencies  (fxy),  as  defined  in  the  text. 


omitting  the  usual  ones  on  behavioral  grounds,  and  using  only  the  total  captures 
for  day  VI  (VIF).  These  values  of  Rxy  are  first  converted  to  relative  recapture 
frequencies  (fxy)  which  correspond  to  what  the  recaptures  would  have  been  if 
100  had  been  marked  and  100  captured  on  each  successive  date.  These  are 
derived  by : 


=    Rxy    X 


100       100 


M3 


X 


We  have  twelve  available  values  (for  f13,  f14,  f24,  f34, 


'• 


ff  £  1 

„„.       iv,,      26»    ^36>    1-46    <"1C 

f56)  and  these  appear  in  brackets  below  the  corresponding  recapture  figures  (Rxy) 
in  the  trellis  of  Table  VII.  Each  vertical  column  with  three  or  more  values  of  fsv 
gives  the  number  recaptured  on  one  day  for  an  estimate  by  Jackson's  negative 
method ;  each  horizontal  row  with  three  or  more  values  of  fxy  represents  the  subse- 
quent recaptures  of  one  set  of  marks  and  provides  one  estimate  by  Jackson's  positive 
method.  Thus  we  have  six  possible  estimates,  three  by  each  method.  We  have 
first  to  calculate  values  of  z  and  z*,  which  are  weighted  ratios  of  relative  recaptures 
(z  for  each  positive,  and  z*  for  each  negative  computation).  Examples  of  these 
weighted  ratios  are : 


f 


z  = 


l4 


f 


l6 


[16 


l3 


'15 


(positive  method),  and 


Z*    = 


4~ 


(negative  method) 


304  W.  RUSSELL  HUNTER  AND  DAVID  C.  GRANT 

These  are  then  employed  in  calculating  a  reciprocal  value  for  the  population  esti- 
mate, which  then  requires  multiplication  by  100  X  100  to  convert  relative  fre- 
quencies back  to  absolute  numbers.  Examples  of  the  formulae  are: 

1Q4(P)-1  =  fl3  +  fl4  +  fl6  -  (f13  +  f14)     (positive  method),  and 

2 
fB6    +    M6    +    fs6    +    126  ,f  £  r      \          /  *.'  ^.U     J\ 

104(p)-i  = _  (f56  -|-  f46  _j_  f36)      (negative  method). 

ft 

From  the  data  of  Table  VII  we  have  positive  estimates  of  P  of  908.3,  1720.9,  and 
1113.0,  and  negative  estimates  of  740.9,  1296.8,  and  1054.4.  The  great  value  of 
the  Jackson  methods  lies  in  their  ability  to  deal  with  changing  population  densities, 
and  these  estimates  above  are  extrapolations  forwards  or  backwards  and  actually 
represent  the  population  estimate  on  a  specific  day.  The  population  of  Polinices 
studied  is  thought  to  have  been  relatively  stable  over  the  short  period  of  this 
experiment.  Another  advantage  of  the  Jackson  computations  is  that  they  are  thus 
applicable  in  conditions  where  a  longer  delay  is  necessary  after  each  release  to 
allow  marked  animals  to  disperse  and  settle  down.  Obviously  they  will  prove 
valuable  with  certain  types  of  littoral  populations  but,  in  the  present  case,  yield  esti- 
mates no  more  valuable  than  the  simpler  ones,  in  spite  of  the  more  complete  utiliza- 
tion of  the  data.  Bailey  (1951,  1952)  has  given  methods  for  an  approximate 
estimate  of  var.  P  for  both  positive  and  negative  methods,  but  these  are  not 
computed  for  the  present  data.  Leslie  (1952;  see  also  Leslie  and  Chitty,  1951; 
Leslie,  Chitty  and  Chitty,  1953)  has  suggested  slight  elaborations  of  the  Jackson 
methods,  less  general  in  application,  but  which  can  be  more  precise  as  a  result  of 
utilizing  a  still  greater  proportion  of  the  information  yielded  by  the  data. 

The  above  may  serve  to  indicate  which  computational  methods  can  be  best 
applied  to  any  future  capture-recapture  work  on  other  marine  benthic  populations. 
Apart  from  behavioral  considerations,  the  sample  size,  and  the  relative  time-scales 
of  sampling  and  of  population  life-cycle  should  be  decisive.  Turning  to  the  results 
of  the  work  on  Polinices  duplicatus,  it  seems  clear  that  the  true  population  level  for 
the  1600  square  meters  at  the  time  of  the  experiment  lay  close  to  the  average  values 
from  maximum-likelihood  estimates  of  1018.4  (simple  calculation),  and  of  986.6 
(Bailey's  modification),  and  from  the  Schumacher-Eschmeyer  computation  of  992.6. 
For  comparative  purposes,  we  can  say  that  a  close  estimate  is  about  1,000  and,  from 
the  different  variance  values  computed,  that  it  was  unlikely  to  lie  outside  850-1150. 
Thus  the  density  is  probably  about  6250  (between  5313  and  7188)  per  hectare,  or 
about  2530  (between  2150  and  2900)  per  acre  of  intertidal  flats. 

The  total  area  of  Barnstable  Harbor  is  approximately  3763  hectares,  or  9298 
acres.  Of  these  55%  are  occupied  by  salt  marsh.  The  remainder  consists  of 
extensive  sand  flats  (26%)  dissected  by  permanent  subtidal  channels  (19%). 
Throughout  the  sand  flats  (approximately  1672  hectares  or  4132  acres)  and  chan- 
nels (approximately  711  hectares  or  1756  acres)  there  are  populations  of  Polinices 
duplicatus.  The  population  densities  vary  greatly  within  this  area.  For  example, 
within  the  permanent  channels  densities  are  relatively  low,  while  along  the  margins 
of  these  channels,  on  areas  of  higher  organic  content,  the  greatest  densities  for 


DENSITY  AND  DISPERSAL  IN  POLINICES  305 

the  harbor  may  be  reached.  On  the  sand  flats  the  greatest  densities  are  found 
around  the  level  chosen  for  the  density  square  in  the  current  study.  Above  and 
below  this  level  the  population  densities  are  usually  lower. 

In  the  central  areas  of  the  harbor  particularly,  it  is  probable  that  the  population 
densities  of  all  moon-snails  have  been  reduced  since  the  time  of  this  study  (summer, 
1962)  by  a  human  agency.  Shellfish  interests  have  supported  extensive  destruc- 
tion of  moon-snails  and  Limulus  in  subsequent  summers.  The  effectiveness  of  this 
program  has  not  been  tested  and  is  questionable. 

One  additional  result  should  be  mentioned  here.  At  the  conclusion  of  the  Vlth 
day's  collections  all  420  marked  and  unmarked  specimens  of  Polinices  duplicatus 
were  taken  to  the  laboratory  where  the  maximum  shell  diameter  was  measured 
with  a  dial-caliper.  The  total  number  of  marked  individuals  recaptured  at  that 
time  was  273,  and  their  size  range  was  18.8  to  53.9  mm.  (mean  =  29.27  mm.,  s.d. 
7.012  mm.).  Individuals  with  double  and  triple  marks  totalled  63,  with  a  size 
range  19.5  to  45.6  mm.  (mean  =  28.98  mm.,  s.d.  5.710  mm.).  Total  unmarked 
captures  (VIF)  was  147,  with  a  size  range  17.6  to  52.2  mm.  (mean  27.95  mm., 
s.d.  6.853  mm.).  Thus  there  is  little  evidence  of  any  size  bias  as  regards  chance 
of  multiple  recapture.  In  addition,  the  mean  values  lie  within  the  range  for  other 
population  samples  from  elsewhere  in  Barnstable  Harbor  (Hunter,  unpublished). 
Further,  it  is  worth  noting  that  the  "undersized"  specimens  of  P.  duplicatus  which 
were  "rejected"  before  marking  had  a  mean  size  of  12.2  mm.,  and  made  up  less  than 
10%  of  each  sample  collected  and  marked  (that  is,  less  than  1.5%  of  the  estimated 
population).  It  is  thus  unlikely  that  their  removal  had  any  significant  effect  on  the 
remaining  adult  population. 

Finally,  it  should  be  pointed  out  that  if  this  work  is  judged  to  be  a  relatively 
successful  demonstration  of  capture-recapture  methods  applied  to  a  marine  benthic 
population,  this  results  from  two  features.  First,  only  two  persons  were  involved 
in  the  field  work,  both  of  whom  had  considerable  experience  of  the  general  biology 
of  Polinices,  including  its  behavior  patterns.  Secondly,  because  of  this  experience 
and  preliminary  survey  work,  it  was  possible  to  set  up  the  density  experiment  with 
the  optimum  time  interval,  sample  size,  and  recapture  rate.  It  is  clear  that  the 
application  of  these  methods  to  a  benthic  population  of  totally  unknown  biology 
could  show  intolerable  bias. 


We  wish  to  thank  Mr.  Homer  P.  Smith  of  the  Marine  Biological  Laboratory, 
and  members  of  the  Supply  Department  staff,  for  making  a  truck  available  for 
the  field  work.  We  are  indebted  to  Myra  Russell  Hunter,  Martyn  L.  Apley, 
William  Charles  Blowers  II,  and  Jay  Shiro  Tashiro  for  checking  calculations,  and 
to  Anthony  Williams  for  assistance  preparing  the  estimate  of  area  for  Barnstable 
Harbor.  We  are  particularly  happy  to  thank  Dr.  Clark  P.  Read,  who  provided 
the  opportunity  to  begin  this  work  at  Woods  Hole. 

SUMMARY 

1.  Capture-recapture  methods  were  used  to  assess  population  density  and 
dispersal  in  Polinices  duplicatus. 


306  W.  RUSSELL  HUNTER  AND  DAVID  C  GRANT 

2.  Preliminary  surveys  indicated  an  area  of  1600  sq.  m.  as  optimal  for  density 
estimations.     This  yielded  capture  rates  averaging  147  per  day  and  the  number 
of  each  mark  recaptured  usually  made  up  7.7%  to  19.3%  of  each  sample. 

3.  Various  computational  methods  are  applied  to  the  data,  and  their  values 
discussed.     These  include  simple  and  modified  maximum-likelihood  estimates  (with 
measures  of  their  variance),  a  serial  census  method  weighted  for  sample  size,  and 
the  classical  Jackson-Fisher  extrapolations  of  relative  recapture  frequencies,  both 
"positive"  and  "negative." 

4.  The  two  maximum-likelihood  methods  yield  average  population  estimates 
of  1018.4  and  1091.2,  and  the  sample-weighted  census  method  yields  a  value  of 
992.6.     With  capture  rates  and  mark  frequencies  as  in  this  study  on  Polinices, 
these  relatively  simple  calculations  are  judged  adequate. 

5.  These  estimates  are  equivalent  to  a  population  density  for  Polinices  dupli- 
catus  of  6250  (5313-7188)  per  hectare,  or  2530  (2150-2900)  per  acre. 

6.  Recommendations  are  made  regarding  the  value  of  different  computational 
methods,  if  capture-recapture  methods  are  applied  to  other  marine  benthic  popula- 
tions.    Approximate  population  density,  sample  size,  and  the  relation  of  sampling- 
rate  to  population  dynamics  should  be  used  to  determine  the  appropriate  procedure. 

LITERATURE  CITED 

ANDREWARTHA,  H.  G.,  1961.  Introduction  to  the  Study  of  Animal  Populations.  Methuen  and 
Co.  Ltd.,  London,  281  pp. 

AYRE,  G.  L.,  1962.  Problems  in  using  the  Lincoln  Index  for  estimating  the  size  of  ant  colonies 
(Hymenoptera :  Formicidae).  /.  N.  Y.  Entomol.  Soc.,  70:  159-166. 

BAILEY,  N.  T.  J.,  1951.  On  estimating  the  size  of  mobile  populations  from  recapture  data. 
Biometrika,  38:  293-306. 

BAILEY,  N.  T.  J.,  1952.  Improvements  in  the  interpretation  of  recapture  data.  J.  Anim.  EcoL, 
21 :  120-127. 

DELURY,  D.  B.,  1958.  The  estimation  of  population  size  by  a  marking  and  recapture  procedure. 
/.  Fish.  Res.  Bd.  Canada,  15:  19-25. 

DOWDESWELL,  W.  H.,  R.  A.  FISHER  AND  E.  B.  FORD,  1940.  The  quantitative  study  of  popula- 
tions in  the  Lepidoptera,  1.  Polvommatus  icarus  Rott.  Ann.  Eugenics,  London,  10: 
123-136. 

DOWDESWELL,  W.  H.,  R.  A.  FISHER  AND  E.  B.  FORD,  1949.  The  quantitative  study  of  popula- 
tions in  the  Lepidoptera,  2.  Maniola  jurtina  L.  Heredity,  3:  67-84. 

EVANS,  F.  C.,  1949.  A  population  study  of  house  mice  (Mns  musculus}  following  a  period  of 
local  abundance.  /.  Mammal.,  30:  351-363. 

FISHER,  R.  A.,  AND  E.  B.  FORD,  1947.  The  spread  of  a  gene  in  natural  conditions  in  a  colony  of 
the  moth  Panaxia  dominula  L.  Heredity,  1:  143-174. 

HUNTER,  W.  RUSSELL,  AND  M.  L.  APLEY,  1965.  A  condition  of  temporary  hyperthermia  in  a 
marine  littoral  snail.  Biol.  Bull.,  129:  408-409. 

JACKSON,  C.  H.  N.,  1933.    On  the  true  density  of  tsetse  flies.    /.  Anim.  EcoL,  2:  204-209. 

JACKSON,  C.  H.  N.,  1937.  Some  new  methods  in  the  study  of  Glossina  morsitans.  Proc.  Zool. 
Soc.  London,  1936:  811-896. 

JACKSON,  C.  H.  N.,  1939.    The  analysis  of  an  animal  population.    /.  Anim.  EcoL,  8:  238-246. 

JACKSON,  C.  H.  N.,  1948.  The  analysis  of  a  tsetse-fly  population.  III.  Ann.  Eugenics, 
London,  14:  91-108. 

LESLIE,  P.  H.,  1952.  The  estimation  of  population  parameters  from  data  obtained  by  means 
of  the  capture-recapture  method,  II.  The  estimation  of  total  numbers.  Biometrika,  39: 
363-388. 

LESLIE,  P.  H.,  AND  D.  CHITTY,  1951.  The  estimation  of  population  parameters  from  data 
obtained  by  means  of  the  capture-recapture  method,  L  The  maximum  likelihood  equa- 
tions for  estimating  the  death-rate.  Biometrika,  38:  269-292. 


DENSITY  AND  DISPERSAL  IN  POL1NICES  307 

LESLIE,  P.  H.,  D.  CHITTY  AND  H.  CHITTY,  1953.  The  estimation  of  population  parameters 
from  data  obtained  by  means  of  the  capture-recapture  method,  III.  An  example  of 
the  practical  applications  of  the  method.  Biometrika,  40:  137-169. 

LINCOLN,  F.  C,  1930.  Calculating  waterfowl  abundance  on  the  basis  of  banding  returns. 
U.  S.  Dcpt.  Agric.  Circ.,  118:  1-4. 

SCHUMACHER,  F.  X.,  AND  R.  W.  ESCHMEYER,  1943.  The  estimate  of  fish  population  in  lakes 
or  ponds.  /.  Tennessee  Acad.  Sci.,  18:  228-249. 

TURNER,  F.  B.,  1960.  Size  and  dispersion  of  a  Louisiana  population  of  the  cricket  frog,  Acris 
gryllus.  Ecology,  41 :  258-268. 


DIURNAL  PATTERNS  OF  METABOLIC  VARIATIONS 

IN  CHICK  EMBRYOS  x 

LELAND  G.  JOHNSON  2 

Department  of  Biological  Sciences,  Northzvestern  University,  Evans  ton,  Illinois 

One  of  the  most  obvious  characteristics  of  living  organisms  is  the  dynamic 
nature  of  their  physiological  constitution.  A  commonly  observed  aspect  of  this 
dynamic  nature  is  cyclical  variation  in  various  physiological  processes.  Many  of 
these  cyclical  variations  disappear  under  laboratory  constant  conditions,  but  others 
are  very  persistent  even  when  the  organism  is  removed  from  obvious  environmental 
timers.  These  cyclical  phenomena  exhibit  periodicities  of  many  different  lengths 
including  approximate  solar  daily,  lunar  daily,  semi-monthly,  monthly,  annual,  and 
longer  term  ones  (Harker,  1958,  1964;  Brown,  1959a;  Webb  and  Brown,  1959; 
Cloudsley-Thompson,  1961;  Bunning,  1964;  Sollberger,  1965;  Aschoff,  1965). 

Since  the  total  physiology  of  any  organism  is  so  broadly  related  to  these  cyclical 
phenomena,  it  is  of  potentially  great  interest  to  consider  the  ontogeny  of  physio- 
logical periodicity.  A  basic  question  is  whether  an  organism's  physiology  possesses 
cyclical  qualities  only  in  a  relatively  mature  state  or  whether  systematic  variability 
can  be  demonstrated  throughout  the  life-history  of  the  organism.  The  former 
seems  to  be  the  case  for  some  overt  patterns  of  cyclical  variability  such  as  the  human 
infant  pulse  rate  and  sleep-wakefulness  cycles  (Hellbrugge,  1960).  However,  since 
these  functions  become  phased  to  obvious  daily  environmental  fluctuations  and  are 
not  observed  in  early  developmental  stages,  it  is  of  interest  to  seek  periodicities 
which  might  exist  under  constant  conditions  and  in  earlier  developmental  stages. 

Hiebel  and  Kayser  (1949)  were  unable  to  detect  a  clear  diurnal  rhythm  of 
movement  of  the  chick  embryo  within  the  egg.  They  were  measuring  movements 
of  the  whole  egg  and  were  seeking  a  clear-cut  nocturnal  reduction  in  activity.  Fol- 
lowing hatching  they  could  detect  no  diurnal  rhythm  in  heat  production  unless  they 
subjected  the  young  chicks  to  alternating  light  and  dark  schedules. 

Aschoff  and  Meyer-Lohmann  (1954),  however,  recorded  motor  activity  of 
newly  hatched  chicks  and  by  summing  all  activity  in  four-hour  periods  could  show, 
under  constant  conditions,  a  clear  rhythmic  variation  in  activity  present  from  the 
time  of  hatching.  This  rhythm  had  an  average  period  of  25.4  hours.  Hoffman 
(1957,  1959)  has  shown  an  activity  rhythm  of  approximately  24  hours  for  newly 
hatched  lizards.  Petren  and  Sollberger  (1953)  found  that  rhythmic  fluctuations 
in  liver  glycogen  content  were  already  established  in  very  young  chickens  (measure- 
ments made  on  days  5-6,  12-13,  and  36-37  post-hatching).  Further  studies 
showed  that  a  periodic  fluctuation  in  glycogen  content  was  already  present  on  the 

1  This  work  is  a  portion  of  a  dissertation  submitted  in  partial  fulfillment  of  the  requirements 
for  the  Ph.D.  degree  from  the  Department  of  Biological   Sciences,  Northwestern  University, 
Evanston,  Illinois. 

2  Present    address :    Department    of    Biology,    Augustana    College,     Sioux    Falls,    South 
Dakota  57102. 

308 


CHICK  EMBRYO  METABOLIC  PATTERNS  309 

first  day  after  hatching  and  that  a  rhythm  of  somewhat  different  character  could  be 
demonstrated  even  if  the  newly  hatched  chicks  were  starved.  In  other  experiments, 
freshly  laid  eggs  were  stored  and  incubated  in  total  darkness  until  the  twentieth 
day  of  incubation  when  liver  glycogen  determinations  were  made.  A  clear  daily 
rhythm  with  a  major  peak  and  a  minor  peak  of  glycogen  content  was  demonstrated. 
Therefore,  the  diurnal  liver  glycogen  rhythm  is  well  established  before  the  time  of 
hatching  even  under  constant  conditions. 

These  data  relate,  however,  to  rather  advanced  stages  of  development.  One 
study  carried  out  in  earlier  stages  of  development  was  the  measurement  of  metabolic 
variations  by  Barnwell  (1960).  He  was  able  to  detect  a  significant  mean  solar 
daily  fluctuation  in  rate  of  oxygen  consumption.  By  measuring  oxygen  consump- 
tion under  constant  conditions,  and  treating  data  to  correct  for  the  increasing  over- 
all rate  of  oxygen  consumption,  such  fluctuations  were  established  to  be  present 
on  the  fifth  through  the  eighth  days  of  incubation  (eggs  were  incubated  at  4  PM 
and  the  day  upon  which  incubation  was  begun  was  designated  as  day  one). 

Barnwell's  work  has  been  criticized  (Heusner,  1963,  1965 ;  Heusner  and 
Zahnd,  1963)  largely  on  the  basis  of  suggested  faults  in  his  techniques.  These  criti- 
cisms appear  to  have  originated  largely  from  misinterpretations  of  experimental 
procedures  and  statistical  techniques  used  by  Barnwell,  including  underestimation 
of  the  sensitivity  of  his  respirometers,  and  have  been  dealt  with  more  extensively 
elsewhere  (Johnson,  1965). 

The  presence  of  a  mean  daily  cycle  of  variation  in  rate  of  oxygen  consumption 
during  these  stages  of  development  raises  some  new  questions.  It  must  be  deter- 
mined whether  this  periodicity  has  a  definite  pattern  of  ontogeny.  The  properties 
of  metabolic  periodicity  in  a  developing  organism  should  be  compared  with  some 
well-known  properties  of  other  rhythmic  systems.  In  order  to  make  meaningful 
comparison,  a  consistent  statistical  treatment  must  be  developed.  Once  such  a 
treatment  is  available,  questions  about  ontogeny  of  periodicity,  seasonal  cycle  form 
differences,  and  temperature  relationships  should  be  answerable. 

METHODS  AND  MATERIALS 

Fertile  White  Leghorn  eggs  were  obtained  from  a  local  commercial  supplier  and 
stored  in  a  cold  chamber  at  approximately  10°  C.  until  used  (some  were  incubated 
directly).  Before  incubation  the  eggs  were  allowed  to  warm  to  room  temperature. 
Incubation  was  initiated  at  9  PM  (CST)  for  all  experiments  described  here  unless 
stated  otherwise.  The  incubator  was  a  forced  draft  model  provided  with  water 
pans  and  set  at  38°  C. 

Respiration  was  measured  using  Brown  automatic  recording  respirometers 
(Brown,  1954,  1957).  The  six  respirometer  units  were  constructed  at  the  begin- 
ning of  the  study. 

In  operation  the  sealed  respirometer  chamber  was  evacuated  to  produce  an 
internal  pressure  of  28.7  inches  Hg  which  then  remained  constant  throughout  each 
recording  period.  Temperature  was  controlled  at  38°  C.  by  a  55-gallon  circulating 
water  bath  which  surrounded  the  respirometer  chamber,  or  barostat.  A  mercury 
switch  and  relay  controlled  a  450-watt  immersion  heater  suspended  in  the  bath. 
Water  temperature  cycled  within  a  range  of  ±0.05°  C.  The  experiments  were 
conducted  in  a  photographic  darkroom  in  which  the  only  light  source  was  a  single 


310 


LELAND  G.  JOHNSON 


ceiling  fixture  controlled  through  a  voltage-regulator.  The  intensity  of  illumination 
reaching  the  interior  of  an  uncovered  barostat  was  less  than  one  foot  candle. 
With  the  glass  cover  in  place,  the  respirometer  in  the  interior  was  provided  with  a 
substantially  lower  level  of  constant  dim  light. 

The  respirometer  flask  itself  was  modified  as  described  by  Barnwell  (1960), 
with  minor  changes.  The  support  frame  which  held  the  egg  in  place  was  a  cylinder 
produced  by  cutting  a  section  out  of  a  glass  vial.  The  cylinder  was  perforated 
at  several  points  to  permit  free  passage  of  air  under  and  around  the  egg.  The 
egg  was  set  on  the  support  frame  with  the  air  chamber  uppermost.  A  KOH  solu- 
tion in  the  bottom  of  the  flask  was  the  CO2  absorbent  and  also  served  to  meet 
humidity  requirements.  A  single  egg  was  suspended  in  the  diver  below  the 
recorder  and  oxygen  consumption  values  were  calculated  in  ml.  consumed  per 
egg  per  hour. 

Control  experiments  for  the  apparatus  were  conducted  at  the  beginning  of  the 
experimental  period  and  at  various  times  thereafter.  Extended  recordings  of  the 

TABLE  I 

Comparison  of  rates  of  Orconsumption  as  ml.  of  Oz/egg/hour  at  several  stages 
of  incubation  as  determined  in  various  studies 


Hrs. 
incub. 

This  study 
(spring) 

I* 

II 

III 

IV 

V 

VI 

VII 

VIII 

72 

0.283±0.055f 

0.266±0.066 

0.253±0.040 

0.216 

96 

0.445±0.105 

0.427±0.110 

0.381±0.100 

0.34±0.11 

0.333 

120 

0.715±0.101 

0.680±0.131 

0.524±0.025 

0.44±0.05 

0.612 

0.628 

144 

1.071±0.199 

0.826±0.178 

0.831±0.134 

0.89±0.07 

0.91  ±0.06 

1.542 

0.922 

0.883 

168 

1.752±0.162 

1.423±0.191 

1.421  ±0.223 

1.19±0.14 

1.19 

2.075 

1.775 

1.168 

192 

2.221±0.145 

1.922±0.173 

1.671±0.173 

1.23±0.26 

1.51±0.03 

1.52 

2.262 

2.428 

1.835 

*I  Barnwell,  1960;  II  Romanoff,  1941a;  III  McLimans,  Siem  and  Scholljegerdes,  1950— 
Series  A;  IV  McLimans,  Siem  and  Scholljegerdes,  1950 — Series  B;  V  McLimans,  Siem,  Mark  and 
Pinska,  1950;  VI  Greiff  and  Pinkerton,  1948;  VII  Hasselbalch,  1900;  VIII  calculated  from 
Murray,  1925,  1926. 

f  Standard  deviation. 

behavior  of  weighted  blank  divers  were  made.  Even  when  runs  were  continued  for 
periods  longer  than  any  of  the  experimental  runs,  no  deviations  of  the  recording 
pens  were  detected.  Since  the  recorders  in  the  control  runs  were  set  at  sensitivities 
equal  to  the  greatest  ones  used  in  the  experiments,  it  could  safely  be  assumed  that  no 
oxygen  loss  which  was  detectable  by  the  methods  used  occurred  during  these 
experiments. 

This  study  was  concerned  more  with  variations  in  rates  of  oxygen  consumption 
than  with  absolute  rates.  However,  comparative  data  concerning  absolute  meas- 
urements made  with  this  apparatus  have  been  compiled  in  Table  I. 

One  problem  in  determining  daily  variations  in  oxygen  consumption  in  chick 
embryos  is  the  increasing  overall  rate  of  consumption.  In  order  to  examine  hourly 
fluctuations  on  a  comparative  basis,  a  correction  for  this  trend  must  be  made.  The 
method  chosen  for  this  study  was  the  construction  of  a  line  of  least  squares  fit  by 
regression  analysis  (Simpson,  Roe  and  Lewontin,  1960).  Regression  analysis  was 
performed  on  every  complete  organism-calendar-day  of  data  collected  during  the 


CHICK  EMBRYO  METABOLIC  PATTERNS 


311 


course  of  the  study.  The  resultant  calculated  values  provided  a  basis  for  deter- 
mining hourly  deviation  from  trend.  Each  recorded  hourly  value  was  divided  by 
the  appropriate  regression  value  to  permit  its  description  as  a  percentage  of  trend 
value.  These  percentage  values  made  plotting  of  daily  values  on  a  non-sloping  line 
possible  and  allowed  more  valid  comparisons  because  all  data  were  reduced  to  a 
common  relationship.  Therefore,  despite  differences  in  total  oxygen  consumption 
among  individual  organisms  on  a  given  day  of  incubation  and  between  different  days 
of  incubation,  meaningful  examinations  of  similarities  and  differences  in  form  of 
individual  patterns  and  mean  patterns  of  groups  could  be  made.  Unless  otherwise 
stated,  all  results  and  discussion  presented  will  relate  to  analysis  of  these  regressional 
relationships. 

Several  devices  were  employed  in  examining  the  daily  patterns.  Weighted  (1, 
2,  1)  moving  means  (Croxton  and  Cowden,  1955)  were  calculated  from  the  hourly 
means  in  some  instances.  Means  for  consecutive  three-hour  periods  were  calculated 
in  other  cases.  Ratios  of  the  number  of  hourly  recorded  values  falling  above  the 
trend  line  to  the  number  falling  below  for  given  periods  were  also  calculated. 

RESULTS 

The  use  of  regression  analysis  and  the  calculation  of  per  cent  of  trend  values 
from  data  such  as  those  collected  in  this  study  reduce  all  data  to  a  common  form 
and  also  make  possible  gross  comparisons  of  relationships  to  the  trend  line.  Since 
this  analysis  corrects  for  the  continually  increasing  overall  rate  of  oxygen  consump- 
tion during  ontogeny,  it  is  possible  to  detect  metabolic  variations  not  accountable 
by  simple  ontogenetic  increase.  As  the  metabolism  of  the  adult  chicken  has  a  strong 
diurnal  variation,  the  per  cent  of  trend  values  were  first  examined  on  the  basis  of 
an  arbitrary  separation  of  nighttime  from  daytime  values  (all  38°  C,  9  PM  incuba- 
tion time  data  were  used).  In  the  1  AM  to  6  AM  data  724  hourly  values  were  found 


1.20      - 


.00 


.80     - 


FIGURE  1.  Ratios  of  the  number  of  hourly  values  greater  than  100%  of  trend  values  to  the 
number  of  values  less  than  100%  of  trend  calculated  for  six-hour  periods.  Closed  circles  repre- 
sent all  of  the  data  gathered  at  38°  C.  from  embryos  which  were  incubated  initially  at  9  PM  (see 
text).  Open  circles  represent  the  same  data  minus  day-seven  and  day-eight  data.  Half-closed 
circles  represent  data  gathered  at  33°  C. 


312 


LELAND  G.  JOHNSON 


to  be  above  the  trend  line  and  836  values  were  below  it.  In  the  7  PM  to  midnight 
data  763  values  were  above  and  797  values  were  below  the  trend  line.  The  twelve 
daytime  hours  (7  AM-6  PM)  showed  a  considerably  different  picture  with  1624 
values  above  100%  of  trend  and  1496  below  it.  Statistical  comparison  of  the  day 
and  night  values  by  use  of  Chi-square  analysis  indicates  a  significant  difference 
(x-  —  12.04,  P<  0.001).  Therefore,  irrespective  of  any  other  complexities  of 
form  in  the  daily  cycles,  there  appears  to  be  a  mean  diurnal  variation  even  at  these 
early  stages  of  development. 

More  refined  examinations  of  these  relationships  can  be  accomplished  in  another 
way.  A  ratio  of  the  number  of  hourly  per  cent  of  trend  values  falling  above  100% 
of  trend  to  those  falling  below  that  level  provides  a  convenient  numerical  value  for 
purposes  of  comparison.  Figure  1  (closed  circles)  shows  the  ratios  calculated 
using  all  hourly  values  for  six-hour  periods  of  the  day. 

The  foregoing  information  is  of  value  only  in  detecting  gross  relationships  and 
it  becomes  necessary  to  use  the  actual  per  cent  of  trend  values  for  more  precise 
examination  of  characteristics  of  this  metabolic  variability.  Ratios  such  as  those 
seen  in  Figure  1  assign  equal  weight  to  all  hourly  values  and  do  not  take  large 
percentage  deviations  into  account.  Separation  of  the  data  into  three-hour  periods 
using  mean  per  cent  of  trend  values  yields  some  interesting  differences  among  the 
days  of  incubation  studied  (Fig.  2).  The  patterns  for  days  four,  five  and  six  show 
considerable  similarities  through  the  pre-dawn  hours,  but  then  a  difference  appears. 
Days  four  (2a)  and  five  (2b)  go  on  to  afternoon  highs  and  evening  lows  but  a 
similar  progression  through  the  day  is  lacking  in  the  day-six  data  (2c)  except  for 


104 


100 


96 


104 


100 


96 


\ 


2A 


B 


\ 


4 


D 


12 


12 


FIGURE  2.  Means  of  hourly  per  cent  of  trend  values  calculated  for  three-hour  periods  of 
the  day.  The  number  of  organism-calendar-days  of  data  involved  in  each  instance  is  indicated 
in  parentheses.  A.  38°  day-four  data  (77).  B.  38°  day-five  data  (90).  C  38°  day-six  data 
(79).  D.  Combined  38°  day-seven  and  day-eight  data  (12  day  sevens  and  7  day  eights). 


CHICK  EMBRYO  METABOLIC  PATTERNS 


313 


the  merest  suggestion.  In  the  day-seven  and  day-eight  data  (2d)  a  new  pattern  is 
emergent.  This  seems  to  be  a  more  emphatic  expression  of  the  gross  pattern  of 
day-night  differences  seen  in  the  embryo  throughout  the  period  investigated.  Since 
the  data  for  days  seven  and  eight  are  included  in  the  ratios  cited  in  Figure  1 


104 


100 


96 


104 


100 


96 


104 


100 


96 


6  12  6 

FIGURE  3.  Hourly  means  of  per  cent  of  trend  values.  A.  Day-four  and  day-five  data  from 
three  seasons.  B.  Day-six  data  from  three  seasons.  C.  Day-seven  (open  circles)  and  day-eight 
(closed  circles)  data.  Bars  indicate  standard  errors  of  means. 


314  LELAND  G.  JOHNSON 

(closed  circles),  the  validity  of  this  statement  might  be  open  to  question.  However, 
the  same  comparison  can  be  made  between  the  day-seven  and  day-eight  pattern  and 
that  seen  in  the  ratios  which  exclude  day-seven  and  day-eight  data  (Fig.  1,  open 
circles). 

Figure  3  permits  examination  of  these  variations  on  an  hour-by-hour  basis. 
Because  of  the  similarities  seen  in  the  patterns  of  variation  in  the  day-four  and  day- 
five  data,  the  values  from  both  days  are  grouped  together  in  Figure  3a.  The 
apparently  unique  pattern  of  variation  around  the  trend  line  seen  in  the  day-six 
data  is  seen  again  in  Figure  3b.  The  data  from  days  seven  and  eight  are  plotted 
separately  in  Figure  3c  to  disclose  their  similarity  to  one  another. 

Another  matter  of  concern  in  determining  the  nature  of  metabolic  variability 
during  development  was  the  possibility  that  the  daily  cycle  might  have  a  different 
form  at  different  times  of  the  year.  For  all  seasons  the  greatest  number  of  unin- 
terrupted respirometer-days  was  obtained  for  the  fifth  day  of  incubation.  This 
day  of  incubation  can  serve  as  an  index  of  seasonal  fluctuation  as  the  data  should 
be  fully  comparable.  Means  of  the  per  cent  of  trend  values  for  three-hour  periods 
of  day  five  are  presented  in  Figure  4  (a,  b  and  c).  Similarities  are  seen  in  the 
early  morning  and  afternoon  relationships,  but  a  striking  inversion  in  the  morning 
values  is  seen  with  the  spring  means  varying  in  a  manner  different  from  the  fall  and 
winter  means.  A  comparable  seasonal  difference  was  evident  for  the  fourth  day 
of  incubation.  In  order  to  show  the  daily  cycle  in  day-five  embryos  in  more  detail, 
three-hour  weighted  (1,  2,  1)  moving  means  of  the  hourly  per  cent  of  trend  values 
are  presented  in  Figure  5  (a,  b  and  c).  Differences  among  the  mean  daily  cycles 
become  obvious  upon  examination  of  this  figure.  The  early  morning  hours  ( 1  AM- 
4  AM  values)  in  all  three  cases  show  a  steady  downward  progression  which  begins 
well  above  the  trend  line.  However,  the  similarity  largely  ceases  here.  In  the 
spring,  one  of  the  highest  values  of  the  day  occurs  at  8  AM  while  a  similar  conspicu- 
ous increase  is  lacking  in  the  fall  and  winter  data.  The  pattern  through  the  mid- 
part  of  the  day  in  the  spring  is  essentially  inverted  relative  to  that  of  the  fall  and 
winter  data.  In  the  fall  and  winter,  a  maximum  occurs  over  the  noon  hour.  A 
common  characteristic  of  the  daily  cycles  is  a  mid-afternoon  low  value  which  is 
seen  in  the  data  for  all  three  seasons.  This  low  value  is  followed  in  all  cases  by  a 
sharp  rise  to  a  late  afternoon  high  value  occurring  at  4,  5,  or  6  PM  depending  upon 
the  season  of  the  year.  The  rate  then  drops  off  into  an  evening  pattern  which  is 
similar  in  all  seasons. 

Experiments  designed  to  reveal  information  about  the  temperature  relationships 
of  this  metabolic  variability  were  conducted  in  the  fall.  Control  respirometers  were 
run  at  38°  C.  while  other  respirometers  were  maintained  at  33°  C.  Eggs  were 
transferred  to  both  the  control  and  the  33°  C.  respirometers  on  the  third  day  of 
incubation.  Data  recorded  on  the  fifth  day  will  be  emphasized  here.  At  the 
beginning  of  day  five  the  embryos  in  the  33°  C.  respirometers  have  already  been 
subjected  to  32  or  more  hours  at  the  depressed  temperature.  Initial  comparisons 
with  control  data  from  day  five  can  be  made  by  comparing  Figures  6a  and  5b. 
The  daytime  patterns  of  O2-consumption  variation  are  quite  similar  (note  6  AM- 
8  PM  values),  but  bear  a  different  relationship  to  the  100%  of  trend  line.  This  is 
due  to  the  fact  that  at  33  °  C.  the  early  morning  and  late  evening  values  are  markedly 
depressed  and  this  depression  is  detected  statistically  in  the  form  of  changed  rela- 


CHICK  EMBRYO  METABOLIC  PATTERNS 


315 


tionships  to  the  trend  line.  This  early  morning  and  late  evening  depression  will  he 
discussed  more  extensively  later,  but  it  should  he  borne  in  mind  when  focusing 
attention  on  the  three-hour  mean  values  (Fig.  4d).  If  the  plots  are  compared  point 
by  point,  it  can  be  seen  that  the  pattern  of  variation  follows  that  of  the  control  data 
(Fig.  4b)  quite  well,  but  the  first  predawn  value  and  the  two  evening  values  are 
lowered  in  relation  to  the  trend  line.  It  should  be  noted  especially  that  the  direction 
of  change  from  one  value  to  the  next  is  parallel  in  all  cases  except  the  one  involving 
the  first  predawn  value.  Another  aspect  of  the  data  taken  at  33°  C.  to  be  noted 
is  the  similarity  of  the  patterns  of  variation  on  day  five  and  day  six  (Figs.  6a  and  b, 
4e  and  d).  Finally,  the  changed  relationships  to  the  trend  line  are  obvious  in  the 
ratios  of  hourly  values  above  100%  of  trend  to  values  below  100%  which  are 
presented  in  Figure  1  (half-closed  circles).  This  is  further  indication  of  the 
relatively  greater  depression  of  the  nighttime  oxygen  consumption  by  the  lower 
temperature. 


104       - 


100 


96      - 


104       _ 


100 


96       - 


104       - 


100 


96      - 


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4A 

D 

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V 

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12 


12 


FIGURE  4.  Means  of  hourly  per  cent  of  trend  values  calculated  for  three-hour  periods  of  the 
day.  The  number  of  organism-calendar-days  of  data  involved  in  each  instance  is  indicated  in 
parentheses.  A.  Spring  38°  day  five  (30).  B.  Fall  38°  day  five  (38).  C.  Winter  38°  day 
five  (22).  D.  Fall  33°  day  five  (33).  E.  Fall  33°  day  six  (35).  F.  Winter  "9  AM"  day 
five  (16). 


316 


LELAND  G.  JOHNSON 


104 


100 


96 


104 


100 


96 


104 


100 


96 


104 


100 


96 


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SA 

\ 

A 

1    ( 

• 

\       A. 

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/                   \ 

xV 

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6  12  6 

FIGURE  5.  Three-hour  weighted  moving  means  (1,  2,1)  of  hourly  per  cent  of  trend  values. 
A.  Spring  38°  day  five.  B.  Fall  38°  day  five.  C.  Winter  38°  day  five.  D.  Winter  "9  AM" 
day  five. 

Data  on  the  nature  of  metabolic  variability  when  incubation  was  initiated  at  a 
different  time  of  the  day  were  obtained  in  winter.  They  are  not  as  extensive  as 
might  be  desired  since  only  two  times  of  incubation  were  considered.  Embryos 
whose  incubation  was  initiated  at  9  AM  were  compared  with  embryos  incubated,  as 
in  all  previous  experiments,  at  9  PM.  Only  16  complete  organism-calendar-days  of 
data  were  obtained  for  the  "9  AM"  group,  but  some  suggestive  conclusions  are 


CHICK  EMBRYO  METABOLIC  PATTERNS 


317 


possible.  Again  using  day-five  data  for  comparison  it  can  be  seen  in  the  three-hour 
mean  plots  (Fig.  4f  and  c)  that  the  pattern  of  variability  is  similar.  For  purposes 
of  general  comparison,  attention  is  also  called  to  the  similarity  between  the  "9  AM" 
day-five  pattern  and  the  overall  mean  day-five  pattern  (Fig.  2b).  The  weighted 
moving  mean  plots  (Fig.  5d  and  c)  show  parallels  in  the  early  morning  hours 
where  the  trend  is  downward.  This  is  followed  by  a  peak  over  6  and  7  AM,  a 
depression  in  mid-morning,  and  a  peak  over  the  noon  hour.  The  remainder  of  the 
dav's  pattern  can  be  followed  in  the  same  way.  No  explanation  other  than  small 


104 

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i 

V 

96 

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— 

100 

/v 

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96 

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6  12  6 

FIGURE  6.     Three-hour  weighted  moving  means  (1,  2,  1)  of  hourly  per  cent  of  trend  values. 

A.  33°  day  five.     B.  33°  day  six. 


sample  size  is  readily  apparent  for  the  altered  relationships  to  the  trend  line,  but 
the  emphasis  is  placed  on  similarity  of  pattern. 

DISCUSSION  AND  CONCLUSIONS 

The  data  gathered  in  this  study  confirm  the  conclusion  of  Barnwell  that  sys- 
tematic metabolic  variations  exist  in  the  chick  embryo  as  early  as  the  first  week  of 
incubation.  The  present  investigation  of  this  phenomenon  involved  study  of  the 
forms  of  the  daily  patterns  of  variation  from  the  fourth  through  the  eighth  days  of 
incubation.  The  pattern  seen  during  days  four  and  five  is  one  in  which  several 
peaks  occur,  but  on  days  seven  and  eight  there  seems  to  be  a  somewhat  simpler 
pattern  while  the  pattern  for  day  six  is  peculiar  to  that  day.  This  could  represent 
an  evolution  toward  the  form  of  the  adult  chicken  metabolic  rhythm  which  has  been 
reported  to  be  strongly  diurnal  (Bacq,  1929;  Barott  ct  «/.,  1938).  The  nature  of 


318  LELAND  G.  JOHNSON 

the  changes  in  the  pattern  of  oxygen  consumption  seen  through  this  period  of 
development  can  only  he  described  and  any  explanations  of  these  changes  must  be 
deferred.  However,  it  may  be  postulated  that  during  this  investigated  period  of 
development  some  overt,  energy-requiring  process  becomes  coupled  into  a  geneti- 
cally determined  relationship  with  the  underlying  rhythmicity  reflected  in  the  early 
basal  metabolic  variability.  If  this  coupling  were  a  gradual  process  rather  than  an 
instantaneous  one,  it  would  provide  one  explanation  of  the  failure  of  the  day-six  data 
to  fit  the  pattern  of  either  the  days  which  precede  or  follow  that  day.  Although 
it  is  possible  that  several  or  many  processes  might  be  involved  in  such  a  coupling, 
it  has  been  reported  that  some  important  neuromuscular  integrations  are  being  estab- 
lished at  about  these  stages  of  development  (Hamburger,  1963;  Hamburger  and 
Balaban,  1963).  Energy-requiring  processes  having  a  periodicity  adopted  from 
underlying  rhythmicity  might  be  related  to  this  neuromuscular  integration.  Limb 
motility  could  be  such  a  process.  This  basic  rhythmicity  is  presumed  to  have 
been  present  from  earliest  stages  of  development  and  is  available  to  the  organism 
as  a  means  of  regulating  various  physiological  processes.  The  organism  appears 
to  function  to  a  certain  extent  as  an  amplifier  and  to  produce  large  scale  differences 
with  periodisms  adopted  from  this  subtler  underlying  rhythmic  system.  The  adult 
may  function  even  more  strongly  as  an  amplifier  system  and  this  rhythmicity  could 
be  utilized  to  time  numerous  physiological  events.  By  the  time  that  the  energy 
requirements  of  all  of  these  processes  have  been  met  it  would  no  longer  be  possible 
to  use  metabolic  variability  as  an  index  of  the  true  character  of  the  basic  rhythmic 
nature  of  the  organism. 

Results  obtained  during  different  seasons  indicate  that  there  is  an  annual  com- 
ponent in  the  expression  of  the  fundamental  metabolic  variability  of  the  chick- 
embryo.  Study  of  a  comparable  stage  of  development  (day  five)  at  different  times 
of  the  year  demonstrates  a  seasonal  variation  in  the  form  of  the  daily  pattern  of 
oxygen-consumption.  It  was  noted  that  the  daily  patterns  were  very  different 
from  one  another  in  spring  and  fall.  Although  the  winter  data  were  more  similar 
to  the  fall  data,  in  some  ways  they  appeared  suggestively  to  be  intermediate  between 
the  spring  and  fall  data.  The  pattern  of  annual  variation  seen  in  the  three-hour 
means  shows  similarities  to  the  pattern  of  annual  variation  reported  for  some  other 
organisms.  One  of  the  most  noticeable  elements  of  seasonal  differences  is  the 
marked  change  of  pattern  over  midday.  Such  a  seasonal  noon  difference  was 
reported  for  the  daily  metabolic  pattern  in  the  potato.  In  that  organism  the  noon 
values  are  relatively  low  points  in  the  daily  cycle  during  the  spring  and  high  points 
in  the  fall  of  the  year  (Brown,  1958,  1960).  The  inversion  of  the  late  morning 
mean  (this  mean  includes  the  noon  value)  which  occurs  between  the  spring  and  fall 
data  produces  a  similar  picture  in  the  chick  embryo.  The  late  afternoon  maximum 
is  very  persistent  in  all  seasons  examined,  just  as  reported  for  the  potato.  In 
organisms  such  as  the  potato  (Brown,  1958)  and  bean  (Lutsch,  1962)  another 
aspect  of  seasonal  differences  is  the  variation  in  mean  overall  rate  of  oxygen  con- 
sumption. Although  very  suggestive  data  indicating  higher  metabolic  rates  in  the 
spring  were  obtained  during  this  study,  this  aspect  of  metabolic  variability  in  the 
chick  embryo  will  require  further  work  and  such  experiments  have  been  planned. 

Study  of  temperature  relationships  of  the  metabolic  fluctuations  produced  some 
interesting  results.  Several  lines  of  evidence  indicate  that  the  period  length  of  the 


CHICK  EMBRYO  METABOLIC  PATTERNS  319 

basic  metabolic  rhythm  is  not  altered  by  lowering  the  temperature  of  the  organism. 
Although  the  temperature  of  the  embryos  had  been  lowered  for  32  hours  or  more 
at  the  beginning  of  day  five,  fluctuations  parallel  those  seen  in  the  38°  C.  organisms. 
This  is  particularly  apparent  in  the  pattern  of  variation  seen  in  the  daytime  hours. 
Another  interesting  parallel  is  seen  upon  comparing  the  patterns  of  variability  seen 
in  the  33°  C.  day-five  and  day-six  embryos  (Fig.  6).  These  embryos  on  succeeding 
days  show  a  remarkably  similar  pattern  of  variation.  A  question  might  be  raised 
about  the  relationship  of  these  data  to  those  presented  on  ontogeny  of  the  pattern 
of  metabolic  variability.  It  must  be  remembered  in  this  connection  that  by  the 
beginning  of  what  is  being  called  day  six  here  the  33°  C.  embryos  have  been  sub- 
jected to  the  lowered  temperature  for  56  hours  or  more  and  it  is  very  difficult  to 
say  to  what  extent  the  previously  discussed  ontogenetic  processes  have  been  retarded 
by  the  lowered  temperature.  The  conclusion  drawn  from  these  data  is  that  the 
period  of  the  metabolic  rhythmicity  seems  to  be  temperature-independent  or,  at  least, 
that  the  O10  value  for  the  period  length  is  so  close  to  1.0  that  any  difference  from 
that  value  is  too  small  to  be  detected  by  the  methods  used  in  this  study. 

A  second  result  of  the  lowered  temperature  is  the  suppression  of  the  rate  of 
oxygen  consumption  in  the  early  and  late  portions  of  the  24-hour  period  (nighttime 
values)  relative  to  the  other  values  (daytime  values).  This  suppression  is  detected 
through  linear  regression  analysis  as  an  altered  relationship  to  the  trend  line.  It  is 
an  interesting  conjecture  that  even  at  these  stages  of  development,  there  might  be  a 
regular  periodic  variation  in  the  sensitivity  of  the  organism's  overall  metabolism 
to  temperature  differences. 

The  similarity  of  the  pattern  of  variation  in  organisms  whose  incubation  was 
initiated  at  different  times  of  the  day  demonstrates  that  there  is  no  triggering  asso- 
ciated with  the  beginning  of  incubation,  but  rather  that  the  characteristic  daily 
pattern  will  find  expression  even  if  the  time  of  incubation  is  different.  The  patterns 
of  variation  shown  by  the  "9  AM"  chicks  were  not  12  hours  out  of  phase  with  the 
"9  PM"  chicks,  but  rather  were  in  phase  with  them  and  with  the  time  of  day  even 
under  laboratory  constant  conditions.  The  differences  observed  between  the  small 
sample  of  "9  AM"  chicks  (16  organism-calendar-days)  and  the  controls  are  probably 
due  to  individual  variation  or  to  the  fact  that  ontogenetic  factors  previously  dis- 
cussed may  obscure  the  results  since  the  "9  AM"  chicks  are  12  hours  younger  in 
incubation  age  than  the  "9  PM"  chicks.  In  this  connection,  attention  is  called  to  the 
overall  pattern  of  variation  in  day-five  embryos  (Fig.  2b)  and  also  to  the  day-four 
pattern  (Fig.  2a). 

The  properties  of  this  metabolic  variability  are  of  interest  in  themselves  because 
of  their  implications  concerning  the  ontogeny  of  periodicity,  but  they  also  have  some 
bearing  on  more  general  aspects  of  the  biological  clock  problem.  A  long-standing- 
controversy  in  this  field  has  been  the  support  by  various  workers  in  the  field  of  two 
different  hypotheses  concerning  the  nature  of  the  timing  mechanism  of  biological 
rhythms.  The  hypothesis  of  exogenous  timing  related  to  geophysical  environmental 
factors  has  been  supported  by  Brown  and  others  (Brown,  1959b,  1960,  1962)  in 
recent  years.  An  alternative  hypothesis  is  that  expressed  by  Pittendrigh  and 
others  (e.g.,  Pittendrigh  and  Bruce,  1957,  1959;  Aschoff,  1963)  which  views  the 
timer  mechanism  of  biological  clocks  as  an  antonomous,  endogenous  oscillator 


320  LELAND  G.  JOHNSON 

system  which  is  physico-chemical  in  nature.  The  merits  of,  and  evidences  for, 
these  viewpoints  have  been  discussed  extensively  in  a  recent  work  by  Brown  (1965). 

The  possession  of  seasonal  differences  in  the  daily  patterns  of  variability  has 
some  bearing  on  this  question.  Chick  embryos  whose  development  is  initiated 
at  one  time  of  the  year  and  others  initiated  at  other  times  of  the  year  show  decidedly 
different  patterns  of  daily  variation  in  oxygen  consumption  and  these  seasonal 
patterns  parallel  those  seen  in  other  organisms.  In  the  case  of  an  autonomous 
internal  timer,  the  genetic  mechanisms  which  must  be  hypothesized  to  account  for 
such  seasonal  differences  would  have  to  be  fantastically  complex.  It  seems  simpler 
and  more  direct  to  hypothesize  a  basic  responding  system  which  when  it  is  examined, 
even  in  embryonic  stages,  shows  seasonal  patterns  of  response  reflecting  seasonal 
differences  in  variations  in  geophysical  factors. 

The  significance  of  the  relative  temperature-independence  of  the  period-length 
of  biological  rhythms  has  been  discussed  extensively  in  the  past.  Proponents  of  the 
autonomous  internal  oscillator  hypothesis  assume  that  such  an  inherent  oscillator 
system  would  be  capable  of  temperature  compensation.  Problems  are  posed  for  this 
viewpoint  because  this  type  of  temperature-independence  is  not  ordinarily  seen  in 
biological  chemical  reactions  where  Q10  values  of  2.0-3.0  and  even  higher  are 
common.  An  alternative  explanation  which  is  consistent  with  the  observed  facts 
could  be  that  a  basic  responding  system  exists  and  that  external  time  cues  continue 
to  be  available  to  the  organism  with  regularity,  no  matter  what  the  temperature  of  its 
immediate  environment. 

The  apparent  lack  of  a  triggering  point  demonstrated  by  the  experiments  where 
incubation  was  initiated  at  different  times  of  the  day  provides  another  piece  of 
evidence  related  to  the  nature  of  the  biological  clocks.  One  group  of  embryos  was 
essentially  12  hours  out  of  phase  with  the  other  group  in  their  relationship  to  the 
actual  time  of  day  at  the  beginning  of  incubation.  The  strikingly  similar  patterns 
of  metabolic  variations  seen  in  the  two  groups  is  again  suggestive  of  an  open 
receptor  system  which  responds  to  an  input  related  to  the  exact  time  of  day  even 
under  laboratory  constant  conditions. 


I  am  grateful  to  Professor  Frank  A.  Brown,  Jr.  for  his  advice  and  constructive 
criticism  during  the  course  of  this  study.  Work  in  Professor  Brown's  laboratory 
was  supported  by  grants  from  the  National  Science  Foundation  (GB-469)  and  the 
National  Institutes  of  Health  (GM  07405)  and  by  a  contract  with  the  Office  of 
Naval  Research  (1228-30).  I  was  supported  by  a  National  Science  Foundation 
Predoctoral  Fellowship. 

SUMMARY 

1.  Continuous  recordings  of  oxygen  consumption  were  made  in  order  to  deter- 
mine ontogenetic,  daily,  seasonal,  temperature,  and  other  relationships  of  metabolic 
variability  in  chick  embryos. 

2.  Statistical  treatment  involving  linear  regression  analysis  facilitated  resolution 
of  daily  cycle  forms  and  allowed  various  comparisons. 

3.  The  pattern  of  metabolic  variation  during  days  four  and  five  has  several 
peaks,  but  by  days  seven  and  eight  a  more  markedly  diurnal  pattern  appears.     Day 
six  appears  to  be  an  intermediate  or  transitional  stage. 


CHICK  EMBRYO  METABOLIC  PATTERNS  321 

4.  Seasonal  differences  in  the  form  of  the  daily  cycle  resemble  those  reported  for 
other  organisms. 

5.  Lowering  the  temperature  5°   C.  does  not  affect  the  period  length  of  the 
metabolic  variations.     A  suppression  of  nighttime  metabolism  relative  to  daytime 
metabolism  suggests  the  expression  of  a  diurnal  variation  in  temperature  sensitivity. 

6.  Initiation  of  incubation  at  different  times  of  day  does  not  result  in  different 
basic  cycle  forms. 

7.  Results  obtained  with  chick  embryos  are  suggestive  of  a  receptor  system 
which  is  responsive  to  external  time  cues. 

LITERATURE  CITED 

ASCHOFF,  J.,  1963.  Comparative  physiology:  diurnal  rhythms.  Ann.  Rev.  Physiol,  25:  581-600. 
ASCHOFF,  J.,  ed.,  1965.  Orcadian  Clocks.  Amsterdam,  North-Holland  Publishing  Company. 
ASCHOFF,  J.,  AND  J.  MEYER-LOHMANN,  1954.  Angeborene  24-Stunden-Periodik  beim  Kiicken. 

Pflilgcrs  Arch.,  260:  170-176. 
BACQ,   Z.    M.,   1929.     Sur  1'existence   d'un   rythme   nycthemeral    de   metabolisme   chez   le   coq. 

Ann.  Physiol.  Physicochimie  Biol.,  5:  497-511. 
BARISTWELL,  F.  H.,   1960.     A  solar  daily  variation  in  oxygen  consumption  of  the  embryonated 

egg.     Proc.  Soc.  Exp.  Biol.  Mcd.,  105:  312-315. 
BAROTT,  H.  G.,  J.  C.  FRITZ,  E.  M.  PRINGLE  AND  H.  W.  TITUS,  1938.     Heat  production  and 

gaseous  metabolism  of  young  male  chickens.     /.  Nutrition,  15:  145-167. 
BROWN,    F.    A.,    JR.,    1954.     Simple,    automatic,    continuous-recording    respirometer.     Rev.    Sci. 

Instr.,  25:415-417. 
BROWN,  F.  A.,  JR.,  1957.     Response  of  a  living  organism,  under  "constant  conditions"  including 

pressure,    to   a   barometric-pressure-correlated,    cyclic,    external    variable.     Biol.    Bull., 

112:288-304. 
BROWN,  F.  A.,  JR.,  1958.     An  exogenous  reference-clock  for  persistent  temperature-independent. 

labile  biological  rhythms.     Biol  Bull,  115:  81-100. 
BROWN,  F.  A.,  JR.,  1959a.     The  rhythmic  nature  of  animals  and  plants.     Aincr.  Scientist.  47: 

147-168. 

BROWN,  F.  A.,  JR.,  1959b.     Living  clocks.     Science,  130:  1535-1554. 
BROWN,  F.  A.,  JR.,  1960.     Response  to  pervasive  geophysical  factors  and  the  biological  clock 

problem.     Cold  Spring  Harbor  Syiup.  Quant.  Biol.,  25:  57-71. 
BROWN,   F.   A.,   JR.,    1962.     Extrinsic   rhythmicity :   a   reference   frame   for   biological    rhythms 

under  so-called  constant  conditions.    Ann.  N.  Y.  Acad.  Sci.,  98:  775-787. 
BROWN,  F.  A.,  JR.,  1965.     A  unified  theory  for  biological  rhythms.     In:   Circadian  Clocks.     J. 

Aschoff,   ed.,   pp.   231-261.     Amsterdam,   North-Holland   Publishing   Company. 
BUNNING,  E.,  1964.     The  Physiological  Clock.     Berlin,  Springer-Verlag. 
CLOUDSLEY-THOMPSON,   J.    L.,    1961.     Rhythmic   activity    in   animal    physiology   and   behavior. 

New  York,  Academic  Press. 
CROXTON,  F.  E.,  AND  D.  J.  COWDEN,  1955.     Applied  General   Statistics.     2nd  ed.,   Englewood 

Cliffs,  New  Jersey,  Prentice-Hall,  Inc. 
GREIFF,   D.,   AND   H.    PINKERTON,    1948.     Effect   of   enzyme    inhibitors   and   activators    on   the 

multiplication  of  typhus  rickettsiae.     III.     Correlation  of  effects  of  PABA  and  KCN 

with  O2-consumption  in  embryonate  eggs.     /.  Exp.  Mcd.,  87:  175-197. 
HAMBURGER,  V.,  1963.     Some  aspects  of  the  embryology  of  behavior.     Quart.  Rev.  Biol.  38: 

342-365. 
HAMBURGER,    V.,    AND    M.    BALABAN,    1963.     Observations    and    experiments    on    spontaneous 

rhythmical  behavior  in  the  chick  embryo.     Dcv.  Biol,  7:  533-545. 
HARKER,  J.  E.,  1958.     Diurnal  rhythms  in  the  animal  kingdom.     Biol.  Rev.,  33:  1-52. 
HARKER,  J.  E.,  1964.     The  Physiology  of  Diurnal  Rhythms.     Cambridge,  Cambridge  University 

Press. 
HASSELBALCH,    K.    A.,    1900.     Ueber    den   respiratorischen    Stoffwechsel    des    Huhnerembryos. 

Skand.  Arch.  Physiol.,  10:  353-402. 


322  LELAND  G.  JOHNSON 

HELLBRUGGE,  T.,  1960.  The  development  of  circadian  rhythms  in  infants.  Cold  Spring  Harbor 
Symp.  Quant.  Biol,  25:  311-323. 

HEUSNER,  A.,  1963.  Analyse  de  la  variation  nycthemeral  du  metabolism  energetique  chez  le 
rat  blanc.  Doctoral  Dissertation,  Strasbourg. 

HEUSNER,  A.,  1965.  Sources  of  error  in  the  study  of  diurnal  rhythm  in  energy  metabolism. 
In:  Circadian  Clocks.  J.  Aschoff,  ed.,  pp.  3-12.  Amsterdam,  North-Holland  Publish- 
ing Company. 

HEUSNER,  A.,  AND  J.  P.  ZAHND,  1963.  Etude  de  la  consommation  d'oxygene  de  1'embryon  de 
poulet  au  cours  du  nycthemere.  C.  R.  Soc.  Biol.,  Paris,  157:  1498-1501. 

HIEBEL,  G.,  AND  C.  KAYSER,  1949.  Le  rythme  nycthemeral  de  1'activite  et  de  la  calorification 
chez  1'embryon  de  poulet  et  le  jeune  poulet  (Light  Sussex).  C.  R.  Soc.  Biol.,  Paris, 
143:864-866. 

HOFFMANN,  K.,  1957.     Angeborene  Tagesperiodik  bei  Eidechsen.     Natitnviss..  44:  359-360. 

HOFFMANN,  K.,  1959.  Die  Aktivitatsperiodik  von  im  18-  und  36-Stunden-Tag  erbrtiteten 
Eidechsen.  Zcitschr.  vcrgl  Phys'wl.,  42 :  422-432. 

JOHNSON,  L.  G.,  1965.  Studies  on  metabolic  variations  in  embryonated  chicken  eggs  at  several 
developmental  stages.  Doctoral  Dissertation,  Northwestern  Univ.,  Evanston,  Illinois. 

LUTSCH,  E.  F.,  1962.  Rhythmic  variations  of  oxidative  metabolism  in  bean  seedlings.  Doctoral 
Dissertation,  Northwestern  Univ.  Evanston,  Illinois. 

McLiMANS,  W.  F.,  R.  A.  SIEM  AND  V.  R.  SCHOLLJEGERDES,  1950.  A  physiological  study  of 
virus  parasitism.  I.  A  method  for  determining  the  oxygen  uptake  of  individual 
embryonated  eggs.  /.  Immunol.,  64:  463-473. 

McLiMANS,  W.  F.,  R.  A.  SIEM,  B.  C.  MARK  AND  E.  PINSKA,  1950.  A  physiological  study  of 
virus  parasitism.  II.  The  effect  of  environmental  temperature  on  the  rate  of  oxygen 
consumption  of  normal  eggs  and  eggs  infected  with  Newcastle  disease  virus.  /. 
Immunol.,  64:  475-485. 

MURRAY,  H.  A.,  JR.,  1925.  Physiological  ontogeny.  A.  Chicken  embryos.  III.  Weight  and 
growth  as  functions  of  age.  J.  Gen.  Physio!.,  9:  39-48. 

MURRAY,  H.  A.,  JR.,  1926.  Physiological  ontogeny.  A.  Chicken  embryos.  XII.  The  metabo- 
lism as  a  function  of  age.  /.  Gen.  Physiol.,  10:  337-343. 

PETREN,  T.,  AND  A.  SOLLBERGER,  1953.  Die  24-Stunden  Rhythmik  des  Leberglykogens  bei 
Hiihnerembryonen  und  Kuken  verschiedenen  Alters  nebst  studien  iiber  die  Unabhangigkeit 
der  Rhythmik  von  ausseren  Faktoren.  Acta.  Med.  Scand.,  145:  suppl.  278,  pp.  54-66. 

PITTENDRIGH,  C.  S.,  AND  V.  G.  BRUCE,  1957.  An  oscillator  model  for  biological  clocks.  In: 
Rhythmic  and  Synthetic  Processes  in  Growth.  Dorothea  Rudnick,  ed.,  pp.  75-109. 
Princeton,  Princeton  Univ.  Press. 

PITTENDRIGH,  C.  S.,  AND  V.  G.  BRUCE,  1959.  Daily  rhythms  as  coupled  oscillator  systems  and 
their  relation  to  thermoperiodism  and  photoperiodism.  In:  Photoperiodism  and  Related 
Phenomena  in  Plants  and  Animals.  R.  B.  Withrow  ed.,  pp.  475-505.  Washington, 
D.  C.,  American  Association  for  the  Advancement  of  Science. 

SIMPSON,  G.  G.,  A.  ROWE  AND  R.   C.  LEWONTIN,   1960.     Quantitative  Zoology.     New   York, 

Harcourt,  Brace  and  Company. 

SOLLBERGER,  A.,  1965.  Biological  Rhythm  Research.  New  York,  American  Elsevier  Publish- 
ing Company,  Inc. 

WEBB,  H.  M.,  AND  F.  A.  BROWN,  JR.,  1959.  Timing  long-cycle  physiological  rhythms. 
Physiol.  Rev.,  39:  127-161. 


THE  ROLE  OF  DNA  SYNTHESIS  IN  THE  DETERMINATION  OF 
AXIAL  POLARITY  OF  REGENERATING  PLANARIANS  x 

D.  M.  KOHL  AND  R.  A.  FLICKINGER 

Department  of  Biology,  State  University  of  New  York,  Buffalo,  New  York  14214 

It  has  been  shown  that  treatment  of  intact  planar ians  with  colcemide  (deacetyl- 
methylcolchicine)  and  chloramphenicol,  followed  by  cutting  the  worms  to  remove 
the  heads  and  tails,  will  cause  the  regeneration  of  bipolar  heads  in  a  significant 
number  of  these  cut  planarians  (Flickinger,  1959).  When  applied  locally  to  the 
prospective  anterior  ends  of  cut  pieces  of  worms,  these  compounds  could  reverse 
the  normal  polarity  of  the  worms  (Flickinger,  1959;  Flickinger  and  Coward,  1962). 
It  was  further  shown  that  both  of  these  compounds  inhibit  the  extent  of  incorpora- 
tion of  C14O2  into  a  trichloroacetic  acid-insoluble  residue,  which  is  primarily  protein. 
The  classical  action  of  colchicine,  or  its  derivatives,  is  to  inhibit  cell  division,  while 
the  action  of  chloramphenicol  in  bacteria  is  to  inhibit  protein  synthesis  without 
significantly  inhibiting  nucleic  acid  synthesis  (Gale  and  Folks,  1953;  Midgley  and 
McCarthy,  1962) .  An  accumulation  of  RNA  in  bacteria  treated  with  chloram- 
phenicol has  been  observed  (Dubin  and  Elkart,  1965)  and  such  RNA  has  some 
similarities  to  messenger  RNA  (Hahn  and  Wolfe,  1962). 

One  purpose  of  this  investigation  was  to  find  the  effects  of  chloramphenicol 
upon  RNA  and  DNA  synthesis  of  the  planarians.  Does  chloramphenicol  stimulate 
or  inhibit  RNA  synthesis?  Is  there  an  inhibition  of  DNA  synthesis  by  chloram- 
phenicol ?  If  so,  what  is  the  per  cent  of  inhibition  of  DNA  synthesis  after  periods 
of  exposure  which  cause  bipolar  head  formation,  compared  to  shorter  times  of 
exposure  which  do  not  affect  polarity  of  the  regenerating  worms  ?  Another  aim  of 
the  present  investigation  was  to  determine  the  temporal  course  of  DNA,  RNA  and 
protein  synthesis  in  the  regenerating  head  and  tail  blastemata  during  the  first  24 
hours  after  cutting  the  worms.  A  study  of  RNA  and  protein  synthesis  in  regener- 
ating planarians  has  been  made  (Coward  and  Flickinger,  1965),  but  these  determi- 
nations were  made  at  daily  intervals  for  a  7-day  period. 

MATERIALS  AND  METHODS 

The  planarians,  Dugcsia  tigrina,  maintained  in  aerated  tap  water,  were  starved 
for  7-10  days  before  being  used  in  an  experiment.  The  effect  of  chloramphenicol 
upon  RNA  and  DNA  synthesis  was  tested  by  exposing  100  worms  for  12  or  24 
hours  to  25  ju.c./ml.  of  uridine-2-C14  or  C14-thymidine  in  1  ml.  of  boiled  tap  water 
containing  chloramphenicol  succinate  (1.5  mg./ml.).  A  similar  number  of  control 
worms  was  cultured  for  similar  periods  in  boiled  tap  water,  containing  the  labeled 
compounds.  At  the  conclusion  of  the  incubation  period  the  worms  were  washed 
five  times  with  boiled  tap  water,  by  which  time  the  final  rinse  water  had  a  level 

1  This  investigation  was  supported  by  a  grant  from  the  National  Science  Foundation. 

323 


324  D.  M.  KOHL  AND  R.  A.  FLICKINGER 

of  activity  similar  to  the  background.  The  worms  were  then  homogenized  in  cold 
5%  trichloroacetic  acid  (TCA)  and  the  pellet  washed  three  times  by  centrifugation, 
which  reduced  the  level  of  isotopic  activity  of  the  wash  to  that  of  the  background. 
The  residue  was  extracted  twice  with  1:1  ethanol-ether,  twice  with  1:4  ethanol- 
ether,  and  once  with  ether  alone  to  remove  lipids.  The  residues  were  then  dried. 
For  the  C14-uridine  experiments  RNA  was  hydrolyzed  with  1  ml.  of  0.3  N  KOH  at 
37°  C.  for  20  hours.  The  hydrolysate  was  acidified  with  0.3  ml.  of  1  N  perchloric 
acid  to  precipitate  the  DNA  and  protein  and  the  supernatant  was  neutralized  with  1 
N  KOH  to  precipitate  the  perchlorate.  The  concentrations  of  RNA  were  deter- 
mined by  the  orcinol  method  (Dische,  1955)  and  the  samples  were  plated  and 
counted  on  a  thin  window  proportional  counter.  The  procedure  for  the  C14- 
thymidine  incubations  was  similar  except  that  both  RNA  and  DNA  were  hydrolyzed 
with  hot  trichloroacetic  acid  (96°  C.)  for  40  minutes  and  the  hydrolysate  was 
extracted  three  times  with  ether  to  remove  the  trichloroacetic  acid.  The  diphenyl- 
amine  method  (Dische,  1955)  was  used  to  determine  DNA  concentration  before 
the  samples  were  plated  and  counted. 

For  the  incubations  with  C14O2  100  cut  worms  were  transferred  to  1  ml.  of 
recently  boiled  tap  water  in  a  small  dish.  A  small  amount  of  lactic  acid  was  added 
to  generate  C14O2  from  150  /j.c.  of  BaC14O3  in  a  center  well  and  the  dish  was  quickly 
sealed  to  prevent  the  escape  of  C14O2.  After  the  incubation,  the  worms  were 
processed  as  before  for  hydrolysis  of  RNA  with  0.3  N  KOH,  and  DNA  of  the 
residue  was  hydrolyzed  with  hot  TCA.  The  protein  residue  from  the  DNA 
hydrolysis  was  dissolved  in  formic  acid,  the  protein  concentration  determined 
(Lowry  et  al.,  1951)  and  then  this  fraction  was  plated  and  counted. 

RESULTS 
Biological  experiments  ivitJi  chloramphenicol 

In  experiments  designed  to  find  the  minimal  concentration  of  chloramphenicol 
that  would  produce  bipolar  heads  in  regenerating  planaria,  50  whole  worms  were 
incubated  in  boiled  tap  water  containing  0.1%  penicillin  and  streptomycin  and  50 
were  placed  in  a  similar  solution  containing  chloramphenicol  succinate  (1.5  mg./ml.). 
This  concentration  of  chloramphenicol  succinate  is  equivalent  to  1  mg./ml.  chloram- 
phenicol. Similar  numbers  of  worms  were  cultured  in  0.75,  0.325  and  0.167 
mg./ml.  of  chloramphenicol  succinate.  After  24  hours  of  incubation  the  worms 
were  each  cut  just  back  of  the  head  and  in  front  of  the  pharynx  and  the  cut 
pieces  were  allowed  to  regenerate  in  boiled  tap  water  containing  0.1%  penicillin  and 
streptomycin.  Thirty  per  cent  of  the  cut  pieces  in  chloramphenicol  succinate  (1.5 
mg./pil.)  subsequently  developed  heads  at  each  cut  end,  but  no  bipolar  heads  were 
observed  in  the  pieces  allowed  to  develop  in  the  boiled  tap  water  alone  or  in  the 
lower  concentrations  of  chloramphenicol.  In  order  to  learn  if  the  polarity  of  the 
regenerating  worms  could  be  affected  by  exposure  to  chloramphenicol  after  the 
worms  were  cut,  60  worms  were  cut  in  a  similar  fashion  to  provide  pre-pharyngeal 
pieces  and  these  were  then  placed  in  chloramphenicol  succinate  (1.5  mg./ml.)  for 
24  hours.  Subsequent  culture  of  these  regenerating  pieces  provided  only  two  cases 
of  bipolar  head  formation. 

To  determine  the  minimal  period  of  exposure  to  chloramphenicol  necessary  to 


DNA  SYNTHESIS  IN  REGENERATION 


325 


produce  bipolar  heads,  groups  of  20  whole  worms  were  incubated  in  chlorampheni- 
col  succinate  (1.5  mg./ml.)  for  periods  of  2,  8,  12,  16,  20  and  24  hours  and  the 
pre-pharyngeal  pieces  were  obtained  by  cutting  the  worms.  These  were  allowed 
to  regenerate  in  boiled  tap  water  containing  0.1  %  penicillin  and  streptomycin  and 
the  water  was  changed  each  day.  No  cases  of  bipolar  heads  occurred  in  worms 
exposed  to  chloramphenicol  succinate  (1.5  mg./ml.)  for  less  than  20  hours.  Two 
of  the  worms  exposed  for  20  hours  and  eight  of  those  exposed  for  24  hours 
regenerated  heads  at  each  end. 

Isotopic  experiments  with  chloramphenicol 

Previous  work  had  shown  that  chloramphenicol  inhibits  the  incorporation  of 
C14O2  into  the  trichloroacetic  acid-insoluble  fraction  of  planarians  (Flickinger, 
1959).  To  ascertain  the  effect  of  chloramphenicol  upon  RNA  synthesis,  100 
intact  worms  were  incubated  separately  in  1  ml.  of  boiled  tap  water  containing 
chloramphenicol  succinate  (1.5  mg./ml.)  and  25  /AC. /ml.  of  uridine-2-C1*  for  24 
hours  at  18°  C.  One  hundred  control  worms  were  incubated  similarly  except  that 
chloramphenicol  was  not  present.  The  RNA  was  hydrolyzed  and  counted  and  the 
results  show  that  worms  in  chloramphenicol  incorporated  less  C14-uridine  into  the 
RNA  fraction  (Table  I).  There  was  a  24.8%  inhibition  based  on  the  activity/100 
worms,  while  there  w^as  a  28.8%  inhibition  on  the  basis  of  specific  activity. 

A  similar  type  of  experiment  was  performed  to  find  the  effect  of  chloramphenicol 
upon  DNA  synthesis.  One  group  of  100  worms  was  incubated  in  boiled  tap 
water  containing  25  /AC. /ml.  of  C14-thymidine  and  chloramphenicol  succinate  (1.5 
mg./ml.)  for  24  hours  at  18°  C.  while  the  incubation  of  the  control  group  of  100 
worms  did  not  contain  chloramphenicol.  Another  two  groups  of  100  worms  were 
incubated  separately  for  12  hours  with  25  /AC. /ml.  of  C14-thymidine,  and  chloram- 
phenicol succinate  (1.5  mg./ml.)  was  present  in  one  of  those  incubations.  From 
the  biological  experiments  it  was  known  that  a  24-hour  exposure  to  this  concentra- 
tion of  chloramphenicol  would  produce  bipolar  heads  in  regenerating  worms,  while 
the  12-hour  exposure  had  no  effect  upon  polarity.  This  experiment  offered  the 
chance  to  compare  the  effect  of  the  biologically  active  and  inactive  doses  of  chloram- 
phenicol upon  the  incorporation  of  C14-thymidine  into  DNA.  The  results  of  this 
experiment  are  given  in  Table  II.  There  was  a  39.1%  inhibition  of  C14-thymidine 
incorporation  into  DNA  with  a  24-hour  exposure  to  chloramphenicol,  and  a  10.3% 
inhibition  following  a  12-hour  exposure. 

TABLE  I 

Effect  of  chloramphenicol  upon  incorporation  of  Cu-uridtne  into  RNA  of  intact  planarians. 

Incubation  of  100  worms  for  24  hours  at  18°  C,  in  boiled  tap  water  containing 

25  fic./ml.  of  uridine-2-Cu,  and  similar  incubation  of  another  100 

•worms  with  chloramphenicol  succinate  (1.5  mg./ml.}  present 


Cpm 

Cpm 

Per  cent  inhibition  of 
specific  activity 

100  worms 

mg.  RNA 

Control  worms 

4378 

8420 

Chloramphenicol  succinate 
(1.5  mg./ml-)  worms 

3303 

6000 

28.8 

326 


D.  M.  KOHL  AND  R.  A.  FLICKINGER 


TABLE  II 

Effect  of  chloramphenicol  upon  incorporation  of  Cu-thymidine  into  DNA  of  intact  planarians. 
Incubation  of  four  groups  of  100  worms  for  12  and  24  hours  at  18°  C.  in  boiled 
tap  water  containing  25  nc./ml.  of  Cu-thymidine.  Chloramphenicol 
succinate  (1.5  mg./ml.)  was  present  in  one  of  the  12- 
hour  and  one  of  the  24-hour  incubations 


Cpm 

Cpm 

Per  cent  inhibition  of 
specific  activity 

100  worms 

mg.  RNA 

Control,  12  hours 

447 

1241 

Chloramphenicol  succinate, 

368 

1115 

10.3 

12  hours 

Control,  24  hours 

598 

1245 

Chloramphenicol  succinate, 

303 

758 

39.1 

24  hours 

DNA,  RNA  and  protein  synthesis  oj  blastemata 

Exposure  of  cut  worms  to  chloramphenicol  for  24  hours  did  not  alter  their 
regeneration  polarity,  while  a  similar  period  of  exposure  of  whole  worms  to  chlor- 
amphenicol, followed  hy  cutting  to  obtain  pre-pharyngeal  pieces,  produced  bipolar 
heads  in  40%  of  the  regenerating  worms.  This  suggested  that  the  mechanisms 
acounting  for  establishment  of  normal  polarity  operate  during  the  first  24  hours 
after  cutting  the  worms.  The  rates  of  DNA,  RNA  and  protein  synthesis  during 
this  period  were  examined  in  the  following  manner.  For  each  experiment  100 
worms  were  cut  transversely  in  front  of  the  pharynx  at  level  X  (Fig.  1)  and  the 
anterior  and  posterior  parts  of  these  worms  were  allowed  to  regenerate  for  2,  4,  6 
or  24  hours  in  boiled  tap  water.  C14O2  was  generated  from  150  /JLC.  of  BaC14O3 
and  the  dish  was  sealed  and  incubated  at  18°  C.  for  three  hours.  The  cut  worms 
were  then  washed  five  times  with  boiled  tap  water,  fixed  in  5%  TCA  and  cut  into 
A2,  A1?  Px,  and  P2  parts,  according  to  Figure  1.  The  head  and  tail  blastemata 
(Pt  and  Ax),  and  the  areas  of  tissues  adjacent  to  the  head  and  tail  blastemata 
(P2  and  A2)  were  homogenized  separately  in  cold  5%  TCA  and  the  centrifugal 
residues  were  washed  four  times  by  centrifugation  and  washing  with  cold  5%  TCA. 
The  DNA,  RNA  and  protein  fractions  were  prepared  according  to  previously 
outlined  methods  and  these  fractions  were  plated  and  counted.  Orcinol  tests 
revealed  the  absence  of  RNA  in  the  DNA  fraction  and  the  diphenylamine  reaction 
showed  there  was  no  DNA  in  the  RNA  fraction.  Examination  of  the  results 
(Table  III)  reveals  only  slight  differences  of  incorporation  of  C14O2  into  DNA, 
RNA  and  protein  of  the  four  areas  of  the  worms  which  had  regenerated  two  hours 
without  label  and  three  hours  with  the  label.  The  blastemata  (P±  and  At)  and 
adjacent  areas  (P2  and  A2)  obtained  from  cut  worms  that  had  regenerated  4  and  6 
hours  without  label,  plus  three  hours  in  labeled  C14O2,  showed  significant  differences 
in  the  levels  of  incorporation  of  label  into  DNA,  RNA  and  protein.  By  this  time 
the  DNA,  RNA  and  protein  fractions  of  regenerating  areas  (Px  and  A:)  had 
higher  isotopic  activities  than  the  non-regenerating  areas  (P2  and  A2).  After  6 
hours  of  regeneration  without  C14O2,  and  three  hours  with  C1462,  the  head  blastema 
fractions  (DNA,  RNA  and  protein)  incorporated  more  label  than  the  tail 


DMA  SYNTHESIS  IN  REGENERATION 


327 


blastema  (Ax)  fractions.  The  non-regenerating  areas  near  the  head  hlastemata  had 
higher  activities  than  similar  areas  near  the  tail  blastemata  (P2  >  A,).  The  great- 
est amount  of  C14O2  incorporation  into  DNA  occurred  at  4  hours  for  the  tail 
blastemata  (AJ  and  the  adjacent  area  (A2),  while  the  incorporation  of  label  into 
both  DNA  and  RNA  of  all  the  other  fractions  was  greatest  at  6  hours.  In  com- 
paring the  levels  of  isotope  incorporation  of  the  worms  allowed  to  regenerate  two 
hours  in  unlabeled  medium  and  three  hours  in  C14O,  with  those  in  which  the  worms 
regenerated  four  or  six  hours  in  unlabeled  medium  and  three  hours  in  C14(X  sig- 
nificant increases  of  incorporation  of  labeled  precursor  into  DNA  and  RNA  were 
observed  in  the  four-  and  six-hour  experiments.  Furthermore,  there  is  a  marked 
stimulation  of  DNA  synthesis  in  the  blastemata  of  worms  that  regenerated  four 
hours  in  unlabeled  medium,  but  no  further  significant  stimulation  of  labeled  pre- 
cursor into  DNA  after  six  hours  of  regeneration  in  unlabeled  medium.  The  extent 
of  stimulation  of  RNA  synthesis,  particularly  in  the  anterior  pieces  (P1  and  P2), 
was  greater  between  four  and  six  hours  of  regeneration  in  unlabeled  medium,  as 
compared  to  the  difference  between  two  and  four  hours  of  regeneration  in  the  un- 
labeled medium.  The  specific  activities  of  the  protein  fraction  were  maximal  after 
24  hours  of  regeneration  in  tap  water  and  three  hours  with  the  label.  The  differ- 
ences in  isotopic  activities  of  the  protein  fraction  between  the  head  and  tail  blastemata 


FIGURE  1.  For  each  experiment  100  worms  were  cut  at  level  x  and  allowed  to  regenerate 
for  2,  4,  6  and  24  hours.  The  blastemata  (A!  and  Pi)  and  adjacent  areas  (A«  and  P->)  were  cut 
and  then  incubated  separately  for  three  hours  in  1  ml.  of  boiled  tap  water  containing  C14O2 
generated  from  150  /*c.  of  BaC14O::. 


328 


D.  M.  KOHL  AND  R.  A.  FLICKINGER 


TABLE  1 1 1 

Incorporation  of  C1402  into  DNA,  RNA  and  protein  of  head  and  tail  blastemata  and  adjacent  regions 

during  the  first  24  hours  of  regeneration.  For  each  experiment  100  worms  were  cut  anterior  to 

the  pharynx  (Fig.  1)  and  the  pieces  allowed  to  regenerate  for  2,  4,  6  or  24  hours  at  18°C. 

The  pieces  (A\,  A2,  Pi,  P2)  were  then  cut  according  to  Fig.l  and  the  pieces  of  each 

kind  were  incubated  for  3  hours  at  18°C.  in  1  ml.  of  boiled  tap  water 

with  C1402  generated  from  150  ^c.  of  BaCu03 


Cpm 

Tissue 

mg. 

Time 

2  hrs. 

4  hrs. 

6  hrs. 

24  hrs. 

DNA 

Tail  blastema    Ai 

14370 

35140 

33900 

18700 

As 

15140 

27290 

25600 

16800 

Head  blastema  PI 

14340 

39780 

40800 

22760 

P, 

13500 

25680 

29400 

13660 

RNA 

Tail  blastema    Ai 

17860 

25350 

32430 

30913 

A2 

15230 

21300 

26955 

22220 

Head  blastema  PI 

18490 

21390 

43250 

35880 

P, 

17640 

17160 

28980 

28500 

Protein 

Tail  blastema    AI 

1370 

1520 

1285 

1820 

A2 

1250 

1088 

1080 

1340 

Head  blastema  PI 

1445 

1863 

1760 

2840 

P-2 

1130 

1187 

1440 

2600 

>  Ai),  the  adjacent  areas  (P2  >  A2),  and  the  regenerating  and  non-regenerat- 
ing areas  (Px  >  P2 ;  Ax  >  A2),  were  also  maximal  at  24  hours. 

DISCUSSION 

The  results  of  this  investigation  show  that  dosages  of  chloramphenicol  succinate 
(1.5  mg./ml.  for  24  hours)  that  can  induce  the  formation  of  bipolar  heads  in 
regenerating  planarians  inhibit  RNA  and  DNA  synthesis  (Tables  I  and  II).  The 
inhibition  of  incorporation  of  C14-uridine  into  RNA  (Table  I)  argues  against  the 
idea  that  the  morphogenetic  activity  of  chloramphenicol  is  due  to  an  accumulation 
of  RNA,  as  can  occur  in  bacteria  (Dttbin  and  Elkart,  1965).  Incubation  of  the 
worms  with  labeled  thymidine  and  chloramphenicol  succinate  (1.5  mg./ml.)  for  24 
hours  revealed  a  severe  inhibition  (39.1%)  of  DNA  synthesis  (Table  II).  Ex- 
posure of  whole  worms  to  this  same  concentration  of  chloramphenicol  for  12  hours 
does  not  alter  the  normal  polarity  during  subsequent  regeneration  and  this  length 
of  exposure  to  chloramphenicol  resulted  in  a  10.3%  inhibition  of  DNA  synthesis 
(Table  II).  The  data  from  these  experiments  show  that  a  marked  inhibition  of 
DNA  synthesis  by  chloramphenicol  is  essential  in  order  to  alter  the  polarity  of  the 
regenerating  worms.  Previous  work  has  shown  that  exposure  of  intact  worms 
to  the  mitotic  poison,  colcemide,  can  affect  the  polarity  of  these  worms  during  their 


DNA  SYNTHESIS  IN  REGENERATION  329 

regeneration  (Kanatani,  1958;  Flickinger,  1959)  and  emphasizes  that  cell  division 
is  a  critical  factor  for  maintenance  of  normal  polarity. 

Exposure  of  cut  worms  to  chloramphenicol  succinate  (1.5  mg./ml.)  for  24  hours 
did  not  alter  their  normal  polarity.  This  agrees  with  the  results  of  Kanatani 
(1958)  who  found  that  colcemide  did  not  alter  polarity  when  applied  to  cut  worms. 
This  suggests  that  the  critical  events  that  maintain  polarity  in  cut  pieces  of 
planarians  are  established  sometime  during  the  first  24  hours  after  the  worms  are 
sectioned.  Investigation  of  C14O2  incorporation  of  head  and  tail  blastemata  (Pj  and 
Ai),  and  adjoining  areas  (P2  and  A2),  revealed  that  DNA,  RNA  and  protein 
synthesis  are  stimulated  7-9  hours  after  cutting  the  worms.  Furthermore,  the  head 
blastema  (Pj  has  a  significantly  higher  incorporation  of  C14Oo  into  these  fractions 
than  the  tail  blastema  (At)  by  this  time.  In  reference  to  the  2-5-hour  levels  of 
incorporation  the  greatest  stimulation  of  DNA  synthesis  occurred  4-7  hours  after 
cutting  and  the  maximal  increase  of  RNA  synthesis  from  6-9  hours  after  cutting, 
while  the  maximal  incorporation  of  C14O2  into  the  protein  fractions  occurred  24-27 
hours  after  cutting.  It  appears  that  the  rate  of  protein  synthesis  is  maximal  at  the 
time  of  actual  differentiation  while  the  rates  of  DNA  and  RNA  synthesis  increase 
during  the  period  of  the  determination  of  axial  polarity.  It  does  seem  that  the 
time  of  greater  stimulation  of  DNA  synthesis  precedes  the  time  of  maximal  stimula- 
tion of  RNA  synthesis,  using  the  two-hour  period  of  regeneration  in  unlabeled 
medium  as  the  reference. 

It  has  been  found  that  the  action  of  chloramphenicol  in  producing  bipolar  heads 
is  correlated  with  a  severe  inhibition  of  DNA  synthesis,  and  that  one  of  the  first 
metabolic  activities  to  show  maximal  stimulation  during  regeneration  is  DNA 
synthesis.  This  suggests  that  cell  division  plays  an  important  role  in  the  establish- 
ment of  axial  polarity  in  planarians.  However,  cytological  data  relating  to  mitotic 
frequency  are  necessary  to  confirm  this  suggestion. 

SUMMARY 

1.  Levels  of  chloramphenicol  which  can  produce  bipolar  heads  in  regenerating 
planarians  were  found  to  inhibit  severely  the  synthesis  of  both  DNA  and  RNA. 
A  shorter  period  of  exposure  to  chloramphenicol,  which  did  not  affect  the  polarity  of 
the  regenerating  worms,  produced  only  a  slight  inhibition  of  DNA  synthesis. 

2.  Incubation  of  blastemata  and  adjacent  areas  of  regenerating  planarians  with 
labeled  CO2  for  periods  of  2,  4,  6  and  24  hours  revealed  a  maximal  stimulation 
of  DNA  at  4-7  hours  and  of  RNA  at  6-9  hours  after  the  worms  were  cut.     It  is  at 
this  time  that  the  blastemata  destined  to  form  heads  attain  a  higher  level  of  DNA 
and  RNA  synthesis  than  the  blastemata  which  will  form  tails.     The  maximal  stimu- 
lation of  protein  synthesis  occurred  24-27  hours  after  cutting  the  worms. 

3.  The  necessity  of  obtaining  a  severe  inhibition  of  DNA  synthesis  with  chlor- 
amphenicol in  order  to  produce  bipolar  heads  in  regenerating  worms,  as  well  as 
the  sequential  nature  of  DNA,  RNA  and  protein  synthesis  in  the  regeneration 
blastemata,   suggests  that  the  stimulation  of  DNA   synthesis   is   involved   in   the 
establishment  of  polarity  of  regenerating  worms.     These  patterns  of  DNA  synthesis 
may  reflect  the  incidence  of  cell  division  during  this  period. 


330  D.  M.  KOHL  AND  R.  A.  FLICKINGER 

LITERATURE  CITED 

COWARD,    S.   J.,   AND   R.   A.   FLICKINGER,    1965.     Axial   patterns    of   protein   and    nucleic    acid 

synthesis  in  intact  and  regenerating  planaria.     Grozvth,  29:  151-163. 
DISCHE,  Z.,  1955.     Color  reactions  of  the  nucleic  acid  components.    In:  The  Nucleic  Acids,  Ed. 

E.  Chargaff  and  J.  Davidson,  Vol.  1,  pp.  285-305,  Academic  Press,  New  York. 
DUBIN,  D.  T.,  AND  A.  T.  ELKART,   1965.     A  direct  demonstration  of  the  metabolic  turnover 

of  chloramphenicol  RNA.     Biochein.  Biophys.  Ada,  103:  355-358. 
FLICKINGER,  R.  A.,   1959.     A  gradient  of  protein  synthesis  in  planaria  and  reversal  of  axial 

polarity  of  regenerates.     Grmvth,  24:  251-271. 
FLICKINGER,    R.   A.,   AND   S.   J.    COWARD,    1962.     The   induction   of   cephalic    differentiation    in 

regenerating    Ditt/csia    dorotocephala    in    the    presence    of    the    normal    head    and    in 

unwounded  tails.     Dcv.  Biol.,  5:  179-204. 
GALE,  E.  F.,  AND  J.  P.  FOLKES,  1953.     The  assimilation  of  amino-acids  by  bacteria.     15.    Action 

of  antibiotics  on  nucleic  acid  and  protein  synthesis  in  Staph\lococcus  aitrcits.     Bioclicm. 

J.,  53 :  493-496. 
HAHN,    F.    E.,    AND    A.    D.    WOLFE,     1962.     Mode    of    action    of    chloramphenicol.     VIII. 

Resemblance    between    labile    chloramphenicol-RNA    and    DNA    of    Bacillus    ccrcus. 

Biochcm.  Biophys.  Res.  Couun.,  6:  464-468. 
KANATANI,  H.,  1958.     Formation  of  bipolar  heads  induced  by  demecolcine   in  the  planarian, 

Dugesia  gonoccphala.    J.  Fac.  Sci.,  Univ.  Tokyo,  Scr.  IV,  8:  253-270. 
LOWRY,  O.  H.,  N.  J.  ROSEBROUGH,  H.  L.  FARR  AND  R.  J.  RANDALL,  1951.     Protein  measurement 

with  the  Folin  phenol  reagent.     /.  Biol.  Chem.,  193:  265-276. 
MIDGLEY,    J.    E.    M.,    AND    B.    J.    MCCARTHY,    1962.     The    synthesis    and    kinetic    behavior    of 

deoxyribonucleic  acid-like  ribonucleic  acid  in  bacteria.     Biochcm.   Bioph\s.   Ada,   61: 

695-717. 


ANALYSIS  OF  SOME  TEMPERATURE  EFFECTS 
ON  DROSOPHILA  PUPAE 

ROGER  MILKMAN  AND  BERTIL  HILLE 

Department  of  Zoology,  Syracuse  University,  Syracuse,  Ncu>  York,  The  Rockefeller  University, 
Nciv  York,  Neiv  York,  and  Marine  Biological  Laboratory,  ll'oods  Hole,  Massachusetts 

Day-old  Drosophila  pupae  respond  to  high  temperature  treatment  in  a  variety 
of  ways.  Three  classes  of  response  have  been  studied  (Milkman,  1962,  1963). 
Adaptive  responses  follow  relatively  gentle  treatments ;  morphogenetic  changes  re- 
sult from  moderate  treatments;  and  more  severe  treatments  lead  to  death.  Even 
when  observations  are  restricted  to  genetically  uniform  D.  melanogaster  pupae, 
raised  under  standard  conditions  at  23°  C.  for  25  hours  after  puparium  formation, 
the  qualitative  diversity  of  response  is  impressive  (Milkman,  1962).  Considering 
the  wings  alone,  reduction  in  size,  anterior  crossvein  defects,  posterior  crossvein 
defects,  holes,  approximation  of  the  third  and  fourth  longitudinal  veins,  and  adventi- 
tious appearance  of  vein  material  are  each  stereotyped  responses  to  certain  treat- 
ments;  and  these  effects  do  not  appear  in  order,  depending  simply  upon  duration 
of  treatment ;  rather,  there  are  specific  and  clear-cut  qualitative  associations  between 
treatment  temperature  and  response.  Death,  too,  appears  in  unexpected  ways; 
for  example,  4^-44  hours  at  37.5°  is  lethal  to  males.  The  same  durations  at  38.0° 
are  not.  Moreover,  the  kinetics  of  the  responses  are  often  unusual :  the  morpho- 
logical response  to  36.5°  reaches  a  peak  at  200  minutes,  then  drops. 

This  welter  of  phenomena  is  forbidding  indeed,  but  there  is  a  body  of  responses 
to  a  large  category  of  treatments  that  is  accessible  to  analysis.  These  responses 
have  already  been  ordered  tentatively ;  the  present  paper  is  largely  to  confirm  the 
order,  to  add  substance  and  detail,  and  to  lay  the  foundation  for  a  quantitative 
scheme  capable  of  defining  the  kinetics  of  the  several  underlying  events  for  a  wide 
range  of  simple  and  complex  temperature  treatments.  The  purpose  of  such  a 
scheme  is  two-fold :  to  serve  as  a  clear  model  of  complex  temperature  responses 
and  to  summarize  the  great  number  of  observations  that  have  been  made  in  the 
present  study. 

We  shall  be  concerned  with  the  production  of  posterior  crossvein  defects  at 
temperatures  above  39.0°,  with  death,  and  with  the  protection  against  these 
responses  conferred  by  exposure  for  certain  durations  to  temperatures  above  27°. 

To  review  the  evidence  and  conclusions  presented  to  date,  we  can  consider  the 
results  of  treatments  of  various  duration  at  40.5°.  It  will  be  useful  to  refer  to 
Figure  1.  Very  short  treatments  (5-10  seconds)  followed  by  an  interval  at  23° 
increase  resistance  to  killing  and  to  the  production  of  crossvein  defects  by  subse- 
quent exposures  to  high  temperatures.  This  resistance  is  transient,  wearing  off  in 
a  couple  of  hours.  We  speak  of  two  transitions,  A  — »  B  at  40.5°  and  B^C 
(transient  protection)  at  23°.  By  comparing  the  durations  at  temperatures  be- 
tween 28°  and  40.5°  which  produce  effects  similar  to  those  of  5  or  10  seconds  at 

331 


332  ROGER  MILKMAN  AND  BERTIL  HILLE 

(a) 


(b)       A  ^ — >  B >  D 


I! 


i 

c' 


FIGURE  1.  The  total  scheme  as  originally  presented  (A)  and  in  its  present  form  (B). 
The  arrows  represent  transitions  considered  possible  between  the  hypothetical  states  represented 
by  letters. 

40.5°,  we  assign  A  —>  B  a  one-degree  temperature  coefficient  (Qt)  of  1.5.     B  —  »  C 
is  independent  of  temperature  in  the  range  observed. 

Similarly,  durations  of  20-120  seconds  at  40.5°  followed  by  an  interval  at 
room  temperature  produce  a  lasting  increase  in  resistance.  The  lasting  nature  of 
the  protection  is  quantitatively  inconsistent  with  the  simple  notion  that  the 
A  —  »  B  —  »  C  process  has  proceeded  to  a  greater  extent  ;  therefore  B  —  •»  D  at  40.5° 
and  D  —  »C'  (lasting  protection)  at  room  temperature  are  postulated.  This  time 
a  Qj  of  about  1.8  is  obtained  for  B  —  »  D.  D  —  >  C'  is  again  temperature-independent 


A  longer  initial  duration  at  40.5°  (on  the  order  of  5  minutes)  enables  a  subse- 
quent treatment  at  temperatures  down  to  32°  to  produce  posterior  crossvein  defects. 
This  is  related  to  a  D  —  >  E  transition.  An  interval  at  23°  still  increases  resistance 
to  subsequent  treatment.  An  E  —  »  C"  step  originally  suggested  has  an  alternative 
for  which  there  is  now  evidence  :  a  reverse  change,  E  —  >  D,  followed  by  D  —  »  C'. 

After  a  longer  period  (20  minutes)  at  40.5°,  an  interval  at  23°  no  longer  confers 
protection,  and  so  we  have  E  —  »  F.  But  the  appearance  of  defects  begins  only  some 
time  after  this  transition  is  essentially  complete,  so  F  —  •»  G,  Thus  it  is  only  in 
the  transition  F  —  »  G,  in  the  present  scheme,  that  heat  treatments  result  in  crossvein 
defects.  The  total  scheme  as  originally  presented  (Milkman,  1963)  is  shown  in 
Figure  la.  In  the  light  of  additional  evidence,  a  modified  form  seems  more 
probable  (Fig.  Ib).  The  individual  transitions  here  are  not  thought  of  as  between 
successive,  discrete  physiological  states,  but  rather  as  overlapping  conversion 
processes  at  a  level  underlying  the  physiological  states.  As  a  working  hypothesis, 
supported  but  improvable  by  kinetics  alone,  it  is  supposed  that  the  transitions  are 
among  several  tertiary  states  of  a  single  protein.  The  letters  in  the  scheme  can  be 
taken  to  stand  for  these  tertiary  states,  and  it  is  possible  to  think  of  several  existing 
at  one  particular  time.  In  any  pupa,  the  number  of  molecules  in  each  tertiary  state 
would  determine  the  observable  physiological  state. 

Naturally,  such  a  set  of  conversions  will  under  many  circumstances  of  time  and 
temperature  present  a  complex  picture  (see  Figure  2  in  Hille  &  Milkman,  1966). 
The  careers  of  various  states  will  be  hard  to  extricate.  So  in  designing  the  experi- 
ments to  be  described,  the  general  purpose  has  been  to  seek  out  the  circumstances 
when  one  particular  process  stands  out,  and  to  study  it  ;  for  with  diverse  temperature 
coefficients  the  various  processes  relate  differently  to  one  another  at  various  tern- 


ANALYSIS  OF  TEMPERATURE  EFFECTS  333 

peratures — being  prominent  in  some  and  obscure  in  others.     How  the  appropriate 
conditions  were  found  will  be  described  for  each  experiment. 

In  this  paper,  it  will  be  impossible  to  ignore  certain  assumptions  relating  the 
transitions:  that  they  connect  the  branched  sequence  of  formal  compartments  (A, 
B,  C,  etc.)  ;  that  they  have  first  order  kinetics ;  and  that  temperature  affects  only  the 
rate  of  individual  processes.  These  assumptions  will,  of  course,  have  some  sig- 
nificance in  what  is  said.  But  the  direct  description  and  test  of  the  set  of  relation- 
ships proposed  is  made  in  another  paper  (Hille  and  Milkman,  1966).  For  now, 
explicit  discussion  will  be  limited  essentially  to  the  individual  steps  and  their 
kinetics. 

MATERIALS  AND  METHODS 

An  inbred  Oregon  R  strain  of  Drosophila  melanogaster  was  used  for  all  the 
experiments.  Flies  were  raised  in  an  incubator  at  23° -25°  until  puparium  forma- 
tion, then  in  a  Precision  water  bath  at  23°  until  treatment.  After  treatment  the 
pupae  were  returned  to  the  23°  water  bath  for  at  least  12  hours  and  allowed  to 
complete  their  development  in  the  incubator  at  23°-25°.  The  temperature  was 
thus  very  closely  controlled  during  the  period  when  it  is  known  to  influence  posterior 
crossvein  formation  and  controlled  fairly  well  throughout  the  life-cycle. 

Water  baths  were  controlled  with  Micro-set  thermoregulators  and  monitored 
from  time  to  time  with  thermometers  calibrated  to  0.01  °.  A  YSI  telethermometer 
was  used  with  a  thermistor  (time  constant  0.8  sec.)  to  confirm  the  thermal  uni- 
formity of  the  various  regions  of  the  bath.  Warmup  time  (room  temperature  to 
within  a  degree  of  40.5°)  for  pupae  in  teabags  was  shown  to  be  less  than  4  seconds. 
Even  this  technique  was  not  sensitive  enough,  so  more  recently,  unambiguous  histo- 
chemical  studies  of  the  thermal  inactivation  of  succinic  dehydrogenase  were  used  to 
show  that  larvae  in  teabags  reach  within  1°  of  64°  in  less  than  2  seconds. 

Treatments  totalling  less  than  40  minutes  were  generally  made  in  teabags ; 
longer  ones  employed  vials,  or  else  pupae  were  treated  first  in  teabags  and  then 
transferred  to  vials  for  completion  of  the  treatment.  Crossvein  defects  were  rated 
from  0  (normal)  to  12  (both  posterior  crossveins  completely  absent)  as  previously 
described  (Milkman,  1963). 

RESULTS 
Early  protection 

We  shall  speak  of  C  and  C'  as  protected  states  because  they  are  off  the  effective 
pathway  from  A  to  the  damaging  state  G.  The  distinction  between  C  and  C',  and 
therefore  between  B  and  D,  rests  on  the  transience  of  protection  due  to  C,  as 
opposed  to  the  lasting  nature  of  protection  due  to  C'.  It  has  been  shown  that  very 
short  exposures  to  40.5°  at  times  between  21  and  24  hours,  followed  by  an  interval 
at  23°,  protect  against  crossvein  defect  production  by  subsequent  exposure  to  40.5°. 
The  effect  of  interval  length  is  distinguishable  from  that  of  the  pupal  age  at  which 
the  protective  treatment  was  given  (Milkman,  1963).  As  C  protection  wears  off, 
the  comparison  in  terms  of  protection  conferred  between  short-interval  and  long- 
interval  treatments  permits  the  resolution  of  C  formation  from  C'  formation.  The 
lasting  protection  found  after  longer  intervals  requires  a  longer  initial  treatment 
at  40.5°.  In  order  to  reconcile  a  long  interval  with  a  test  treatment  at  the  time 


334 


ROGER  MILKMAN  AND  BERTIL  HILLE 


of  peak  sensitivity,  25  hours,  the  pretreatments  were  originally  given  at  21  hours 
(Milkman,  1963).  More  recently,  in  anticipation  of  making  a  unifying  model  for 
the  events  at  25  hours,  these  experiments  were  repeated  in  detail  with  pretreatments 
at  24  hours  and  test  treatments  at  25^  hours.  The  results  were  essentially  identi- 
cal. Therefore,  although  the  kinetics  of  at  least  one  later  step  in  the  overall 
sequence  must  be  different  at  the  two  ages  (see  the  age-response  curve  for  defect 
production  [Milkman,  1962]),  the  kinetics  through  D  formation  are  similar. 

TABLE  I 

Onset  and  decline  of  quick  protectability  at  40.5°;  y  seconds  at  40.5°  +  x  minutes 
at  23°  +  IS  minutes  at  41.5°  data  expressed  in  rating  units 


X 

1 

i 

2 

3 

4 

5 

6 

8 

10 

y 

0 

M 

5.7 

5.8 

5.4 

5.6 

4.3 

3.8 

5.1 

3.4 

1.8 

F 

6.5 

6.7 

6.9 

6.5 

6.2 

6.6 

6.2 

7.2 

3.2 

5 

M 

5.4 

5.5 

4.8 

4.5 

4.1 

1.7 

2.0 

0.3 

0.0 

F 

6.1 

6.4 

6.1 

5.9 

6.7 

5.0 

3.0 

1.4 

0.3 

10 

M 

4.5 

5.1 

4.1 

3.9 

2.4 

2.1 

1.0 

0.1 

0.1 

F 

5.2 

6.3 

6.4 

5.8 

5.2 

4.6 

1.9 

0.9 

0.3 

15 

M 

4.8 

4.5 

4.6 

2.1 

2.5 

2.1 

0.5 

0.1 

0.3 

F 

5.8 

5.9 

5.9 

4.3 

4.4 

2.9 

2.0 

0.6 

0.7 

20 

M 

4.9 

5.1 

5.0 

4.1 

2.4 

1.6 

0.4 

0.1 

0.0 

F 

6.1 

5.6 

6.1 

5.3 

4.9 

3.6 

1.5 

0.2 

0.6 

30 

M 

4.8 

5.7 

4.5 

4.7 

2.7 

1.5 

0.3 

0.2 

0.1 

F 

5.3 

5.1 

4.7 

4.9 

4.4 

3.5 

1.8 

0.4 

0.1 

60 

M 

4.6 

3.8 

2.9 

4.6 

1.9 

1.2 

0.1 

0.3 

0.1 

F 

5.1 

4.7 

4.5 

4.9 

3.9 

3.0 

2.5 

1.4 

0.1 

120 

M 

3.6 

5.4 

3.5 

4.2 

2.7 

2.3 

1.2 

0.6 

0.2 

F 

4.4 

5.5 

4.2 

4.6 

3.5 

4.1 

3.1 

1.7 

0.8 

180 

M 

5.0 

5.6 

5.6 

5.2 

5.4 

3.3 

2.0 

0.8 

0.3 

F 

4.7 

4.9 

6.9 

6.2 

6.4 

3.9 

3.9 

3.2 

1.4 

240 

M 

5.8 

6.4 

6.5 

6.2 

5.5 

5.5 

4.4 

3.4 

1.6 

F 

7.0 

7.1 

7.8 

7.9 

7.0 

6.0 

4.6 

4.4 

1.7 

300 

M 

6.5 

8.0 

6.0 

6.5 

6.8 

6.8 

7.2 

5.4 

2.4 

F 

6.0 

7.5 

8.0 

6.8 

8.0 

7.7 

7.0 

7.3 

3.1 

360 

M 

— 

— 

— 

— 

— 

— 

— 

5.2 

4.8 

F 

— 

.  — 

— 

— 

— 

— 

— 

8.0 

dead 

480 

M 

— 

.  — 

— 

— 

— 

— 

— 

— 

7.0 

F 

— 

— 

— 

— 

— 

— 

— 

— 

dead 

Although  the  onset  at  40.5°  of  protectability  (presence  of  a  protectable  state) 
is  rapid,  its  decline  is  slower.  Moreover  we  may  distinguish  the  relatively  fast 
conversion  of  B  and  D  to  protected  states  from  the  much  slower  conversion  of  E. 
The  data  in  Table  I  show  the  rapid  onset  and  slower  decline  of  what  we  may  call 
"quick  protectability."  Reading  across,  the  ratings  fall  as  the  interval  (x) 
increases.  As  the  duration  of  the  first  treatment  (y)  increases,  changes  in  protect- 
ability are  best  seen  by  reading  down  the  right  hand  columns.  For  example,  with 
6-minute  intervals  protectability  is  great  after  only  10  seconds  at  40.5°,  while 


ANALYSIS  OF  TEMPERATURE  EFFECTS  335 

several  minutes  at  40.5°  are  required  to  progress  beyond  this  stage.  Here  the 
protection  against  crossvein  defects  is  much  more  dependent  on  interval  length  than 
on  pretreatment  duration  over  a  large  range.  Were  it  not  for  the  previously 
established  distinctions  between  transient  and  lasting  protection  and  between  the 
Q/s  of  1.5  and  1.8,  these  data  would  suggest  simply  a  rapid  buildup  and  slow 
conversion  of  a  rapidly  protectable  state.  As  things  stand,  however,  it  appears 
more  likely  that  two  states,  B  and  D,  exist  which  are  indistinguishable  in  this 
experiment. 

The  disappearance  of  D  coincides  with  the  formation  of  E,  and  the  decline  of 
rapid  protectability  thus  coincides  with  the  onset  of  increased  morphogenetic  sensi- 
tivity to  temperatures  below  39°.  Two  types  of  attempt  have  accordingly  been 
made  to  measure  specifically  the  D  -H»  E  transition ;  one  measures  the  decline  in 
protectability,  and  the  other  measures  the  increase  in  response  to  a  selected  tempera- 
ture below  39°. 

We  shall  first  describe  the  type  of  experiment  which  measures  the  decline  in 
protectability.  As  we  shall  see,  in  one  form  it  is  used  to  measure  the  disappearance 
of  D,  and  in  another  form  it  is  used  to  measure  the  disappearance  of  E.  This  type 
of  experiment,  called  a  split  series,  involves  a  treatment  of  fixed  length  split  into 
two  parts  by  an  interval.  The  variable  is  the  placement  of  the  interval.  In  either 
case  a  total  exposure  to  40.5°  (or  a  nearby  temperature)  is  chosen  which  ordinarily 
produces  severe  crossvein  defects  but  little  reduction  in  survival.  An  interval  at 
a  lower  temperature  is  interposed  at  each  of  various  times  in  the  treatment.  To 
the  extent  that  the  flies  are  protectable  by  the  interval,  the  final  defect  production  will 
be  reduced.  Figure  2  presents  such  data.  Ten  minutes  at  37.5°  are  sufficient  to 
convert  a  large  amount  of  any  D  present  to  C.  B  could  also  be  converted  to  C, 
but  in  fact  the  B  is  essentially  gone  after  even  the  shortest  first  treatment  used 
here.  The  10-minute  interval  at  37.5°  will  not  convert  a  significant  amount  of  E 
to  a  protected  state.  Thus  this  split  series  with  a  10-minute  interval  at  37.5°  is 
used  to  chronicle  the  disappearance  of  D. 

An  interval  at  23°  will  convert  B,  D  and  E  to  C  or  C'.  In  principle,  such  an 
interval  can  be  used  to  monitor  the  amount  of  E,  which  of  course  remains  substantial 
for  some  time  after  the  disappearance  of  D.  Figure  3  contains  the  results  of  such 
experiments.  Notice  by  comparing  these  data  to  those  of  Figure  2  that  23° 
protection  is  indeed  still  seen  after  first  treatments  too  long  to  be  mitigated  by  a 
37.5°  interval.  To  maximize  this  23°  protection,  one  would  like  to  use  a  60-  to 
90-minute  interval,  since  conversion  of  E  to  C  takes  a  long  time.  But  intervals  of 
this  length  force  either  the  early  or  the  late  part  of  the  high  temperature  treatment 
out  of  the  maximally  sensitive  period,  and  so  30  minutes  is  used  as  the  best 
compromise. 

Although  temperatures  other  than  40.5°  can  be  used  for  the  treatment,  the 
number  of  events  taking  place  makes  it  impossible  to  calculate  temperature  coeffi- 
cients for  single  transitions  by  this  method.  Note  that  teabags  are  used  whenever 
possible  to  minimize  warmup  time ;  even  when  the  total  elapsed  time  from  beginning 
to  end  of  treatment  is  too  great  for  a  teabag  to  be  used  exclusively,  it  can  be  used 
for  20  minutes  at  40.5°,  after  which  no  protection  at  23°  is  observed,  and  the 
pupae  are  then  transferred  to  vials.  The  transfer  period  has  no  mitigating  effect 
at  this  time. 


336 


ROGER  MILKMAN  AND  BERTIL  HILLE 


To  return  to  D  decay,  the  data  in  Figure  2  show  that,  at  40.5°,  D  is  essentially 
gone  after  8  minutes.  Further  displacement  of  the  interval  results  in  no  great 
increase  in  ratings,  indicating  no  further  loss  in  37.5°  protectability.  At  higher 
temperatures,  particularly  42.5°,  the  overall  process  of  crossvein  defect  production 
(appearance  of  G  in  amounts  exceeding  a  threshold)  takes  place  so  rapidly  that  one 
cannot  monitor  the  total  disappearance  of  D.  At  these  temperatures  a  substantial 
amount  of  D  is  still  present  when  enough  G  has  been  formed  to  cause  serious  cross- 
vein  defects,  and  even  death.  This  is  consistent  with  the  observation  that  the 
overall  process  leading  from  A  to  G  has  a  Qj  of  2.3,  while  D  formation  has  a  Ql 
of  1.8  and  the  conversion  of  D  to  E,  though  not  accessible  to  exact  measurement, 
seems  also  to  have  a  Q^  well  below  2.  Thus  the  conversion  of  a  certain  amount 


8 


41.5° 
=  20 


o    o 


37.5° 


41.5° 
40.5° 

23° 


0  5  10  15 

x(MINUTES) 


20 


FIGURE  2.  Disappearance  of  D  as  measured  by  split  series  experiment  with  interval  at 
37.5°  C.  Interval  begins  x  minutes  after  start  of  treatment;  y  =  total  duration  (minutes)  at 
40.5°  or  41.5°.  As  D  disappears,  the  protective  effect  of  37.5°  interval  declines;  rating  thus 
rises  as  onset  of  interval  becomes  later.  Open  circles,  females ;  filled  circles,  males.  Tempera- 
ture sequence  and  reactions  associated  with  each  step  diagrammed  at  right.  Dashed  horizontal 
line,  variable  duration ;  solid  horizontal  line,  fixed  duration. 

of  D  into  E  will  be  followed  by  its  subsequent  conversion  to  G  before  all  the  D 
disappears.  And  this  amount  of  G  is  enough  to  cause  extreme  responses.  Why 
the  latter  part  of  the  sequence — why  indeed  the  overall  sequence — has  such  a  high 
temperature  coefficient  will  be  explained  in  terms  of  the  D  branch  point  to  be 
discussed  shortly. 

The  other  measure  of  the  D  — »  E  transition  monitors  E  formation  rather  than 
D  disappearance ;  in  other  words,  it  follows  the  increase  in  sensitivity  to  lower 
temperatures  rather  than  the  loss  of  protectability.  This  kind  of  experiment  in- 
volves a  variable  first  treatment  at  40.5°  (or  nearby)  and  a  constant  second  treat- 
ment at  a  temperature  below  39°.  The  rationale  here  is  that  with  successively 


ANALYSIS  OF  TEMPERATURE  EFFECTS 


337 


longer  first  treatments,  the  second  treatment  should  become  more  and  more 
effective  in  causing  crossvein  defects  because  in  terms  of  our  scheme,  as  more  E  is 
made,  more  F  can  be  made  at  the  lower  temperature.  Thus  the  response  would  rise 
sharply  with  increased  first  treatment  length,  reflecting  E  formation  primarily. 
After  the  E  is  all  made  (essentially),  a  further  increase  in  first  treatment  duration 
would  increase  the  total  response  much  less,  merely  in  relation  to  the  lengthening  of 
the  total  treatment ;  we  would  see,  in  a  simple  case,  something  approaching  a  broken 


0 


0 


0 


i        r 


425° 
41.5° 
40.5° 


F-*G 


42.5° 
41  .5° 
40.5° 


23' 


23' 


23° 


42.5° 
25hrs. 


40.5° 
24  1/4  hrs. 


12 


16  20  24 

(x)    MINUTES 


28 


32 


36 


FIGURE  3.  Disappearance  of  E  as  measured  by  split  series  experiment  with  interval  at 
23°  C.  Interval  begins  x  minutes  after  start  of  treatment.  Total  duration  at  40.5°  =  35  minutes ; 
at  41.5°,  18  minutes;  at  42.5°,  8  minutes.  As  E  disappears,  the  protective  effect  of  23°  interval 
declines;  rating  thus  rises  as  onset  of  interval  becomes  later.  Females:  40.5°,  circles;  41.5° 
and  42.5°,  open  triangles  and  squares.  Males  :  remaining  symbols. 


curve.  In  practice,  as  might  be  predicted  (from  the  apparent  overlap  of  the 
various  transitions,  and  from  the  necessarily  unequal  total  treatment  durations), 
the  results  do  not  form  such  a  simple  pattern.  Accordingly,  the  split  series  experi- 
ments are  the  experiments  of  choice.  Nevertheless,  the  broken  curve  experiments 
have  been  of  some  value  (Milkman,  1963),  and  the  data  obtained  fit  the  predictions 
made  with  our  analog  computer  (Hille  and  Milkman,  1966). 

The  conversion  of  E  to  F,  too,  has  a  measurable  course  at  40.5°  but  not  at 


338 


ROGER  MILKMAN  AND  BERTIL  HILLE 


42.5°.  The  dose  which  produces  death  limits  the  length  of  time  over  which 
one  can  follow  D  decay  or  E  decay.  Once  more,  it  appears  possible  (at  42.5°)  to 
produce  a  lethal  amount  of  the  terminal  form  before  completing  the  transitions  in 
the  middle  of  the  sequence.  This  illustrates  the  overlaps  which  we  must  consider. 
The  protection  of  E  has  been  studied  in  terms  of  a  response  vs.  interval  length 
experiment.  Because  of  the  length  of  time  required  to  protect  E,  such  an  experi- 
ment runs  the  risk  of  extending  beyond  the  sensitive  peak  period.  However, 
short  intervals  produce  enough  information  to  permit  some  conclusions.  A  simple 
hypothesis  is  an  E  — -»  C"  transition,  as  was  previously  proposed  (Milkman,  1963). 
One  would  expect  a  great  amount  of  protection  with  a  short  interval  and  a  progres- 
sive reduction  in  added  protection  as  interval  length  increased.  An  alternative 
notion  of  comparable  simplicity  would  involve  the  reversal  of  the  D  — •»  E  transition, 
with  E  going  back  to  D,  which  will  then  go  to  C',  as  we  have  already  described. 
Here  one  might  expect  (depending  on  the  actual  quantitative  characteristics)  a 
lag  first,  then  protection  as  one  increased  the  interval.  The  kinetics  of  protection 

TABLE  II 

Kinetics  of  E  protection;  40.5°  (10  min.}  +  34.0°  (x  min.)  +  40.5°  (y  mm.). 

Data  expressed  in  rating  units 


Males 

Females 

y 

X 

25 

30 

37 

25 

30 

37 

0 

8.0 

d 

d 

8.9 

d 

d 

10 

8.6 

10.3 

(10.0) 

8.3 

9.7 

d 

20 

7.2 

9.2 

(9.8 

7.0 

9.1 

d 

30 

5.4 

8.2 

8.8 

5.8 

7.4 

8.8 

40 

4.4 

6.4 

8.0 

4.6 

6.3 

7.7 

50 

3.5 

4.1 

5.3 

4.3 

5.4 

5.1 

60 

1.8 

3.2 

3.7 

3.0 

3.8 

4.0 

would  not  be  first  order,  even  if  each  process  were  a  monomolecular  event.  The 
experimental  results  are  shown  in  Table  II.  They  favor  the  indirect  E  — »  D  — *  C' 
path,  since  there  is  a  lag  of  10  or  20  minutes,  after  which  protection  increases 
linearly  with  interval.  Accordingly,  we  presently  believe  that  protection  of  E 
involves  its  conversion  back  to  D  and  then  to  C'. 

After  about  20  minutes  at  40.5°,  essentially  all  the  A  has  gone  either  to  F  or 
to  the  protected  states.  No  further  protection  is  possible.  This  is  true,  not  only 
for  25-hour  pupae  but  for  younger  and  older  ones  as  well.  The  evidence  for  this 
comes  from  a  comparison  between  two  kinds  of  age-response  curves.  The  first 
relates  responses  to  35  minutes  at  40.5°  with  respect  to  pupal  age.  The  second 
deals  with  split  treatments :  20  minutes  at  40.5°  are  administered  at  a  given  age,  and 
the  rest  at  25  hours,  the  age  of  peak  sensitivity  (Milkman,  unpublished).  These 
curves  are  essentially  parallel  (the  split  treatment  curve  shows  greater  response,  due 
to  the  contribution  of  the  treatment  at  peak  sensitivity),  but  treatments  much 
shorter  than  20  minutes  at  any  age,  followed  by  room  temperature,  confer  protection. 


ANALYSIS  OF  TEMPERATURE  EFFECTS 


339 


Since  the  20-minute  first  treatment  is  the  significant  variable  when  administered 
at  or  before  25  hours,  and  since  the  F  — >  G  transition  is  not  important  until  later, 
it  appears  that  one  or  more  of  the  early  steps  is  age-dependent,  resulting  in  a 
variable  proportion  of  A  going  to  a  protected  state  in  the  first  20  minutes. 

After  25  hours,  however,  the  latter  part  of  the  treatment  is  the  important  vari- 
able, indicating  either  that  F  — »  G  slows  down  with  increasing  age,  or  that  the  effect 
of  G  production  on  posterior  crossvein  formation  decreases  with  time.  The  cross- 


TABLE  III 

Results  of  treatment  completion  at  lower  temperatures ;  first  treatment:  25  minutes  at  40.5 ' 


Second  treatment 

r 

Temperature 

Duration 

(°C.) 

(min.) 

<* 

9 

32.5 

250 

10.4 

9.9 

200 

8.8 

8.5 

150 

6.2 

6.5 

100 

4.3 

6.3 

50 

2.7 

4.4 

40 

1.6 

3.2 

31.5 

240 

8.8 

8.7 

120 

3.8 

5.6 

30.5 

240 

7.9 

7.1 

150 

4.4 

6.3 

120 

2.5 

5.1 

90 

2.5 

4.5 

60 

1.8 

4.1 

29.5 

240 

7.9 

7.9 

28.5 

240 

4.5 

6.7 

27.5 

240 

4.0 

5.0 

26.5 

240 

2.6 

4.5 

24.5 

240 

0.5 

1.5 

22.5 

240 

0.1 

1.6 

18.5 

240 

0.0 

1.1 

vein  is  being  laid  down  at  this  time  (Mohler  and  Swedberg,  1964;  Waddington, 
1940). 

The  last  step  in  the  sequence,  F  — »  G,  proceeds  much  more  slowly  than  the 
others,  and  it  has  been  the  easiest  to  study  with  respect  to  its  temperature  coefficient. 
Merely  by  treating  the  pupae  for  25  minutes  at  40.5°,  one  reaches  a  stage  where 
essentially  only  this  reaction  is  going  on.  Previous  data  (Milkman,  1963)  on  the 
amount  of  subsequent  time  at  various  temperatures  needed  to  produce  a  certain 


340 


ROGER  MILKMAN  AND  BERTIL  HILLE 


average  defect  have  indicated  a  Ql  of  1.4-1.5  down  to  33.5°.  Further  experiments 
have  extended  this  relationship  down  to  26.5°,  below  which  temperature  the  dura- 
tions required  would  he  excessive.  The  new  data  are  given  in  Table  III  and 
illustrated,  together  with  the  previous  data,  in  Figure  4.  These  results,  incidentally, 
point  up  the  importance  of  controlling  temperature  after  treatments. 

Although  the  temperature  coefficients  are  accessible  to  study,  the  actual  rate  of 
the  process  is  not  easy  to  describe  in  terms  other  than  average  amount  of  defect 
produced  per  unit  time.  If  F  — >  G  is  a  first  order  process,  it  is  very  slow  indeed, 
since  the  relationship  between  dose  and  response  has  a  linear  appearance.  Only 


2.5  - 


2.0 


co 


1.5 


1.0 


UJ 


0.5 


0.0 


40.5          38.5  36.5  34.5  32.5 

TEMPERATURE     °C 


30.5 


28.5 


26.5 


FIGURE  4.  Determination  of  the  temperature  coefficient  of  the  F  — >  G  transition.  Ordinate  : 
log  duration  at  a  given  temperature  which,  when  added  to  a  25-minute  exposure  to  40.5°  C., 
produces  a  criterion  response  (sum  of  male  and  female  ratings  =  7).  Visually  plotted  line 
corresponds  to  Qi  ^  1.4. 


the  early  part  of  a  first  order  reaction  appears  linear.  This,  in  turn,  would  imply 
that  very  little  loss  of  the  hypothetical  functional  protein  would  lead  to  morpho- 
logical defects  and  that  very  little  further  loss  would  lead  to  death.  This  is  not 
unreasonable  in  a  living  organism,  particularly  since  we  have  sought  out  this 
developmental  process  on  the  basis  of  its  sensitivity.  There  is  no  present  basis 
for  further  speculation,  however. 

The  measurements  of  overall  temperature  coefficients  at  higher  temperatures, 
difficult  in  vials  because  of  the  significant  warmup  period,  have  been  extended 
through  the  use  of  teabags.  The  durations  at  temperatures  from  40.0°  to  42.5° 


ANALYSIS  OF  TEMPERATURE  EFFECTS 


341 


required  to  produce  a  criterion  response  (sum  of  male  and  female  ratings  =  6.0) 
have  been  compared  and  are  plotted  in  Figure  5.  These  data  yield  an  overall  Ql 
of  2.0,  somewhat  lower  than  the  value  previously  obtained  (2.3)  but  still  higher 
than  the  O,  for  any  individual  step. 

The  paradox  thus  remains  that  the  overall  Ql  appears  to  be  between  2.0  and  2.3, 
while  the  individual  steps  appear  to  have  Q/s  no  higher  than  1.8,  with  the  Qx  of  the 
slowest  reaction,  F  —>  G,  being  1.4-1.5.  It  was  proposed  (Milkman,  1963)  that 
the  overall  Ql  represented  a  product  of  two  kinds  of  temperature  functions.  One, 
the  temperature  coefficient  of  the  rate,  is  familiar,  but  the  other  is  less  obvious.  It 
is  the  proportion  of  a  form  proceeding  to  one  of  two  possible  subsequent  forms. 
We  illustrate  with  D  because  it  appears  in  practice  to  be  the  only  relevant  case  at 


CO 
UJ 


50 
40 

30 

20 

15 

10 
8 


40.0        41.0        42.0 
TEMPERATURE    °C 


43.0 


FIGURE  5.  Determination  of  the  overall  temperature  coefficient.  Ordinate  :  duration  at  a 
given  temperature  which  produces  a  criterion  response  (sum  of  male  and  female  ratings  =  6). 
Visually  plotted  line  corresponds  to  Qi  =  2.0. 

the  temperatures  we  are  considering.  Since  D  can  go  to  C'  and  also  to  E,  and  since 
D  —>  C'  has  a  temperature  coefficient  of  1  (is  temperature-independent)  and 
D  — •»  E  has  a  temperature  coefficient  in  the  neighborhood  of  1.7,  we  can  imagine  a 
low  temperature  at  which  all  the  D  goes  to  C,  a  high  temperature  at  which  all  the 
D  goes  to  E,  and  an  intermediate  range  over  which  the  proportion  of  D  going  to 
E  is  a  sensitive  function  of  temperature.  In  a  first  order  system,  the  rate  of 
formation  of  F  is  kEF  [E],  and  since  kEF  and  [E]  are  both  temperature-dependent, 
the  rate  must  depend  on  the  product  of  these  temperature-dependent  functions. 
Similarly  the  rate  of  formation  of  F  must  have  a  two-factor  temperature  dependence 
even  if  we  treat  the  pupae  at  a  single  temperature  from  beginning  to  end ;  and 
dG/dt  must  share  this  dual  dependence.  Thus  the  overall  temperature  coefficient, 


342 


ROGER  MILKMAN  AND  BERTIL  HILLE 


Qx  —  2.0-2.3,  could  be  factored  into  the  product  of  1.4—1.5  (Qx  FG,  F  -»  G  being  the 
slowest  reaction  in  sequence)  times  1.3-1.6,  which  we  attribute  to  the  temperature 
dependence  of  the  proportion  of  D  going  to  E. 

Experimental  support  for  this  hypothesis  comes  from  examining  the  response 
to  39.5°  of  pupae  which  have  largely  reached  the  F  stage  at  one  of  a  number  of 
experimental  temperatures.  Clearly  the  rate  constant  of  F  -»  G  at  39.5°,  kFG,  is 
fixed.  But  if  the  total  amount  of  E  made,  and  therefore  the  total  amount  of  F 
made,  is  temperature-dependent,  then  the  dose-response  relationship  at  39.5°  will 
depend  upon  the  temperature  at  which  the  early  part  of  the  treatment  was  given. 
The  results  are  illustrated  in  Figure  6,  supporting  qualitatively  the  hypothesis 
that  the  nature  of  the  Ql  of  2.0-2.3  is  compound.  The  slopes  of  the  dose-response 
curves  show  the  relationship  between  rate  of  G  synthesis  at  39.5°  and  the  tempera- 
ture at  which  E  was  made.  The  increase  in  slope  is  about  1.3  per  degree,  within 
the  range  1.3-1.6  predicted  crudely  here,  and  in  good  quantitative  agreement  with 


13   Mm  at  41  5° 
+  39  5° 


12 


Mm.  at  40.5° 
+  39.5° 


I0r 


395°    all 
the     way 


10  20  30  40  50 

Mmutes    at  39  5° 


60 


70 


80 


FIGURE  6.  Dependence  of  dose-response  relationship  at  39.5°  upon  temperature  of  first  part 
of  treatment.  Examples  selected  for  purposes  of  effective  display.  Data  from  additional  ex- 
periments at  41.5°  and  40.5°  where  first  treatments  have  different  durations  show  similar  slopes. 
Lines  drawn  from  computer  calculations  (Hille  and  Milkman,  1966). 


our  computer  predictions,  which  can  and  must  take  a  number  of  additional  factors 
into  account  (Hille  and  Milkman,  1966). 

It  is  noteworthy  that,  no  matter  what  the  assortment  of  treatments  within 
the  present  range  (and  specifically  excluding  4  hours  at  37.5°  reported  previously 
[Milkman,  1963]  and  described  as  not  capable  of  inclusion  in  the  present  scheme), 
an  average  defect  rating  of  9  or  more  is  always  accompanied  by  high  mortality  and 
11  is  virtually  unattainable,  apparently  because  of  death.  It  is  possible  to  protect 
against  crossvein  defects  so  that  death  comes  first ;  but  it  is  not  possible  to  produce 
extreme  crossvein  defects  and  yet  retain  good  survival.  Thus  it  seems  that  the 
hypothetical  protein  involved  in  crossvein  formation  is  also  vital — presumably  else- 
where in  the  organism — and  there  it  has  a  slightly  greater  reserve  which  can  be 
destroyed  before  measurable  effects  appear.  When  death  occurs  in  pupae  protected 
against  crossvein  defects,  it  requires  a  longer  treatment  than  usual  and  is  presum- 
ably due  to  an  event  which  ordinarily  is  preceded  by  death  due  to  the  destruction 
of  the  crossvein  protein. 


ANALYSIS  OF  TEMPERATURE  EFFECTS  343 

One  can  calculate  first  order  rate  constants  for  each  of  the  transitions,  on  the 
basis  of  the  relationship  k  =  0.69/tj,  where  t»  is  the  time  for  the  reaction  to  proceed 
halfway.  One  might  expect  tertiary  structure  transitions  in  proteins  to  have 
first  order  kinetics,  since  many  denaturation  processes  do.  The  rate  constants  have 
thus  been  calculated  for  all  the  processes  that  reach  completion  ( essentially).  For 
F  — »  G,  which  comes  nowhere  near  completion,  not  even  enough  curvature  can  be 
seen  in  its  time  curve  to  permit  a  calculation  of  a  rate  constant.  This  fact  does  set 
an  upper  limit,  though,  which  enables  us,  rather  speculatively,  to  set  the  rate 
constant  in  the  range  of  0.01.  Table  IV  lists  all  the  first  order  rate  constants. 

An  heuristic  aspect  of  setting  rate  constants  in  this  way — based  on  the  individual 
processes — is  that  we  can  try  to  see  whether  in  concert  they  are  consistent  with  a 

TABLE  IV 

First  order  rale  constants  for  the  individual  steps* 

Rate  constant  (min."1) 
Transition  at  40.5°C. 

A  -»  B  10 

B  ->  C  0.15 

B  ->  D  4.0 

D-*C'  0.15 

D->E  0.10 

E-»F  0.15 

F->G  0.01** 

*  Estimates  are  given  for  the  forward  steps  only;  for  complete  list  of  constants,  see  Hille  and 
Milkman,  1966. 

**  Very  rough  estimate;  see  text. 

wide  variety  of  results  at  various  single  temperatures  and  combinations  of  tempera- 
tures.    Such  an  analysis  is  to  be  found  in  another  paper  (Hille  and  Milkman,  1966). 

DISCUSSION 

The  work  described  in  this  paper  is  largely  confirmatory  and  raises  no  new 
issues  (cf.  Milkman,  1963).  The  minor  changes  in  the  overall  scheme  are  the 
dropping  of  one  tertiary  state  (C")  and  the  introduction  of  a  reverse  arrow  1  jet  ween 
D  and  E.  It  appears  from  the  body  of  information  now  accumulated  that  the  vari- 
ous transitions  are  valid,  both  in  themselves  and  in  relation  to  the  others.  More- 
over the  practical  limit  seems  to  have  been  reached  in  terms  of  the  experimental 
dissection  of  steps  in  the  overall  process.  There  are  certain  temperatures  at  which 
some  transitions  are  prominent ;  the  E  — >  F  transition,  however,  is  not  resolvable 
over  a  broad  enough  range  to  calculate  a  temperature  coefficient. 

The  notion  that  the  transitions  are  related  in  a  sequence  undercuts  the  calcula- 
tion of  an  original  amount  of  A,  relative  to  a  threshold  and  relative  to  crossvein 
defect  rating  units,  by  the  simple  extrapolation  of  G  formation.  Similarly, 
the  branch  ratio  at  D  cannot  be  considered  in  isolation,  as  one  factor  of  a  two- 
factor  product,  if  one  is  to  systematize  the  several  events  and  their  outcome  over  a 
broad  temperature  range.  Xow  that  the  best  estimates  obtainable  from  these 


344  ROGER  MILKMAN  AND  BERTIL  HILLE 

experiments  are  at  hand  for  the  time  course  of  each  transition,  and  since  the 
postulated  tertiary  structure  changes  suggest  first  order  kinetics  and  easily  calculated 
rate  constants,  it  is  of  theoretical  and  practical  value  to  place  the  calculated  values 
in  the  postulated  scheme.  By  so  doing,  one  can  attempt,  with  the  aid  of  an  analog 
computer,  to  predict  the  results  of  an  array  of  simple  and  complex  treatments  that 
would  otherwise  be  difficult  to  approach.  This  is  done  in  another  paper  (Hille  and 
Milkman,  1966). 

Very  little  conclusive  information  has  been  added  lately  with  respect  to  the 
mechanism  of  temperature  adaptation  and  other  temperature  effects,  although  such 
effects  are  being  studied  in  a  number  of  theatres,  with  findings  that  are  interesting 
in  themselves  and  as  leads  (Ushakov,  1964;  Prosser,  1966).  Since  Northrop's 
work  (1920)  on  temperature  adaptation  in  Drosophila,  which  was  conveyed  from 
one  generation  to  the  next  via  the  egg,  the  observations  on  this  animal  and  others 
have  been  intriguing  but  neither  explained  nor  thoroughly  generalized.  Mohler's 
(1965)  finding  that  not  all  parts  of  the  posterior  crossvein  respond  identically  to 
high  temperatures  in  flies  of  certain  genotypes  makes  it  clear  once  again  that  the 
present  scheme  holds  in  a  restricted  theatre  at  best. 

The  idea  of  tertiary  structure  change  has  little  direct  experimental  support. 
One  of  us  (R.  M.)  has  attempted  using  both  Drosophila  extracts  and  pure  proteins 
(bovine  serum  albumin,  hemoglobin)  to  demonstrate  heat-induced  deviant  tertiary 
structure  states  as  expressed  by  altered  migration  rates  in  acrylamide  gel  disc 
electrophoresis.  After  numerous  failures,  recent  efforts  with  larval  extracts  treated 
at  55°  have  been  successful  (Milkman,  in  Prosser,  1966).  These  experiments  are 
still  preliminary  and  we  know  of  no  similar  work  ;  yet  it  is  logical  at  present  to 
allow  for  the  transition  of  some  proteins  among  various  stable  or  meta-stable 
tertiary  structure  states,  rather  than  restricting  them  to  a  single-structured  condition 
whose  only  alteration  would  be  denaturation  to  a  disordered  state.  For  further 
discussion  of  this  subject,  see  Milkman  in  Prosser  (1966). 

Finally,  it  should  be  emphasized  that  the  collected  evidence  presented  here 
strongly  supports  a  single  branched  sequence,  whatever  the  fundamental  mechanism, 
because  of  the  indissociability  of  the  steps  from  one  another  and  the  impossibility 
of  rearranging  them. 


The  assistance  of  Mary  Ann  Cady,  Tonja  Knapp  and  Maren  Brown  is  acknowl- 
edged with  thanks. 

This  work  was  supported  by  National  Science  Foundation  Grant  G-24023. 

SUMMARY 

1.  A  previously  presented  scheme  unifying  a  variety  of  high  temperature  effects 
on  day-old  Drosophila  inclanogastcr  pupae  has  been  confirmed  substantially  by  a 
large  body  of  new  data.     Several  minor  modifications  have  been  made. 

2.  New  experiments  confirm  the  validity  of  analyzing  heat-induced  temperature 
adaptation    over    a    several-hour    period    at    the    end    of    the    first    day    of    pupal 
development. 

3.  Several  new  types  of  experiments  have  been  described  which  involve  com- 
binations of  exposures  to  more  than  one  temperature  and  which  provide  information 
on  the  transitions  between  several  intermediate  stages  in  the  scheme. 


ANALYSIS  OK  TKM  I'KKATURK  EFFECTS  345 

4.  A  double  temperature-dependence  is  demonstrated  in  which  both  the  amount 
of  a  precursor  and  the  rate  of  its  conversion  vary  with  temperature  in  a  particular 
range.     In  this  range,  the  temperature  coefficient  of  crossvein  defect  induction  is  the 
product  of  two  components.     This  property  inheres  in  branched  pathways. 

5.  Rate  constants  and  temperature  coefficients  have  been  calculated   for   the 
individual  steps  on  the  basis  of  data  from  specific  experiments. 

6.  The  scheme  incorporates  the  information   from  a   large   array   of  detailed 
experiments  and  is  capable  of  generalization  over  a  broad  temperature  range. 

LITERATURE  CITED 

HILLE,  B.,  AND  R.  MILKMAN,   1966.     A  quantitative  description  of  some  temperature  effects 

on  Drosophila.    Biol.  Bull,  131 :  346-361. 
MILKMAN,  R.,   1962.     Temperature  effects  on  day  old  Drosophila   pupae.       /.   Gen.  Phvsiol., 

45:  777-799. 
MILKMAN,  R.,  1963.     On  the  mechanism  of  some  temperature  effects  on  Drosophila.      J.  Gen. 

Physiol,  46:  1151-1170. 
MOHLER,   J.   D.,    1965.     The   influence   of   some   crossveinless-like   genes    on    the    crossveinless 

phenocopy  sensitivity  in  Drosophila  melanogaster.     Genetics,  51:  329-340. 
MOHLER,  J.  D.,  AND  G.  S.  SWEDBERG,  1964.     Wing  vein  development  in  crossveinless-like  strains 

of  Drosophila  melanogaster.     Genetics,  50:  1403-1419. 

NORTHROP,  J.  H.,  1920.     Concerning  the  hereditary  adaptation  of  organisms  to  higher  tempera- 
ture.   /.  Gen.  Physiol.,  2:  313-318. 
PROSSER,  C.  L.,  ed.,  1966.     Molecular  Mechanisms  of  Temperature  Adaptation.     Amer.  Assoc. 

Adv.  Sci.  (in  press). 
USHAKOV,  B.,  1964.     Thermostability  of  cells  and  proteins  of  poikilotherms  and  its  significance 

in  speciation.     Physiol.  Rev.,  44:  518-560. 
WADDINGTON,    C.    H.,    1940.     The    genetic    control    of    wing    development    in    Drosophila.    J. 

Genetics,  41 :  75-139. 


A  QUANTITATIVE  DESCRIPTION  OF  SOME  TEMPERATURE 

EFFECTS  ON  DROSOPHILA 

BERTIL  HILLE  AND  ROGER  MILKMAN 

The  Rockefeller  University,  Neiv  York,  Nciv  York,  Department  of  Zoology,  Syracuse  University, 
Syracuse,  New  York,  and  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts 

The  appearance  of  the  posterior  crossvein  of  the  wing  of  adult  Drosophila  is  a 
delicate  measure  of  physicochemical  processes  underlying  wing  vein  development 
during  early  pupal  life.  Previous  studies  of  the  disturbance  of  posterior  crossvein 
formation  by  temperatures  from  30°  C.  to  42.5°  C.  (Milkman,  1961,  1962,  1963) 
have  led  to  the  outlining  of  a  complex  hypothesis  which  seemed  capable  of  explain- 
ing a  large  number  of  experimental  results  (Milkman,  1963).  It  is  the  purpose  of 
this  paper  to  cast  the  hypothesis  in  a  completely  quantitative  form  and  to  show  that 
the  predictions  of  the  model  do  indeed  agree  with  published  and  hitherto  unpub- 
lished experiments. 

The  experimental  background  of  these  studies  starts  with  the  basic  observation 
that  heat  damage  to  the  development  of  the  posterior  crossvein  is  a  threshold 
phenomenon.  At  temperatures  from  39.5°  to  41.5°  the  time  to  reach  a  threshold 
response,  or  any  more  severe  disturbance,  decreases  by  a  one-degree  temperature 
coefficient  (Oj)  as  large  as  2.3.  Subthreshold  effects  are  readily  revealed  by 
additional  heat  treatments  to  consist  of  a  series  of  related  processes  which  precede 
the  process  leading  to  damage.  Nearly  threshold  pretreatments  are  definitely 
damaging  as  they  are  additive  with  a  second  treatment.  Briefer  pretreatments, 
however,  would  in  certain  conditions  lead  to  a  rapid  temperature  adaptation  which 
protected  the  pupa  against  the  damaging  effects  of  additional  treatments.  In  some 
cases  the  protection  was  transient  and  in  others  it  persisted  for  many  hours.  The 
wealth  of  effects  emerging  from  these  experiments  led  to  the  original  kinetic  scheme 
(Milkman,  1963).  It  was  suggested  that  high  temperatures  induced  a  sequence 
of  tertiary  structure  changes  in  some  hypothetical  protein  required  for  crossvein 
formation  in  the  pupa.  One  could  explain  the  various  effects  of  pretreatments  by 
the  properties  of  the  predominant  tertiary  structure  state  remaining  after  the  pre- 
treatment.  The  sole  and  yet  compelling  evidence  that  a  protein's  tertiary  structure 
was  being  changed  was  the  very  high  temperature  coefficient  of  some  of  the 
individual  steps. 

POSTULATES 

The  full  details  of  the  original  scheme  can  be  incorporated  into  four  postulates 
which  may  be  regarded  as  the  hypothesis  to  be  demonstrated  by  this  paper : 

1.  Normal  development  of  the  posterior  crossvein  requires  a  protein  A  within  a 
few  hours  after  a  biological  age  equivalent  to  25  hours  of  pupal  development 
at  23°  C. 

346 


INTEGRATION  OF  TEMPERATURE  EFFECTS  347 

2.  At  elevated  temperatures  A  is  channel  to  states   I',  C,  C',  D,  E,  F  and  G 
as  in  the  reaction  sequence  below  : 

A?±B-*D^±E-*F-*G 

H      L 
c  — c' 

The  individual  steps  of  the  conversion,  represented  by  arrows,  are  first  order  in  the 
reactant  and  may  be  assigned  rate  constants  and  temperature  coefficients. 

3.  The  algebraic  sum  of  the  amounts  of  protein  in  all  the  states  except  G  linearly 
determines  the  ultimate  length  of  the  posterior  crossvein  within  a  defined  range. 
Thus  defects  result  from  treatments  which  produce  G,  the  terminal  inactive  state. 

4.  The  adjustable  parameters  of  the  model :  rate  constants,  temperature  coeffi- 
cients, initial  amount  of  A,  and  the  function  relating  posterior  crossvein  length  to 
the  sum  of  the  effective  states,  are  themselves  functions  of  the  age,  raising  tempera- 
ture, sex,  and  genome  of  the  organism. 

MATERIALS  AND  METHODS 

Because  of  postulates  1  and  4,  in  this  paper  we  evaluate  the  parameters  only 
for  female  Oregon  R  Drosopliila  inclanogastcr  25-hour  pupae  raised  at  23°  C. 
This  age  happens  to  be  the  time  of  maximum  heat  sensitivity  of  posterior  crossvein 
formation  (Milkman,  1961,  1962). 

The  equations  of  the  model  were  solved  on  a  ten-amplifier  Dormer  3500  analog 
computer  using  standard  techniques  for  linear  systems.  Initial  conditions  were  set 
with  a  Tektronix  161  pulse  generator.  The  time  course  of  the  solutions  was 
photographed  on  Polaroid  projection  film  from  the  face  of  a  Tektronix  502  oscillo- 
scope. The  curves  were  enlarged  on  graph  paper  by  tracing  the  projected  image 
under  an  enlarger.  We  often  took  advantage  of  the  additivity  of  solutions  of  linear 
systems  when  curves  for  experiments  involving  split  treatments  were  to  be 
calculated.  In  a  two-temperature  experiment,  for  example,  the  quantities  of  all 
the  intermediates  after  the  first  treatment  were  calculated  in  a  straightforward 
manner  and  then  the  separately  calculated  fates  of  each  intermediate  in  the  second 
treatment  were  added  to  get  the  final  solution.  The  maximum  error  was  less  than 
5%.  This  error  will  be  seen  to  correspond  to  an  error  of  less  than  one  crossvein 
rating  unit  in  the  predictions. 

The  fruit  flies  used  in  these  experiments  were  a  highly  inbred  strain  of  Oregon 
R  Drosopliila  inclanogastcr  raised  by  standard  techniques  in  an  incubator  at  23°. 
Pupae  were  placed  in  shell  vials  within  one  hour  after  puparium  formation,  to  be 
aged  in  a  23°  regulated  water  bath.  Depending  on  the  duration  of  treatment,  high 
temperature  heat  treatments  were  started  sometime  after  the  24th  hour  of  develop- 
ment and  were  completed  by  the  26th  hour.  Unfortunately,  even  during  this  period, 
the  response  of  the  flies  changes  sufficiently  rapidly  to  affect  the  agreement  between 
experiment  and  predictions  (Milkman,  1961,  1962).  Short  experiments  whose 
total  treatment  could  be  given  close  to  the  25th  hour  were  therefore  considered 
to  be  of  the  greatest  importance.  The  high  temperature  water  baths  were  regulated 
to  within  ±0.05°,  but  their  absolute  temperature  can  have  differed  by  ±0.15°  from 
one  year's  experiments  to  the  next.  This  variation  of  temperature  would  lead  to  a 
variation  in  experimental  data  approximately  equivalent  to  a  W%  change  in  the 
time  axis.  For  all  short  treatments  the  pupae  were  in  a  teabag.  Teabags  or 


348  BERTIL  HILLE  AND  ROGER  MILKMAN 

vials  were  used  in  longer  experiments.  While  the  results  of  identical  treatments  in 
teabags  and  vials  are  different  at  times,  we  have  made  no  effort  to  distinguish  them 
in  this  paper.  We  have  also  ignored  any  possible  warm-up  time  for  glass  vial 
treatments.  At  least  five  days  later,  after  the  flies  had  emerged,  their  posterior 
crossveins  were  rated  on  a  scale  from  zero  (normal)  to  twelve  (none  remaining) 
according  to  the  total  length  of  posterior  crossvein  remaining  on  the  two  wings  and 
regardless  of  how  the  remaining  fragments  were  distributed.  The  ratings  (r) 
of  about  50  flies  were  averaged  for  each  data  point.  The  details  of  these  methods 
have  been  published  before  (Milkman,  1961,  1962).  Almost  all  of  the  data  used 
here  have  been  published  already  (Milkman,  1962,  1963;  Milkman  and  Hille, 
1966). 

The  figures  of  this  paper  contain  points,  lines,  and  a  summary  of  the  tempera- 
ture sequences  used.  The  points  are  always  experimentally  determined  data 
points  referring  to  large  numbers  of  flies.  The  lines  are  theoretically  calculated 
using  the  explicitly  stated  postulates,  the  rate  constants,  and  the  conversion  factor 
between  G  units  and  crossvein  rating  units.  There  are  no  additional  adjustable 
parameters,  so  that  to  the  degree  that  the  lines  and  dots  in  all  examples  agree,  the 
unified  scheme  is  satisfactory.  The  temperature  sequences  have  been  indicated 
diagrammatically  by  a  series  of  rectangular  steps.  If  the  duration  at  a  particular 
temperature  is  variable,  the  horizontal  line  has  been  dotted ;  otherwise  the  line 
is  solid. 

RESULTS 

To  explain  how  our  hypothesis  fits  experimental  data  we  must  first  examine 
certain  mathematical  properties  of  the  model  system  without  reference  to  any 
experiments.  According  to  postulate  2,  first-order  differential  equations  for  the 
rate  of  change  of  each  state  may  be  written  in  the  conventional  form  of  chemical 
kinetics : 

dA 

~T~  =  — kAsA  +  ICBAB 


~r~  =  IvAfiA  -  -  (kuA  +  kBC  +  kBo)B  +  KceC 
~77  =  kscB  -  -  (kcB 

—77-  =  kcc'C  +  IvDC' 

dD 

dt 

dE 

"dT 

dF 

d^  =  kE] 

dG 


-  -  (koc'  ~r~  KDE)!)  -)- 

-  -  (kEo  +  kEF)E 


INTEGRATION  OF  TEMPERATURE  EFFECTS 


349 


10(1.5) 


0.10(1.0) 


0.15(1.0) 


^ 

D 


4.0(1.8) 


0.15(1.0) 


1.0(1.8) 


0.10  (1.8) 


0.015  (1.0) 


0.15(1.0) 


0.15(1.5)       _      0.01(1.5) 

wl 


-c' 


FIGURE  1.  The  parameters  of  the  model.  The  first  number  by  each  arrow  is  the  first  order 
rate  constant  in  mm.'1  for  that  step.  The  second  number,  in  parentheses,  is  the  one-degree 
temperature  coefficient,  Qa,  of  the  rate  constant. 

This  linear  system  of  simultaneous  differential  equations  may  be  solved  analytically 
to  give  an  answer  in  closed  form,  but  this  type  of  solution  is  impractical  because 
of  the  tedious  calculations  required  to  make  quantitative  predictions.  For  this 
reason  we  used  an  analog  computer,  an  ideal  instrument  for  linear  systems,  to  see 
solutions  displayed  directly  on  an  oscilloscope  screen  where  the  effect  of  any  adjust- 
ment in  the  parameters  was  immediately  observable. 

Figure  1  shows  the  rate  constants  estimated  for  40.5°.  Figure  2  is  a  photograph 
of  a  solution  using  these  rate  constants  and  starting  with  100  units  of  A  and  zero 
units  of  all  other  states.  In  a  fraction  of  a  minute  A  is  gone.  The  subsequent 
intermediates  rise  to  a  peak  and  fall  again  until  by  25  minutes  the  only  reaction 
proceeding  at  a  significant  rate  is  the  conversion  of  F  to  G.  It  will  be  a  helpful 
rule  to  remember  that  even  in  this  complicated  system  most  reactions  will  have 
half-times  given  in  minutes  by  0.7  times  the  reciprocal  of  the  rate  constant  k. 
In  the  presentation  we  shall  speak  loosely  of  the  time  when  a  certain  reaction  is 
complete.  We  shall  really  refer  to  the  time  when  the  reaction  is  80%  to  90% 
complete. 


too 


15 


20 


Minutes 


FIGURE  2.  The  time  courses  of  the  states  of  the  model  at  40.5°  photographed  from  the 
face  of  the  oscilloscope.  The  solution  was  started  with  100  units  of  A  (four  boxes  on  the 
reticle)  and  zero  units  of  all  other  states.  As  indicated,  some  of  the  curves  are  recorded  at 
higher  gain  for  better  resolution.  C  formation  is  negligible.  C'  formation  is  60  units  at  20 
minutes.  Neither  state  is  recorded  here.  At  other  temperatures  the  temporal  relationships 
are  different. 


350  BERTIL  HILLE  AND  ROGER  MILKMAX 

As  the  temperature  is  raised  many  of  the  reactions  of  the  model  speed  up  and 
the  intermediate  states  come  and  go  more  rapidly.  Figure  1  gives  one-degree 
temperature  coefficients  (Q,)  to  he  used  in  the  following  relation  to  calculate  the 
rate  constants  at  any  temperature  (T)  : 


-40.: 


k(T)==k(40.5")Ql' 

A  consequence  of  the  general  speeding  up  with  temperature  may  be  seen  in  Figure  3 
where  the  family  of  solid  curves  represents  the  time  course  of  G  formation  at  differ- 
ent temperatures.  These  curves  behave  as  though  they  were  generated  by  a 
process  having  a  Qt  greater  than  2.0.  This  is  surprising  at  first  because  QtFc  is 
only  1.5  and  no  other  reaction  has  a  O,  higher  than  1.8.  The  extra  multiplicative 
factor  of  more  than  1.3  is  explained  as  follows: 

There  are  two  terminal  states  in  the  model,  C'  and  G.  In  general  we  may  say 
that  the  more  C'  is  made,  the  less  material  can  ever  become  G  and  the  slower  is  G 
formation  at  any  time.  Thus  we  could  call  C'  a  "protected"  state  because  its 
formation  prevents  the  formation  of  G,  the  damaged  state.  At  40.5°  the  pattern 
of  rates  of  the  reaction  favors  the  production  of  C'  over  the  production  of  G.  At 
higher  temperatures  there  is  a  different  pattern  and  more  material  is  destined  for  G. 
Tt  is  this  temperature-dependence  of  the  amount  of  G  to  be  formed  that  provides  the 
additional  factor  which  accounts  for  the  very  high  Ql  of  G  formation. 

The  amount  of  C'  formed  depends  on  four  important  pathways  of  protection : 
directly  from  D,  directly  from  C,  indirectly  from  B  z'ia  C,  and  indirectly  from  E 
via  D.  Each  of  these  assumes  importance  at  different  temperatures.  Let  us  first 
consider  the  direct  route  from  D.  Some  D  goes  to  E  and  some  to  C'.  Thus  D  may 
be  said  to  be  at  a  "branch  point."  The  velocities  of  E  and  C'  formation  from  D  are 
in  the  same  proportion  as  the  rate  constants  kDE  and  kDC-.  We  therefore  define  the 
concept  of  "D  branch  ratio"  as  kDE  divided  by  kDE  +  kI)C'.  In  words  it  is  the 
fraction  of  D  going  to  E,  but  we  must  bear  in  mind  that  some  of  the  E  produced 
may  be  returning  to  D  at  the  same  time.  Similarly  the  B,  C,  and  E  branch  ratios 
may  lie  defined  as  in  Figure  4.  Larger  branch  ratios  mean  less  C'  and  more  G 
formation.  "We  shall  see  presently  how  they  are  useful. 

One  rate  constant  in  each  branch  ratio  is  temperature-sensitive  and  one  insensi- 
tive, making  the  ratio  a  strong  function  of  temperature,  as  can  be  seen  in  Figure  4. 
Notice  that  the  only  significant  contribution  to  C'  formation  in  the  range  from 
38.5°  to  42.5°  is  at  the  D  branch  point  and  that  its  variation  with  temperature  is 
sufficient  to  give  the  required  factor  of  1.3. 

If  the  reactions  of  the  model  are  interrupted  after  some  time  and  temperature 
when  there  is  a  large  amount  of  D.  to  be  continued  at  a  temperature  at  which  the 
D  branch  ratio  is  very  small,  then  almost  all  the  D  will  be  channelled  to  C'.  In 
this  case  there  is  protection  against  G  formation  at  high  temperatures.  Similarly 
with  E,  if  the  temperature  is  lowered  to  where  both  the  E  and  D  branch  ratios  are 
small,  E  can  be  diverted  via  D  to  C'.  Protection  from  B  is  only  slightly  more 
complex  in  that  the  B  and  C  branch  ratios  are  never  simultaneously  very  low. 
Therefore  a  low  temperature  is  first  required  to  make  C  followed  by  a  high  tem- 
perature to  make  C'.  Because  C  readily  forms  C'  at  high  temperatures,  we  shall  also 
use  "protected"  to  describe  C.  Thus  the  curves  of  Figure  4  are  useful  for  selecting 
temperatures  at  which  particular  pathways  of  C'  formation  will  be  operant. 


INTEGRATION  OF  TEMPERATURE  EFFECTS 


351 


10 


10 

c 

3 
I 


A 


42  5 


2  - 


10 


20 


20 


40 


100 

Minutes 


200 


FIGURES  3A  AND  3B.  The  time  courses  of  G  formation  at  various  temperatures  starting 
with  100  units  of  A  at  zero  time.  In  this  and  all  figures  after  Figure  4  the  solid  lines  are  the 
expectations  of  the  model  and  the  points  are  the  average  response  of  a  large  number  of  female 
flies.  The  two  always  reflect  the  linear  relation  between  crossvein  rating  units  (r)  and  G  forma- 
tion derived  in  the  text.  The  points  in  this  figure  are  from  Figure  Ib  of  an  earlier  paper 
(Milkman,  1962)  plus  a  few  unpublished  data.  Except  at  39.0°  and  below,  the  lines  run  close  to 
the  data  points.  The  three  points  at  39.0°  are  below  the  line  and  the  three  points  at  38.5° 
are  down  at  3  G  units,  corresponding  to  no  crossvein  defects  (r  =  0).  No  points  are  shown 
from  38.0°.  Whole  degree  points,  filled  circles.  Half  degree  points,  open  circles. 

Applying  the  model 

We  are  now  prepared  to  apply  the  model  to  experiments.  The  first  step  is  to 
determine  the  linear  relation  between  crossvein  rating  units  and  G  formation  as  in 
postulate  3.  We  shall  choose  the  original  (Milkman,  1961)  dosage-response 
experiment  at  40.5°  as  our  standard.  The  threshold  for  defect  production  was 
around  20  minutes  and  average  ratings  of  10.0  were  produced  by  40  minutes.  Thus 
the  range  of  G  values  from  20  to  40  minutes,  namely  from  3.0  units  to  8.5  units, 


352 


BERTIL  HILLE  AND  ROGER  MILKMAN 


corresponds  to  crossvein  ratings  from  0.0  to  10.0.  The  linear  scale  is  established 
by  the  following  relation : 

rating  =  (G  units  —  3.0)  X  1.8 

On  this  basis  the  experimental  dosage-response  data  points  previously  reported 
(Milkman,  1961,  1962)  and  some  unreported  data  have  been  drawn  in  Figure  3. 
The  model  fits  the  data  fairly  well.  We  believe  that  the  actual  ratings  at  low 
temperatures  are  less  than  the  predictions  because  the  lengthy  treatments  required 
necessarily  pass  out  of  the  period  of  maximum  heat  sensitivity  which  we  have  chosen 
to  describe.  We  have  already  explained  why  the  model  with  no  Qa  larger  than  1.8 
fits  dosage-response  data  with  a  much  higher  temperature  coefficient. 

Uninterrupted  single-temperature  treatments  cannot  reveal  the  many  postulated 
intermediate  states.  Two-  and  three-part  treatments  form  the  crux  of  the  basis  of 
the  model.  Many  experiments  were  based  on  the  following  reasoning.  The  pre- 
treatment  at  an  elevated  temperature  such  as  4  minutes  at  40.5°  brought  the  pupae 
to  some  combination  of  intermediate  states  which  in  our  example  can  be  read  from 
Figure  2.  Then  an  interval  at  a  lower  temperature  diverted  material  at  the  three 
branch  points  from  the  path  to  G  to  the  protected  states  C  and  C'.  The  interval  was 
followed  by  a  treatment  at  an  elevated  temperature  designed  to  assess  how  much 
material  had  been  diverted  at  the  branch  points.  Suitable  selection  of  the  interval 
temperature  and  duration  permitted  a  partial  resolution  of  the  three  branch  points. 
Such  experiments  established  a  schedule  for  the  reactions  at  40.5°  published  in  a 
previous  paper  (Milkman,  1963,  Table  VI),  where  the  reactions  A  — >  B,  B  — »  D, 
D  — »  E,  and  E  — >  F  were  shown  to  be  near  completion  at  10  seconds,  30  seconds,  5 
minutes,  and  12  minutes,  respectively.  This  schedule  sets  stringent  conditions  on 
the  values  of  the  rate  constants.  In  Figure  2  the  same  four  conversions  in  the 
model  are  half  complete  by  4  seconds,  15  seconds,  3  minutes,  and  10  minutes, 
respectively. 

We  have  shown  that  our  model  agrees  both  with  the  overall  dosage-response 
experiments  and  with  the  general  schedule  at  40.5°.  Now  we  shall  examine  in 
detail  its  capabilities  in  more  specific  experiments  designed  to  test  the  individual 
rate  constants  and  temperature  coefficients  over  a  wide  range  of  temperatures. 


tn 
O 


C 

o 

t_ 

CD 


35 


40°  45° 

Temperature 


50C 


FIGURE  4.  The  branch  ratios  as  a  function  of  temperature.  The  ratios  are  defined  on  the 
right  and  explained  in  the  text.  In  general,  the  larger  the  ratio  at  a  branch  point,  the  more 
material  can  get  from  that  branch  point  to  G. 


INTEGRATION  OF  TEMPERATURE  EFFECTS 


353 


-   40.5° +  23° +  40. 5° 


10  20 

Sec.  at  40.5° 


+  40.5<> 


1234 

Min.  at  36.5° 


0  10  20  30       0 

Min.  at  32.5°  or  32.0° 


10  20 

Min.  at  28.0° 


40.5° 
36.5° 
32.5° 
28.0° 

23° 


B 
D 


40.5e 


23' 


23' 


FIGURE  5.  Transient  and  lasting  rapid  temperature  adaptation  at  different  temperatures. 
The  pretreatment  temperature  and  duration  are  indicated.  Treatments  were  37  minutes  at 
40.5°.  Transient  adaptation  (open  circles)  was  achieved  with  a  5-minute  interval  at  23°. 
These  data  from  previously  published  experiments  (Milkman,  1963,  Table  II).  Lasting  adapta- 
tion (solid  circles)  used  a  60-  to  90-minute  interval,  depending  on  duration  of  pretreatment 
(at  24  hours).  These  data  are  unpublished  but  similar  to  published  experiments  (Milkman, 
1963,  Table  IV)  where  pretreatments  started  at  21  hours.  The  ratings  decrease  as  protection 
increases. 

B,  C,  and  D  formation 

The  first  three  states  are  normally  in  an  equilibrium  mixture  of  75%  A,  6%  B, 
and  19%  C  at  23°.  When  the  temperature  is  raised,  kAB  is  increased,  more  A  goes 
to  B,  and  the  equilibrium  shifts,  B  and  C  becoming  re-equilibrated  in  about  10 
minutes.  If  the  temperature  is  so  high  that  the  B  branch  ratio  is  large,  then  most 
of  the  B  will  continue  to  D,  never  to  equilibrate  with  C.  Returning  the  tempera- 
ture to  23°  at  any  moment  will  let  the  B  — »  C  reaction  equilibrate  to  one  part  B  to 
three  parts  C  in  10  minutes.  At  the  same  time  B  is  returning  to  A  until  after  40 


354 


BERTIL  HILLE  AND  ROGER  MILKMAN 


minutes  the  original  23°  equilibrium  ratios  are  nearly  restored.  The  actual  amount 
of  material  remaining  at  A,  B,  or  C  will  be  reduced  if  some  D  has  formed  at  the 
high  temperature.  Any  D  existing  on  return  to  23°  will  be  converted  to  C'  in  10 
minutes. 

These  properties  of  the  model  explain  the  phenomena  of  transient  and  lasting 
rapid  temperature  adaptation  which  have  been  described  earlier  (Milkman,  1961, 
1962).  Figure  5  shows  the  response  to  37  minutes  of  40.5°  after  the  indicated 
pretreatments  and  an  interval  at  23°.  The  5-minute  interval  used  to  demonstrate 
transient  rapid  temperature  adaptation  (open  circles)  was  sufficient  to  bring  ap- 
proximately half  the  B  and  half  the  D  to  the  protected  states  C  and  C'.  As  the 
major  factor  in  leading  to  predictability  in  this  experiment  is  the  increased  A  — >  B 


10  sec 


25 


30  35  40  45 

Minutes  at  40.5° 


30  sec. 


90  sec. 


4  min. 


50 


55 


A  -*  B  -*  D  -*•  E  -*  F  -*  G 


B 


36.5' 


B 


40.5' 


23' 


23' 


23° 


FIGURE  6.  Transient  rapid  temperature  adaptation.  Dosage-response  at  40.5°  following 
pretreatments  at  36.5°  with  a  5-minute  interval  at  23°.  As  A  is  converted  to  B,  the  23° 
interval  diverts  more  B  to  a  protected  state.  The  protection  is  manifested  as  a  change  in  both 
intercept  and  slope.  Unpublished  data. 

reaction,  we  take  the  agreement  of  the  data  (open  circles)  with  the  theory  (curves) 
over  an  8.5°  range  to  confirm  our  choice  of  kAs  and  QIAB-  The  long  interval  used 
to  demonstrate  lasting  rapid  temperature  adaptation  (solid  circles)  was  sufficient 
completely  to  protect  D  as  C'  and  to  reverse  the  formation  of  B  and  the  protected 
state  C  by  re-establishing  the  23°  equilibrium  ratios.  The  agreement  between  data 
and  theory  over  8°  confirms  the  choice  of  kBD  and  QIBD-  A  second  kind  of  experi- 
ment showing  that  transient  rapid  temperature  adaptation  leads  to  the  predicted 
lowering  of  the  40.5°  dosage-response  in  slope  as  well  as  response  to  37  minutes  is 
illustrated  in  Figure  6.  This  experiment  suggests  that  rather  than  just  creating 
a  delay  in  G  production,  protection  diverts  material  from  the  pathway  to  G  as  we 
have  postulated. 


INTEGRATION  OF  TEMPERATURE  EFFECTS 


355 


E  and  F  formation 

As  can  be  seen  from  the  D  branch  ratio  in  Figure  4,  protection  of  D  by  C' 
formation  is  nearly  complete  at  temperatures  below  38°.  This  step  takes  about 
10  minutes  (kDC>  =  0.15  min.'1).  Protection  of  E  via  D  formation  is  slower  (kED 
=  0.015)  and  is  not  large  above  35°.  Therefore  a  10-minute  interval  at  37.5°,  by 
selectively  protecting  D  and  not  E,  serves  to  measure  the  time  course  of  the  D  to  E 
transition.  A  time  course  of  this  transition  is  recorded  in  Figure  2.  Figure  7 
shows  an  experiment  where  a  10-minute  interval  at  37.5°  was  intercalated  between 
40.5°  treatments  totalling  32  minutes  (open  circles)  or  between  41.5°  treatments 


r 

10 


41  5 


40.5°*  37° +  40.5° 


4  6  8  10  12  14  16  18 

Time  of  interruption  of  treatment  in  minutes 


20 


41.5° 
40.5° 


23° 


-*B-»-D— E 


D  ~~  ^ 

D-»C' 

HU.3 

37.5° 

23' 


FIGURE  7.  The  disappearance  of  D.  The  response  to  a  20-minute  treatment  at  41.5°  (solid 
circles)  or  a  32-minute  treatment  at  40.5°  (open  circles),  each  with  a  10-minute  interval  at  37.5° 
intercalated  at  one  of  various  times  (time  of  interruption).  As  D  is  converted  to  E,  the 
interval  at  37.5°  becomes  less  effective  in  lowering  the  rating.  Data  from  Figure  3  of  Milkman 
and  Hille  (1966). 

totalling  20  minutes  (solid  circles).     The  time  and  rate  of  decrease  of  protectability 
as  D  goes  to  E  confirms  our  choice  of  kDE  and  QIDE- 

Because  the  preceding  experiment  covered  only  a  1°  range,  it  is  desirable  to 
determine  QIDE  over  a  wider  range.  This  can  be  done  by  designing  experiments 
to  test  the  D  branch  ratio  at  different  temperatures,  for,  as  we  have  discussed,  the 
temperature  dependence  of  this  ratio  is  a  consequence  of  QIDE-  The  branch  point 
ratio  may  IDC  measured  by  first  exposing  pupae  to  different  high  temperatures  until 
all  the  D  has  been  converted  to  E  and  C'.  Then  the  slopes  of  subsequent  dosage- 


356 


BERTIL  HILLE  AND  ROGER  MILKMAN 


r 
10 

8 
6 
4 
2 


13  min. 
41. 5°+ 39 


12  min.  6  min. 

40.5  +  39.5°     40.5°  +  39.5° 


10  20  30  40  50 

Minutes    at  39.5° 


60 


70 


80 


39.5°,40.5°,4l.5c 


395' 


23' 


23' 


FIGURE  8.  Response  to  39.5°  immediately  after  exposure  to  indicated  temperatures  for 
indicated  times.  Slopes  reflect  temperature-dependence  of  D  branch  ratio.  At  lower  tempera- 
ture more  A  goes  to  C'  and  less  to  F,  so  the  rate  of  G  production  (slope)  is  smaller. 
Unpublished  data,  in  part.  See,  also,  Figure  6,  Milkman  and  Hille  (1966) . 

response  curves  at  a  single  temperature  will  measure  what  fraction  of  the  D  actually 
proceeded  to  E.  Figure  8  shows  such  an  experiment.  In  our  theory  the  D  branch 
ratios  at  39.5°,  40.5°,  and  41.5°  are  27%,  40%,  and  55%,  respectively.  The  data 
show  that  the  expected  ratios  of  slopes  1:1.5:2  are  obtained. 

E  was  previously  shown  (Milkman,  1963)  to  be  a  state  which  was  protectable 
only  by  long  intervals  at  temperatures  below  35.5°.  This  is  the  last  manifesta- 
tion of  rapid  temperature  adaptation.  The  E  — »  F  reaction  is  revealed  by  the 
disappearance  of  this  mode  of  protection.  Very  few  data  are  available,  but  the 
points  on  Figure  9  suggest  that  we  have  chosen  an  approximately  correct  time 
course.  The  dashed  curve  in  the  figure  shows  the  consequence  of  removing  the 
reaction  which  protects  E  (kED  =  0).  The  difference  between  this  line  and  the 
points  shows  the  contribution  of  E-protection.  The  solid  line  is  the  expectation 
from  the  model  as  it  stands.  We  must  now  determine  QIEF-  As  with  QIDE  for 
the  D  branch  ratio,  QIEF  governs  the  temperature  dependence  of  the  E  branch 
ratio.  If  we  take  QIED,  kED,  and  kEp  at  their  postulated  values  and  try  three  values : 
1.4,  1.5,  and  1.7  for  QIEF,  we  find  that  the  E  branch  point  ratios  fall  to  50%  at 
33.5°,  35.0°,  and  36.3°,  respectively.  At  present  our  choice  is  1.5  simply  to  have 
E-protection  below  35.5°. 

G  formation 

After  25  minutes  at  40.5°  the  only  reaction  still  proceeding  is  the  conversion 
of  F  to  G.  As  has  been  illustrated  (Milkman,  1963),  this  is  the  best  time  to 


INTEGRATION  OF  TEMPERATURE  EFFECTS 


357 


measure  QIFG-  The  curves  in  Figure  10  show  that  1.5  is  a  good  temperature  coeffi- 
cient over  a  6°  range.  It  was  this  unequivocal  demonstration  of  a  QIFG  of  1.5 
which  forced  us  to  seek  a  further  explanation  of  the  2.3  temperature  coefficient  of 
pure  dosage-response  curves  (Fig.  3).  A  multiplicative  factor  was  discovered  in 
the  effect  of  the  D  branch  point,  as  we  have  proven  by  the  experiment  of  Figure  8. 
In  Figure  3  we  saw  that  QIFG  and  the  factor  from  the  D  branch  point  are  sufficient 
to  explain  the  temperature  dependence  of  the  data. 

So  far  we  have  not  discussed  how  we  chose  kPc-  At  40.5°  the  estimated  half- 
time  of  this  reaction  is  70  minutes.  None  of  our  experiments  was  long  enough  to 
reach  the  half-time  of  the  F  to  G  reaction  at  any  temperature.  Recall  that  at 
times  much  shorter  than  the  half-time,  an  experimental  curve  is  nearly  linear  so 
that  all  our  predictions  are  based  on  a  nearly  linear  production  of  G  once  all  the 
reactions  leading  to  F  have  ceased.  Suppose  we  were  to  decrease  kFc  by  a  factor 
of  100 :  the  predictions  of  the  model  are  unchanged.  This  surprising  result  is  under- 
stood when  we  remember  that  all  G-production  curves  will  be  reduced  100-fold 


r 
12 

10 
8 
6 
4 
2 
0 


-   40.5° 


40.5° 


8  10  12  14 

Pretreatment:  min.  at   40.5° 


16 


40.5' 


32 


23' 


23° 


FIGURE  9.  Response  to  25  minutes  at  40.5°  after  stated  pretreatment  and  1  hour  at  32°. 
Rapid  rise  reflects  in  part  the  disappearance  of  E,  whose  protectability  depends  on  the  reversal 
of  the  D  — »  E  reaction.  Were  this  reaction  irreversible,  the  dashed  line  would  represent  the 
prediction. 


358 


BERTIL  HILLE  AND  ROGER  MILKMAN 


and  hence  the  values  of  G  selected  to  correspond  to  posterior  crossvein  ratings  from 
0  to  10  will  also  be  reduced.  For  this  reason  any  choice  of  kPG  smaller  than  the 
present  one  will  give  equally  satisfactory  predictions.  If  we  increased  kFG  3-fold, 
the  40.5°  half-time  would  be  reduced  to  23  minutes,  and  all  the  dosage-response 
productions  of  Figure  1  would  curve  noticeably.  As  the  agreement  would  no  longer 
be  satisfactory,  we  cannot  increase  kFG.  Thus  our  selection  of  0.01  for  kFG  is  the 
maximum  permissible.  The  arbitrariness  of  this  choice  could  be  eliminated  if 


r 
10 


8 


6 


40.5°       40.5°+ 38. 5° 


10 


20 


30  40  50  60  70 

Minutes  at  second   temperature 
—>F  F-*-G 


80 


110 


40.5C 


23' 


40.5° 
38.5° 
36.5° 
34.5° 

23° 


FIGURE  10.  Response  to  various  temperatures  after  exposure  to  40.5°  for  25  minutes.  At 
this  time,  sole  reaction  is  F  — >  G.  Slopes  reflect  temperature  coefficient  of  this  reaction. 
Data  from  Table  IV  of  Milkman  (1963). 

we  could  find  a  way  of  measuring  the  dosage-response  behavior  after  treatments 
long  enough  to  produce  a  bend  in  the  curve.  In  experiments  using  protection  to 
permit  extending  the  exposure  to  40.5°  (Fig.  6)  the  treatment  has  not  been  long 
enough  yet  to  show  marked  deviation  from  our  nearly  straight  prediction. 

The  temperature-insensitive  reactions 

The  five  reactions  with  a  Qi  of  1.0  are  primarily  concerned  with  the  different 
kinds  of  temperature  adaptation.  Their  rates  can  be  determined  from  the  length 


INTEGRATION  OF  TEMPERATURE  EFFECTS  359 

of  the  low  temperature  interval  required  to  achieve  a  certain  amount  of  protection. 
As  the  interval  temperature  from  low  temperatures  up  to  34°  does  not  affect  the  rate 
of  protection  we  have  chosen  Qi's  of  1.0.  Experiments  depending  on  some  of  these 
relations  have  been  published  (Milkman,  1963,  Tables  VIII  and  IX,  Figure  1). 
A  more  thorough  demonstration  appears  in  another  paper  (Milkman  and  Hille, 
1966).  In  new  experiments  protection  of  E  is  found  to  be  more  rapid  in  the 
second  10  minutes  of  interval  than  in  the  first.  It  is  because  of  this  lag  in  E 
protection  that  we  have  chosen  to  let  E  revert  to  D  before  reaching  a  protected  state, 
rather  than  having  E  form  a  protected  state  directly,  as  has  been  suggested  before 
(Milkman,  1963). 

DISCUSSION 

We  shall  reconsider  the  four  postulates  of  the  model.  The  existence  of  the 
required  protein  A  of  postulate  1  is  known  only  through  defects  produced  after 
heat  shocks.  It  would  be  desirable  to  demonstrate  A  in  some  other  manner. 
Most  meaningful  of  all  would  be  the  chemical  isolation  of  a  substance  with  the 
properties  of  A.  Less  direct  and  somewhat  in  the  spirit  of  this  investigation  would 
be  the  analysis  of  genetic  factors  or  chemical  treatments  which  produce  effects 
interacting  with  heat  shock  effects.  So  far  none  of  these  lines  has  been  pursued 
successfully. 

The  kinetic  sequence  of  postulate  2  is  a  very  complex  explanation  for  responses 
of  pupae  to  heat  shocks.  Nevertheless,  we  are  certain  that  we  would  fail  to 
describe  all  the  data  if  any  single  arrow  were  eliminated,  regardless  of  how  the  other 
constants  were  changed.  It  is  possible  that  some  entirely  different  network  of 
reactions  could  be  as  satisfactory  as  ours,  but  we  do  not  believe  that  it  could  be 
simpler.  One  essential  feature  would  be  to  have  a  branching  scheme  in  order  to 
get  rapid  temperature  adaptation  by  a  protected  state.  Possibly  some  reactions 
could  be  of  a  different  order  from  first,  but  this  is  beyond  the  range  of  our 
computer  to  test. 

The  kinetic  scheme  of  postulate  2  is  also  a  complex  description  of  the  heat 
denaturation  of  a  protein  in  a  living  organism.  The  conclusion  that  A  is  a  protein 
rests  on  the  now  firm  establishment  of  reactions  with  Qi's  of  1.5  and  1.8,  corre- 
sponding to  activation  energies  of  about  75  and  110  kcal.  per  mole.  Steps  in  other 
protein  denaturations  have  been  shown  to  become  rapid  at  temperatures  from  40° 
to  60°,  to  have  activation  energies  from  35  to  200  kcal.  per  mole  to  be  reversible, 
to  have  temperature-insensitive  reverse  reactions,  and  to  have  branching  mechanisms 
(Chase,  1950;  Johnson,  Eyring  and  Polissar,  1954;  Kunitz,  1948). 

The  generality  of  temperature  adaptation  and  heat  prostration  in  plants  and 
animals  (Alexandrov,  1964;  Precht,  Christophersen  and  Hensel,  1955;  Prosser 
and  Brown,  1961 ;  Ushakov,  1964)  suggests  to  us  that  branching  mechanisms  of 
protein  structural  change  might  be  a  general  explanation.  Especially  striking  are 
the  temperature  adaptation  and  heat  immobilization  of  streaming  in  epidermal  cells 
of  many  plants  (Alexandrov,  1964).  These  phenomena  require  practically  the 
same  temperatures  and  durations  as  those  used  here  and  might  well  yield  to  a 
similar  analysis. 

Postulate  3  is  striking  because  it  assumes  that  many  tertiary  structure  states  of  a 
protein  have  full  biological  activity.  In  the  experiments  discussed,  only  four  states 


360  BERTIL  HILLE  AND  ROGER  MILKMAN 

remained  in  significant  quantity  by  the  26th  hour  of  pupal  development :  A,  C,  F, 
and  G.  In  the  present  scheme  D  could  never  remain  longer  than  10  minutes  and 
E  never  longer  than  60  minutes  after  treatment,  each  going  to  C'.  B  and  C  could 
be  maintained  in  high  equilibrium  concentration  by  keeping  the  pupae  at  the  tem- 
peratures around  30°.  Undoubtedly  the  shift  of  the  A-B-C  equilibrium  has  an 
important  role  in  making  pupae  whose  entire  pupal  life  was  spent  at  a  higher 
temperature  more  resistant  to  temperature  effects.  At  any  rate  we  have  shown 
that  A,  C'  and  F  are  active  states  in  posterior  crossvein  development. 

The  relationship  between  crossvein  formation  and  G  production  shows  the 
extreme  sensitivity  of  this  developmental  process  to  changes  in  the  amount  of  active 
protein.  Defects  follow  on  a  3%  (or  less,  depending  on  the  choice  of  kFG)  loss 
of  active  substance.  This  is  not  to  say  that  A  is  a  factor  required  only  in  posterior 
crossvein  development.  It  might  well  be  common  and  essential  to  many  other 
processes  whose  sensitivity  to  small  concentration  changes  is  negligible.  Indeed, 
it  seems  to  be  essential  for  life  in  that  flies  never  live  to  achieve  expected  average 
ratings  of  11  and  12  (about  9  G  units).  Other  causes  of  heat  death  are  also 
operating,  so  that  in  some  kinds  of  heat  experiments  viability  was  zero  even  with 
very  little  G  production.  We  should  say  also  that  other  causes  of  posterior  crossvein 
disturbance  are  also  operating  because  in  very  long  treatments  at  36°  to  38°, 
defects  are  produced  when  there  has  been  almost  no  G  production  (Milkman,  1961, 
1962).  These  phenomena  remain  unexplained.  We  find  it  remarkable  that  the 
wide  range  of  temperature  effects  treated  here  can  be  described  by  the  fate  of  a  single 
substance  so  that  the  existence  of  other  processes  comes  as  no  surprise. 

The  last  postulate,  saying  that  the  parameters  of  the  model  are  functions  of  the 
age,  sex,  genome,  etc.,  has  been  adequately  documented  (Milkman,  1961,  1962). 
It  opens  the  road  to  studying  the  development  of  protein  A,  its  denaturation  proper- 
ties, and  its  translation  into  posterior  crossvein.  Hopefully  by  the  time  these 
properties  are  known  we  will  also  know  protein  A's  chemical  and  developmental 
function. 


The  invaluable  instructions  on  computer  technique  of  Dr.  Frederick  Dodge  and 
Dr.  Charles  Stevens;  the  facilities  provided  by  Mr.  John  Hervey;  the  technical 
assistance  of  Mary  Ann  Cady  and  Tonja  Knapp;  and  the  clerical  assistance  of 
Maren  Brown  are  gratefully  acknowledged. 

Part  of  this  work  was  done  at  the  Marine  Biological  Laboratory,  Woods  Hole. 
This  work  was  supported  by  National  Science  Foundation  Grant  G-24023  to  R.  M. 

SUMMARY 

1.  A  complex  array  of  high  temperature  effects  on  Drosophila  melanogaster 
pupae  is  described  in  terms  of  a  quantitative  hypothesis.     A  branched  series  of 
reactions,  first  order  in  the  reactant,  provides  a  unifying  basis  for  a  set  of  adapta- 
tional,  morphogenetic,  and  lethal  effects. 

2.  The  temperature  coefficients  of  some  of  the  reactions  suggest  that  they  may 
be  specific,  serial  tertiary  structure  changes  in  an  otherwise  undescribed  protein. 


INTEGRATION  OF  TEMPERATURE  EFFECTS  361 

LITERATURE  CITED 

ALEXANDROV,  V.  YA.,  1964.     Cytophysiological  and  cytoecological  investigation  of  heat  resist- 
ance of  plant  cells  toward  the  action  of  high  and  low  temperature.     Quart.  Rev.  Biol., 

39:  35-77. 
CHASE,  A.  M.,  1950.     Studies  on  cell  enzyme  systems.     IV.    The  kinetics  of  heat  inactivation 

of  Cypridina  luciferase.     /.  Gen.  Physiol.,  33:  535-546. 
JOHNSON,  F.  H.,  H.  EYRING  AND  M.  J.   POLISSAR,   1954.     The   Kinetic   Basis   of   Molecular 

Biology.     John  Wiley  &  Sons,  Inc.,  New  York. 
KUNITZ,  M.,  1948.     The  kinetics  and  thermodynamics  of  reversible  denaturation  of  crystalline 

soybean  trypsin  inhibitor.    /.  Gen.  Physiol.,  32 :  241-260. 
MILKMAN,  R.  D.,  1961.     The  genetic  basis  of  natural  variation.     III.    Developmental  lability 

and  evolutionary  potential.     Genetics,  46:  25-38. 
MILKMAN,   R.,   1962.     Temperature  effects   on   day   old  Drosophila   pupae.     J.   Gen.   Physiol., 

45:777-799. 
MILKMAN,    R.,    1963.     On    the    mechanism    of    some    temperature    effects    on    Drosophila.    J. 

Gen.  Physiol,  46:  1151-1170. 
MILKMAN,    R.,   AND   B.    HILLE,    1966.     Analysis   of   some    temperature   effects    on   Drosophila 

pupae.    Biol.  Bull,  131:  331-345. 
PRECHT,  H.,  J.  CHRISTOPHERSEN  AND  H.  HENSEL,  1955.     Temperatur  und  Leben.     Springer- 

Verlag,  Berlin. 
PROSSER,    C.    L.,    AND   F.    A.    BROWN,    JR.,    1961.     Comparative    Animal    Physiology.     W.    B. 

Saunders,  Philadelphia. 
USHAKOV,  B.,  1964.     Thermostability  of  cells  and  proteins  of  poikilotherms  and  its  significance 

in  speciation.     Physiol.  Rev.,  44:  518-560. 


THE  EFFECTS  OF  HYPOPHYSECTOMY  AND  BOVINE  PROLACTIN 

ON  SALT  FLUXES  IN  FRESH-WATER-ADAPTED 

FUNDULUS  HETEROCLITUS 

W.  T.  W.  POTTS  AND  D.  H.  EVANS 

Department  of  Zoology  and  Comparative  Physiology,  University  of  Birmingham,  Birmingham, 
England,  and  Department  of  Biology,  Stanford  University,  Stanford,  California  94305 

Some  of  the  characteristics  of  salt  balance  in  the  euryhaline  killifish  Fundnlus 
heteroclitus  have  been  outlined  by  us  elsewhere  (Potts  and  Evans,  1966).  One  of 
the  major  features  of  osmotic  regulation  in  this  fish  is  a  marked  reduction  in  the 
sodium  and  chloride  fluxes  on  adaptation  to  fresh  water.  A  similar  reduction  has 
been  observed  in  several  euryhaline  teleosts  including  the  stickleback  Gasterosteus 
aculeatus  (Mullins,  1950),  the  rainbow  trout  Salmo  gairdneri  (Gordon,  1962), 
Fundulus  kansae  (Fleming  and  Kamemoto,  1963),  a  blenny  Blennius  pholis 
(House,  1963)  and  the  flounder  Platichthys  flessus  (Motais  and  Maetz,  1964). 
Measurements  of  the  drinking  rate  in  Fundulus  heteroclitus  (Potts  and  Evans, 
1966)  and  in  Platichthys  flessus'  (Motais  and  Maetz,  1964)  show  that  the  larger 
part  of  the  influx  in  sea  water  takes  place  through  the  body  surface,  not  through 
the  gut.  The  site  of  this  influx  is  uncertain  but  may  well  be  the  gills.  Whatever 
the  site  it  is  clear  that  the  body  surface  of  sea-water-adapted  fish  is  relatively 
permeable  to  ions  but  the  low  fluxes  found  in  fresh-water-adapted  fish  show  that 
they  are  relatively  impermeable  to  ions. 

Burden  (1956)  first  demonstrated  that  hypophysectomized  killifish  were  unable 
to  survive  in  fresh  water  although  they  could  survive  in  saline  solutions.  Later 
Pickford  and  Phillips  (1959)  showed  that  ovine  prolactin  promoted  survival  of 
hypophysectomized  killifish  in  fresh  water.  Similar  results  have  been  obtained 
with  several  other  genera  including  Mollienesia  (Mollies)  (Ball,  1962),  Poecilia 
(Ball  and  Olivereau,  1964),  Xiphophorus  (platyfish  and  sword-tails)  (Schreibman 
and  Kallman,  1962)  and  Tilapia  (Handin,  Nandi  and  Bern,  1964).  In  addition 
Schreibman  and  Kallman  (1962)  and  Ball  et  al.  (1965)  have  demonstrated  that 
pituitary  transplants  enable  hypophysectomized  fish  to  survive  indefinitely  in  fresh 
water.  Schreibman  and  Kallman  (1966)  have  recently  reviewed  the  problem  of 
hypophysectomy  and  survival  in  fresh-water  fishes. 

In  the  light  of  this  evidence  of  the  influence  of  prolactin  on  the  osmoregulatory 
ability  of  teleosts  the  effects  of  hypophysectomy  and  prolactin  on  the  salt  fluxes  of 
Fundulus  have  been  examined. 

MATERIALS  AND  METHODS 

The  fish  used  were  Fundulus  heteroclitus,  weighing  between  2  and  5  gm., 
collected  by  the  Supply  Department  of  the  Woods  Hole  Marine  Biological  Labora- 
tory. Sea-water-adapted  fish  were  kept  in  running  sea  water  which  varied  slightly 

362 


PROLACTIN  AND  SALT  FLUXES  IN  FISH  363 

in  salinity  during  the  course  of  the  work,  from  between  420  to  435  mM  Na/L., 
30.7-31.8/^c  salinity,  and  between  19°  and  21°  C.  in  temperature.  Fish  adapted  to 
40%  sea  water,  approximately  isosmotic  with  the  blood,  and  to  fresh  water  were 
kept  in  aerated  plastic  tanks  which  stood  in  trays  of  running  sea  water  so  that  the 
temperature  of  the  tanks  was  approximately  that  of  the  sea  water.  Artificial  fresh 
water  was  prepared  by  diluting  sea  water  to  1  mM  Na/L.  with  distilled  water. 
The  fish  were  fed  every  two  or  three  days  with  chopped  clam. 

Sodium  fluxes  were  measured  by  the  methods  described  elsewhere  (Potts  and 
Evans,  1966).  The  fish  were  loaded  by  placing  them  in  a  solution  containing 
24Na  for  two  hours.  The  fish  were  then  washed  and  the  active  solution  replaced 
by  an  inactive  solution.  The  efflux  was  measured  after  two  hours  in  the  inactive 
solution.  All  handling  was  kept  to  a  minimum.  The  influx  is  obtained  in  absolute 
terms,  i.e.,  the  amount  of  sodium  which  has  entered  the  fish/hr.,  but  the  efflux  is 
obtained  in  terms  of  the  rate  constant,  i.e.,  that  fraction  of  the  activity  which 
leaves/unit  time. 

Hypophysectomy  was  performed  by  a  method  similar  to  that  of  Abramowitz 
( 1937)  and  Handin  ct  al.  ( 1964) .  The  fish  were  anaesthetized  by  1 : 5000  MS  222. 
The  parasphenoid  was  cut  by  iridectomy  scissors  and  the  pituitary  removed  by 
gentle  suction.  The  animals  were  allowed  to  recover  in  40%  sea  water  and  were 
used  for  experiment  after  normal  feeding  had  been  resumed,  usually  after  one  to  two 
weeks.  Bovine  prolactin  (NIH-P-BI,  13  International  units/mg.)  was  provided 
by  the  NIH  Pituitary  Hormone  Distribution  Program.  Prolactin  was  injected 
intraperitoneally  in  isotonic  NaCl  containing  168  mM  NaCl/L.,  and  500  mg. 
prolactin/L. 

Unless  otherwise  stated  fish  were  injected  with  20  y  prolactin/fish  (ca.  5  y/gm. 
fish)  48  hours  before  the  experiment  and  injected  again  with  20  y/fish  24  hours 
before  the  experiment.  After  the  second  injection  the  fish  were  transferred  to  fresh 
water.  Each  injection  was  equal  to  about  1%  of  the  weight  of  the  fish.  This  is 
equivalent  to  only  half  an  hour  of  normal  drinking  in  40%  sea  water  (Potts  and 
Evans,  1966)  or  to  one  hour  of  urine  production  in  fresh  water  (Stanley  and 
Fleming,  1964).  The  injections  are  unlikely  to  alter  water  balance  significantly 
24  hours  later. 

RESULTS 

After  recovery  from  hypophysectomy  the  fish  survived  for  at  least  several 
weeks  in  40%  sea  water  but  after  transfer  to  fresh  water  the  fish  became  aesthenic 
in  a  few  hours  and  the  majority  died  within  24  hours.  Burden  found  that 
hypophysectomized  Fundulns  would  survive  for  several  days  in  fresh  water  at 
15°  C.  The  more  rapid  deterioration  of  our  fish  may  have  been  due  to  the  higher 
temperature.  Control  fish  survived  indefinitely  in  the  fresh  water. 

Sodium  and  bromide  fln.vcs  in  hypophysectomised  Fundulus 

The  rate  constants  of  sodium  and  bromide  efflux  from  hypophysectomized 
Fundulus  in  the  two  hours  following  transfer  from  40%  sea  water  to  fresh  water  are 
shown  in  Table  I.  Salt  loss  is  clearly  much  higher  in  hypophysectomized  fish. 

The  rate  of  influx  of  sodium  into  hypophysectomized  fish  in  fresh  water  was 
similar  to  that  into  normal  fish.  The  mean  rate  of  influx  into  a  small  series  of 


364 


W.  T.  W.  POTTS  AND  D.  H.  EVANS 


four  hypophysectomized  fish  in  the  two  hours  following  transfer  from  40%  sea 
water  was  0.50  pM/gm./hr.  (range  0.63-0.31).  In  normal  fish  fully  adapted  to 
fresh  water  the  influx  averages  0.58  ±  0.10  juM/gm./hr.  (N  =  16)  (Potts  and 
Evans,  1966).  It  is  clear  that  the  hypophysectomized  fish  is  at  a  disadvantage  in 
fresh  water.  As  the  sodium  content  of  the  fish  will  decline  during  the  experiment 
the  rate  of  loss  will  vary  with  time.  However,  if  the  initial  sodium  content  of  the 
fish  were  60  p,M  Na/gm.  (Potts  and  Evans,  1966)  and  the  efflux  rate  constant 
were  invariable,  the  initial  rate  of  loss  would  be  about  12  /xM/gm./hr.  while  the 
influx  would  be  only  0.5-0.6  pM  Na/gm./hr. 

In  normal  fish  adapted  to  fresh  water  the  greater  part  of  the  sodium  loss  takes 
place  through  the  body  surface  (ca.  0.36  /xM/gm./hr.)  while  the  remainder  (ca.  0.22 
/xM/gm./hr.)  is  lost  through  the  kidney  and  gut  (Potts  and  Evans,  1966;  Meier 

TABLE  I 

Rate  of  constants  of  sodium  and  bromide  efflux  from  normal  and  hypophysectomized 

Fundulus  in  various  media;  h~l  20° C. 


Sea  water 

40%  sea  water 

Fresh  water  (during 
2  hours  following 
transfer  from  40% 
sea  water) 

Fresh  water 
(adapted  fish) 

Sodium 
Hypophysectomized  fish 

Normal  fish 

Bromide 
Hypophysectomized  fish 

Normal  fish 

0.446  +  0.041 
(7) 
0.462  ±  0.024 
(19) 

0.209  db  0.07 
(4) 
0.175  db  0.027 
(14) 

0.192  ±  0.080 

(H) 
0.050  ±  0.013 

(6) 

0.255  ±  0.032 
(17) 
0.134  ±  0.023 
(18) 
Mean  ±  S.E. 

0.0114  ±  0.003 

(8) 

0.492  ±  0.027 

(9) 

0.087  ±  0.007 
(27) 

0.037  ±  0.004 
(14) 
(No.  of  dels.) 

and  Fleming,  1962;  Stanley  and  Fleming,  1965).  In  an  attempt  to  isolate  the 
site  of  the  increased  loss  of  salt,  hypophysectomized  fish  were  transferred  to  fresh 
water  four  hours  after  the  anus  and  excretory  opening  had  been  ligated.  The  mean 
rate  constant  of  the  sodium  efflux  during  the  two-hour  period  after  transfer  was 
then  0.208  ±  0.039  (N  =  9).  Hence  the  greater  part  of  the  loss  probably  takes 
place  through  the  body  surface. 

The  effect  of  prolactin 

Hypophysectomized  fish  which  received  20  y  of  prolactin  every  two  days 
survived  well  in  fresh  water.  The  mortality  that  occurred,  ca.  10%/week,  may 
be  attributed  to  the  handling  associated  with  the  injections.  The  mean  rate 
constant  of  6  fish  maintained  in  fresh  water  for  24  hours  after  receiving  the  second 
20  y  injection  of  prolactin  was  0.026  ±  0.006.  Three  hypophysectomized  controls 
which  had  received  no  prolactin  were  all  dead  after  24  hours.  In  comparison 
normal  fish,  fully  adapted  to  fresh  water,  have  an  efflux  constant  of  0.0114  ±  0.003 
(N  =  8)  (Potts  and  Evans,  1966). 


PROLACTIN  AND  SALT  FLUXES  IN  FISH  365 

Normal  fish  adapted  to  fresh  water  contain  about  50  /*M  Na/gm.  If  the 
hypophysectomized  fish  receiving  prolactin  contained  the  same  quantity  an  efflux 
rate  constant  of  0.026  Ir1  would  correspond  to  a  sodium  loss  of  1.3  /xM/gm./hr. 
The  mean  measured  influx  into  the  four  hypophysectomized  fishes  receiving  pro- 
lactin was  1.0  p.M  Na/gm./hr.  As  the  fish  survived  almost  indefinitely,  a  balance 
must  be  struck  between  efflux  and  influx.  How  this  is  achieved  requires  further 
investigation.  If  the  efflux  is  initially  greater  than  the  influx  the  blood  concen- 
tration will  decline.  This  may  stimulate  the  uptake  system  above  its  normal  levels. 
Alternatively  as  the  total  body  sodium  declines  the  absolute  loss  will  decline  in 
proportion  even  if  the  rate  constant  does  not  change. 

DISCUSSION 

The  ability  of  prolactin  to  prolong  the  survival  of  hypophysectomized  fish  in 
fresh  water  has  been  known  for  seven  years  but  the  physiological  basis  of  this  action 
has  not  been  clear.  A  priori  two  simple  explanations  present  themselves.  Pro- 
lactin might  facilitate  salt  uptake  in  fresh  water  and/or  it  might  reduce  salt  loss. 
In  the  latter  case  the  loss  might  be  reduced  either  at  the  body  surface  and/or  in  the 
kidney.  The  experiments  reported  above  show  that  prolactin  acts  primarily  in 
reducing  salt  loss.  In  the  absence  of  prolactin  the  blood  concentration  will  fall  as 
loss  exceeds  uptake.  Pickford,  Pang  and  Sawyer  (1966)  have  shown  that  when 
hypophysectomized  Fundulus  heteroclitus  fails  in  fresh  water  the  blood  concentra- 
tion lies  in  the  region  of  0.25-0.29  M/L.  compared  with  a  normal  concentration 
of  0.37  M/L.  The  effect  of  hypophysectomy  on  salt  uptake  requires  further 
investigation.  The  survival  of  hypophysectomized  fish  for  more  than  a  few  hours 
suggests  that  some  compensation  for  the  large  losses  has  taken  place.  Any  decline  in 
blood  concentration  might  be  expected  to  stimulate  salt  uptake  to  some  extent,  but 
any  such  effect  will  be  secondary. 

The  ligation  experiments  suggest  that  the  greater  part  of  the  salt  loss  for  hypo- 
physectomized fish  occurs  at  the  body  surface.  The  difference  observed  between 
ligated  and  non-ligated  hypophysectomized  fish,  0.208  ±  0.039  and  0.235  ±  0.089, 
respectively,  is  not  significant.  Stanley  and  Fleming  (1960)  have  shown  that 
prolactin  does  affect  renal  function.  In  the  plains  killifish,  F.  kansae,  prolactin  was 
found  to  increase  urine  flow  but  to  reduce  urine  sodium  concentration  in  fish 
adapted  to  fresh  water.  It  is  not  clear  whether  or  not  the  overall  renal  sodium 
loss  was  increased  or  decreased. 

The  relationship  between  the  effects  of  prolactin  in  these  experiments  and  the 
normal  function  of  the  pituitary  in  the  control  of  osmoregulation  must  now  be 
considered.  It  has  been  shown  that  adaptation  to  fresh  water  by  euryhaline  teleosts 
is  associated  with  a  marked  reduction  in  the  extrarenal  loss  of  sodium  (Motais  and 
Maetz,  1964;  Potts  and  Evans,  1966).  This  is  brought  about  in  Fundulus  by  a 
reduction  of  the  permeability  of  the  body  surface  to  ions  (Potts  and  Evans)  although 
in  other  species  exchange  diffusion  plays  an  important  part  in  the  apparent  reduc- 
tion of  sodium  loss  (Maetz,  personal  communication).  In  Fundulus  a  significant 
reduction  in  permeability  may  be  induced  by  the  immersion  of  sea-water-adapted 
fish  in  fresh  water  for  only  10  minutes.  Once  induced  the  reduced  permeability 
is  maintained  for  several  hours.  It  is  tempting  to  suggest  that  this  reduction  of 
permeability  is  brought  about  by  the  release  of  a  prolactin-like  hormone  from  the 


366  W.  T.  W.  POTTS  AND  D.  H.  EVANS 

fish  pituitary.  It  is  significant  in  this  respect  that  hypophysectomized  fish  survived 
well  in  sea  water  and  the  rate  constants  of  sodium  exchange  were  similar  to  those 
in  normal  fish  (Table  I).  Similarly  the  rate  constant  of  hypophysectomized  fish 
in  fresh  water,  and  hence  their  permeability,  was  similar  to  that  of  fish  adapted  to 
40%  sea  water. 

A  prolactin-like  hormone  has  been  identified  in  Funduhts  and  in  the  fresh-water 
carp  but  not  in  the  marine  cod  (Cadus)  or  hake  (Urophycls}  (Grant  and  Pickford, 
1959).  This  teleost  prolactin-like  hormone  resembles  mammalian  prolactin  but  is 
not  identical  with  it.  The  teleost  prolactin-like  hormone  possesses  red  eft  water 
drive  activity  (Grant  and  Pickford,  1959)  but  lacks  pigeon  crop  sac  stimulating 
activity  (Nicoll  and  Bern,  1964).  Similarly  frog  prolactin  shows  some  but  not 
all  the  characteristics  of  mammalian  prolactin  (Chad wick,  1966). 

A  prolactin-like  hormone  is  not  essential  for  the  survival  of  all  teleosts  in  fresh 
water.  Both  the  eel  Anguilla  anguilla  and  the  golfish  Carasshis  aiiratus  will 
survive  in  fresh  water  following  hypophysectomy  (Fontaine,  Callamand  and 
Olivereau,  1949;  Schreibman  and  Kallman,  1966),  although  the  eel  at  least  also 
produces  a  prolactin-like  hormone  (Ball  and  Olivereau,  1964).  In  the  case  of  the 
eel  survival  may  be  due  to  its  very  low  permeability.  Normal  silver  eels  will 
survive  "readily"  in  glass-distilled  water  for  three  months  and  the  rate  of  sodium 
loss  in  fresh  water  is  of  the  order  of  1 1  /^M/kg./hr.  compared  with  580  /iM/kg./hr. 
in  Fimdulus  (Chester  Jones,  Henderson  and  Butler,  1965).  At  low  temperatures 
even  hypophysectomized  Fimdulus  will  survive  in  fresh  water  for  several  days 
(Burden,  1956)  so  it  must  be  very  close  to  salt  balance  in  these  conditions. 

Attempts  by  the  authors  to  induce  a  state  of  low  permeability  by  the  injection  of 
prolactin  into  sea-water-adapted  Fimdulus,  comparable  with  that  induced  by  a  short 
treatment  with  fresh  water,  were  not  successful.  This  may  indicate  that  further 
stimuli  are  required  to  bring  about  a  state  of  low  permeability  in  addition  to  prolac- 
tin alone.  On  the  other  hand  more  success  might  be  obtained  with  ovine  prolactin 
or  better  still  with  a  fish  prolactin-like  hormone.  Ovine  prolactin  supports  hypo- 
physectomized Fundulus  rather  better  than  bovine  ( Pickford,  Robertson  and 
Sawyer,  1965). 

Pickford,  Pang  and  Sawyer  (1966)  have  recently  suggested  that  prolactin 
prolongs  survival  in  fresh  water  by  its  action  on  the  fish  mucous  cells.  The 
greater  part  of  the  extrarenal  sodium  exchange  probably  takes  place  through  the 
gills  rather  than  through  the  general  body  surface.  Mucus  might  reduce  loss  over 
the  gills  by  increasing  the  thickness  of  the  non-stirred  layer  but  a  direct  action  on 
the  gill  epithelium  is  also  possible. 

Olivereau  (1966)  found  that  prolactin  had  a  thyroid-stimulating  action  in  hypo- 
physectomized eels  (Anguilla)  but  thyrotropin  does  not  prolong  survival  in 
hypophysectomized  platyfish  (Xiphoplwrits  maculatus)  in  fresh  water  (Schreib- 
man and  Kallman,  1966). 

There  is  some  evidence  that  hypophysectomy  reduces  urine  flow  in  fresh  water 
while  prolactin  increases  it  again.  Chester  Jones  et  al.  (1965)  found  that  urine  flow 
in  hypophysectomized  eels  was  less  than  half  normal  while  Stanley  and  Fleming 
(1965)  found  that  hypophysectomized  F.  kansae  had  low  rates  of  urine  production 
which  were  increased  by  prolactin.  Their  results  may  imply  that  prolactin  increases 
water  permeability  but  a  low  permeability  to  water  would  not  be  a  disadvantage 
in  fresh  water. 


PROLACTIN  AND  SALT  FLUXES  IN  FISH  367 

This  work  was  supported  by  N.I.H.  grant  no.  GM  1030-03.  We  are  also 
indebted  to  the  N.I.H.  Endocrinology  Study  Section  for  the  supply  of  the  prolactin. 

SUMMARY 

Hypophysectomized  Fundulus  in  fresh  water  lost  sodium  several  times  as 
rapidly  as  normal  fish.  The  greater  part  of  the  loss  takes  place  extrarenally. 
Prolactin  reduces  the  loss  almost  to  normal  levels,  thus  prolonging  survival.  The 
possible  functions  of  prolactin  in  osmoregulation  in  normal  fish  are  discussed. 

LITERATURE  CITED 

ABRAMOWITZ,  A.  A.     1937.     The  opercular  approach  to  the  pituitary.     Science,  85:  609. 
BALL,  J.  N.,  1962.     Brood  production  after  hypophysectomy  in  the  viviparous  teleost  Mollienesia 

latipinna.    Nature,  194:  787. 
BALL,  J.  N.,  AND  M.  OLIVEREAU,  1964.     Role  de  la  prolactin  dans  la  survie  en  eau  douce  de 

Poecilia  latipinna  hypophysectomise.     C.  R.  Acad.  Sci.  Paris,  259:  1443-1446. 
BALL,  J.  N.,  M.  OLIVEREAU,  A.  M.  SLICKER  AND  K.  D.  KALLMAN,  1965.     Functional  capacity  of 

ectopic  pituitary  transplants   in  the  telost   Poecilia  formosa.   Phil.    Trans.   Roy.   Soc. 

Ser.B,2W:  69-99. 
BURDEN,    C.    E.,    1956.     The    failure    of    hypophysectomized    Fundulus    to    survive    in    fresh 

water.    Biol.  Bull,  110:  8-28. 
CHADWICK,  A.,  1966.     Prolactin-like  activity  in  the  pituitary  gland  of  the  frog.     /.  Endocrin., 

34:  247-255. 
FLEMING,  W.  R.,  AND  F.  I.  KAMEMOTO,   1963.     The  site  of  sodium  outflux  from  the  gill   of 

Fundulus  kansac.     Comp.  Biochem.  Physiol.,  8:  263-269. 
FONTAINE,   M.,   O.   CALLAMAND   AND   M.   OLIVEREAU,    1949.     Hypophyse   et   euryhalinite   chez 

Anguille.     C.  R.  Acad.  Sci.  Paris,  228:  513-514. 
GORDON,  M.  S.,  1962.     Chloride  exchanges  in  the  rainbow  trout  (Sahno  gairdneri)   adapted  to 

different  salinities.     Biol.  Bull.,  124:  45-54. 
GRANT,  W.  C.,  AND  G.  E.  PICKFORD,  1959.     Water  drive  factor  in  teleosts.     Biol.  Bull.,  116: 

429-435. 
HANDIN,  R.  I.,  J.  NANDI  AND  H.  A.  BERN,  1964.     The  effect  of  hypophysectomy  on  survival 

and  on  thyroid  and   interrenal   histology   of   the   cichlid   teleost    Tilapia   mossambica. 

J.  Exp.  Biol.,  157:  339-344. 
HOUSE,  C.  R.,  1963.     Osmotic  regulation  in  the  brackish  water  teleost  Blennius  pholis.    J.  Exp. 

Biol.,  40:  87-104. 
JONES,  CHESTER  I.,  I.  W.  HENDERSON  AND  D.  G.  BUTLER,  1965.     Water  and  electrolyte  flux  in 

the  European  eel  (Anguilla  anguilla').    Arch.  Anat.  Micr.  Morph.  Exp.,  54:  453-469. 
A^EIER,  A.  H.,  AND  W.  R.  FLEMING,  1962.     The  effect  of  Pitocin  and  Pitressin  on  water  and 

sodium    movement    in    the    euryhaline    killifish    Fundulus    kansac.     Comp.    Biochem. 

Physiol,  6:  215-231. 
MOTAIS,  R.,  AND  J.  MAETZ,  1964.     Action  des  hormones  neurohypophysaires  sur  les  echanges  de 

sodium  (measures  a  1'aide  du  radiosodium  24Na)  chez  teleosteen  euryhalin  Platichthys 

flessus.    Gen.  Comp.  Endocrin.,  4:  210-224. 
MULLINS,  L.  J.,  1950.     Osmotic  regulation  in  fish  as  studied  with  radioisotopes.     Acta  Physiol. 

Scand.,21:  303-314. 
NICOLL,   C.   S.,  AND  H.   A.   BERN,    1964.     'Prolactin'   and  the  pituitary   glands   of   fish.     Gen. 

Comp.  Endocrin.,  4:  457-471. 
OLIVEREAU,  M.,  1966.     Action  de  la  Prolactine  chez  1' Anguille  intacte  et  hypophysectomisee. 

I.     Systeme  hypophyso-thyroidien  et  pigmentation.     Gen.  Comp.  Endocrin.,  6:  130-143. 
PICKFORD,  G.  E.,  AND  J.  G.   PHILLIPS,   1959.     Prolactin,  a  factor  promoting  the   survival   of 

hypophysectomized  killifish  in  fresh  water.     Science,  130:  453. 
PICKFORD,  G.  E.,  P.  K.  T.  PANG  AND  W.  H.  SAWYER,  1966.     Prolactin  and  serum  osmolarity 

in  hypophysectomised  Fundulus  heteroclitus  in  fresh  water.     Nature,  209:  1040-1040, 


368  W.  T.  W.  POTTS  AND  D.  H.  EVANS 

PICKFORD,  G.  E.,  E.  E.  ROBERTSON  AND  W.  H.  SAWYER,  1965.     Hypophysectomy  replacement 

therapy    and    the    tolerance    of    the    euryhaline    killifish    Fundulus    heteroditus    to 

hypotonic  media.     Gen.  Comp.  Endocrin.,  5:  160-180. 
POTTS,  W.   T.  W.,  AND  D.   H.   EVANS,   1966.     Sodium  and  chloride   balance   in   the  killifish 

Fundulus  heteroditus.      (Unpublished.) 
SCHREIBMAN,  M.  P.,  AND  K.  D.  KALLMAN,   1962.     Functional  pituitary  grafts  in  freshwater 

teleost  Xiphophorus.    Amer.  Zool.,  4:  417. 
SCHREIBMAN,  M.  P.,  AND  K.  D.  KALLMAN,  1966.     Endocrine  control  of  freshwater  tolerance 

in  teleosts.     Gen.  Comp.  Endocrin.,  6:  144-155. 
STANLEY,  J.  G.,  AND  W.  R.  FLEMING,  1960.    The  effects  of  ACTH  and  prolactin  on  the  salt 

and  water  metabolism  of  Fundulus  kansae.    Amer.  Zool.,  3 :  502-503. 
STANLEY,   J.    G.,   AND   W.   R.    FLEMING,    1964.     Excretion   of  hyper  tonic    urine   by   a   teleost. 

Science,  144:  63-64. 
STANLEY,  J.  G.,  AND  W.  R.  FLEMING,  1965.     Sodium  metabolism  in  Fundulus  kansae  in  fresh 

water  and  during  adaptation  to  sea  water.    Amer.  Zool.,  5:  688. 


THE  MECHANISM  OF  BURROWING  IN  THE  POLYCHAETE 
WORM,  ARENICOLA  MARINA  (L.) 

E.  R.  TRUEMAN 
Zoology  Department,  The  University,  Hiill,  England 

New  techniques  of  recording  pressure  changes  and  activity  have  increased  our 
knowledge  of  the  fluid  dynamics  of  burrowing  in  Arenicola  marina  (L.)  (Trueman, 
1966a).  The  new  information,  together  with  an  understanding  of  the  habits  of  the 
lugworm,  derived  from  the  extensive  researches  of  Wells  (1961),  allow  an  assess- 
ment of  the  mechanism  of  burrowing  to  be  made. 

Initial  entry  of  a  worm  into  sand  is  brought  about  by  the  eversion  of  the 
proboscis  at  comparatively  low  coelomic  pressures  (2-6  cm.  water  pressure)  and 
when  several  segments  have  passed  beneath  the  surface  a  series  of  pressure  peaks 
commence,  each  of  about  two  seconds  duration.  They  occur  at  intervals  of  5-7 
seconds,  and  as  burrowing  progresses  increase  in  amplitude  up  to  110  cm.  During 
burrowing,  waves  of  peristaltic  contraction  pass  along  the  trunk  from  the  posterior 
segments  to  the  anterior  buried  region  of  the  worm  where  they  appear  to  develop 
into  high  pressure  peaks  by  the  synchronous  contraction  of  the  longitudinal  muscles 
of  all  or  part  of  the  trunk  segments.  The  fluid  of  the  essentially  single  trunk 
coelom  acts  in  a  hydraulic  system  which  allows  the  force  produced  by  the  longi- 
tudinal muscles  of  the  posterior  trunk  to  be  transferred  to  the  anterior  end,  there  to 
be  utilized  in  burrowing.  The  principal  function  of  the  high  pressure  is  to  anchor 
the  anterior  end  during  the  contraction  of  the  longitudinal  muscles.  Each  time  the 
pressure  increases  the  anterior  end  is  pressed  firmly  against  the  substrate,  while  the 
posterior  trunk  is  pulled  into  the  burrow. 

The  purpose  of  this  article  is  to  consider  further  observations  of  the  movements 
made  by  Arenicola  during  burrowing  and  to  compare  the  mechanism  with  that  of 
other  animals,  in  particular  bivalve  molluscs. 

MATERIAL  AND  METHODS 

Observations  were  made  of  the  burrowing  of  Arenicola  of  15-20  cm.  length  both 
at  Hull  and  at  the  Marine  Biological  Laboratory,  Millport,  using  specimens  which 
would  burrow  rapidly.  Direct  visual  observation  of  burrowing  into  sand  could  be 
made  from  above  or  from  the  side,  through  the  glass  of  an  aquarium  tank.  The 
latter  was  largely,  but  not  always,  unrewarding  as  even  when  the  worm  was  close 
to  the  glass  a  thin  layer  of  sand  could  obstruct  detailed  observation.  Accordingly 
a  technique  (Trueman,  1966a)  of  continuously  recording  the  pressure  imparted 
to  the  sand  by  a  burrowing  worm  was  further  developed  by  use  of  a  more  sensitive 
pressure  transducer  (Statham,  Model  P  23  BB,  maximum  sensitivity  0.4  cm. 
pressure/cm,  pen  deflexion)  which  was  coupled  to  a  multichannel  pen  recorder. 
Both  instruments  were  obtained  from  E.  &  M.  Instrument  Company  Inc. 

369 


370 


E.  R.  TRUEMAN 


Worms  were  allowed  to  burrow  over  a  glass  tube  (3  mm.  bore)  buried  in  the 
sand  with  its  external  opening  covered  by  a  coarse  nylon  mesh  to  prevent  entry  of 
sand  grains.  This  was  connected  by  pressure  tubing  to  the  transducer,  and  pressure 
applied  to  the  adjacent  sand  either  by  a  plunger  or  by  an  Arenicola  burrowing 
caused  a  negative  response.  The  explanation  of  this  may  lie  in  the  dilatant  proper- 
ties of  the  sand  (Chapman  and  Newell,  1947),  for  the  applied  pressure  disturbs 
the  packing  of  the  sand-water  system  and  tends  to  cause  water  to  be  drawn  in.  In 
a  full  account  of  this  technique  (Hoggarth  and  Trueman,  1966)  it  is  emphasized  that 
all  recordings  must  be  interpreted  by  direct  visual  observations  but  that  with  this 
proviso  it  serves  as  a  useful  method  of  determining  the  activity  of  an  animal, 
invisible  beneath  the  sand  yet  without  any  obstruction  by  electrodes.  Although  bur- 
rowing was  recorded  in  this  manner  for  about  50  worms,  direct  observations  of 
burrowing  movements  were  only  satisfactorily  made  on  5  occasions  when  the  events 
were  marked  on  the  recording  by  means  of  a  manually  operated  key.  Coelomic 
pressures  during  burrowing  were  recorded  as  previously  with  a  Bourdon  transducer 
obtained  from  the  E.  &  M.  Instrument  Company  Inc.  (Trueman,  1966a). 

EXPERIMENTAL  RESULTS 

The  recording  of  external  pressures  derived  from  an  Arenicola  burrowing  in 
sand  covered  by  several  cm.  of  water  consists  of  a  series  of  negative  pressures 
whose  amplitude  varies  with  the  distance  of  the  worm  from  the  recording  device. 
These  pressures  were  observed  to  correspond  to  the  swelling  of  the  anterior  seg- 
ments and  to  a  marked  increase  in  the  turgidity  of  the  entire  trunk  region  (Fig.  la). 


i      i — I — i — I — i — i — i 


-1  5s 


FIGURE  1.  Recordings  of  the  pressures  produced  by  Arenicola  in  sand  (external)  and  in  the 
coelom  during  burrowing,  a,  sequence  from  the  commencement  of  burrowing  (extreme  left) 
showing  gradual  increase  in  amplitude  of  the  negative  swings  as  penetration  proceeds  (at  X) 
and  the  reduction  of  their  frequency  (at  Y)  after  6  branchial  segments  have  passed  into  the 
sand.  Visual  observations  of  the  swelling  and  turgidity  of  the  anterior  segments  are  indicated 
above  the  time  trace,  b,  simultaneous  recordings  of  coelomic  and  external  pressures,  com- 
mencing with  four  anterior  segments  beneath  the  sand.  Lower  amplitude  of  the  external 
pressures  due  to  greater  distance  from  the  recording  device ;  flat  tops  of  the  coelomic  pressure 
trace  caused  by  saturation  of  the  recorder. 


MECHANISM  OF  BURROWING  IN  ARENICOLA 


371 


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FIGURE  2.  Recording  of  the  pressures  produced  in  sand  by  Arcnicola  when  burrowing 
against  glass  with  the  trunk  almost  completely  beneath  the  surface.  Flanging  (F)  and  proboscis 
eversion  (P)  were  marked  by  direct  observation  of  the  anterior  segments. 

Synchronous  recordings  demonstrate  that  the  negative  pressures  correspond  to  the 
pressure  peaks  in  the  coelom  (Fig.  Ib).  At  the  commencement  of  burrowing  the 
latter  increase  in  amplitude  with  penetration  (Trueman,  1966a;  Fig.  6)  and  a 
similar  feature  occurs  in  respect  of  the  negative  pressures  (Fig.  la,  X).  This 
indicates  that  until  a  firm  anchorage  is  obtained,  maximum  pressures  are  not  exerted 
on  the  substrate. 

The  external  pressure  recorded  fluctuates  continuously  between  the  negative 
peaks  and  it  has  not  been  possible  to  interpret  these  changes  in  detail.  The  most 
marked  positive  peak,  following  immediately  after  the  negative  pressure,  may  be  due 
to  the  dilatant  properties  of  the  sand-water  system.  Proboscis  eversion  and  the  oc- 
currence of  flanging  were  marked  by  visual  observations,  through  the  side  of  a 
glass  tank.  Flanging  (Fig.  3b)  was  most  clearly  observed  on  the  first  three  trunk- 
segments  where  the  fleshy  parapodial  ridges  each  form  an  annulus  which  may  be 
raised  suddenly  into  a  sharply  projecting  flange  (Wells,  1944,  1961).  Flanging  and 
proboscis  eversion  were  never  observed  at  peak  pressures  but  occurred  between 
these  (Fig.  2).  Conversely  the  anterior  4  or  5  segments  became  very  dilated  when 
the  maximum  pressures  were  recorded  (Fig.  3a).  High  pressures  were  clearly  not 
synchronized  with  proboscis  extrusion  nor  was  there  any  apparent  forward  move- 
ment of  the  head  of  the  worm  at  this  phase  of  digging  activity.  It  was  previously 
(Trueman,  1966a)  considered  likely  that  the  high  coelomic  pressure  contributed  to 
forward  movement  of  the  head  of  the  worm  but  in  the  light  of  the  present  observa- 
tions this  appears  to  be  incorrect. 

After  Arenicola  has  burrowed  for  1  to  1^  minutes  the  frequency  of  the  negative 
pressures  often  shows  a  marked  reduction  (Fig.  la,  Y).  When  recording  the 
coelomic  pressure  a  similar  feature  was  observed  and  was  interpreted  as  being  due 
to  the  lack  of  development  of  the  full  muscular  power  of  the  worm,  possibly  because 
of  fatigue  (Trueman,  1966a).  Waves  of  peristaltic  contraction  pass  forward  along 
the  trunk  during  burrowing  and  develop  into  pressure  peaks  upon  reaching  the 
anterior  segments.  As  burrowing  proceeds  each  peristaltic  wave  does  not  produce 
high  coelomic  pressures  or  dilation  and  accordingly  the  negative  pressures  also 
occur  less  frequently.  Proboscis  extrusion  and  flanging  continue,  however, 
throughout  the  interval  between  maximum  pressures.  The  proboscis  effects  the 
initial  entry  of  the  worm  into  the  sand  by  a  scraping  action  (Wells,  1961)  and  very 
likely  continues  to  scrape  away  the  substrate  when  more  deeply  burrowed.  Re- 


372 


E.  R.  TRUEMAN 


peated  proboscis  extrusion  between  pressure  peaks  is  thus  probably  related  to  the 
progression  of  the  worm.  The  resistance  of  the  substrate  to  penetration  increases 
with  depth  of  burial  (Trueman,  Brand  and  Davis,  1966b)  and  the  longer  interval 
between  pressure  peaks  allows  time  for  more  extrusions  of  the  proboscis  and 
extension  of  the  head.  During  studies  of  burrowing  by  bivalves  similar  observations 
have  been  made,  indicating  that  the  amount  of  probing  by  the  foot  increases  with 
depth  of  burial  (Trueman,  Brand  and  Davis,  1966a). 


FIGURE  3.  Diagram  of  two  successive  stages  of  burrowing  of  Arenicola.  a,  shows  the 
anterior  segments  dilated  to  form  an  anchor  (arrowheads),  allowing  the  worm  to  penetrate  the 
burrow  (solid,  tailed  arrow)  on  contraction  of  the  longitudinal  muscles  (double  arrow,  L).  b, 
shows  the  flange  anchor  (arrowheads)  and  eversion  of  the  proboscis  (solid  arrow),  b,  im- 
mediately follows  a,  at  the  fall  of  coelomic  pressure,  and  involves  elongation  by  the  contraction  of 
circular  muscles  (double  arrow,  C).  Provided  the  flanges  afford  an  anchorage,  e.g.,  at  F,  on 
the  second  chaetigerous  annulus,  the  head  pushes  forward  and  the  posterior  trunk  segments 
retract  from  the  burrow  (solid,  tailed  arrows).  Movement  between  a  and  b  is  also  indicated  by 
broken  lines  drawn  between  comparable  parts  of  the  worm  and  by  the  numbering  of  the  segments. 


BURROWING  ACTIVITY  OF  ARENICOLA 

On  the  basis  of  recordings  (Figs.  1  and  2)  and  direct  observations  it  has  been 
determined  that  two  principal  conditions  of  the  anterior  trunk  segments  occur 
successively  during  burrowing  activity.  These  are  first,  dilation,  caused  by  the 
high  coelomic  pressures  and  secondly,  flanging,  accompanied  by  proboscis  eversion 
(Fig.  3).  Both  of  these  conditions  involve  the  anchorage  of  some  part  of  the 
anterior  region  of  the  worm  and  in  a  normal  burrowing  sequence  the  type  of 
anchorage  alternates  between  the  dilation  anchor  (Fig.  3a)  and  the  flanging 
anchor  (Fig.  3b). 

The  peristaltic  wave  passing  forwards  along  the  trunk  forces  coelomic  fluid  into 
the  head  with  the  contraction  of  the  circular  muscles  posterior  to  segment  7  (Fig. 


MECHANISM  OF  BURROWING  IN  ARENICOLA  373 

3a).  This  causes  some  increase  in  coelomic  pressure,  partial  dilation  of  the  most 
anterior  segments  (Trueman,  1966a)  and  is  immediately  followed  by  the  contrac- 
tion of  the  longitudinal  muscles  of  the  trunk.  This  brings  about  maximum  dilation 
and  a  firm  anchorage  of  the  head,  so  allowing  the  posterior  of  the  trunk  to  be  pulled 
forward  into  the  burrow.  The  effect  of  the  head  segments  exerting  pressure  on  a 
dilatant  substrate  is  to  make  the  sand-water  mixture  more  resistant  so  that  the 
chaetae  can  grip  and  the  body-wall  adhere  to  form  a  firm  anchor.  This  stage 
of  burrowing  is  equivalent  to  that  described  by  Wells  (1961)  as  an  "anti-seagull" 
reflex,  which  he  demonstrated  by  allowing  a  worm  to  burrow  down  the  stem  of 
a  large  glass  filter  funnel,  the  stem  being  closed  by  rubber  tubing  and  a  clamp. 
When  the  Arenicola  was  halfway  into  the  stem,  pulling  the  hinder  end  backwards 
caused  the  dilation  of  the  anterior  segments  and  resulted  in  a  tenacious  grip.  This 
experiment  has  been  repeated  with  a  pressure  transducer  attached  to  the  coelom  and 
gave  rise  to  high  internal  pressures  which  persisted  while  the  hind  end  was  being 
pulled. 

In  normal  burrowing  the  high  coelomic  pressure  and  the  dilation  anchor  are 
sustained  by  the  contraction  of  the  longitudinal  muscles  for  not  more  than  2  seconds, 
being  followed  by  the  relaxation  of  these  muscles  and  the  contraction  of  the  circular 
fibers  so  causing  elongation  of  the  worm.  This  elongation  appears  on  the  surface 
of  the  sand  as  a  re-emergence  of  the  posterior  trunk  segments  from  the  burrow  as 
the  pressure  drops  (Trueman,  1966a).  Contraction  of  the  circular  muscles 
completely  eliminates  the  dilation  anchor  but  this  is  replaced  by  the  flanging 
anchor  (Fig.  3b).  This  anchorage  allows  the  second  or  third  chaetigerous 
segment  to  remain  static  at  elongation  of  the  worm  so  that  the  segments  behind 
will  be  pushed  backwards  from  the  burrow  and  those  in  front  forwards  into  the 
substrate  as  the  proboscis  everts.  This  condition  is  shown  diagrammatically  in 
Figure  3b  where  the  second  annulus  is  arbitrarily  taken  as  a  fixed  point  about 
which  movement  backwards  and  forwards  occurs.  Wells  (1954),  in  a  detailed 
account  of  the  mechanism  of  proboscis  movement,  considered  that  the  head  of 
Arenicola  narrows  and  lengthens  during  the  first  stage  of  proboscis  extrusion.  This 
is  in  accord  with  the  observation  that  eversion  takes  place  when  the  dilation  anchor 
is  lost  as  the  worm  elongates. 

The  burrowing  activity  of  Arenicola  consists  of  the  following  stages:  (1)  Prob- 
ing forward  by  the  head,  proboscis  eversion  obtaining  initial  penetration  into  the 
sand  with  no  large  pressures  recorded.  (2)  Several  segments  buried,  allowing  a 
dilation  anchor  to  form  (Fig.  3a)  with  accompanying  high  pressures  and  the  pulling 
forward  of  the  worm  into  the  burrow.  (3)  Relaxation  of  longitudinal  and  contrac- 
tion of  circular  muscles,  resulting  in  the  lengthening  of  the  worm  and  the  produc- 
tion of  the  flange  anchor  (Fig.  3b).  Further  penetration  of  the  substrate  is  then 
obtained  by  proboscis  extrusion.  (4)  The  second  and  third  stages  are  repeated 
cyclically  until  burial  is  complete. 

High  coelomic  pressures  correspond  to  stage  (2)  but  as  the  worm  elongates 
during  the  third  stage,  the  pressure  drops  sharply  to  the  equivalent  of  little  more 
than  2  cm.  of  water.  The  function  of  the  high  pressure  is  both  to  obtain  an 
anchorage  and  to  compact  the  sides  of  the  burrow.  External  pressure  recordings 
of  Arenicola  in  normal  U-shaped  burrows  show  occasional  strongly  negative  pres- 
sures, comparable  to  those  recorded  during  digging,  which  suggest  that  high 


374 


E.  R.  TRUEMAN 


coelomic  pressures  are  used  in  the  natural  habitat,  possibly  to  consolidate  the 
burrow  wall.  Somewhat  similar  observations  have  been  made  in  respect  of 
burrowing  in  the  earthworm  (Roots  and  Phillips,  1960). 

DISCUSSION 

The  digging  activity  of  bivalve  molluscs  that  burrow  into  soft  substrates,  such  as 
Cardium,  Donax,  Anodonta  or  Ensis  (Trueman,  19661) ;  Trueman  et  al.,  1966a), 
makes  an  interesting  comparison  with  that  of  Arenicola,  for  all  are  well  adapted  for 


FIGURE  4.  Diagram  of  two  successive  stages  of  burrowing  of  a  generalized  bivalve  mollusc 
showing  pedal  (PA)  and  shell  (SA)  anchorages  (arrows),  a,  valves  adducted  (double  arrow, 
AM)  producing  pedal  dilation  and  a  cavity  (C)  in  the  sand  around  the  valves.  Contraction 
of  the  retractor  muscles  (RM)  then  causes  the  shell  to  be  pulled  down,  b,  valves  reopened  and 
pressed  against  the  sand  by  the  elasticity  of  the  ligament  (solid,  double  arrow,  L)  holding  the 
shell  fast  when  contraction  of  the  protractor  (P)  and  transverse  muscles  (T)  causes  pedal 
protraction.  AM,  adductor  muscle;  H,  pedal  haemocoele;  M,  mantle  cavity. 


MECHANISM  OF  BURROWING  IN  ARENICOLA 


375 


burrowing.  The  latter  has  an  essentially  single  coelomic  system  in  contrast  to 
the  double  system  of  the  bivalves,  which  consists  of  the  haemocoele,  the  hydro- 
dynamic  equivalent  of  the  coelom  in  Arenicola,  and  the  pallial  system.  Many 
bivalves  thus  have  the  advantage  of  being  able  to  eject  water  from  their  mantle 
cavity  during  digging  to  loosen  the  sand  adjacent  to  the  shell  (Fig.  4a,  C).  In  both 
the  thrust  used  in  initial  penetration  is  limited  by  the  weight  of  the  animal  since 
any  force  in  excess  of  the  weight  causes  the  animal  to  be  pushed  back  from  the 
sand.  Further  penetration  of  a  bivalve  consists  of  a  series  of  step-like  movements, 
each  termed  a  "digging  cycle,"  which  were  well  recorded  by  Quayle  (1949)  in  a 
study  of  the  digging  movements  of  Venerupis.  Each  digging  cycle  involves  the 
integration  of  adduction  and  the  reopening  of  the  valves  with  retraction  and  pro- 
traction of  the  foot.  Adduction  causes  high  pressure  in  the  haemocoele  and  as  a 
consequence  the  foot  becomes  swollen  to  form  a  pedal  anchor  (Fig.  4a)  (Trueman, 
1966b).  Immediately  after  adduction  the  retractor  muscles,  equivalent  to  the 
longitudinal  muscles  of  Arenicola,  contract,  pulling  the  shell  down  and  sustaining 


E 

u 

~  1 

V 

3 

in 

P 

in 

y 

9 

a° 

r~      ~r~  i^  '  5s 

FIGURE  5.  Recording  of  the  pressures  produced  in  sand  by  the  burrowing  of  Mactra 
subtruncata.  At  the  commencement  the  bivalve  is  lying  on  the  sand  and  the  foot  penetrates 
by  probing  (P).  A  succession  of  adductions  of  the  valves  and  pedal  retractions  (AR)  follow, 
giving  first  (left)  negative  swings,  when  the  foot  alone  is  beneath  the  sand,  to  be  replaced  by 
large  positive  pressures  (right)  when  the  valves  have  entered  the  sand.  These  are  caused  by 
ejection  of  water  from  the  mantle  cavity  at  adduction. 

the  pedal  pressure  and  anchorage.  Subsequently  the  opening  moment  of  the 
hinge  ligament  opens  the  valves  and  presses  them  against  the  substrate  (Fig.  4b)  so 
forming  a  shell  or  secondary  anchor.  This  holds  the  animal  firmly  whilst  the  foot 
extends  by  contraction  of  the  protractor  and  transverse  pedal  muscles,  which  in  this 
respect  are  the  equivalent  of  the  circular  muscles  of  the  body  wall  of  Arenicola. 
The  pedal  and  shell  anchors  of  a  bivalve  correspond  to  the  dilation  and  flange 
anchors  of  Arenicola,  respectively. 

The  entire  pattern  of  burrowing  activity  of  a  bivalve  as  recorded  by  the  external 
pressures  (Fig.  5)  is  very  similar  to  that  of  the  lugworm.  It  consists  of  a  series 
of  digging  cycles  each  of  which  produces  high  pressure  at  adduction-retraction 
(AR).  These  are  seen  as  negative  pressures  at  the  commencement  of  burrowing 
(Fig.  5,  left)  as  in  recording  of  Arenicola,  but  as  depth  of  penetration  increases  (to 
the  right,  Fig.  5)  and  the  shell  enters  the  sand,  water  is  ejected  from  the  mantle 
cavity  into  the  substrate  and  causes  the  succession  of  positive  pressures  (Hoggarth 
and  Trueman,  1966).  Between  the  "AR"  peaks,  when  the  shell  anchor  is  applied, 
numerous  probes  (P)  are  made  by  the  foot.  These  correspond  to  the  pushing 
forward  of  the  head  of  a  worm  from  the  flange  anchor  which  occurs  between  each 


376  E.  R.  TRUEMAN 

negative  pressure  recorded.  Although  the  structures  used  in  digging  in  Arenicola 
and  in  bivalves  are  anatomically  quite  different  there  is  a  fundamental  similarity 
in  the  mechanism  that  they  employ. 

Whilst  discussing  the  burrowing  of  worms  Clark  (1964)  considered  that  the 
method  of  burrowing  used  by  all  soft-bodied  animals  is  essentially  the  same.  He 
suggested  that  part  of  the  body  wall  is  first  dilated  to  form  an  anchor  while  the 
head  is  forced  into  the  substrate  by  contraction  of  the  circular  muscles,  and  that 
secondly  the  anterior  end  of  the  worm  dilates  to  form  a  new  anchor  while  the  body 
is  drawn  downwards  by  contraction  of  the  longitudinal  muscles.  These  two 
anchorages  correspond  respectively  to  the  flanging  and  dilation  anchors  of  Arenicola 
or  to  the  shell  and  pedal  anchors  of  a  bivalve.  Essentially  the  same  mechanism  is 
used  by  other  soft-bodied  animals  to  burrow  into  sand,  e.g.,  Nephtys  (Clark  and 
Clark,  1960),  Urechis  (Fisher  and  MacGinitie,  1928).  Detailed  knowledge, 
derived  from  continuous  recordings  of  activity  and  internal  pressures,  is  so  far 
limited  to  Arenicola  and  members  of  the  Bivalvia.  It  is  hoped  to  extend  these 
observations  in  the  near  future. 

SUMMARY 

1.  The  burrowing  activity  of  Arenicola  has  been  studied  by  means  of  direct  ob- 
servations and  recordings  of  pressure  changes  both  internally  and  in  the  adjacent 
sand. 

2.  Maximum  coelomic  pressures  correspond  to  the  swelling  of  the  anterior  seg- 
ments  to   form   an   anchor    (dilation   anchor)    which   allows   the   posterior   trunk 
segments  to  be  pulled  into  the  sand  and  the  sides  of  the  burrow  to  be  compacted. 
This  condition  occurs  alternately  with  the  occurrence  of  flanges  on  the  anterior 
segments  as  the  worm  elongates  by  contraction  of  the  circular  muscles. 

3.  The  flanges  tend  to  form  an  anchor    (flange   anchor)    from  part  of  the 
anterior  region  so  that  lengthening  forces  the  head   into  the   substrate,   as   the 
proboscis  everts.     At  the  same  time  the  posterior  trunk  region  undergoes  some 
retraction  from  the  burrow. 

4.  Essentially  the  same  method  is  used  by  all  soft-bodied  animals  to  dig  into 
sand,  notably  in  bivalve  molluscs.     In  this  group  a  pedal  anchor  is  formed  by  the 
foot  becoming  swollen  by  the  hydrostatic  pressure  derived  from  adduction  of  the 
valves  immediately  before  the  shell  is  pulled  down  by  the  pedal  retractor  muscles. 
The  shell  is  subsequently  held  still  by  the  opening  of  the  valves  against  the  substrate 
(shell  anchor)  while  the  foot  is  protracted  by  the  intrinsic  pedal  musculature. 

LITERATURE  CITED 

CHAPMAN,  G.,  AND  G.  E.  NEWELL,  1947.     The  role  of  the  body-fluid  in  relation  to  movement 

in    soft-bodied    invertebrates.     I.      The    burrowing    of    Arenicola.    Proc.    Roy.    Soc. 

London,  Ser.  B,  134:  431-455. 

CLARK,  R.  B.,  1964.     Dynamics  in  Metazoan  Evolution.     Clarendon  Press,  Oxford,  313  pp. 
CLARK,  R.  B.,  AND  M.  E.  CLARK,  1960.     The  ligamentary  system  and  the  segmental  musculature 

of  Nephtys.     Quart.  J.  Micr.  Sci.,  101 :  149-176. 
FISHER,  W.  K.,  AND  G.  E.  MACGINITIE,   1928.     The  natural  history  of  an  echiuroid  worm. 

Ann.  Mag.  Nat.  Hist.,  Ser.  X,  1:  204-213. 
HOGGARTH,  K.  R.,  AND  E.  R.  TRUEMAN,  1966.     Techniques  for  recording  the  activity  of  aquatic 

invertebrates.     Nature,  in  press. 


MECHANISM  OF  BURROWING  IN  ARENICOLA  377 

QUAYLE,    D.    B.,    1949.     Movements    in    Vencrupis    (=Paphia}    pullastra    (Montagu).    Proc. 

Malac.  Soc.  London,  28:  31-37. 
ROOTS,  B.  I.,  AND  I.  I.  PHILLIPS,  1960.     Burrowing  and  the  action  of  the  pharynx  in  earthworms. 

Med.  Biol.  Illust.,  10:28-31. 
TRUEMAN,  E.  R.,  1966a.     Observations  on  the  burrowing  of  Arcnicola  marina   (L.).    J.  Exp. 

Biol.,  44:93-118. 
TRUEMAN,    E.    R.,    1966b.     Bivalve    mollusks :    fluid    dynamics    of    burrowing.     Science,    152: 

523-525. 
TRUEMAN,  E.  R.,  A.  R.  BRAND  AND  P.  DAVIS,  1966a.     The  dynamics  of  burrowing  of  some 

common  littoral  bivalves.    /.  Exp.  Biol.,  44:  in  press. 
TRUEMAN,  E.  R.,  A.  R.  BRAND  AND  P.  DAVIS,  1966b.     The  effect  of  substrate  and  shell  shape 

on  the  burrowing  of  some  common  bivalves.     Proc.  Malac.  Soc.  London,  37:  in  press. 
WELLS,  G.  P.,  1944.     The  parapodia  of  Arenicola  marina  L.    Proc.  Zool.  Soc.  London,  114: 

100-116. 
WELLS,  G.  P.,  1954.     The  mechanism  of  proboscis  movement  in  Arenicola.     Quart.  J.  Micr. 

Sci.,  95:251-270. 
WELLS,  G.  P.,  1961.     How  lugworms  move.    In:  The  Cell  and  the  Organism,  J.  A.  Ramsay 

and  V.  B.  Wigglesworth,  Eds.,  University  Press,  Cambridge,  pp.  209-233. 


ABSTRACTS  OF  PAPERS  PRESENTED  AT 
THE  MARINE  BIOLOGICAL  LABORATORY 

1966 

ABSTRACTS  OF  SEMINAR  PAPERS 

JULY  26,  1966 

Effects  of  ultraviolet  radiation  with  special  reference  to  racial  differences  in  colora- 
tion. GEORGE  SZABO. 

Single  and  repeated  exposures  of  ultraviolet  radiation  of  erythemal  doses  were  delivered 
on  human  subjects  of  Caucasian  (redhead  and  Mediterranean),  Negro  and  Mongoloid  races, 
to  compare  the  response  of  exposed  areas  (forearm)  with  that  of  the  unexposed  regions  (hip  or 
abdomen)  of  the  same  individual. 

There  was  a  marked  difference  in  response  between  various  races  and  between  different 
regions  of  the  integument  of  the  same  individual.  The  same  erythemal  dose  provoked  no 
visible  reddening  in  the  unexposed  (hip)  areas  of  Negroes  or  Mongoloids,  whereas  visible 
erythema  developed  in  redheaded  Caucasians.  There  was  no  visible  erythema  in  Caucasians 
or  in  other  races  when  the  forearm  was  exposed  to  ultraviolet.  The  melanocyte  population  did 
not  show  a  clear  tendency  toward  increase  after  single  exposure,  but  in  the  case  of  the  hip, 
three  subjects  out  of  6  showed  an  increase  10  days  after  radiation.  In  the  case  of  the  forearm, 
after  fluctuation  at  5th  day  post-radiation,  there  was  no  such  increase  in  melanocyte  frequency. 
After  multiple  doses  of  radiation,  there  was  an  increase  in  melanocyte  frequency  both  in  the 
forearm  and  on  the  abdomen.  In  the  forearm,  this  increase  was  much  smaller  than  in  the 
abdomen.  After  the  cessation  of  multiple  radiation,  the  elevated  melanocyte  population  showed 
a  tendency  to  revert  to  its  original  lower  density.  Electron  microscopical  studies  have  revealed 
that  non-irradiated  melanocytes  contain  mostly  pre-melanosomes,  although  the  neighboring 
Malpighian  cells  may  be  full  of  melanized  melanosomes.  After  radiation,  however,  the  melano- 
cytes are  full  of  melanized  melanosomes,  and  the  number  of  melanosomes  inside  Malpighian  cells 
also  increases. 

The  work  was  supported  by  grants  CA  05401-04-06,  N.I.H.,  and  USPHS  Career  Develop- 
ment Award,  K3-GM-14,987. 

Microtubules  and  morphogenesis.  The  role  of  microtubules  in  the  development  of 
the  primary  mesenchyme  in  the  sea  urchin  embryo.  LEWIS  G.  TILNEY  AND 
JOHN  R.  GIBBINS. 

In  sea  urchin  embryogenesis  the  complex  and  species-specific  skeleton  of  the  pluteus  larva 
is  produced  exclusively  in  the  cells  of  the  primary  mesenchyme.  During  their  development 
the  cells  of  the  primary  mesenchyme  undergo  a  predetermined  sequence  of  changes  in  shape 
which  result  finally  in  an  oriented  syncytium.  It  is  in  this  syncytium  that  the  skeleton  is 
deposited.  At  each  stage  examined  cytoplasmic  microtubules  are  disposed  in  patterns  which 
correlate  with  the  shape  of  the  cell  at  that  stage.  The  shape  of  the  cells  during  their  develop- 
ment then  may  be  controlled  by  the  microtubules  and  therefore  the  microtubules  could  be 
responsible  ultimately  for  the  orientation  and  configuration  of  the  skeleton.  To  test  this 
hypothesis  developing  embryos  were  exposed  to  two  types  of  agents  known  to  affect  the 
microtubules  of  the  mitotic  spindle  as  well  as  cytoplasmic  microtubules.  Agents  of  a  type 
which  produce  tubule  breakdown,  colchicine  and  hydrostatic  pressure,  inhibited  the  development 

378 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         379 

of  the  primary  mesenchyme  and  resulted  in  the  loss  of  microtubules.  The  cells  tended  to 
spherulate  and  no  skeleton  was  produced.  D2O,  an  agent  which  "freezes"  or  stabilizes  micro- 
tubules,  also  stopped-  development  but  the  microtubules  remained  and  the  cells  retained  their 
asymmetric  form.  It  would  appear,  then,  that  the  sequential  disassembly  and  reassembly  of 
cytoplasmic  microtubules  into  new  patterns  is  essential  for  the  form  differentiation  of  the  cells 
of  the  primary  mesenchyme.  Disruption  of  normal  development  results  from  interference  with 
this  system  or  with  its  control. 

Research  supported  by  USPHS  Grant  #2G-707-05  to  Dr.  Keith  R.  Porter. 

Cilia  regeneration  in  tlie  sea  urchin  embryo.     WALTER  AUCLAIR  AND  BARRY  W. 
SIEGEL. 

Late  gastrulae  of  Paracentrotus  lividus  regenerated  cilia  following  deciliation  in  hypertonic 
sea  water.  The  rate  of  growth,  following  a  ten-minute  lag  period,  was  1  /J./5  mm.  for 
approximately  two  hours.  The  rate  then  slowed  down  for  the  next  2-3  hours  until  the  cilia 
reached  a  final  length  of  24—25  microns,  equal  to  that  of  the  original  cilia.  The  apical  tuft 
cilia  did  not  regenerate  to  their  original  length  of  70-75  microns. 

Neither  dactinomycin  (25  and  50  ^g./ml.)  nor  puromycin  (50  and  200  yug./ml.)  affected 
the  rate  of  growth  of  the  regenerating  cilia.  Dactinomycin  (25  /tig. /ml.)  also  did  not  affect 
incorporation  of  Cu-l-leucine  and  C"-l-glutamic  acid  (each  at  concentrations  of  0.1  ^c./ml.) 
into  the  proteins  of  regenerated  cilia  during  the  regeneration  phase,  but  total  embryo  protein 
incorporation  was  reduced.  Puromycin  (50  /ig./ml.)  inhibited  both  total  embryonic  and  ciliary 
protein  synthesis  drastically. 

The  data  indicate  that  ciliary  protein  synthesis  is  continuous  and  under  the  control  of  a 
pre-existing,  long-lived  messenger  RNA  template.  In  addition,  the  data  suggest  that  there  is 
present  an  intracellular  pool  of  pre-formed  ciliary  proteins  that  aggregate  into  cilia  even  when 
protein  synthesis  is  stopped. 

Supported  by  Office  of  Scientific  Research,  Office  of  Aerospace  Research,  U.  S.  Air  Force, 
Grant  no.  964-66. 

AUGUST  9,  1966 

The  effect  of  15°  C.  on  the  stai/cs  of  normal  development  of  Funditlus  heteroclitus. 
D.  R.  SHANKLIN. 

Selection  of  morphological  stages  for  a  developmental  series  is  partially  arbitrary,  although 
some  phases  of  embryogenesis  warrant  separate  designations.  Further,  functional  integration 
and  morphogenetic  movements  require  a  reasonable  synchrony  which  is  a  prerequisite  for 
comparative  staging.  The  Armstrong-Child  series  for  Fundulus  (Bio!.  Bull.,  128:  143-168, 
1965)  qualifies  as  a  base  sufficiently  detailed  for  quantitative  study.  Their  series  was  con- 
structed at  20°  C.  In  early  June,  1966,  the  running  sea  water  at  the  M.B.L.,  Woods  Hole, 
was  15°  C.  Seven  batches  were  fertilized  and  followed  at  15°  C.  Two  were  returned  to  20°  C. 
at  gastrulation  (stages  12  &  15)  and  two  at  onset  of  circulation  (stage  25)  ;  all  were  followed 
until  hatching  (stage  34).  Ordinary  plots  of  stages  irrsus  total  time  were  hyperbolic;  semi- 
logarithmic  plots  were  biphasic  linear,  with  a  steeper  slope  after  about  12  hours.  The  15° 
curve  lay  to  the  right  on  the  time  plot  and  on  the  rectangular  plot  gradually  moved  further  to 
the  right.  The  changes  in  the  intervals  between  stages  were  examined.  This  revealed  a  great 
variability  in  the  ratio  of  interval  times  (15°/20°)  which  ranged  from  1.04  to  3.0  up  to  stage  33; 
the  interval  ratio  33-34  exceeded  4.72.  When  the  interval  ratios  were  plotted  against  time, 
with  stages  marked  in,  a  fluctuant  curve  resulted  which  alternates  great  delay  with  little  delay. 
This  suggests  a  critical  effect  of  cooling  at  certain  points,  which,  when  relieved  metabolically 
or  morphokinetically,  affects  stage  achievement  only  slightly  until  the  next  critical  period  is 
reached.  The  phases  of  great  delay  were :  mid-cleavage,  preblastula  cell  multiplication,  gastru- 
lation, growth  and  organodifferentiation  prior  to  general  circulation,  and  the  acquisition  of 
axial  linearity  and  motile  strength  preceding  hatching. 

This  work  was  supported  by  The  John  A.  Hartford  Foundation,  New  York,  New  York. 


380          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Rate  of  hatching  of  Fundulus  heteroclitus  at  20°  C.  and  the  effect  of  prior  exposure 
at  15°  C.     D.  R.  SHANKLIN. 

Hatching  of  Fundulus  results  from  two  phenomena  related  insofar  as  is  known  only 
temporally :  release  of  an  enzyme  from  the  oropharynx  and  strong  muscular  action  of  the  tail. 
With  temperature  maintained  at  15°  C.,  Armstrong-Child  stage  34  is  reached  in  about  400 
hours  as  compared  to  228  hours  at  20°  C.  (Biol.  Bull,  128:  143-168,  1965).  Even  though  a 
full  range  of  axial  movement  occurs  and  both  the  mouth  and  gill  slits  are  open  and  show  a 
gentle  rhythmic  action,  no  hatching  or  activities  seen  immediately  prior  thereto  (e.g.,  shrinkage 
of  yolk  sac)  were  observed  even  up  to  136  additional  hours  at  stage  34,  over  5  times  the 
interval  33-34  usually  required  at  20°  C.  This  great  delay  was  previously  noted  by  Gabriel 
(/.  Exp.  ZooL,  95:  105-147,  1944).  Certain  writings  imply  that  hatching  is  a  reasonably 
simultaneous  process,  interrupting  only  transiently  the  course  of  development.  A  small  group 
of  eggs  maintained  at  20°  C.  took  191  hours  for  hatching  to  reach  100%.  Two  groups  of  108 
and  112  eggs,  respectively,  required  164  hours  (these  were  at  15°  C.  until  stage  25)  and  when  the 
per  cent  hatched  is  plotted  against  time  an  S-shaped  curve  results  with  good  linearity  between 
30%  and  90%.  Several  groups  were  put  at  20°  C.  after  various  preselected  stages  were  reached. 
These  also  showed  a  significant  spread  of  total  hatching  time  (0-100%)  at  stage  34.  The  plot 
of  total  exposure  time  at  15°  C.  against  rate  of  hatching  yields  a  rough  but  reasonable  direct 
relationship  whose  approximate  average  slope  is  log  1/t  =  0.74  +  0.00125d,  where  t  =  time  in 
hours  to  reach  100%  hatching  once  it  has  begun  and  d  =  duration  of  exposure  at  15°  beginning 
at  fertilization.  The  study  does  not  show  which  hatching  action  is  affected  by  the  treatment 
or  whether  both  are. 

This  work  was  supported  by  The  John  A.  Hartford  Foundation,  New  York,  New  York. 

Fine  structure  of  tight  junctions.     JEAN-PAUL  REVEL  AND  MORRIS  J.  KARNOVSKY. 

After  fixation  in  the  presence  of  neutralized  lanthanum  nitrate,  we  have  observed  that  the 
intercellular  space  becomes  filled  with  an  intensely  electron-opaque  material.  Presumably  a 
form  of  lanthanum  hydroxide  acts  as  tracer  and  penetrates  in  the  tissues  via  these  spaces.  In 
the  mouse,  the  most  intensively  studied  species,  we  have  successfully  delineated  the  extracellular 
space  in  heart,  liver,  kidney,  intestinal  epithelia,  smooth  muscle,  transitional  epithelium  and  the 
intermediate  line  of  nerve  myelin.  The  lanthanum  compound  also  stains  the  intermediate  line 
of  tight  junctions  and  we  describe  here  in  particular  the  results  obtained  in  the  intercalated  disc 
of  the  mouse  heart  and  in  the  liver.  The  stained  intermediate  line  is  wider  than  in  unstained 
preparations,  but  the  total  width  of  the  tight  junctions  is  unchanged.  Seen  in  oblique  view  the 
tight  junctions  show  a  series  of  striations  with  a  periodicity  of  90  A,  while  in  face-on  view 
one  observes  a  hexagonal  pattern  with  a  center-to-center  distance  of  90  A.  The  appearance 
of  the  pattern  does  not  change  as  the  temperature  is  varied  from  0°  C.  to  37°  C.  The  patterns 
observed  are  identical  to  those  reported  by  Robertson  at  the  electrical  synapse  of  the  Mauthner 
cell  in  the  goldfish  brain,  but  the  use  of  lanthanum  allows  one  to  view  the  hexagonal  pattern 
with  much  better  contrast.  Similar  patterns  have  also  been  reported  in  negatively  stained, 
isolated  liver  cell  membranes  by  Emmelot  and  Benedetti.  Species  variations,  and  the  physio- 
logical implications  of  the  hexagonal  pattern,  were  discussed. 

Supported  by  grants  HE  09125  and  GM  11380  from  the  NIH,  USPHS.  MJK  was  the 
recipient  of  a  Lederle  Medical  Faculty  Award,  and  JPR  of  a  Research  Career  Development 
Award. 

Fine-structural  basis  for  chemical  and  electrotonic  transmission  in  a  parasympathetic 
ganglion.     A.  ].  DARIN  DE  LORENZO  AND  GERALD  R.  BARNETT. 

The  ciliary  ganglion  of  the  newly-hatched  and  adult  chicken  contains  synapses  of  a  unique 
type.  Preganglionic  nerve  fibers  enter  the  ganglion  and  terminate  in  calyciform  or  cup-like 
endings  upon  postganglionic  nerve  cells.  Since  the  postsynaptic  cells  have  no  dendrites  the 
endings  are  entirely  axo-somatic  and  they  embrace  as  much  as  65%  of  the  cell  surface.  Electro- 
physiological  studies  of  these  synapses  have  demonstrated  both  chemical  and  electrical 
transmission. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         381 

Examination  of  these  junctions  with  the  electron  microscope  has  revealed  the  following 
organization.  Postsynaptic  cells  are  surrounded  with  a  myelin  sheath  which  covers  the  synapses 
and  the  perikaryon  except  at  the  axon  hillock  region.  The  synaptic  membranes  of  the  calyx 
are  separated  from  the  postsynaptic  cell  by  a  cleft  about  250-300  A  wide.  Structural  specializa- 
tions seen  in  chemical  synapses  consisting  of  accumulations  of  synaptic  vesicles,  thickened 
membranes  and  clusters  of  mitochondria  are  seen  throughout  the  calyx.  Only  in  the  area  of 
the  axon  hillock  do  the  synaptic  membranes  exhibit  tight  junctions  characteristic  of  electrotonic 
transmission  sites  seen  elsewhere.  At  these  sites  synaptic  vesicles  are  conpicuously  absent.  The 
synaptic  cleft  is  obliterated  by  a  fusion  of  the  inner  components  of  the  unit  membrane  compris- 
ing the  synaptolemma.  Cross-striations  are  often  resolved  in  the  fusion  sites.  Of  particular 
interest  is  the  occurrence  of  structural  characteristics  implicating  both  chemical  and  electrotonic 
transmission  in  the  same  synaptic  membrane  separated  by  distances  measuring  less  than  a 
micron.  Hexagonal  structures  seen  in  other  tight  junctions  have  not  as  yet  been  demonstrated. 

Supported  by  USPHS  Grant  NB  02173. 

The  fine  structure  of  vesicles  associated  with  excitatory  and  inhibitory  junctions. 
G.  D.  PAPPAS  AND  M.  V.  L.  BENNETT. 

Our  previous  studies  on  spinal  motoneurons  which  innervate  the  swimbladder  muscle  of 
Opsanus  tau  indicate  that  excitatory  transmission  to  these  cells  is  electrotonic  and  mediated  at 
axo-somatic  junctions  where  the  apposing  membranes  are  fused.  Synchronous  firing  of  the 
motoneurons  is  probably  aided  by  dendro-somatic  junctions  where  the  apposing  membranes  are 
also  fused.  On  the  other  hand,  typical  axo-somatic  synapses  where  a  synaptic  gap  of  about 
200  A  occurs  provide  a  morphological  basis  for  hyperpolarizing  inhibitory  postsynaptic  potentials 
which  must  be  chemically  mediated  (Ann.  New  York  Acad.  Sci.,  137:  495,  1966).  In  both 
classes  of  axo-somatic  junction,  the  axon  terminals  contain  similar  vesicles,  although  clustering 
of  vesicles  to  the  pre-junctional  membrane  is  more  common  in  junctions  where  there  is  no 
membrane  fusion. 

Uchizono  (Nature,  207:  642,  1965)  and  Bodian  (Science,  151:  1093,  1966)  suggest  that  in 
electron  micrographs  of  formalin-  or  glutaraldehyde-fixed  tissue  inhibitory  synapses  are 
characterized  by  synaptic  vesicles  with  ellipsoid  profiles.  In  contrast,  the  vesicles  in  excitatory 
synapses  appear  uniformly  round.  After  glutaraldehyde  fixation,  two  distinct  classes  of  vesicles 
cannot  be  differentiated  in  the  toadfish  swimbladder  nucleus.  Axon  terminals  showing  regions 
of  fusion  contain  a  spectrum  of  vesicular  profiles.  Terminals  where  an  extra-cellular  space 
is  maintained  also  contain  round  to  ellipsoid  vesicles.  In  addition,  both  kinds  of  terminals 
contain  tubular  elements  more  frequently  than  after  fixation  in  osmic  acid. 

AUGUST  16,  1966 

Chemical  studies  of  directin.     ANDREW  F.  HEGYELI. 

A  biodynamic  agent  was  found  in  extracts  from  human  urine,  which  is  identified  by  its 
induction  of  directional  growth  of  malignant  and  not  of  normal  cells  in  vitro,  and  was  therefore 
tentatively  named  directin  (D). 

The  chemical  separation,  purification,  properties,  and  possible  biological  significance  of  D 
were  discussed.  The  properties  of  D  were  presented  under  the  following  headings :  stability, 
enzyme  degradation  studies,  molecular  weight,  group  reagent  reactions,  chemical  analyses,  and 
chromatographic  studies.  Evidence  was  presented  that  D  has  a  molecular  weight  around  600 
and  is  a  hydrolyzable  phosphate  derivative,  possibly  containing  a  sugar  moiety.  The  loss  of 
directional  growth  activity  is  related  to  the  splitting  off  of  one  or  more  phosphates. 

The  effect  of  D  on  D-treated  rats  carrying  Walker  256  tumors  can  only  be  explained  by 
postulating  that  D  is  involved  in  the  energy  transformation  processes  in  the  cell.  When 
unstained  fixed  histological  sections  of  the  tumors  were  excited  by  fluorescent  light  at  450 
Atmicrons,  the  tumor  cell  nuclei  emit  a  rapidly  quenched  light. 

ADP,  ATP,  and  inorganic  phosphate  are  known  to  be  important  in  energy  transformations 
in  the  cell.  ATP  and  ADP  have  about  the  same  molecular  weight  as  D,  and  contain  hydrolyz- 
able phosphate  groups.  They  were  therefore  investigated  in  the  same  biological  assay  used  for 


382         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

D.  (This  was  done  at  the  suggestion  of  R.  Poirier.)  Both  ATP  and  ADP  induced  directional 
growth  of  KB  cells  in  vitro.  AMP  and  inorganic  pyrophosphate  were  inactive,  while  ortho- 
phosphate  gave  borderline  activity.  Evidence  was  presented  that  D  is  not  identical  with  either 
ATP  or  ADP.  It  has  a  different  solubility  pattern  and  100  times  less  N  content  for  the  same 
biological  activity.  The  negative  test  with  AMP  suggests  that  the  active  site,  responsible  for 
the  directional  growth  activity  in  both  ATP,  ADP,  and  D,  is  an  easily  hydrolyzable  phosphate 
group. 

This  research  was  supported  by  Battelle  Memorial  Institute. 

In  vitro  and  in  vivo  studies  of  directin.     RUTH  JOHNSSON-HEGYELI  AND  ANDREW 
HEGYELI. 

A  biodynamic  substance  was  discovered  last  year  in  extracts  from  human  urine  and 
tentatively  named  directin  (D)  because  of  its  induction  of  directional  growth  of  malignant  cells 
and  not  of  normal  cells  in  vitro.  Chemical  evidence  obtained  to  date  indicates  that  D  is  a 
hydrolyzable  phosphate  derivative,  possibly  a  sugar  of  about  600  molecular  weight. 

This  paper  reported  on  further  biological  studies  of  D.  These  were  illustrated  by  slides  of 
cell  cultures  and  histological  sections,  followed  by  a  brief  movie  of  D-treated  KB  cells. 

The  action  of  D  was  considered  under  three  headings :  cells,  medium,  and  substrate.  Its 
action  on  the  following  types  of  cells  was  discussed :  solid  tumor  cell  lines,  virus  leukemia 
strain,  normal  cell  lines,  normal  cell  strains,  and  primary  cultures  from  14-day-  and  7-day-old 
chick  embryos.  Directin  has  three  microscopically  detectable  effects  in  malignant  tissue 
cultures:  (1)  It  is  growth-retarding,  (2)  it  changes  the  morphology  of  the  cells,  and  (3)  it 
changes  the  growth  pattern  of  the  cells.  After  16  to  20  hours'  exposure  to  D  the  cells  become 
bipolar,  there  is  increased  communication,  and  formation  of  tight  junctions  between  neighboring 
cells,  and  polarization  of  growth.  Immediately  following  mitosis  the  nuclei  move  to  opposite 
poles  of  the  two  cells  and  the  nucleoli  are  also  lined  up  in  a  row  at  this  time.  The  D  effect 
with  cell  cultures  grown  in  different  media  and  on  conducting  metal  surfaces  rather  than 
on  insulating  glass  was  discussed. 

Experiments  with  D-treated  rats  carrying  Walker  256  solid  tumors  showed  that  D  has  three 
effects  on  tumor  cells  in  vivo:  (1)  It  changes  the  morphology  of  the  cells,  particularly  the 
nuclei,  (2)  it  induces  areas  of  parallel  orientation  of  tumor  cells,  and  (3)  the  nuclei  of 
treated  unstained  tumor  cells  emit  rapidly  quenched  light  when  excited  by  light  at  450  /^microns 
in  fluorescent  microscope. 

This  research  was  supported  by  Battelle  Memorial  Institute. 

The  effects  of  sonic  inhibitors  on  the  temperature-dependent  component   of  the 
lobster  axon  resting  potential.     JOSEPH  P.  SENFT. 

The  resting  membrane  potential  of  the  lobster  axon  increases  5-8  mV  when  the  tempera- 
ture of  the  perfusion  solution  is  increased  10°  C.  This  potential  change  is  about  twice  that 
predicted  if  the  axon  membrane  potential  followed  that  expected  for  a  potassium  ion  electrode 
potential.  When  the  inhibitors  2,4-dinitrophenol,  cyanide,  and  azide  were  added  separately  to 
the  axon  perfusion  medium  the  potential  change  was  reduced  to  about  1.4  times  that  predicted 
for  a  potassium  ion  electrode  potential.  Assays  of  axons  exposed  to  these  inhibitors  showed 
that  ATP  levels  were  reduced  to  about  one-fourth  that  obtained  for  control  axons.  Ouabain 
added  to  the  perfusion  medium  reduced  the  potential  change  to  that  expected  for  a  potassium 
ion  electrode  potential.  These  results  suggest  that  the  lobster  axon  resting  potential  changes 
with  temperature  as  a  result  of  the  activity  of  an  electrogenic  ion  pump. 

Supported  in  part  by  NSF  grant  GB-332. 

Studies  on  a  major  protein  from  isolated  sea  urchin  egg  cortex.     R.  E.  STEPHENS 
AND  R.  E.  KANE. 

Isolated  cortex  was  prepared  by  the  method  of  Sakai,  involving  lysis  of  eggs  of  the  sea 
urchins  Colobocentrohis  atrattis,  Tripneustes  gratillia,  and  Arbacia  punctnlata  in  0.1  M 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         383 

MgCh,  followed  by  light  homogenization  and  differential  centrifugation  to  remove  cytoplasmic 
contaminants.  Analytical  ultracentrifugation  revealed  that  distilled  water  extracts  of  the  cortex 
were  composed  of  only  a  few  components,  the  most  prominent  of  which  was  a  hypersharp  7S 
material,  identified  as  a  calcium-precipitable  protein  originally  described  by  Kane  and  Hersh 
as  a  component  of  whole  egg  extracts.  Essentially  all  of  the  7S  protein  recovered  from  whole 
egg  lysates  could  be  obtained  from  isolated  cortex  alone.  The  protein  constituted  2-4%  _  of 
the  total  cell  protein  and  50-95%  of  the  soluble  cortical  extracts,  depending  upon  the  species. 

The  protein  was  only  sparingly  soluble  in  solutions  of  ionic  strength  greater  than  0.2, 
producing  a  particle  with  a  sedimentation  constant  in  the  range  of  10-12  Svedbergs.  The 
Yphantis  short  column  sedimentation  equilibrium  method  indicated  that  the  7S  protein  from 
C.  atratus  had  an  average  molecular  weight  of  725,000  at  PH  7.5,  386,000  at  pH  11.5,  277,000 
in  8  M  urea,  and  299,000  in  8  M  urea  plus  2%  mercaptoethanol ;  strong  association  was  evident 
in  all  cases  and,  hence,  these  values  should  be  considered  tentative.  The  pH  and  urea  subunits 
were  reversible  to  the  original  protein  by  dialysis  into  neutral  buffer. 

The  amino  acid  composition  showed  no  significant  species  variation  and  was  characterized 
by  high  glutamic  acid,  valine,  and  proline  contents.  Optical  rotatory  dispersion  measurements 
indicated  virtually  no  a-helix  to  be  present,  a  finding  consistent  with  the  proline  content. 

Divalent  cation-precipitated  fibers  of  the  7S  protein  were  highly  birefringent  but 
lengthened  and  lost  birefringence  upon  treatment  with  0.001  M  EDTA;  addition  of  divalent 
cations  caused  the  fibers  to  shorten  and  regain  birefringence. 

Supported  by  Public  Health  Service  Grant  GM  14363  from  the  Division  of  General 
Medical  Sciences. 

GENERAL  SCIENTIFIC  MEETINGS 
AUGUST  22-25,  1966 

Abstracts  in  this  section  are  arranged  alphabetically  by  authors.  Author  and 
subject  references  will  be  found  also  in  the  regular  volume  index,  appearing  in  the 
December  issue. 

Squid  lens  development  in  compounds  that  affect  microtubules.     JOHN  M.  ARNOLD. 

The  squid  lens  develops  by  fusion  of  elongate  cellular  processes  (lentigenic  processes) 
which  grow  out  of  a  specialized  group  of  cells  (lentigenic  cells)  in  the  front  of  the  developing 
optic  vesicle.  These  processes  wrap  about  each  other  to  form  an  "onion  bulb-like"  structure 
(lens  primordium)  in  which  elaboration  of  an  electron-dense  lens  material  takes  place.  The 
lens  primordium  increases  in  size  by  continued  application  of  lentigenic  processes  from  the 
lentigenic  cells.  Eventually  the  elaboration  of  this  dense  lens  material  obliterates  the  cytoplasm 
of  the  lentigenic  processes.  During  the  phase  of  active  outgrowth  the  lentigenic  processes  con- 
tain many  microtubules,  Golgi-derived  vesicles,  mitochondria,  and  ribosomes.  Fixation  with 
glutaraldehyde  and  post-osmication  demonstrates  electron-dense  areas  (probably  a  precipitation 
product)  in  the  lentigenic  cells  during  the  time  of  lens  material  elaboration  but  not  in  other 
tissues  of  the  eye. 

Treatment  of  the  stage  25  embryo  (Arnold,  1965)  with  10"3  M  or  10"4  M  colchicine  for 
24  hours  causes  the  microtubules  to  disappear  and  the  lentigenic  cells  to  undergo  a  very 
dense  accumulation  of  ribosomes  and  an  increase  in  size  of  the  electron-dense  areas.  Washing 
the  treated  embryos  for  an  additional  24  hours  in  normal  sea  water  causes  a  reversal  to  the 
typical  appearance  and  a  reappearance  of  the  microtubules.  Colchicine  treatment  of  the  stage 
22  embryos  prevents  outgrowth  of  the  lens  primordium.  Treatment  of  the  stage  22  or  stage  24 
embryo  with  0.1  M  mercaptoethanol  apparently  allows  the  lentigenic  processes  to  continue  their 
outgrowth  but  they  no  longer  fuse  to  form  a  lens  primordium.  Mercaptoethanol  also  causes  the 
microtubules  to  become  solid,  irregular,  and  uniformly  electron-dense. 

It  is  therefore  indicated  that  microtubules  may  play  a  role  in  transport  of  materials  to 
the  developing  primordium.  However,  their  specific  tubular  structure  is  not  essential  in  this 
transportive  role. 

Aided  by  a  grant  from  the  National  Science  Foundation,  GB-3202. 


384         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 
Ribonucleic  acid  in  membranes  of  developing  cells.     ARYA  K.  BAL,  PAUL  L.  KRUPA 

AND    GlLLES    H.    COUSINEAU. 

Previous  work  on  RNase-treated  membrane  systems  in  Allium  cepa  L.  root-meristem 
cells  showed  removal  of  a  ribonuclease-sensitive  material  from  unit  membranes.  In  the  present 
studies  possible  contamination  of  ribonuclease  with  lipase  and  proteases  (which  might  have 
led  to  similar  findings  in  the  initial  investigation)  was  circumvented  by  the  use  of  d)  boiled 
ribonuclease,  or  (2)  lipase  and  trypsin. 

The  tissues  were  fixed  in  a  mixture  of  5%  glutaraldehyde-paraformaldehyde  at  23°  C. 
After  repeated  washing,  the  tissues  were  incubated  with  each  enzyme  (dissolved  in  distilled 
water)  at  37°  C.  for  6  to  18  hours  (pH  6.5).  The  concentrations  of  lipase  and  trypsin  were 
1  mg./ml.  Ribonuclease  (0.5  nig./ml.)  was  boiled  for  5  minutes  and  cooled  to  37°  C.  before  use. 
Controls  were  run  concurrently  in  distilled  water.  The  enzyme  treatment  was  followed  by 
post-osmication  and  embedding  in  Epon  in  the  usual  manner. 

In  root-meristem  cells  treated  with  lipase  and  trypsin  there  was  no  appreciable  change  in 
cell  structure  compared  with  the  controls.  On  the  other  hand,  boiled  ribonuclease  was  effective 
in  removal  of  electron-dense  material  between  the  leaflets  of  the  lipoprotein  membranes. 

These  results  were  confirmed  in  gastrula  cells  of  Arbacia  punctulata.  After  treatment 
with  boiled  ribonuclease,  the  bilaminar  organization  of  the  unit  membranes  became  more 
evident,  due  to  removal  of  the  ribonuclease-labile  component.  This  was  seen  in  the  nuclear 
envelope,  endoplasmic  reticulum,  Golgi  complex,  plasma  membrane,  and  in  the  membranes 
surrounding  pigment  granules. 

Thus,  under  these  experimental  conditions,  it  is  very  likely  that  RNA  is  present  (in 
association  with,  or  as  an  integral  part  of)  the  membrane  systems  of  the  embryonic  cells  in 
the  tissues  studied. 

This  work  was  supported  by  grants-in-aid  of  research  from  the  Damon  Runyon  Memorial 
Fund  for  Cancer  Research  (grant  #DRG-918),  the  National  Research  Council  of  Canada 
(grant  #731-741),  and  the  Society  of  the  Sigma  Xi. 

Investigations  of  the  subunit  structure  of  Limit-Ins  hemocyanin.     FRANK  C.  BAN- 
CROFT, ROBERT  C.  TERWILLIGER  AND  K.  E.  VAN  HOLDE. 

Preliminary  studies  have  involved  the  determination  of  conditions  for  obtaining  the  various 
subunits  of  hemocyanin,  and  the  characterization  of  the  subunits  by  sedimentation  coefficient 
(s'ao,  w)  and  molecular  weight.  The  Yphantis  sedimentation  equilibrium  method  was  used 
for  molecular  weight  determinations.  Buffers  were  of  ionic  strength  0.1.  Whole  blood  was 
allowed  to  clot ;  it  was  then  centrifuged  and  filtered  to  yield  stock  solutions  of  hemocyanin. 
These  stock  solutions  were  of  pH  7.3,  and  contained  about  36  mg./ml.  hemocyanin,  mostly  as  a 
62S  component,  with  some  16S  component  also  present.  Dilution  with  pH  6.6  phosphate  to 
7.0-0.36  mg./ml.  resulted  in  a  progressive  dissociation  of  the  62S  component  to  a  40S  compo- 
nent with  dilution,  suggesting  a  protein  concentration-dependent  equilibrium.  However,  passage 
through  Sephadex  G-25  equilibrated  with  pH  6.6  phosphate,  or  dialysis  against  this  buffer, 
caused  dissociation  to  40S  at  7  mg./ml.,  suggesting  involvement  of  a  dialyzable  component  of 
the  blood.  Dilution  of  the  stock  to  0.36  mg./ml.  with  either  pH  6.6  phosphate  +  0.01  M  Mg++ 
(I),  or  pH  6.6  cacodylate  ±  0.01  M  Mg++  or  Ca++  (II)  yielded  the  60S  component.  Incubation 
of  these  solutions  for  24  hours  at  25°  C.  caused  dissociation  in  I,  whereas  dissociation  was 
prevented  in  II.  Dilution  of  the  stock  with  pH  10.6  bicarbonate  yielded  a  5.2S  component. 
Under  various  conditions  a  16S  component  was  observed.  Dilution  of  the  stock  with  pH  4.6 
acetate  yielded  a  25S  component,  which  dissociated  largely  to  the  5.2S  component  after  24  hours 
at  25°  C.,  or  overnight  dialysis  vs.  pH  4.6  acetate,  ±  0.01  M  Mg++.  Molecular  weights  of 
6.0  X  10*  and  1.94  X  10"  were  obtained  for  the  5.2S  and  40S  components,  respectively,  assuming 
partial  specific  volumes  of  0.710  and  0.740,  respectively.  A  tentative  model  for  the  subunit 
structure  has  been  formulated.  Electron  micrographs  by  Van  Bruggen  suggest  the  16S  compo- 
nent is  composed  of  eight  5.2S  components.  The  relative  sedimentation  coefficients  of  the 
larger  subunits  suggest  that  the  25,  42,  and  60S  components  are  dimers  of  the  16,  25,  and 
42S  components,  respectively.  The  molecular  weights  obtained  are  consistent  with  this 
model. 

This  work  was  supported  in  part  by  NIH  Grant  #5  Tl  GN25608. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         385 

The  fine  structure  of  the  eye  of  the  scallop,  Pecten  irradians.     ALLEN  L.  BELL. 

Available  electrophysiological  evidence  indicates  that  the  proximal  retina  of  the  scallop  eye 
gives  an  "on"  response  to  light  while  the  distal  retina  gives  an  "off"  response.  It  is  not 
known  whether  the  distal  retina's  response  is  a  case  of  primary  inhibition  or  is  a  result  of 
inhibition  by  the  proximal  retina. 

Preliminary  studies  of  the  fine  structure  of  both  retinas  show  processes  close  to  the  distal 
retinal  cells  which  contain  clear  vesicles  600  to  700  A  in  diameter.  This  suggests  a  mechanism 
for  modification  of  the  response  of  the  distal  retina  by  some  other  cell.  Studies  are  in  progress 
to  determine  the  origin  of  these  processes. 

All  of  the  surface  epithelial  cells  of  the  scallop  eye  have  microvilli  at  their  surface.  The 
surface  epithelium  proximal  to  the  pigmented  region  of  the  eye  is  thrown  into  folds  which  bring 
the  microvilli  of  opposing  cells  into  a  configuration  resembling  a  rhabdomere.  If  these 
rhabdomere-like  structures  were  photoreceptors,  one  would  expect  to  find  nerves  or  processes 
passing  centrally  from  them.  None  have  been  seen  up  to  this  point  in  the  investigation. 

Other  tissues  of  the  eye  which  were  examined  include :  the  cornea,  the  pigmented  surface 
epithelium,  and  the  tapetum.  A  full  description  of  these  tissues  will  be  given  in  future  reports 
of  this  work. 

Further  observations  on  the  thermodynamics  of  the  living  rnitotic  spindle.     ROBERT 
M.  CAROLAN,  HIDEMI  SATO  AND  SHINYA  INOUE. 

With  the  polarizing  microscope  the  birefringence  (measured  as  retardation)  of  the  mitotic 
spindle  of  Pcctinaria  goiildi  oocytes  was  measured  at  temperatures  from  5°  C.  to  38°  C.  in  both 
artificial  H2O  sea  water  and  artificial  42%  D2O  sea  water.  Below  21°  C.  increased  temperature 
enhanced  spindle  retardation;  the  plot,  log  B/(A0  — B)  versus  I/temp.  (°K)  follows  a  van't 
Hoff  relationship  giving  a  straight  line.  B  is  spindle  retardation  and  A0  the  asymptote  B 
approaches.  AH  in  H2O  was  82  ±  8.5  and  in  D2O  59  ±  12  kcal.  AS  in  H2O  was  286  ±  29  and 
in  D2O  208  ±  40  eu.  Both  differences  are  significant  to  the  0.001  level.  The  ratios  of  the  H2O 
and  D2O  values  are  identical  to  those  reported  previously  although  the  absolute  values  are 
greater,  due  presumably  to  technical  improvements. 

These  data  are  consistent  with  the  hypothesis  that  spindle  retardation  reflects  the  reversible 
association  of  protein  "monomers"  into  linearly  aggregated  polymers.  At  17°  C.  the  equilibrium 
constants  of  the  D2O  and  H2O  reactions  are  equal,  meaning  the  ratios  of  effective  "monomer" 
concentration  to  effective  polymer  concentration  are  equal  at  this  temperature.  Since  D«O 
increases  the  spindle  retardation  and  presumably  the  polymer  concentration,  this  suggests  D2O 
increases  the  pool  of  "monomers"  available  for  polymerization.  In  addition  D2O  must  have 
some  direct  effect  on  the  polymerization  reaction  itself  since  it  alters  its  thermodynamic 
parameters. 

Above  21°  C.  increased  temperature  reduced  spindle  retardation.  A  van't  Hoff  relation- 
ship exists  here  also  but  with  negative  AH's  and  AS's.  These  values  appear  less  negative 
in  D2O  than  H2O. 

After  exposing  the  cells  to  temperatures  as  high  as  36°  C.  normal  bipolar  spindles  re- 
appeared at  room  temperature.  After  exposure  to  37°  C.  tripolar,  tetrapolar  and  other 
anomalous  spindles  appeared.  After  exposure  to  38°  C.  no  spindles  could  be  recovered.  These 
effects  are  identical  in  both  D2O  and  H2O  even  though  in  H2O  spindle  birefringence  becomes 
undetectable  around  34°  C.  while  in  D2O  it  persists  to  37°  C. 

Aided  by  grants  from  the  National  Cancer  Institute  CA  10171  and  the  National  Science 
Foundation,  GB-5120. 

Ultra-structural  relationships  between  the  developing  oocyte  and  auxiliary  cells  in 
adult  Artcmia  salina.     REV.  JOSEPH  D.  CASSIDY,  O.P. 

Fine-structure  associations  were  surveyed  in  ovarian  germ  plasm,  nurse  cells  and  follicular- 
like  epithelium  of  Artemia  salina.  Stock  #3  females  were  cultured  in  filtered  sea  water  supple- 
mented with  50  g./liter  NaCl.  Dissected  ovaries  were  fixed  for  2  hours  in  5%  glutaraldehyde 
prepared  in  Millonig's  buffer  at  pH  7.4  with  10%  sucrose,  stained  with  1%  OsO4  and  saturated 


386          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

uranyl  acetate,  dehydrated  in  ethanol  and  embedded  in  Maraglas.  Thin  sections  were  examined 
in  the  Siemens  Elmiskop  IB.  Adjacent  1  /j,  sections  were  taken  for  cytochemical  reactions  and 
studies  with  Zeiss  phase  contrast  illumination.  Differentiated  regions  of  the  trophocyte-oocyte 
complex  were  analyzed  in  longitudinal  view. 

Spherical  nurse  cell  nuclei  are  highly  polyploid  with  prominent  nucleoli  rich  in  RNA. 
The  nuclear  envelope  is  studded  with  closely  packed  annuli.  Cytoplasmic  profiles  of  numerous 
Golgi,  ellipsoidal  mitochondria  and  polysomes  are  interpreted  as  morphological  concomitants 
of  synthetic  activity.  A  true  syncytium  is  lacking,  as  evidenced  by  extensive  membrane 
systems  which  separate  nurse  cell  nuclei.  The  nurse  cell-oocyte  interface  consists  of  a  border  of 
interdigitating  microvillar  projections.  These  cortices  of  nurse  cells  show  a  disorganized 
array  of  microtubular  elements,  free  RNP  particles,  and  scattered  filaments  of  endoplasmic 
reticulum.  Droplets,  particles  and  intact  mitochondria  appear  in  transit  via  micropynocytotic 
vesicles  extending  from  the  oocyte  border.  At  the  cortex  of  the  oocyte  distinct  types  of  yolk 
spheres  and  lamellae  are  observed  in  close  contact.  The  germinal  vesicle  gives  a  lobate  appear- 
ance in  transverse  and  tangential  view.  A  previously  unreported  cell  type  enveloped  part  of 
the  oocyte  boundary.  It  has  a  distinctively  elongate  nucleus,  an  irregular  acellular  outer  wall 
and  could  occupy  the  position  of  a  follicle  cell  if  one  is  present.  Along  its  attachment  to  the 
oocyte  border  are  pale  lipid  vacuoles.  Submorphology  and  distribution  suggest  a  possible 
role  in  lipogenesis  of  the  developing  oocyte. 

Induction  of  the  shell  gland  by  transplanted  polar  lobes  in  Ilyanassa.     JAMES  N. 
GATHER. 

To  determine  the  cell  lineage  of  the  shell  gland  in  Ilyanassa  the  2d  and  2c  micromeres 
were  marked  with  carbon  granules.  The  2d  micromere  derivatives  form  the  shell  gland  but  2c 
derivatives  are  later  incorporated  into  the  mantle.  Deletion  of  2d  causes  shell  abnormalities, 
but  in  about  10%  of  the  cases  the  shell  appears  normal  but  somewhat  small.  Deletion  of  2c  also 
causes  characteristic  shell  abnormalities.  After  deletion  of  both  2d  and  2c  no  shell  forms. 
When  2c  is  marked  and  2d  deleted  the  shell  gland  is  marked.  Any  1/4  embryo  can  form  internal 
shell  masses.  In  1013  isolated  ectoblasts  (-3A,  -3B,  -3C,  -3D)  no  shell  material  formed. 
Ectoblast  with  any  single  macromere  formed  shell  with  good  morphogenesis,  as  did  ectoblast 
plus  4d.  Posterior  ectoblast  plus  4d,  equivalent  in  size  to  isolated  whole  ectoblast,  formed 
external  shell.  Properly  oriented  ectoblast  grown  in  contact  with  a  polar  lobe  formed  shell 
in  up  to  70%  of  the  cases ;  in  one-half  of  these  the  shell  was  external.  Deletion  of  the  macro- 
mere  from  an  ectoblast  plus  3C  series  of  embryos  at  intervals  through  the  first  day  resulted  in 
embryos  which  did  not  form  shell.  Ectoblast  in  contact  with  polar  lobes  for  6  days,  then 
separated,  developed  shell  on  the  seventh  or  eighth  day.  Thus:  (1)  Neither  ectoderm  nor 
endoderm  in  isolation  will  form  shell  but  any  ectoderm-endoderm  combination  has  the  histo- 
genetic  ability  to  form  shell  material ;  (2)  the  endoderm  exerts  its  influence  between  the  second 
and  sixth  days  of  development;  (3)  only  those  combinations  including  or  in  contact  with 
polar  lobe  cytoplasm  through  the  third  quartet  stage  undergo  normal  morphogenesis ; 
(4)  transplanted  polar  lobes  can  induce  ectoderm  to  form  shell;  (5)  the  histogenetic  ability 
to  form  shell  is  suppressed  or  unactivated  when  polar  lobe  cytoplasm  is  present,  except  in 
the  D  quadrant. 

This  work  was  supported  by  NSF  Grant  GB-1035. 

Respiration  studies  with  the  shark:  biochemical  aspects.     C.  LLOYD  CLAFF,  ARMAND 
A.  CRESCENZI  AND  ARTHUR  P.  RICHMOND. 

The  shark  lives  in  an  environment  of  sea  water  with  a  pH  of  8.0  to  8.6.  Its  gills  are 
laved  with  this  alkaline  fluid.  It  is  likely  that  he  extracts  oxygen  from  his  environment  more 
easily  than  we  humans  do  from  air.  It  is  interesting  to  note,  in  passing,  that  we  humans  for 
the  first  nine  months  live  in  the  uterine  fluid  of  a  pregnant  woman  with  a  pH  of  7.9  to  8.0,  the 
calculated  pH  of  the  Pre-Cambrian  era  sea  water. 

I  suggested  the  use  of  the  shark  as  an  experimental  animal  in  connection  with  our  work 
on  the  Pulsatile  Pressure  Membrane  Oxygenator  for  open-heart  surgery.  I  did  this  because 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         387 

the  shark  gets  rid  of  some  of  its  nitrogenous  waste  of  metabolism  through  its  gills  as  well  as 
its  cloaca.  Therefore,  its  blood  is  always  loaded  with  waste  material. 

Dr.  Crescenzi  and  I  spent  four  months  in  1964  at  the  Lerner  Marine  Laboratory,  Bimini, 
Bahamas,  on  this  work.  Our  first  attempts  were  failures,  although  we  were  using  a  cellophane 
membrane  on  the  dialyzing  side  of  our  instrument  and  Teflon  membrane  on  the  oxygen  gas  side. 

A  native  boy  helping  us  suggested  dialyzing  with  sea  water,  and  our  success  was  immediate. 
Dr.  Crescenzi  noticed  that  we  were  getting  an  alkaline  shift  in  our  blood  passing  through  the 
oxygenator.  It  was  the  typical  "Bohr"  effect  of  the  dissociation  curve  of  blood  oxygen  to  the 
right  with  increased  CO»  and  alkaline  pH  of  7.5. 

This  was  first  pointed  out  by  Niels  Bohr  over  50  years  ago.  Those  of  us  who  concerned 
ourselves  with  designing  oxygenators  had  completely  ignored  this  important  phase  of 
respiration. 

The  biochemistry  of  respiration  is  as  important  as  the  physical  diffusion  factors  and  must 
be  incorporated  into  any  artificial  membrane  device  for  maximum  efficiency. 

Basic  research  can  and  does  provide  answers  for  clinical  medicine  and  public  health. 

Supported  by  the  Single  Cell  Research  Foundation,  Inc.,  5  Van  Beal  Road,  Randolph, 
Massachusetts. 

Cleavage  and  differentiation  of  the  vegetal  half  of  the  Ilyanassa  egg  after  removal 
of  most  of  the  yolk  by  centrifugal  force.     ANTHONY  C.  CLEMENT. 

By  holding  Ilyanassa  eggs  "upside  down"  on  the  centrifuge  in  a  gelatin-sea  water  gel,  yolk 
was  driven  into  the  animal  hemisphere ;  the  lipid  zone,  clear  zone,  and  pronuclei  were  forced 
into  the  vegetal  hemisphere.  Eggs  showing  this  reversal  of  the  usual  pattern  of  stratification 
were  freed  from  the  gel  and  centrifuged  in  a  raffinose  solution  until  they  separated  into  light  and 
heavy  halves.  Nucleated  light  halves  derived  from  the  vegetal  hemisphere,  and  free  of  most 
of  the  yolk  normally  contained  in  the  vegetal  hemisphere,  formed  a  polar  lobe  and  cleaved 
unequally  in  the  normal  pattern.  Many  such  yolk-poor  vegetal  fragments  differentiated  lobe- 
dependent  structural  features  (eyes,  shell,  foot,  etc.),  and  some  developed  into  small  but  well- 
proportioned  veliger  larvae.  The  morphogenetic  influence  of  the  polar  lobe  region  is  thus  not 
displaced  by  centrifugal  force  of  the  strength  employed  in  these  experiments  (2000  g  for  about 
six  minutes),  nor  is  it  dependent  upon  the  presence  of  the  normal  yolk  complement. 

This  work  was  supported  by  NSF  grant  GB-1572. 

Electron  microscopy  of  the  gas-secreting  gland  of  Portuguese  man-of-war.     D. 
EUGENE  COPELAND. 

Portuguese  man-of-war  (Physalia  physalis)  secretes  carbon  monoxide  as  the  gas  to  inflate 
the  float  or  pneumatophore.  An  oval  patch  of  epithelium  (ectodermal  in  origin)  at  the  base 
of  the  inner  layer  of  the  float  is  believed  responsible  for  the  gas  production.  Based  on  a  study 
of  animals  collected  off  Gay  Head  (Martha's  Vineyard,  Mass.),  it  was  previously  reported  that 
the  gas-secreting  epithelium  possessed  no  mitochondria  or  recognizable  Golgi  material.  Vesicles 
at  the  free  surface  could  be  the  gas-release  mechanism.  An  entirely  different  picture  is  now 
presented,  based  on  Physalia  collected  in  the  Gulf  of  Mexico  off  the  Mississippi  Delta.  It  is 
now  obvious  that  the  gas-secreting  epithelium  is  extremely  sensitive  to  adverse  environmental 
conditions  (in  this  case,  temperature).  The  gas-secreting  epithelium  contains  large,  closely 
packed  mitochondria  in  the  end  of  the  cell  toward  the  gas  surface.  The  mitochondria  have  few, 
short  cristae ;  a  dense  matrix  occupies  the  internum  of  the  mitochondrion.  Well  developed  Golgi 
and  a  large  population  of  lysosomes  are  found  in  the  general  area  between  the  mitochondria 
and  the  nucleus.  Mitochondria  are  seen  incorporated  into  autophagic  vacuoles  and  appear 
to  then  degenerate  into  lysosomes.  The  nuclei,  previously  reported  as  surrounded  by  a  satellite 
of  vesicles,  are  instead  surrounded  by  extensive  double  membranes  that  enclose  flattened  cisternal 
spaces.  The  free  surface  of  the  cell  is  thrown  into  irregular  projections  of  various  lengths. 
There  is  no  obvious  sign  of  gas  release  by  vesicles.  The  vesicles  previously  reported  un- 
doubtedly arise  by  collapse  and  reordering  of  the  surface  projections. 

Support  provided  by  National  Science  Foundation  (GB-676)  and  USPHS,  National 
Institutes  of  Health  (GM  06836). 


388          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  contribution  of  the  abdominal  nerve  cord  to  the  chromatic  physiology  oj  the 
prawn,  Palaemonetes  vitlgaris.  ERNEST  F.  COUCH,  MILTON  FINGERMAN  AND 
EDWARD  W.  STOOL. 

The  ratio  of  red  pigment-dispersing  hormone  to  concentrating  hormone  in  the  abdominal 
nerve  cord  of  Palaemonetes  is  higher  than  for  any  other  organ  of  this  prawn.  Experiments 
were  designed  to  determine  the  role  of  this  structure  in  the  color  change  process  as  compared 
with  the  function  of  the  neuroendocrine  structures  in  the  cephalothorax.  When  both  eyestalks 
were  removed  from  animals  whose  nerve  cord  was  cut  between  the  thorax  and  abdomen,  they 
darkened  as  is  typical  of  eyestalkless  prawns  but  slower  than  animals  with  intact  cords.  Dark- 
adapted  eyed  prawns  injected  with  an  extract  of  tritocerebral  commissure  to  induce  blanching 
re-dark-adapted  more  slowly  if  their  ventral  nerve  cord  was  cut.  These  findings  are  readily 
explained  by  assuming  that  the  prawns  with  cut  nerve  cords  had  less  darkening  hormone 
available  to  them.  However,  eyed  prawns  with  cut  cords  adapt  to  a  black  background  at  the 
normal  rate,  showing  that  sufficient  hormone  for  this  function  to  occur  at  the  normal  rate  can 
be  released  from  the  cephalothorax.  The  nerve  cord  was  divided  into  three  portions  and 
assayed  on  pale  prawns  to  determine  the  linear  distribution  of  red  pigment-dispersing  hormone. 
Cords  from  freshly  collected  animals  and  those  maintained  for  six  days  with  cut  cords  were 
compared.  No  increase  in  activity  was  found  in  the  anterior  end  of  the  cord  from  the  prawns 
kept  six  days.  The  lack  of  accumulation  in  the  anterior  part  of  the  cord  strongly  suggests 
that  hormone  is  not  carried  forward  through  the  cord  for  release  in  the  cephalothorax.  Heat 
and  electrical  stimulation  of  the  cord  caused  darkening  in  isolated  abdomens,  demonstrating 
thereby  direct  release  of  darkening  hormone  from  the  cord.  Light  microscopy  revealed 
neurosecretory  cells  within  the  ganglia  of  the  abdominal  nerve  cord  which  could  be  the 
source  of  chromatophorotropins. 

Supported  by  Grant  GB-5236  from  the  NSF. 

The  challenge  oj  actinomycin   D   on   early  development  oj  sea   urchin   embryos. 

GlLLES  H.  COUSINEAU,  PAUL  L.  KRUPA  AND  ARYA  K.  BAL. 

Eggs  of  the  sea  urchin  Arbacia  punctulata  were  fertilized  and  allowed  to  develop  at  23°  C. 
with  gentle  agitation.  Aliquots  were  then  taken  every  30  minutes  and  placed  into  flasks 
containing  actinomycin  D  at  a  final  concentration  of  50  /ug./ml.  At  330'  post-fertilization  (p.f.) 
the  embryos  were  pulsed  for  one  hour  with  either  uracil-2-C"  or  DL-leucine-1-C14.  Samples 
of  unfertilized  eggs  with  and  without  actinomycin,  and  fertilized  eggs  without  and  in  continuous 
contact  with  the  drug  were  also  pulsed  with  the  labeled  precursors  at  that  time.  Embryos 
challenged  with  the  antibiotic  during  development  incorporate  leucine-Cu  into  protein.  Setting 
the  incorporation  in  the  330-minute  control  at  100,  relative  incorporation  rates  for  the  actino- 
mycin-treated  embryos  were  computed  as  follows,  0',  59;  30',  55;  60',  60;  120',  63;  150',  65; 
180',  78;  210',  77;  240',  88;  270',  98;  300',  99.  A  40%  decrease  in  incorporation  into  protein  of 
Cu-leucine  was  also  observed  between  6  and  7  hours  p.f.  in  bulk  labeling  experiments,  although 
the  actinomycin  embryos  in  earlier  stages  had  consistently  greater  activity.  Incorporation  of 
uracil-C14  into  RNA  gave  the  following  results:  0',  10;  30',  21 ;  60',  29;  90',  30;  120',  33;  150',  60; 
180',  62;  210',  65;  240',  79;  270',  80;  300',  82;  control  330',  100.  Embryos  challenged 
during  the  first  two  hours  of  development  continued  to  divide  but  failed  to  differentiate,  while 
those  treated  between  150  and  210  minutes  p.f.  showed  increasing  orderly  differentiation ; 
nevertheless  formation  of  normal  blastulae  was  only  observed  with  those  embryos  challenged 
later  than  210  minutes  p.f.  The  consistently  greater  RNA  synthesis  obtained  from  all 
challenged  embryos,  and  certainly  the  increase  observed  in  the  30-  and  60-minute  samples, 
would  indicate  that  once  initiation  of  DNA-dependent  RNA  synthesis  has  occurred,  such 
activity  is  in  some  way  resistant  to  actinomycin  D,  even  when  given  in  massive  doses  over  long 
periods.  Further  work  is  in  progress  to  characterize  the  RNA  produced. 

This  work  was  supported  by  grants-in-aid  of  research  from  the  Damon  Runyon  Memorial 
Fund  for  Cancer  Research  (grant  #DRG-918),  the  National  Research  Council  of  Canada 
(grant  #731-741),  and  the  Society  of  the  Sigma  Xi. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         389 

Phytoplankton  sources  of  the  eicosapentaenoic  and  docosahe.raenoic  fatty  acids 
characteristic  of  marine  mctazoa.  JOYCE  E.  DUNHAM,  GLENN  W.  HARRING- 
TON AND  GEORGE  G.  HOLZ,  JR. 

The  most  characteristic  polyunsaturates  of  marine  invertebrates  and  vertebrates  are  the 
€20:5,  A6'  *•  u-  "• 1T  and  the  C±>:8,  A4- 7>  10'  "• 19' 19  acids.  They  are  members  of  the  a-linolenic  acid 
series,  characteristic  of  plants,  and  are  synthesized  via  a  pathway  in  which  methyl  group- 
oriented  desaturation  of  linoleic  acid  is  a  feature.  Their  biological  origin  has  been  a  matter 
of  concern  to  those  interested  in  the  sources  and  fates  of  metabolic  products  in  marine  food 
chains.  Whales,  fishes,  copepods  (Calamts}  and  euphausids  (Ruphasia)  contain  large  amounts 
of  these  acids  but  are  unable  to  carry  out  their  Ac  twvo  synthesis.  They  can  incorporate  them 
intact  from  food,  and  can  form  them  by  chain  elongation  and  desaturation  of  dietary  18  carbon 
unsaturates. 

It  has  been  assumed  that  components  of  the  phytoplankton,  the  primary  producers,  synthesize 
C»o:5  and  €22:0  but  direct  proof  has  been  offered  in  the  literature  only  for  7  diatom  species 
(rich  in  CMS)  and  one  dinoflagellate  (rich  in  both  acids). 

We  have  analyzed  a  variety  of  ecologically  important  primary  producers,  chrysomonads, 
coccolithophorids  and  dinoflagellates,  grown  axenically  in  a  fatty  acid-free  medium  and  have 
found  that  they  synthesize  the  polyunsaturates  as  follows  (%  total  fatty  acids)  :  chrysomonads— 
Isochrysis  galbana  CM*  trace  and  €22:8  5.3% ;  Monochrysis  hithcri  3.8%  and  0.8% 
coccolithophorids — Cricosphacra  carteri  3.2%  and  6.8%;  Coccolithus  huxleyi  2.7%  and  7.3% 
dinoflagellates — Gyrodinium  cohnii  trace  and  35% ;  Gymnodimmn  sp.  20%  and  20% 
Amphidinium  carteri  22%  and  22%;  Exuviella  sp.  2.5%  and  7.9%. 

From  these  results  it  is  clear  that  the  primary  producers  can  be  important  sources.  The 
pool  of  C2o:s  and  Cz2-.s  in  marine  fishes  and  in  whales,  then,  is  made  up  of  molecules  passed  intact 
along  food  chains,  and  acids  formed  by  conversion  of  simpler  unsaturates  by  primary  consumers 
( zooplankton)  and  by  the  vertebrates  themselves. 

Supported  by  grant  AI  05802  from  the  National  Institute  of  Allergy  and  Infectious  Diseases 
to  G.  G.  Holz,  Jr. 

Ionic  fluxes  in  crayfish  muscle  fibers  before  and  after  swelling  of  the  TTS. 
PHILIP  B.  DUNHAM,  JOHN  P.  REUBEN  AND  PHILIP  W.  BRANDT. 

Many  studies  have  implicated  the  transverse  tubular  system  (TTS)  of  muscle  cells  in 
excitation-contraction  coupling.  Studies  on  crayfish  and  crab  muscle  fibers  have  shown  that 
the  TTS  can  be  greatly  swollen  by  inducing  an  efflux  of  Cr.  The  terminal  portion  of  the 
membrane  of  the  TTS  is  thought  to  have  a  relatively  high  permeability  to  Cl~.  By  comparing 
the  permeability  properties  of  normal  and  TTS-swollen  fibers  of  crayfish  muscle,  further  infor- 
mation on  the  properties  and  function  of  the  TTS  could  be  obtained.  Extracellular  space  was 
measured  using  H3-inulin  simultaneously  with  the  other  isotopes  with  every  preparation.  In 
the  control  fibers,  inulin  equilibrated  with  11.4%  of  the  volume  in  two  minutes;  it  equilibrated 
with  a  slower  compartment  over  the  next  8  minutes,  giving  a  total  inulin  space  of  16.8%  at 
steady-state.  The  TTS-swollen  preparations  were  equilibrated  with  inulin  within  one  minute 
with  a  space  of  19.9%.  The  more  rapid  entry  of  inulin  and  the  slightly  larger  inulin  space  were 
expected  for  the  TTS-swollen  fibers  on  the  basis  of  their  morphology.  The  explanation  for 
the  large  (6.4%)  slow  inulin  compartment  of  control  fibers  is  not  clear.  Rates  of  entry  of  K" 
into  fibers  in  steady-state  were  measured  for  control  preparations,  TTS-swollen  preparations, 
and  preparations  equilibrated  in  Cl~-free  (propionate)  medium.  The  rate  of  K+  flux  was  the 
same  for  all  three  conditions.  However,  when  rates  of  entry  of  Cl36  were  compared  for  control 
and  TTS-swollen  fibers,  the  rate  of  entry  of  Cl~  was  greater  for  the  TTS-swollen  fibers.  The 
structural  change,  data  on  tension,  and  now  the  data  on  Cl~  permeability  all  suggest  a  role  of  Cl~ 
movement  in  the  function  of  the  TTS. 

Supported  by  NSF  grant  GB-1615  and  NIH  grant  GM  11441-03  to  Dr.  Dunham;  NIH 
grant  NB  05910-01A1  to  Dr.  Brandt;  NIH  grants  NB  03728-04  and  NB  03270  and  NSF  grant 
GB-2940  to  Dr.  H.  Grundfest ;  Dr.  Reuben  holds  an  NIH  Career  Development  Award. 


390         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Analysis  of  the  melanin-dispersing  and  red  pigment-dispersing  hormones  of  the 
praivn,  Palaemonetes  vulgaris,  and  the  fiddler  crab,  Uca  pugilator,  by  means  of 
gel  filtration.  MILTON  FINGERMAN,  ERNEST  F.  COUCH  AND  EDWARD  W. 
STOOL. 

Extracts  of  eyestalks  from  Uca  pugilator  and  Palaemonetes  vulgaris,  prepared  in  distilled 
water,  were  passed  through  a  column  containing  G-25  Sephadex.  One-half-  and  one-milliliter 
fractions  were  collected,  mixed  with  an  equal  volume  of  200%  sea  water,  and  then  assayed  on 
eyestalkless  Uca  for  red  pigment-dispersing  and  melanin-dispersing  activity.  Some  of  the 
fractions  were  also  assayed  on  intact  Uca  for  red  pigment-concentrating  hormone.  With 
respect  to  the  fractions  obtained  with  Palaemonetes  eyestalks,  although  the  eyestalks  contained 
virtually  no  red  pigment-dispersing  hormone  for  Palaemonetes,  they  had  a  large  dispersing  effect 
on  the  red  and  black  pigments  of  Uca.  Furthermore,  although  both  the  red  pigment-dispersing 
hormone  and  the  melanin-dispersing  hormone  were  retained  by  the  G-25  Sephadex,  the  peak  of 
melanin-dispersing  activity  never  occurred  in  the  same  fraction  as  that  which  contained  the 
greatest  quantity  of  red  pigment-dispersing  hormone.  Assays  for  red  pigment-concentrating 
hormone  revealed  that  this  difference  in  peaks  could  not  have  been  due  to  suppression  of  the 
red  pigment-dispersing  hormone  by  the  red  pigment-concentrating  hormone  in  the  fractions 
examined.  Therefore,  the  hormones  from  Palaemonetes  that  dispersed  the  red  and  black 
pigments  in  Uca  must  have  been  different  substances.  In  contrast  to  the  eyestalks  of 
Palaemonetes,  with  eyestalks  of  Uca  the  same  fraction  produced  the  largest  response  with  both 
pigments.  However,  some  of  the  other  fractions  had  a  large  quantity  of  melanin-dispersing 
hormone  but  had  virtually  no  effect  on  the  red  pigment.  Assays  for  the  red  pigment-concentrat- 
ing hormone  confirmed  that  in  Uca  also,  as  in  Palaemonetes,  the  red  pigment-dispersing  hormone 
and  melanin-dispersing  hormone  were  not  the  same  substance. 

Supported  by  Grant  GB-5236  from  the  NSF. 

Collagen  from  the  cuticles  of  marine  worms.     Louis  FISHMAN  AND  MILTON  LEVY. 

The  compositions  of  the  structural  proteins  of  the  cuticles  of  several  species  of  marine 
worms  were  investigated.  The  nemertean  Ccrebratulus  lacteus  showed  no  evidence  of  a  cuticle. 
Among  the  annelid  polychaetes,  Arenicola  cristata  has  a  secreted  coat  of  non-collagenous 
material  with  no  discernible  cuticle  beneath.  Nereis  pclagica  has  a  cuticle  which  is  difficult  to 
remove  due  to  the  setae.  This  collagen  could  be  extracted  with  0.5  M  NaCl  and  precipitated 
with  ammonium  sulfate.  A  partially  purified  collagen  from  Nereis  was  hydrolyzed  and  the 
amino  acid  composition  determined.  Calculated  as  residues  per  1000  residues  it  contained :  gly 
234,  pro  43,  hypro  44,  p-ala  30,  and  cyst/2  16.  The  rest  of  the  amino  acids  were  compatible 
with  most  collagens. 

The  sipunculate  Phascolosoma  gouldi  has  a  firm  cuticle  which  is  almost  all  collagen. 
The  collagen  was  not  extracted  by  0.5  M  acetic  acid,  pH  4.3  citrate,  or  0.5  M  NaCl.  It  was 
partially  dissolved  by  0.5  M  CaCU.  These  extracts  did  not  show  a  denaturation  temperature 
(To)  nor  did  the  whole  cuticle  show  a  shrink  temperature  but  stretched  at  53°  C.  An  HC1 
hydrolysate  gave  the  following  amino  acid  composition  given  as  residues  per  1000  residues : 
gly  315,  pro  42,  hypro  64,  asp  46,  thre  48,  ser  56,  glu  128,  ala  95,  val  13,  meth  6,  isol  10,  leu  13, 
tyr  5,  p-ala  7,  lys  10,  hist  6,  arg  90,  cyst/2  8. 

The  structural  proteins  of  worms  should  be  classified  as  collagens  because  they  contain 
hydroxyproline  and  are  about  £  glycine  although  some  of  the  physical  properties  are  not 
characteristic. 

We  feel  that  the  cysteine  disulfide  bonds  are  structurally  important  since  0.2  M  mercapto- 
ethanol  in  0.5  M  CaCl2  completely  gelatinizes  Phascolosoma  cuticles. 

This  work  was  supported  by  NSF  funds  through  New  York  University. 

Reversible  changes  in  the  birefringence  of  the  squid  giant  axon  with  temperature. 
DAVID  S.  FORMAN. 

Studies  of  changes  in  the  birefringence  of  nerve  fibers  may  provide  information  about  the 
properties  of  the  oriented  molecules  of  the  axoplasm.  The  squid  giant  axon  is  a  favorable 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         391 

preparation  for  studying  this  birefringence  because  its  large  retardation  can  be  measured  easily. 
The  effect  of  temperature  on  this  birefringence  was  studied  in  nine  axons  using  a  rectified 
polarizing  microscope.  Several  millimeters  were  dissected  free  of  surrounding  small  fibers,  and 
the  axon  was  placed  in  sea  water  on  a  temperature  slide.  Retardations  were  measured  with 
a  calibrated  quartz  wedge,  and  diameters  were  measured  from  photographs  and  with  a  calibrated 
fine  focus.  All  measurements  were  made  at  a  single  region  of  the  axon. 

In  the  range  of  5°  to  40°  C,  lowering  the  temperature  decreased  the  birefringence,  and 
this  change  was  completely  reversible.  The  coefficient  of  birefringence,  uncorrected  for  the 
contribution  of  the  sheath,  was  an  approximately  linear  function  of  temperature.  The  change 
in  coefficient  of  birefringence  per  degree  varied  between  axons,  and  averaged  +1.5  X  10~*  per 
degree,  with  a  range  of  0.9  X  10~9  to  2.6  X  10~8  per  degree.  The  coefficient  of  birefringence 
averaged  +9.5  X  10~5  at  25°  and  +6.9  X  10"5  at  10°.  After  a  change  in  temperature,  birefringence 
reached  a  new  equilibrium  value  in  from  5  to  15  minutes.  Good  preparations  gave  stable  values 
for  several  hours.  Volume  changes  were  negligible  and  were  not  correlated  with  changes  in 
birefringence.  Resting  potentials  were  measured  in  four  axons  at  the  end  of  the  experiments. 
These  averaged  53  mv,  although  action  potentials  were  found  in  only  one  of  two  externally 
stimulated  axons. 

The  dependence  of  the  birefringence  of  the  axon  on  temperature  is  reminiscent  of  the 
effect  of  temperature  on  microtubular  systems,  such  as  the  mitotic  apparatus. 

Aided  by  grants  from  the  National  Cancer  Institute,  CA  10171,  the  National  Science 
Foundation,  GB-5120,  and  by  a  National  Science  Foundation  Graduate  Fellowship. 

Destruction  of  the  male  gonophores  of  Tubularia  larynx  by  a  hytnenostome  dilate  of 
the  genus  Parana phrys.  MAURA  GEENS,  MARGARET  JAMES  AND  GEORGE  G. 
HOLZ,  JR. 

A  Paranophrys  has  been  found  parasitizing  the  gonophores  of  the  hydroid  Tubularia  larynx. 
It  was  located  most  often  in  male  gonophores  between  the  epidermis  and  the  gastrodermal  wall 
of  the  spadix  where  it  fed  on  germ  cells.  It  was  found  much  less  frequently  in  female  gonophores 
and  actinula  larvae.  Apparently  it  can  attack  the  gastrodermis  and  destroy  the  spadix  while 
the  epidermis  remains  intact,  since  gonophores  without  internal  structure  were  found  packed 
with  ciliates. 

Ciliates  actively  divided  in  infected  gonophores  and  all  the  individuals  in  a  single  gonophore 
were  Paranophrys. 

The  route  of  infection  and  the  physiological  condition  of  the  host  at  the  time  of  infection 
are  unknown,  but  heavily  infected  gonophores  were  found  adjacent  to  uninfected  ones  which 
displayed  normal,  periodic  muscular  contractions. 

Paranophrys  in  this  circumstance  is  probably  a  facultative  parasite  of  the  hydroid.  We 
have  been  able  to  culture  the  ciliate  in  a  casein-yeast  autolysate-sea  water  medium  with  a 
marine  bacterium. 

Freshly  isolated  from  infected  gonophores  the  ciliate  was  pear-shaped,  had  a  sharply- 
pointed  anterior  end  and  was  28-65  p  long  and  17-28  ^  wide.  Cultured  ciliates  were  smaller ; 
17-38  n  long  and  5-18  ^  wide,  and  had  a  rounded  anterior  end.  Both  isolated  and  cultured 
types  had  12  ciliary  meridians,  in  contrast  to  the  9  of  the  type  species  of  the  genus,  Paranophrys 
marina  Thompson  &  Berger  1965. 

We  are  attempting  axenic  culture  so  that  the  biochemistry  of  the  ciliate  can  be  studied. 
Such  a  DNA-rich  diet  as  the  gonophore  form  favors  may  reflect  some  interesting  enzymatic 
qualities  and/or  capacities  with  respect  to  nucleotide  catabolism. 

Supported  by  a  grant,  GB-3447,  from  the  National  Science  Foundation  to  the  Department 
of  Invertebrate  Zoology,  Marine  Biological  Laboratory,  and  by  grant  AI  05802  from  the 
National  Institute  of  Allergy  &  Infectious  Diseases  to  G.  G.  Holz,  Jr. 

The  incorporation  of  C-14  lysine  and  C-14  phenylalanine  into  embryonic  (1-hr.) 
cell-free  Arbacia  punctulata  preparations.  ALBERT  GROSSMAN  AND  WALTER 
TROLL. 

The  incorporation  of  C-14  lysine  into  a  TCA-tungstate-insoluble  fraction  was  several-fold 
greater  than  that  of  C-14  phenylalanine.  However,  C-14  lysine  incorporation  did  not  decrease 


392          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

in  the  absence  of  Poly  A  as  did  C-14  phenylalanine  incorporation  in  the  absence  of  Poly  U. 
In  vivo  studies  showed  tht  both  amino  acids  were  taken  up  into  acid-insoluble  material  to  the 
same  extent  during  a  one-hour  incubation  period  immediately  following  fertilization.  To  test 
whether  the  cell-free  system  was  capable  of  synthesizing  a  substance  similar  to  Poly  A,  various 
concentrations  of  Poly  U  were  added  to  the  reaction  mixtures  to  complex  with  any  high 
adenylic  acid-containing  compounds  which  might  have  been  formed.  The  addition  of  Poly  U 
prior  to  the  one-half-hour  incubation  period  at  37°  C.  inhibited  markedly  C-14  lysine  incorpora- 
tion. It  has  also  been  shown  that  ATP  is  incorporated  into  an  acid-insoluble  fraction  which 
is  independent  of  other  nucleotide  triphosphates.  It  is  likely,  therefore,  that  cell-free  extracts 
of  one-hour-old  sea  urchin  embryos  are  capable  of  synthesizing  Poly  A  or  polynucleotides 
similar  to  Poly  A.  Although  the  function  of  this  polymer  remains  obscure,  it  could  code  for 
lysine  oligopeptides  which  would  aid  in  the  regulation  of  RNA  and  DNA  synthesis,  as  has  been 
suggested  for  other  polyamines  in  bacterial  systems. 

The  cell-free  sea  urchin  system  was  also  found  to  be  sensitive  to  exogenous  sources  of 
S-RNA.  The  addition  of  yeast  S-RNA  (3.7  mg./ml.)  stimulated  C-14  lysine  incorporation 
but  inhibited  C-14  phenylalanine  uptake.  When  E.  coli  S-RNA  was  used  the  reverse  occurred ; 
C-14  lysine  incorporation  was  inhibited  while  C-14  phenylalanine  uptake  was  stimulated.  The 
sensitivity  of  this  system  to  exogenous  sources  of  S-RNA  suggests  that  a  change  of  a 
single  S-RNA  species  might  be  a  rate-limiting  step  in  overall  protein  synthesis  and,  therefore, 
a  possible  regulator  of  such  synthesis. 

A.  G.  is  Senior  Postdoctoral  Fellow  of  the  New  York  Heart  Association.  This  work 
was  supported  by  Grants  71145115  and  03-013276  from  the  National  Institutes  of  Health. 

Isolation  of  surface  membranes  of  Strongylocentrotus  drobachiensis  eggs.  RAY- 
MOND L.  HAYS  AND  ALBERT  I.  LANSING. 

Repeated  attempts  at  application  of  variations  of  Neville's  technique  for  isolation  of  cell 
membranes  have  proven  unsatisfactory  when  applied  to  some  embryonic  tissues  and  marine  ova. 
We  report  herein  a  procedure  for  the  isolation  of  surface  membranes  which  are  structurally 
intact,  of  high  purity  and  of  good  yield.  Eggs  of  Strongylocentrotus  drobachiensis  are  harvested 
by  stimulation  with  1.0%  KNO3  and  suspended  in  an  equal  volume  of  4.0%  methylcellulose  in 
0.001  M  NaHCO3,  pH  7.6.  After  1-2  hours  exposure  to  this  solution  at  4°  C.,  the  cells 
are  washed  three  times  in  2.0%  methylcellulose  and  homogenized  by  10-20  strokes  of  a  loose- 
fitting  Bounce  homogenizer.  The  ruptured  cells  are  then  washed  three  times  in  2.0% 
methylcellulose  and  the  resulting  pellet  is  layered  over  a  discontinuous  sucrose  gradient  (d  1.16, 
d  1.18,  d  1.20).  Membranes  are  recovered  at  the  1.18-1.20  sucrose  interface  and' washed  twice 
with  0.001  M  NaHCOa. 

The  membranes  obtained  from  this  procedure  are  smooth,  homogeneous  in  normal  section 
and  of  200-300  A  thickness.  These  membranes  are  intact  and  appear  as  rolled  sheets  or  sacs. 
Ultrastructural  characteristics  of  membranes  recovered  from  fertilized  or  unfertilized  eggs  are 
similar.  No  unit  membrane  structure  is  observed  in  either  these  isolated  preparations  or  in 
intact  eggs.  The  isolated  membranes  are  of  the  same  thickness  as  those  of  intact  eggs. 
Similar  isolations  with  1.0%  sialic  acid  yield  comparable  results.  In  both  instances,  the 
membranes  recovered  are  intact  and  uncontaminated  by  other  cytoplasmic  organelles.  We 
believe  that  methylcellulose  may  preserve  the  integrity  of  the  surface  membrane  during  the 
isolation  procedure  as  does  sialic  acid  in  vivo. 

Quantitative  aspects  of  early  life-history  in  the  salt-marsh  pulnwnate  snail, 
Melampus  bidentatns,  and  their  evolutionary  significance.  W.  RUSSELL 
HUNTER  AND  MARTYN  L.  APLEY. 

Few  studies  on  growth  in  natural  populations  of  invertebrates  involve  measures  of 
actual  organic  biomass.  However,  a  colorimetric  method  of  "wet-oxidation"  now  allows 
assessments  of  total  organic  carbon  in  molluscs.  Determinations  of  shell-lengths  (L),  live 
wet  weights  (WW),  dry  weights  (DW),  tissue  dry  weights  ("ash-free")  (TDW),  shell 
calcium  carbonate  (Ca),  and  organic  C  content  (C),  were  made  for  growth  stages  of  the 
salt-marsh  pulmonate,  Melampus  bidentatns.  This  snail,  an  ellobiid,  retains  a  free-swimming 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         393 

planktonic  veliger,  and  is  of  considerable  evolutionary  interest,  being  almost  certainly  related 
to  the  stem-group  of  both  land  snails  and  fresh-water  pulmonates. 

In  Melampus,  egg-masses  averaging  850  small  eggs  are  laid  after  spring  tides  between 
early  June  and  mid-July.  Mean  values  for  individual  eggs  are  :  WW  =  4.72  /*g.,  DW  =  354 
nano-g.,  organic  C  =  109  nano-g.  Free-swimming  veligers  emerge  after  about  13  days,  and 
mean  values  per  individual  are :  L  =  127  /*,  WW  =  494  nano-g.,  DW  =  129  nano-g.,  TDW 
=  116  nano-g.,  Ca  <  13  nano-g.,  organic  C  =  33.4  nano-g.  Spat  snails  resettle  into  salt- 
marshes  after  about  6  weeks,  and  mean  values  are  then :  L  =  675  /j.,  WW  =  65  fig.,  DW  =  23.2 
^g.,  TDW  =  11.3  /teg.,  Ca  =  11.9  Mg-,  organic  C  =  5.03Mg-  Comparative  mean  values  for  two 
size  groups  of  adult  Melampus  are:  L  =  5.8  mm.,  WW:=36.1  mg.,  DW  =  17.4  mg.,  TDW 
=  4.3  mg.,  Ca  =  13.1  mg.,  organic  C  =  1.86  mg. ;  and  L  =  10.1  mm.,  WW  =  176  mg.,  DW  =  81 
mg.,  TDW  =  18.2  mg.,  Ca  =  62.8  mg.,  organic  C  =  7.4  mg.  Individuals  can  grow  to  L  =  12.3 
mm.,  WW  =  312  mg.,  and  organic  C  =  14.6  mg.  The  ratio  of  organic  C  in  Mg-.per  mg. 
wet  wt.  changes  from  23.1  (egg),  67.6  (veliger),  77.4  (spat)  to  51.5  through  42.1  (in 
adult  growth). 

In  Melampus.  using  any  real  measure,  such  as  total  organic  C  or  TDW,  growth  extends 
through  three  orders  of  magnitude  in  the  first  three  months  of  life,  and  through  nearly  6  in 
the  3-4-year  life-span.  In  contrast,  most  fresh-water  or  land  pulmonates  hatch  from  relatively 
large  eggs  and  increase  only  2  to  3  orders  of  magnitude  in  their  life-span  (i.e.,  Physa 
heterostropha  from  36  /xg.  to  5.3  mg.  organic  C). 

Discussions  of  significant  reproductive  adaptations  in  non-marine  pulmonates  usually 
emphasize  "need  to  suppress  the  free  larval  stage,"  but  these  data  suggest  that  selection 
pressures  to  reduce  the  temporal  extent  of  immature  growth  have  influenced  evolution  of  larger 
eggs  in  non-marine  environments. 

Supported  by  Grant  GM  11693  from  the  National  Institutes  of  Health. 

Histo-incoinpatibility    and    stolon    overgrowth    between    interbreeding    strains    of 
Hydractinia  echinata.     FRANCES  SHAPIRO  IVKER. 

Hydractinia  echinata  is  normally  found  as  a  mat  of  tissue  with  protruding  hydranths, 
growing  flat  on  snail  shells  inhabited  by  hermit  crabs.  Ten  strains  were  isolated  from  different 
shells  and  grown  on  glass  slides,  two  strains  per  slide,  in  all  possible  combinations.  Colonies 
grew  in  all  directions  and  random  contact  was  eventually  made,  at  which  time  one,  or,  in  two 
cases,  both  strains  produced  a  tangled  mass  of  stolons,  rising  as  high  as  5  mm.  from  the 
substrate.  This  stolon  overgrowth  eventually  overran  the  feeding  hydranths  of  the  opposing 
colony.  Only  after  the  death,  by  starvation,  of  the  underlying  colony  did  feeding  hydranths 
appear  on  the  stolon  mass,  indicating  one  or  several  interacting  inductive  systems.  During  the 
period  when  a  colony  was  actively  engaged  in  producing  overgrowing  stolons,  further  growth 
and  development  (sexuality)  in  other  areas  was  sharply  reduced  or  halted.  In  two  cases, 
a  tangled  stolon  mass  was  produced  between  two  colonies,  but  neither  overran  or  destroyed 
the  other.  In  all  the  other  cases,  there  was  a  hierarchy  of  stolon  production  potency. 

Several  strains  were  sexually  crossed  and  40%-60%  of  the  larvae  metamorphosed  on  the 
bottom  of  the  container.  The  offspring  displayed  either  fusion  with  sibling  colonies  or  histo- 
incompatibility  and  stolon  overgrowth  with  other  sibling  colonies.  Further  tests  are  under  way 
to  determine  the  extent  of  incompatibility  between  siblings,  and  between  parents  and  offspring. 
Crude  extract  of  colony,  and  media  in  which  incompatible  strains  were  grown  failed  to  produce 
overgrowth  in  a  known  inducible  strain,  suggesting  that  a  surface-bound  substance,  or  a  heavy, 
low  concentration  compound  is  responsible  for  this  histo-incompatibility. 

Chroniatographic  studies  on   cardioactive   compounds  extracted  from  Mercenaria 
mercenaria  hearts.     DAVID  JACOBOWITZ  AND  MORRIS  A.  SPIRTES. 

Hearts  of  clams  (M.  mercenaria}  were  homogenized  in  0.1  N  HC1,  ultracentrifuged 
(39,500  rpm)  for  1  hour  and  lyophilized.  Such  heart  extracts  tested  on  the  isolated  clam 
hearts  caused  inhibition.  After  benzoquinonium  (Mytolon)  10  /ug./cc.  the  heart  extract  caused 
excitation.  The  heart  extracts  were  run  on  thin  layer  chromatograms,  butanol/acetic  acid/ 
water  (4:1:2),  and  dried  overnight.  The  chromatograms  were  examined  under  UV  light 


394         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

and  a  portion  was  sprayed  with  ninhydrin.  The  remaining  regions  were  eluted  and  tested  on  the 
clam  heart.  Two  regions  with  average  Rf  0.35  and  0.55  (ninhydrin-positive)  showed  cardio- 
excitatory  effects  (increase  in  amplitude  and  frequency).  The  Rf  0.35  region  contained  a 
whitish  fluorescent  band.  The  Rf  0.55  component  was  inhibited  by  methysergide  (10  /ig./cc.) 
while  the  Rf  0.35  eluate  was  not.  The  lower  region  was  inhibited  by  trypsin  (1  /xg./cc.) 
incubation  (H  hrs.  at  37°  C.).  The  Rf  for  standard  serotonin  was  0.72.  Preliminary 
spectrophotofluorimeter  studies  indicate  that  serotonin  was  not  contained  within  the  Rf  0.55 
component.  A  third  region  with  an  average  Rf  0.22  contained  an  inhibitory  substance.  This 
region  was  identical  to  the  acetylcholine  Rf.  Benzoquinonium  caused  a  reversal  of  effect  to  that 
of  excitation.  Incubation  (10  minutes,  37°  C.)  of  this  fraction  with  washed  human  red  blood 
cells  did  not  alter  the  inhibitory  action  while  the  acetylcholine  effect  was  completely  inhibited 
by  this  treatment.  Because  of  this  it  cannot  be  concluded  that  this  heat-stable  inhibitory 
substance  is  acetylcholine.  There  therefore  appear  to  be  at  least  two  cardioexcitatory  sub- 
stances extracted  from  the  clam  heart,  one  of  which  may  be  a  peptide,  and  an  inhibitory 
substance,  the  chemical  nature  of  which  remains  unknown. 

The    stalk    conducting -system    mediating    behavioral    inhibition    in    the    hydroid 
Tubularia.     R.  K.  JOSEPHSON  AND  J.  F.  UHRICH. 

A  Tubularia  polyp  contains  two  principal  pacemaker  systems,  one  localized  in  the  distal 
stalk  (the  neck  system)  and  the  other  in  the  hydranth  (the  hydranth  system).  Repetitive 
stimulation  of  the  stalk  reduces  the  frequency  of  spontaneous  electrical  pulses  produced  by 
these  pacemaker  systems.  Three  conducting  systems  are  known  for  the  stalk  of  Tubularia, 
the  slow  system  (SS),  the  distal  opener  system  (DOS)  and  the  triggering  system  (TS). 
The  SS,  which  has  the  highest  threshold  of  the  three,  is  apparently  not  involved  in  polyp 
inhibition,  for  clear  inhibition  is  obtained  with  stalk  stimuli  well  below  the  SS  threshold. 
Similar  elimination  of  the  TS  and  DOS  has  not  been  possible,  for  their  thresholds  are  very 
close  and  somewhat  variable.  Two  other  approaches,  however,  indicate  that  the  TS  is  also  not 
involved  in  polyp  inhibition  during  stalk  stimulation.  (1)  Tubularia  often  forms  small  colonies 
of  2-3  polyps  with  tissue  connections  between  them.  The  TS,  which  is  common  throughout  the 
colony,  is  activated  each  time  the  neck  pacemaker  system  of  any  one  of  the  connected  polyps  fires. 
Thus  a  polyp  of  a  colony  receives  a  continuous,  low  frequency  input  from  the  TS.  But 
isolating  a  polyp  from  a  colony  does  not  result  in  an  increase  in  spontaneous  activity  as  would 
be  expected  if  the  tonic  TS  input  were  inhibitory.  (2)  By  stimulating  the  distal  tentacles 
it  is  possible  to  activate  the  DOS  without  exciting  the  TS.  Activating  the  DOS  alone  does 
inhibit  spontaneous  activity,  and  distal  tentacle  stimulation  (DOS  without  the  TS)  more  fully 
inhibits  the  neck  pacemaker  than  does  stalk  stimulation  (exciting  the  DOS  and  TS). 

Supported  by  USPHS  grant  NB  06054  and  grant  GB-3447  from  the  National  Science 
Foundation  to  the  Department  of  Invertebrate  Zoology,  Marine  Biological  Laboratory. 

Intracellular  ionic   concentrations   determined   by   ultra-micro   flame   photometry. 
GEORGE  M.  KATZ. 

In  standard  flame  photometry,  an  atomizer  is  used  to  introduce  the  sample  solution  into 
the  flame.  If,  instead,  the  sample  is  placed  on  a  platinum  holder  and  inserted  directly  into  the 
flame,  the  integral  of  the  brief  light  emission  will  be  a  measure  of  the  total  number  of  moles 
of  the  salt  on  the  platinum.  This  technique  results  in  a  thousand-  to  a  million-fold  improvement 
in  sensitivity  over  standard  flame  photometry.  An  integrative  ultra-micro  flame  photometer 
has  been  developed  which  can  detect  between  10~13  and  10"14  moles  of  Na  and  K  simultaneously 
from  the  same  sample.  The  accuracy  and  reliability  of  the  method  depend  upon  the  size 
and  shape  of  the  platinum  holder,  the  nature  of  the  associated  anions,  interfering  cations,  and 
chemical  reactions  of  the  salts  in  the  sample  which  appear  to  involve  platinum.  An  analysis 
of  these  various  perturbing  factors  has  been  made  and  techniques  have  been  developed  to 
minimize  their  effects.  Thus,  an  accuracy  of  2.5%  can  be  achieved  even  in  the  most  sensitive 
ranges  of  the  instrument.  The  integrative  flame  photometer  has  been  used  to  determine 
intracellular  ions  in  two  single  cell  preparations — crayfish  muscle  fibers  and  lobster  axons. 
Single  isolated  muscle  fibers  were  digested  in  0.5  ml.  of  0.3  M  HAc  for  the  determination  of 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         395 

intracellular  K.  The  calculated  concentration  was  130  ±  19  mM/Kg.  fiber  water.  One  to 
5  cm.  of  a  lobster  circumesophageal  giant  axon  was  placed  on  a  platinum  pan  and  ashed  to 
remove  the  organic  matrix  prior  to  insertion  into  the  flame.  The  K  concentration  was 
348  ±  59  mM/Kg.  fiber  water  and  the  Na  concentration  was  61  ±  18.6  mM/Kg.  fiber  water. 
Work  supported  by  USPHS  grants  NB  03728-04  and  NB  03270-05  from  the  National 
Institute  of  Neurological  Diseases  and  Blindness  and  by  grant  GB-2940  from  the  National 
Science  Foundation  to  Dr.  H.  Grundfest. 

Morphological  comments   on   blood   pressure   relationships   in   Squalus   branchial 
arteries.     RUDOLF  T.  KEMPTON. 

While  it  is  quite  typical  to  find  a  large  drop  in  blood  pressure  during  transit  of  a 
capillary  bed,  Burger  and  Bradley  found  in  the  spiny  dogfish  a  decrement  of  only  1  to  5  mm. 
of  mercury  between  afferent  and  efferent  branchial  arteries.  Anatomical  features  of  these 
gills  have  been  examined  for  an  explanation  of  this  aberrant  behavior. 

There  appears  to  be  a  low  resistance  to  flow  due  to  a  number  of  factors.  (1)  Blood  is 
brought  into  the  vicinity  of  the  secondary  lamellae  by  an  artery  which  is  relatively  short  and 
wide.  (2)  Distribution  from  the  artery  to  the  secondary  lamella  is  through  a  peculiar 
flattened  non-elastic  chamber,  which  extends  the  entire  length  of  the  filament  and  is  much 
larger  in  diameter  than  the  parallel  artery.  Each  millimeter  of  this  chamber  has  approximately 
20  connections  with  the  artery  and  supplies  20  pairs  of  secondary  lamellae.  This  chamber 
is  traversed  by  columns  which  create  turbulence  and  which  are  covered  with  phagocytic 
cells.  (3)  Blood  from  this  large  channel  flows  into  a  thin  space  within  each  secondary 
lamella,  one  whose  dimensions  are  approximately  1.5  X  0.5  X  0.01  mm.  The  limiting  membranes 
of  this  space  are  maintained  in  a  stable  position  by  connecting  pilaster  cells,  as  described  by 
Keys  and  Killmer  for  the  teleost.  (4)  The  large  chamber  and  the  thin  but  wide  spaces 
permit  blood  flow  with  much  reduced  friction  and  without  the  pressure-consuming  hindrance  of 
tubes  partially  blocked  by  piled-up  red  cells.  (5)  There  is  a  channel  along  the  outer  edge 
of  the  lamella  which  tends  to  distribute  blood  evenly  over  the  entire  lamella.  (6)  The  total 
length  of  flow  along  the  lamella  is  probably  not  more  than  1.5  mm.  (7)  There  are 
approximately  300,000  lamellae  per  animal.  (8)  The  lamellae  drain  into  an  artery  which 
courses  along  the  distal  end  of  the  filament  accompanied  by  many  nerve  fibers  of  uncertain 
function. 

The  fine  structure  of  the  redia  of  the  trematode,  Cryptocotyle  lingua.     PAUL  L. 
KRUPA,  ARYA  K.  BAL  AND  GILLES  H.  COUSINEAU. 

Rediae  of  Cryptocotyle  lingua,  isolated  from  the  snail  host  Littorina  littorea,  were  studied 
with  the  electron  microscope.  The  larvae  were  fixed  (5%  glutaraldehyde-paraformaldehyde 
mixture  in  sea  water),  post-osmicated,  and  embedded  in  Epon  in  the  usual  manner. 

Numerous  microvilli  are  found  to  extend  from  the  epithelial  surface  of  the  intestine  into 
the  lumen.  These  measure  about  2.3  X  0.02  ^  near  the  anterior  end  of  the  gut,  but  they  become 
shorter  (0.5  /u)  and  fewer  at  the  blind  end.  The  external  surface  of  the  redia  is  also  covered 
by  many  microvilli  which  are  located  0.06  to  0.08  /j,  apart  from  each  other  at  their  proximal 
ends.  These  integumentary  projections  are  about  0.04  ^  thick  and  some  are  2.0  /j.  long;  a 
unit  membrane  (0.01  fj.  thick)  covers  them.  Vesicles  and  granules  measuring  up  to  1.05  X  0.6  ^ 
and  mitochondria  are  clearly  visible  between  the  surface  membrane  and  basement  lamina. 
The  latter  is  a  fine-textured  layer  of  low  density,  sometimes  thrown  into  undulations,  and 
measuring  0.1  to  0.2  /it  across.  Directly  beneath  the  basement  lamina  are  found  outer  circular 
and  inner  longitudinal  muscles.  Other  constituents  in  the  redial  body  wall  are  glycogen  deposits 
in  the  form  of  single  granules  (beta  particles)  and  rosettes  (alpha  particles). 

At  one  stage  in  its  development,  the  integument  of  the  immature  cercaria,  within  the 
redia,  is  characterized  by  numerous  irregular  undulations  which  increase  the  surface  area. 
Some  of  the  projections  may  represent  spine  production,  but  others  suggest  that  pinocytosis  or 
phagocytosis  may  take  place. 

The  presence  of  subsurface  mitochondria  and  integumentary  projections  in  this  parasite 
supports  the  observations  of  others  that  the  integument  of  trematodes  is  metabolically  active. 


396         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Preliminary  autoradiographic  observations  with  leucine-C"  show  that  incorporation  into 
protein  occurs  in  specific  sites  of  the  developing  germ  balls  and  cercariae.  Further  electron 
microscopic  and  biochemical  studies  of  the  redia  and  the  other  life-cycle  stages  are  in  progress. 
This  work  was  supported  by  grants-in-aid  of  research  from  the  Damon  Runyon  Memorial 
Fund  for  Cancer  Research  (grant  #DRG-918),  the  National  Research  Council  of  Canada 
(grant  #731-741),  and  the  Society  of  the  Sigma  Xi. 

Further  characteristics  of  lenticular  gamma  crystallin.     SIDNEY  LERMAN,  WILLIAM 
F.  FORBES  AND  SEYMOUR  ZIGMAN. 

Gamma  crystallin  is  the  cryoprotein  responsible  for  the  cold  cataract  phenomenon  observed 
in  the  lenses  of  young  animals.  In  the  dogfish  lens  the  cold  cataract  phenomenon  is  normally 
prevented  because  of  a  relatively  high  concentration  of  urea  (0.3  M)  but  when  the  urea  is 
removed  from  the  lens  by  means  of  dialysis,  an  in  vitro  cold  precipitation  phenomenon  can  be 
demonstrated.  Amino  acid  analyses  of  dogfish  gamma  crystallin  reveal  that  it  is  similar  in 
composition  to  rat  gamma  crystallin  but  that  it  has  a  higher  concentration  of  tyrosine.  Gamma 
crystallin  from  these  two  species  does  not  contain  tryptophan  (as  determined  by  amino  acid 
analyses  following  basic  hydrolysis,  and  by  the  N-bromsuccinamide  reaction).  Electronic 
absorption  studies  reveal  an  unusually  high  E\  value  (approximately  27  for  dogfish  gamma 
crystallin  and  22  for  gamma  crystallin  derived  from  the  rat  lens).  This  hyperchromic  effect 
appears  to  be  due  to  the  tyrosine  residues  within  the  molecule.  Approximately  one-half  of  these 
residues  can  be  readily  ionized.  It  is  postulated  that  some  of  the  tyrosyl  residues  in  gamma 
crystallin  are  closely  held  together  in  the  native  molecule,  permitting  a  special  type  of 
electronic  interaction. 

Tritium-hydrogen  exchange  studies  on  gamma  crystallin  reveal  that  this  protein,  or  some 
fraction  thereof,  contains  a  relatively  large  number  of  hydrogen  atoms  (approximately  J  of 
the  total)  which  exchange  very  slowly.  This  might  indicate  a  relatively  tight  structure  in  at 
least  a  significant  portion  of  the  molecule.  These  studies  also  indicate  that  the  tightest 
structure  of  gamma  crystallin  is  at  pH  4.5-4.8.  Tritium-hydrogen  exchange  data  on  gamma 
crystallin  derived  from  the  older  animal  show  a  decrease  in  the  number  of  slowly  exchangeable 
hydrogen  atoms. 

Supported  by  USPHS  grant  NB  03081  and  MBL  ONR  research  grant. 

Collagens  of  echinoderms.     MILTON  LEVY  AND  Louis  FISH  MAN. 

Collagens  of  several  echinoderms  were  converted  to  gelatins  and  these  were  analyzed  for 
amino  acid  contents  after  hydrolysis.  The  purpose  was  to  compare  the  compositions  with  those 
of  other  more  familiar  collagens.  To  prepare  the  materials,  eviscerated  sea  urchins  were  wet- 
ground  with  mortar  and  pestle.  Large  amounts  of  crystalline  calcium  carbonate  became 
suspended  and  could  be  poured  off  with  the  supernatant  after  minimal  settling.  After  each 
grinding  several  washings  were  done.  The  grinding  was  repeated  four  times  until  some 
tendency  of  the  residual  material  to  float  made  difficulties.  The  residues  were  then  suspended 
in  water  and  6  N  HC1  added,  with  mechanical  stirring  to  keep  the  pH  at  1.3-1.4.  After  the 
addition  of  about  20  millimoles  of  acid  the  insoluble  material  was  collected  on  a  cloth  filter, 
washed  with  water,  resuspended  and  the  addition  of  acid  restarted.  This  process  was  continued 
until  no  acid  was  required  to  keep  the  pH  in  the  range  indicated  during  two  hours.  The  final 
solution  is  neutralized  to  pH  4,  filtered  on  cloth,  wrung  "dry"  and  suspended  in  4  times  its 
volume  of  water.  The  suspension  is  heated  to  120°  for  3  hours,  filtered  and  evaporated  at  90° 
in  a  petri  dish.  The  dark  gelatin  is  scraped  out  for  analysis.  Body  walls  of  starfish  could 
not  be  ground  in  this  way  but  were  sufficiently  softened  by  acid  (5%)  to  allow  blending.  After 
further  acid  treatment  to  the  endpoint  used  for  urchins  the  material  was  suspended  in  water  and 
gelatinized  at  120°  for  12  hours.  White  fenestrated  ossicles  were  in  the  residue  and  H2S  was 
evident.  Filtration,  dialysis  and  evaporation  gave  a  glassy  brown  residue.  The  analyses 
showed  per  1000  residues  85-93  hydroxyproline,  86-94  prolines  and  300-320  glycines.  These 
are  expected  values.  Glutamic  acid,  92-108,  is  above  vertebrate  levels  while  lysine  at  10-11 
is  about  40%  of  vertebrate  gelatins. 

This  work  was  supported  by  NSF  funds  through  New  York  University. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         397 

Transglutaminase  and  blood  clotting.     L.   LORAND,  J.  BRUNER-LORAND,  H.   H. 
ONG,  N.  G.  RULE  AND  T.  URAYAMA. 

Clotting  of  fibrinogen  in  vertebrates  is  controlled  by  two  enzymes.  Hydrolysis  by  thrombin 
uncovered  glutamine  sidechains  to  which  e-amino  groups  of  lysine  (and  probably  not  a-amines 
of  glycine  as  previously  believed)  from  a  neighboring  molecule  can  subsequently  crosslink  by 
transpeptidation.  By  contrast,  in  the  invertebrate  Homanis,  transpeptidation  alone  is  sufficient 
for  clotting.  Thus  crosslinking  by  transpeptidation  seems  to  be  the  original  reaction.  Hydro- 
lytic  activation  of  fibrinogen  in  vertebrates  merely  provides  an  extra  control. 

Transglutaminase  preparations  (guinea  pig  liver)  can  substitute  both  for  the  transpeptidase 
(but  not  for  thrombin)  of  vertebrate  plasma  and  for  the  clotting  enzyme  of  Homarus.  Since 
transglutaminase  substrates  (benzyloxycarbonylglutaminylglycine  (7)  or  histamine)  inhibit 
clotting  activities  of  the  preparations,  true  transglutaminase  specificity  is  involved. 

Fluorescence  micromethods  were  developed  using  mono(l-dimethyl-aminonaphthalene-5- 
sulfonyl)pentamethylenediamine  (//)  for  testing  transpeptidases.  For  example,  transglutami- 
nase, incubated  with  30  mM  of  /  and  5  mM  of  //  (pH  8.5 ;  20  mM  calcium  chloride  and 
glutathione)  produced  two  additional  fluorescent  derivatives  (A  and  B)  which  could  be 
identified  by  several  techniques.  In  paper  electrophoresis  (1%  v/v  pyridine-acetate,  pH  5.4) 
A  stayed  at  the  origin,  B  migrated  to  the  anode  and  //  to  the  cathode.  Using  thin-layer 
carboxymethylcellulose  chromatography  (above  pyridine  solvent),  A  was  stationary,  B  and  // 
moved  with  Rt's  of  about  0.4  and  0.1.  On  Sephadex  G-10  column  (water  solvent),  A  was 
eluted  first,  then  B;  II  was  greatly  retarded.  Excitation  and  emission  spectra  of  A  were 
different  from  those  of  B  and  //;  the  latter  two  were  identical.  Acid  hydrolysis  of  B  yielded 
glutamic  acid  and  glycine,  equimolar  to  the  naphthalene  chromophore  present  (computed  from 
absorptivity  at  326  m,u).  Properties  of  B  were  those  expected  from  the  7-amide  of  /  with  //. 
Product  A  was  the  result  of  incorporating  //  into  the  proteins  of  the  enzyme  preparation. 

Aided  by  USPHS  Research  Career  Award. 

Vinblastine  and  griseofulvin  reversibly  disrupt  the  living  mitotic  spindle.     STEPHEN 
E.  MALAWISTA  AND  HIDEMI  SATO. 

Studies  of  particular  stages  of  mitosis  are  hampered  by  the  rapid  passage  of  most  dividing 
cells  through  the  mitotic  cycle.  However,  freshly  spawned,  unfertilized  oocytes  of  the  marine 
annelid,  Pectinaria  gonldi,  persist  for  several  hours  at  the  first  meiotic  metaphase.  The  content 
of  oriented  spindle  material  in  these  oocytes  can  be  measured  in  polarized  light  as  retardation 
induced  by  spindle  birefringence.  We  have  studied  spindle  effects  of  some  metaphase-arresting 
agents  that  are  of  special  medical  interest. 

Perfusion  with  the  chemotherapeutic  Vine  a  alkaloid,  vinblastine  (Velban,  1  X  10~5  M), 
resulted  in  a  decrease  in  birefringence  and  size  of  the  spindle  and,  within  6  to  12  minutes 
at  24°  to  25°  C,  complete  dissolution.  On  subsequent  perfusion  with  artificial  sea  water, 
recovery  of  spindles  began  in  about  20  minutes  and  was  complete  by  40  to  50  minutes.  This 
reversible  effect  was  repeatable  in  the  same  preparation,  and  was  at  least  as  efficient  as  that  of 
N-desacetyl-N-methylcolchicine  (Colcemid,  1  X  10~5  M).  Neither  dissolution  nor  recovery  was 
affected  by  glutamate,  1  X  10"3  M.  Dissolution  by  vinblastine  was  retarded  in  40%  D^O-sea 
water,  but  recovery  was  not  hastened  by  D2O-sea  water.  At  1  X  10~6  M,  vincristine  (Oncovin), 
a  Vinca  alkaloid  of  similar  structure,  did  not  abolish  spindle  birefringence.  At  1  X  10~4  M, 
dissolution  required  24i  minutes,  and  recovery  was  incomplete,  with  many  small  tri-  and 
tetrapolar  spindles.  Podophyllotoxin  (IX  10~7  M)  produced  spindle  dissolution  in  about  20 
minutes  ;  recovery  was  incomplete. 

Perfusion  with  the  fungistatic  antibiotic,  griseofulvin  (Grisactin,  1  X  10"5  M),  resulted  in 
a  decrease  in  spindle  birefringence  and  size,  disappearance  in  3i  to  6i  minutes,  and  complete 
recovery  in  51  to  11  minutes.  This  reversible  dissolution  could  be  carried  out  repeatedly  in  a 
single  oocyte ;  recovery  was  complete  even  with  a  10-fold  increase  in  concentration  of 
griseofulvin. 

These  agents,  then,  are  useful  additions  to  the  colchicine  analogues  for  studying  the 
molecular  architecture  of  the  mitotic  spindle.  Furthermore,  the  shift  of  emphasis  from 


398          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

"mitotic  arrester"   to  "spindle   poison"   will   hopefully   stimulate   the    investigation   of   possible 
effects  of  such  substances  on  gelated  structures  in  resting  cells. 

Aided  by  grants  from  the  National  Cancer  Institute,  CA  10171,  the  National  Science 
Foundation,  GB-5120,  and  the  National  Institute  of  Arthritis  and  Metabolic  Diseases, 
AM  10493.  Dr.  Malawista  is  a  Senior  Investigator  of  the  Arthritis  Foundation. 

Inland  culture  of  Botryllus  schlosseri;  genetic  crosses.  ROGER  MILKMAN  AND 
JUDITH  PEDERSON. 

Botryllus  schlosseri  has  been  cultured  in  beakers  of  still  Instant  Ocean  containing  initial 
daily  concentrations  of  0.5-2.2  X  105  cells/ml,  of  Cyclotclla  nana,  a  centric  diatom  about  5  micra 
in  diameter.  Asexual  reproduction  is  rapid,  with  zooids  often  producing  4  buds  apiece,  all  of 
which  become  functional  zooids.  Tadpoles  give  rise  to  egg-producing  colonies  in  less  than 
H  months ;  once  begun,  egg  production  has  improved  with  each  asexual  generation.  Colonies 
must  be  kept  vertical,  upside  down,  or  in  between. 

Techniques  for  in  vitro  fertilization  and  embryo  culture,  necessary  for  the  control  required 
in  the  genetic  crossing  of  self-compatible  hermaphroditic  organisms,  have  been  refined.  Up  to 
100%  of  eggs  nested  in  a  dense  layer  of  sperm  are  fertilized ;  good  yields  of  metamorphosing 
larvae  are  obtained  when  embryos  are  placed  on  filter  paper  in  fingerbowls  and  transferred 
daily.  Crosses  to  date  support  Sabbadin's  conclusion  that  double  pigment  bands  are  associated 
with  a  simple  dominant  gene;  in  one  locale  (Eel  Pond)  such  a  gene's  frequency  is  calculated  to 
be  0.4.  The  work  preliminary  to  the  extensive  investigation  of  the  developmental  genetics  of 
Botryllus  is  now  complete. 

Supported  by  Research  Grant  GM  07810  from  the  National  Institute  of  General 
Medical  Sciences. 

Hyperbaric  oxygen  and  succinic  dehydrogenase  in  the  embryology  of  Tubularia. 
JAMES  A.  MILLER,  JR.,  DAVID  L.  DESHA,  PAUL  M.  HEIDGER  AND  FAITH  S. 
MILLER. 

Increases  in  the  activity  of  succinic  dehydrogenase  accompany  each  visible  step  in 
morphogenesis  in  Tubitlaria  (Miller,  Hegab  and  Miller,  1964).  Since  oxygen  stimulates  regen- 
eration (Barth,  1938)  but  hyperbaric  oxygen  inactivates  succinic  dehydrogenase  (Stadie  and 
Haugaard,  1954),  a  study  was  made  to  determine  whether  hyperbaric  oxygen  was  inhibitory 
or  stimulatory  in  the  embryonic  development  of  Tubularia  and  what  its  effects  were  on  succinic 
dehydrogenase  activity.  Hyperbaric  oxygen  at  all  levels  tended  to  protect  embryos  and  larvae 
from  death  in  standing  sea  water.  Oxygen  at  li  atmospheres  absolute  did  not  affect  embryonic 
development  but  at  2,  2J,  3  and  4  atmospheres  blocked  development  at  early  actinula  stages. 
Blocked  embryos  were  unable  to  secrete  adherent  material  from  their  adhesive  organs  or  produce 
perisarc  and  thus  were  unable  to  transform  into  polyps.  The  block  in  differentiation  developed 
between  24  and  48  hours  and  was  completely  reversible  even  after  72  hours  at  4  atmospheres. 

Succinic  dehydrogenase  was  estimated  histochemically  by  a  modification  of  the  Nachlas 
method.  It  showed  a  reduction  in  overall  activity  in  embryos  subjected  to  hyperbaric  oxygen 
and  the  failure  to  develop  the  localized  zones  of  high  activity  which  were  associated  in  controls 
with  differentiation  of  tentacles,  gonophore  buds,  perisarc-secreting  region  and  adhesive  organ. 
Since  hyperbaric  oxygen  does  not  inactivate  a  variety  of  enzymes  including  cytochrome 
oxidase  but  does  reversibly  inactivate  those  containing  SH  groups  including  succinic 
dehydrogenase,  our  experiments  suggest  that  enzymes  with  SH  groups  are  concerned  with 
differentiation  in  Tubularia  and  that  additional  studies  along  these  lines  will  further  elucidate 
the  energetics  of  development  in  this  species. 

The  effect  of  phenylthiourea  on  melanogenesis  in  the  embryo  of  Fundulus  hetero- 
clitus.  BEVERLY  S.  MITCHELL  AND  GEORGE  SZABO. 

Phenylthiourea  in  a  concentration  of  8  X  10~3  M  was  observed  to  inhibit  melanin  formation 
in  the  yolk  sac  epithelium,  on  the  body  surface  and  in  the  retinal  pigment  epithelium  of 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         399 

Fundulus  embryos.  Organ  differentiation  continued  in  those  embryos  developing  in  PTU, 
although  the  growth  rate  was  impaired  and  maximal  survival  time  was  nine  days. 

Electron  microscopic  studies  of  the  pigment  layer  of  the  normally  developing  retina  showed 
a  number  of  fully  melanized  granules  (melanosomes)  surrounded  by  a  single,  closely  applied 
membrane.  In  contrast,  the  retinal  pigment  layer  of  embryos  which  developed  in  PTU  for 
three  days  prior  to  fixation  and  in  which  macroscopic  deposition  of  melanin  had  been  inhibited, 
showed  very  few  melanosomes,  mostly  in  pre-melanosome  stage.  There  were  also  large  vacuo- 
lated  areas  containing  some  pigment,  presumably  early  pre-melanosomes.  In  addition,  there 
were  large  numbers  of  glycogen  granules  present.  Cell  divisions  were  observed  to  be  taking 
place  after  PTU  treatment  as  well  as  in  the  controls. 

Preliminary  studies  on  the  incorporation  of  tritiated  thymidine,  valine  and  DOPA  into 
embryos  developing  normally  and  in  PTU  were  carried  out.  The  effect  of  PTU  on  thymidine 
and  valine  incorporation  was  variable,  giving  little  indication  of  the  overall  effect  on  growth 
and  development.  PTU  was  consistent,  however,  in  decreasing  the  incorporation  of  tritiated 
DOPA  into  the  entire  embryo  and  into  the  developing  eye. 

The  morphogenetic  evidence,  in  conjunction  with  these  biochemical  studies,  indicates  that 
PTU  interferes  with  the  melanization  of  the  preformed  melanosomes.  This  is  consistent  with 
the  view  that  PTU  is  acting  directly  on  the  tyrosine-tyrosinase  system. 

Supported  by  a  Student  Fellowship  of  Harvard  Medical  School  and  by  a  Research  Grant 
#CA  050401-07,  National  Cancer  Institute,  USPHS  and  a  Career  Development  Award 
#Ke-GM-14.987  USPHS. 

Distribution   and   responses   to   salinity    of   larval   chironomids   from    the   upper 
Pocasset  River.     SAMUEL  C.  MOZLEY. 

The  sandy-bottomed  upper  120  meters  of  the  Pocasset  River  harbor  the  larvae  of  eight 
species  in  three  sub-families  of  the  dipteran  family  Tendipedidae  (=Chironomidae).  Salinities 
in  this  region  vary  from  0.5  to  26  ppt.  Salinities  of  interstitial  water  also  vary  tidally  to  a 
depth  of  9-10  cm.,  where  they  remain  at  4-8  ppt.  The  tidal  salinity  range  decreases  steadily 
to  this  depth.  Springs  affect  this  pattern  at  some  sites. 

The  two  most  abundant  species  are  Tcndipes  modestus  (Say)  and  Polypedilum  scalaemim 
(Schrank).  The  former  is  denser  subtidally,  and  the  latter,  intertidally.  These  and  four 
other  species  extend  to  the  edge  of  silty  bottoms  downstream,  but  two  species'  larvae  are 
restricted  to  the  upper  30  meters.  T.  modestus  occurs  as  deep  as  4  cm.  into  the  sediment,  but 
is  most  abundant  in  the  top  2  cm.  Larvae  of  this  species  build  sand-and-mucus  tubes,  which 
they  irrigate  periodically  by  undulating.  In  the  laboratory,  intervals  between  undulation  are 
longer  at  high  salinities.  A  greater  percentage  of  undulation  time  is  spent  pumping  interstitial 
water  out  through  the  tube  opening  with  increasing  salinity  to  19  ppt.,  by  larvae  in  tubes  with 
only  one  opening. 

The  LD-50  time  for  T.  modestus  at  42°  C.  is  not  affected  by  salinity.  At  41°,  salinities 
greater  than  6  ppt.  decrease  the  survival  time,  up  to  32  ppt.  Here  the  LD-50  time  is  the 
same  as  at  42°.  At  40°,  a  salinity  of  48  ppt.  is  required  to  reduce  the  LD-50  time  to  this  point. 
Reduction  in  available  oxygen  decreases  tolerance  to  high  salinities. 

This  work  was  supported  by  NASA  training  grant  NsG(T)  123-64  and  NIH  grant 
2TIGM  535-06  to  the  Marine  Ecology  Course  of  the  Marine  Biological  Laboratory.  Special 
appreciation  is  due  Dr.  Howard  L.  Sanders  for  regular  advice  during  this  study. 

The   development   of   an   ordered   array   of   collagen   in   Fundulus.     JOSEPH    B. 
NADOL,  JR. 

The  skin  of  Fundulus  hetcroditus  possesses  a  highly  ordered  array  of  collagen  fibrils, 
called  the  basement  lamella.  Fibrils  are  arranged  in  alternate  layers  nearly  parallel  to  the 
surface  and  crossing  each  other  at  105-110°.  Previous  investigators  have  drawn  an  analogy 
between  the  basement  lamella  of  amphibian  skin  and  the  structure  of  plywood,  and  have 
suggested  that  each  fibril  layer  is  exactly  parallel  to  the  basement  membrane.  They  have 
further  proposed  that  during  growth  the  basement  lamella  is  established  by  polymerization  of 
collagen  fibrils  at  the  basement  membrane  in  one  orientation  and  that,  by  some  repeated 


400         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

switching  mechanism,  each  subsequent  layer  is  laid  down  at  approximately  right  angles  to  the 
previous  (and  now  underlying)  layer. 

Our  observations  in  Funduhts  do  not  support  this  "plywood  theory."  Instead  collagen 
layers  descend  at  a  slight  angle  from  the  basement  membrane.  The  name  "scindulene"  has 
been  adopted  to  describe  this  shingle-like  arrangement  of  layers.  As  a  mechanism  of  establish- 
ing the  orthogonal  array  of  the  basement  lamella,  it  is  proposed  that  collagen  fibrils  are  inserted 
at  end  points  into  the  basement  membrane  and  are  polymerized  in  one  orientation  only  in  one 
area  of  the  basement  membrane,  and  at  105-110°  to  this  first  orientation  in  adjacent  areas, 
thus  obviating  the  necessity  of  a  switching  mechanism  required  by  the  plywood  theory. 
Thickening  of  the  basement  lamella  with  growth  is  accounted  for  by  expansion  of  these  "areas 
of  insertion"  of  fibrils  into  the  basement  membrane.  As  a  result  the  number  of  fibril  rows  in 
each  layer,  but  not  the  number  of  layers,  increases.  This  corresponds  exactly  with  the 
observed  morphological  changes  with  growth  in  Fundulus. 

Other  morphological  findings  in  Funihilus,  such  as  the  appearance  of  "crossover  fibrils" 
continuous  from  one  layer  to  the  next  in  the  same  orientation,  and  the  gradual  tapering  and 
disappearance  of  the  basement  lamella  toward  the  fin  tip,  are  more  easily  explained  by  the 
scindulene  theory  than  by  the  plywood  theory. 

This  work  was  supported  by  Grant  5T1-GM-707  from  the  National  Institutes  of  Health  to 
Dr.  Keith  R.  Porter  and  by  Grant  GY  108  from  the  National  Science  Foundation  to  Harvard 
University  and  the  author. 

The  diurnal  nature  of  the  tidal  migration  rhythm  of  the  diatom,  Hantzschia.  JOHN 
D.  PALMER  AND  FRANK  E.  ROUND. 

The  inter  tidal  benthic  diatom,  Hantzschia,  lives  buried  in  the  sand  at  night  and  during  high 
tide,  but  emerges  onto  the  surface  sediments  during  daytime  low  tides.  The  cells  accumulate 
in  such  vast  numbers  on  the  surface,  that  the  exposed  sediments  are  colored  a  golden  brown. 

Intact  Haiitsschia -bear ing  sand  samples  were  returned  to  the  laboratory  where  the 
rhythmic  movements  could  be  studied  under  controlled  conditions.  In  constant  temperature, 
continuous  illumination  of  a  constant  intensity,  and  away  from  the  influence  of  the  tide,  the 
rhythm  was  found  to  persist  for  as  long  as  11  days.  During  this  time  the  diatoms  appeared  on 
the  surface  in  approximate  synchrony  with  the  feral  cells  on  the  sand-flats.  A  similar 
laboratory  population  behaved  in  a  like  manner  in  alternating  light  and  dark  conditions  (14.5 
hrs.  light ;  9.5  hrs.  dark) . 

In  nature,  as  the  time  of  low  tide  moves  to  the  late  afternoon,  the  following  low  tide 
overlaps  with  sunrise  the  next  day ;  thus  twice  each  month  the  sand-flats  are  exposed  both  in 
the  morning  and  late  afternoon.  During  these  days  the  diatoms  gradually  abandon  the 
afternoon  phase  of  their  rhythm  and  now  appear  on  the  surface  during  the  early  morning 
exposure.  To  examine  this  phase  change  in  detail,  cells  were  collected  during  late  afternoon 
tides  and  placed  in  either  alternating  light-dark  conditions,  or  under  constant  light  in  the 
laboratory.  Over  the  next  three  days,  in  both  conditions,  the  rhythm  rephased  to  the  morning 
hours,  indicating  that  this  abrupt  phase  change  is  also  under  clock  control. 

Using  these  results  a  working  hypothesis  was  developed,  postulating  that  the  rhythm  is  a 
manifestation  of  an  interacting  dual-clock  system :  one  clock  running  at  a  speed  of  24.8  hours 
per  day  and  responsible  for  a  bimodal  migration  rhythm ;  and  a  second  solar-day  clock 
responsible  for  the  suppression  of  the  night-time  supra-surface  phase  of  the  migration  rhythm. 

This  work  was  supported  by  National  Science  Foundation  grant  GB-5045  to  JDP. 

Intracellular  distribution  of  malic  dehydrogenase  isozytnes  in  developing  red  and 
white  halves  of  sea  urchin  eggs.  GRANT  PATTON,  LAURENS  METS  AND 
CLAUDE  VILLEE. 

Whole  unfertilized  eggs  of  the  sea  urchin,  Arbacia  punctulata,  have  five  to  seven  electro- 
phoretically  distinct  forms  of  L-MDH,  which  decrease  to  three  in  the  64-cell  stage.  Centrifugal 
separation  of  these  embryos  into  large  and  small  blastomeres  revealed  two  forms  of  the 
enzyme  in  the  former  and  three  in  the  latter  fraction.  Since  inhibition  of  protein  synthesis 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         401 

with  actinomycin  and  puromycin  did  not  alter  this  change  it  was  of  interest  to  investigate 
other  cytoplasmic  influences  present  at  fertilization. 

Red  and  white  cell  halves  were  obtained  by  centrifuging  whole  unfertilized  eggs  in  a  sucrose 
gradient.  The  lighter,  white  half,  containing  nucleus,  microsomes,  mitochondria  and  a  little 
yolk,  had  the  full  complement  of  MDH  isozymes  identified  by  polyacrylamide  gel  electro- 
phoresis  of  whole  eggs.  At  the  two-cell  stage  following  normal  fertilization  this  number 
was  reduced  to  four.  The  heavier  red  halves,  which  contain  yolk,  pigment,  some  mitochondria 
and  soluble  enzymes,  contained  five  of  the  seven  isozymes  found  in  whole  eggs  and  white  halves. 
Ninety  minutes  after  fertilization  red  halves  had  lost  two  forms  of  MDH  and  showed  increased 
concentration  of  the  early  cathodal  band.  The  average  ratio  of  the  rate  of  reaction  of  MDH 
with  acetyl  pyricline  adenine  dinucleotide  (APAD)  compared  to  NAD  was  0.55  in  white  halves 
and  increased  to  0.61  in  two-cell  embryos.  In  unfertilized  red  halves  the  ratio  was  0.68, 
increasing  to  0.99  in  the  samples  90  minutes  after  fertilization. 

Soluble  and  particulate  MDH  was  prepared  by  differential  centrifugation  at  22,000  g  for 
20  minutes.  Four  soluble  isozymes  (analogue  ratio  0.55)  and  four  particulate  isozymes 
(analogue  ratio  1.51)  were  found;  only  one  of  the  particulate  forms  coincided  with  the 
soluble  isozymes.  The  seven  isozymes  of  the  egg  thus  reflect  a  synthesis  of  soluble  and 
particulate  forms. 

Arbacia  sperm  revealed  three  forms  of  L-MDH,  one  identical  with  the  darkest  anodal 
particulate  isozyme  and  two  intermediate  bands  characteristic  of  the  soluble  fraction ;  the 
analogue  ratio  of  the  total  sperm  sample  was  2.30. 

Autoradiographic  studies  of  DNA  synthesis  in  cultures  of  peripheral  blood  from 
the  smooth  dogfish,  Mustelus  cams.    THORU  PEDERSON  AND  SEYMOUR  GELFANT. 

We  have  designed  a  culture  method  for  peripheral  white  blood  cells  from  the  smooth 
dogfish.  Whole  blood  is  drawn  from  the  caudal  vein  and  allowed  to  sediment  in  the 
syringe  at  37°  C.  The  white  blood  cell-rich  supernatant  plasma  is  extruded  into  a  sterile  tube 
and  a  cell  count  is  made.  From  50  ml.  of  whole  blood  this  method  routinely  yields  around 
5  X  10s  cells.  The  cultures  are  seeded  at  2  X  10"  cells/ml.  The  culture  medium  is  elasmobranch 
Ringer's  without  Ca++  (to  prevent  agglutination  in  vitro)  and  with  0.01  gm./L.  phenol  red  as  a 
pH  indicator  (pH  adjusted  to  7.8  with  0.1  N  HC1  or  NaOH).  Just  before  use  the  medium 
is  supplemented  with  penicillin  (0.06  gm./L.),  streptomycin  (0.13  gm./L.)  and  autologous 
plasma  (20%  v/v).  Cultures  are  put  up  in  screw-cap  vials  and  are  kept  stationary  at  20°  C. 
With  this  method,  cells  remain  viable  for  at  least  25  days. 

The  basic  question  we  sought  to  answer  was  whether  or  not  these  cells  move  through 
the  cell  division  cycle  in  vitro.  No  mitoses  were  observed  in  any  of  several  experimental 
situations,  including  the  use  of  factors  which  are  mitogenic  for  human  lymphocyte  cultures. 
Nevertheless,  dogfish  white  blood  cells  do  synthesize  DNA  and  RNA  in  vitro,  as  measured  by 
autoradiography  of  cells  treated  with  TdR-H3  or  UR-H3,  respectively  (at  2  /^c./ml.).  Hema- 
cytoblasts  and  small  lymphocytes  are  most  active  in  these  respects.  However,  in  vivo  experi- 
ments using  pulse  administrations  of  TdR-H3  (at  1  /uc./gm.  body  wt.)  indicated  that  peripheral 
white  blood  cells  are  not  engaged  in  DNA  synthesis. 

These  results  suggest  that  dogfish  peripheral  white  blood  cells  are  in  the  d  period  of 
the  cell  cycle  in  vivo,  are  stimulated  to  enter  the  S  period  in  vitro  and  then  become  arrested 
in  the  G2  period. 

This  work  supported  by  research  grant  GB-2803  from  the  National  Science  Foundation. 

The  effects  of  NaCl  on  respiration  of  Squalns  acanthias  rectal  gland   in  vitro. 
JACOB  RAAB. 

Burger  (1960)  showed  that  Sqitahts  acanthias  rectal  gland  has  an  ion-regulating  function. 
The  following  results  indicate  a  NaCl-induced  humoral  control  of  rectal  gland  metabolism. 

Blood  was  drawn  from  the  caudal  vein  of  a  normal  fish  and  immediately  10  ml.  of  1,  2,  or 
4  M  NaCl  were  injected  into  the  same  fish  and  10  ml.  of  blood  were  again  drawn  from 
the  fish  after  one  hour.  The  sera  obtained  were  used  for  incubation  media.  Rectal  gland 
tissue  was  prepared  by  slicing  the  gland  freehand  with  a  razor  on  Ringer-moistened  filter 


402          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

paper  at  0°  C.  and  quickly  transferring  slices  to  Ringer's  (Na,  250  mM ;  K,  10  mM ;  Ca, 
5  mM;  Mg,  5  mM ;  Cl,  270  mM;  SO4,  5  mM;  glucose,  10  mM;  Tris,  10  mM;  malic  acid, 
4.4  mM;  urea,  400  mM)  at  0°  C.  Constant  pressure  respirometers  were  shaken  at  about  140 
oscillations  per  minute  in  a  stirred  water  bath  that  varied  from  18°  to  21°  C.  The  tissue  and 
incubating  fluid,  under  air,  were  allowed  to  equilibrate  for  one  hour ;  readings  were  taken  for 
the  next  two  hours. 

Respiration  (in  microliters  of  oxygen  consumed  per  hour  per  mg.  dry  weight  of  tissue)  in 
Ringer's  was  1.79  (18  determinations),  2.32  (21)  in  normal  serum,  and  2.72  (15)  in  serum 
from  the  salt-loaded  fish.  These  incubation  media  had  little  effect  on  tissue  levels  of  Na,  K, 
or  Cl.  Incubation  in  serum  from  salt-loaded  fish  in  the  presence  of  10~4  ouabain  led  to 
increased  tissue  Na  and  decreased  tissue  K,  and  resulted  in  an  oxygen  consumption  of  2.31  (7). 
Raising  the  NaCl  and  osmotic  pressure  of  the  control  serum  to  that  of  the  serum  from  salt- 
loaded  fish  led  to  a  respiration  level  of  2.35  (7).  Enhanced  respiration  occurred  with  serum 
from  an  injected  fish  which  had  the  same  K  level  as  control  serum.  Little  difference  was  found 
in  the  respiration  level  of  spleen  tissue  slices  incubated  in  serum  from  control  or  salt-injected  fish. 

Supported  by  USPHS  Grant  GM  10542. 

Fine  structure  of  intercellular  contacts  in  the  sponge,  Microciona  prolifera.     JEAN- 
PAUL  REVEL. 

While  much  information  on  cellular  adhesion  has  been  gained  by  the  study  of  the 
reaggregation  of  sponge  cells,  there  are  as  yet  few  data  on  the  ultrastructural  aspects  of  cell- 
to-cell  relationships  in  these  organisms.  In  the  preliminary  work  outlined  here,  we  describe 
the  intercellular  junctions  in  the  intact  sponge.  Microciona  was  most  successfully  preserved  by 
fixation  in  mixtures  of  glutaraldehyde  and  formaldehyde  followed  by  osmium  tetroxide.  The 
cell  membrane  of  the  cells  of  Microciona  presents  a  very  clear  "unit  membrane"  appearance. 
We  could  find  no  evidence  for  desmosome-like  structures,  nor  for  the  presence  of  septate  junc- 
tions. There  are,  however,  areas  where  cell  membranes  of  adjacent  cells  run  an  approximately 
parallel  course,  separated  by  a  space  rarely  wider  than  1000  A.  In  sponges  fixed  under 
conditions  producing  extreme  shrinkage,  as  judged  by  the  distortion  of  the  histological  organi- 
zation, one  finds  short  pseudopodia  extending  from  one  cell  toward  its  neighbor.  There 
seems  to  be  a  focal  area  of  contact  between  the  adjacent  membranes,  a  small  macula  occludens, 
at  the  apex  of  the  pseudopod.  In  sponges  where  the  histological  appearance  is  more  normal, 
the  blunt  cytoplasmic  projections  are  rare,  and  one  finds  instead  wide  areas  of  contact  with 
the  typical  appearance  of  a  tight  junction:  there  is  a  complete  obliteration  of  the  extracellular 
space  which  results  in  the  formation  of  pentalaminar  junctions.  One  also  finds  areas  where  a 
narrow  gap,  only  10  A  or  so  wide,  is  present  between  the  facing  outer  leaflets  of  opposing 
membranes.  Both  types  of  junctional  specializations  are  found  between  similar  and  dissimilar 
cells.  A  single  examination  of  reaggregating  sponge  cells  shows  cell  contact  specializations 
similar  to  those  observed  in  the  intact  organism.  It  would  thus  appear  that  structures  similar 
or  identical  to  the  tight  junctions  of  vertebrates  are  already  present  in  organisms  that  are  still 
at  the  "cellular  level  of  organization." 

Supported  by  grant  GM  11380  of  the  USPHS. 

Determination  of  the  quantum  efficiency  of  the  human  eye  by  a  new  method. 
GEORGE  T.  REYNOLDS. 

The  quantum  efficiency  of  the  human  eye  has  been  measured  over  a  period  of  years  with 
contradictory  results.  The  efficiency  has  usually  been  determined  by  comparing  the  performance 
of  a  human  eye  with  that  of  an  ideal  device  in  which  all  of  the  light  entering  the  eye  results 
in  information  processed  for  the  performance  of  the  prescribed  task  (Rose,  1942,  1946;  Jones, 
1957;  Barlow,  1962).  The  quantum  efficiency  of  the  eye  is  then  given  by  the  ratio: 

~  _  Quantity  of  light  required  by  ideal  device  to  perform  task 
Quantity  of  light  required  by  human  eye  to  perform  task    - 

The  value  of  Q  is  found  to  depend  upon  the  level  of  light  to  which  the  eye  is  adapted, 
and  may  also  depend  upon  the  task  to  be  performed. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         403 

The  present  determination  was  made  by  comparing  the  performance  of  the  eye  with  that 
of  an  image  intensifier  cathode  of  known  quantum  efficiency  (Reynolds,  1964).  The  task 
was  to  detect  a  pattern  in  a  background  of  noise  (Rose,  1946).  In  one  case  the  eye  was 
required  to  detect  the  pattern  unaided,  and  in  the  other  case  was  required  to  detect  the  pattern 
at  the  output  of  the  image  intensifier  system.  The  ratio  of  the  light  levels  required  in  the  two 
cases  provides  a  value  for  Q.  Measurements  were  made  on  five  observers,  with  eyes  adapted 
for  fluxes  ranging  from  0.5  X  10"  to  4  X  10"  quanta  sec.'1  deg.~2,  under  conditions  in  which  103  to 
10s  quanta  entered  the  eye  during  the  integrating  time.  Results  were  in  excellent  agreement 
with  those  obtained  by  Barlow  (1962)  under  similar  conditions,  but  by  an  entirely  different 
method,  and  gave  reproducible  values  for  Q  of  between  10"3  and  10~4. 

Supported  by  AEC  Contract  AT  (30-1) -3406. 

The  identity  and  photon  yield  of  scintillons  of  Gonyaulax  polyedra.     GEORGE  T. 
REYNOLDS,  J.  W.  HASTINGS,  HIDEMI  SATO  AND  A.  RANDOLPH  SWEENEY. 

Light  emission  in  Gonyaulax  polyedra  originates  from  particles  which  are  obtained  in  active 
form  by  gentle  rupture  of  cells  in  pH  8.2  buffer.  These  light-emitting  bodies,  termed  scintillons, 
emit  a  brief  (0.1  sec.)  flash  of  light  in  vitro,  when  the  pH  is  simply  lowered  to  5.7. 

In  the  experiments  reported  here  we  have  used  the  image  intensifier,  together  with  the 
polarizing  microscope,  to  observe  and  record  the  in  -vitro  flash  from  individual  scintillons  and 
we  have  determined  that  each  emits  about  500  photons.  This  confirms  the  conclusion  that  the 
scintillon  is  to  be  identified  with  the  birhombohedral  crystalline  structure  which  occurs  in  the 
purest  preparations,  for  previous  measurements  of  the  average  photon  yield  per  crystal  have 
also  been  about  500.  These  measurements  thereby  exclude  the  possibility  that  the  scintillon 
could  be  some  other  structure  emitting  many  times  as  much  light  per  particle,  but  occurring 
less  commonly.  The  results  also  exclude  the  possibility  that  only  a  small  percentage  of  the 
structures  in  purified  preparations  are  active  scintillons. 

In  order  to  observe  the  light  from  individual  scintillons,  they  must  be  kept  virtually 
immobile  while  the  buffer  is  rapidly  replaced  by  another  at  a  lower  pH.  This  was  achieved 
by  taking  advantage  of  the  fact  that  scintillons  strongly  adhere  to  a  Butvar  film  on  a  coverslip. 
A  scintillon  suspension  was  placed  in  a  1  mm.  space  between  slide  and  coverslip  for  4 
minutes  and  then  repeatedly  flushed  with  pH  8.2  buffer,  and  the  adhered  scintillons  were 
counted  as  strongly  birefringent  bodies.  While  appropriately  positioned,  on  the  microscope 
stage,  0.03  N  HAC  was  rapidly  flushed  under  the  coverslip  utilizing  a  pneumatically  driven 
syringe.  The  scintillons  remained  affixed  during  this  treatment.  The  flashes  were  observed 
through  the  microscope  and  image  intensifier  and  recorded  photographically. 

Supported  in  part  by  AEC  Contract  AT  (30-1) -3406. 

Behavioral  sequences  in  the  feeding  response  of  Hydra  littoralis.     NORMAN  B. 

RUSHFORTH  AND  FLORENCE  HoFMAN. 

The  capture  and  engulfment  of  a  single  Artemia  nauplius  by  H.  littoralis  consists  of  a  series 
of  complex  behavioral  sequences:  (1)  nematocyst  discharge,  (2)  tentacular  movements,  (3) 
mouth  opening,  creeping  over  the  prey  and  closure,  and  (4)  inhibition  of  column  contractions. 
We  wish  to  report  some  factors  involved  in  tentacular  movements  and  inhibition  of  column 
contractions. 

On  attachment  of  the  prey  to  a  tentacle  by  nematocysts  there  is  a  latent  period  (mean 
2.9,  s.d.,  3.0  sec).  Then  the  portion  of  the  tentacle  proximal  to  the  Artemia  contracts,  sometimes 
accompanied  by  oral  bending  or  inward  spiraling  when  the  prey  attaches  to  the  basal  or  distal 
tentacle  regions,  respectively.  The  latency  is  independent  of  the  position  of  attachment  or  the 
type  of  tentacular  movement.  This  implies  that  neither  conduction  time  nor  the  time  for  the 
diffusion  of  chemical  factors  to  hypostomal  regions  is  a  predominant  component  of  the  latent 
period. 

As  the  prey  nears  the  mouth  on  tentacle  contraction,  the  surrounding  tentacles  concertedly 
flex  orally  (a  concert).  Concerts  are  frequent  during  and  following  engulfment  of  the  prey, 
while  tentacle  and  column  contractions  are  inhibited.  They  are  highly  coordinated  movements 
unlike  tentacle  writhing  which  is  also  frequently  observed  when  hydra  feeds.  Concerts  are 


404         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

a  component  of  endogenous  behavior,  whose  frequency  is  markedly  enhanced  by  exposure  of 
hydra  to  homogenates  of  Artemia  or  reduced  glutathione  (GSH).  Concert  frequency  increases 
approximately  linearly  with  log  GSH  concentration,  over  the  range  1  X  10~10  to  5  X  10~8  M 
GSH,  above  which  tentacle  writhing  is  induced.  No  electrical  correlates  of  concerts  have 
been  observed. 

Endogenous,  compound,  electrical  pulses  (approx.  30  mV,  and  100  msec,  duration)  and 
their  associated  column  contractions  are  inhibited  when  hydra  is  exposed  to  Artemia, 
homogenates  of  Artemia  or  reduced  glutathione.  Contractions  and  associated  electrical  pulses 
recorded  from  excised  tentacles  are  also  inhibited  by  these  stimuli. 

Supported  in  part  by  grants  from  the  National  Science  Foundation  and  the  National 
Institutes  of  Health. 

Recovery  from  radiation-induced  mitotic  delay   in  sea  urchin   eggs.     RONALD  C. 

RUSTAD. 

Sea  urchin  eggs  were  cut  into  halves.  Following  fertilization  with  normal  sperm  the 
whole  cells  and  the  diploid  half-cells  divided  at  the  same  time,  while  the  haploid  half-cells 
were  delayed  from  5  to  15  minutes.  The  mitotic  delay  arising  from  -/-irradiation  of  the  sperm 
was  greatest  in  diploid  half-eggs,  less  in  diploid  whole  eggs,  and  least  in  haploid  half-eggs. 
The  mitotic  delay  in  each  type  of  cell  was  dose-dependent  over  the  range  of  10  to  50  Kr. 

Radiation-induced  mitotic  delay  may  depend  not  only  on  the  initial  damage  but  also  on 
the  amount  of  recovery  that  can  occur  before  a  critical  mitotic  stage.  Other  studies  in  our 
laboratory  have  demonstrated  that  haploid  eggs  remain  U.V.-sensitive  longer  than  diploid 
ones.  Therefore,  the  critical  radiation-sensitive  mitotic  stage  appears  to  be  delayed  in 
haploid  cells.  Such  a  delay  would  allow  more  time  for  recovery  in  haploid  cells  than  in 
diploid  ones.  If  the  rate  of  recovery  is  dependent  upon  the  cytoplasmic  volume,  diploid 
half-cells  would  be  expected  to  divide  later  than  diploid  whole  cells.  The  volume  dependence 
may  be  associated  with  the  capacity  for  protein  synthesis.  The  rate  of  prefertilization  recovery 
of  7-ir radiated  eggs  has  previously  been  shown  to  be  reduced  by  exposure  to  puromycin  (an 
inhibitor  of  protein  synthesis).  Since  unfertilized  Arbacia  eggs  do  not  normally  synthesize 
proteins,  the  puromycin  effects  suggested  that  radiation  activates  protein  synthesis.  Preliminary 
autoradiographic  evidence  indicates  that  7-irradiation  stimulates  the  incorporation  of  HMeucine 
into  the  proteins  of  unfertilized  eggs. 

These  data  are  compatible  with  the  hypothesis  that  7-ray-induced  mitotic  delay  is  inversely 
proportional  to  the  time  available  for  a  recovery  process,  the  rate  of  which  is  dependent  on 
cytoplasmic  volume  and  perhaps  specifically  on  protein  synthesis. 

These  studies  were  supported  by  the  U.  S.  Atomic  Energy  Commission  and  the  Office 
of  Naval  Research. 

An  analysis  of  living  squid  sperm  head  fine  structure  through  polarized  UV  micro- 
beam  irradiation.     HIDEMI  SATO  AND  KENNETH  J.  MULLER. 

Living,  mature  sperm  heads  of  the  squid  Loligo  pcalcii  were  irradiated  after  the  method 
of  Inoue  and  Sato  with  a  UV  microbeatn  plane-polarized  at  various  angles  to  the  main  sperm 
axis.  With  reduction  of  birefringence  upon  irradiation,  a  regionally  constant  shift  of  the 
optic  axis  toward  the  polarized  microbeam's  E-vector  was  observed.  Measured  optic  axis 
shifts  were  8°  in  the  anterior  and  posterior  2  p  and  12°  in  the  middle  3.5  fj.  of  the  sperm 
head.  The  angles  between  the  optic  axis  and  main  axis  of  the  sperm  in  the  three  regions 
are  —3°,  +2°,  and  +8°  to  +20°  progressing  posteriorly,  viewed  with  maximum  head  curvature. 
DNA  absorbs  energy  differentially  with  the  UV  E-vector  polarized  in  and  normal  to  base 
planes.  From  our  data  we  obtain  an  arrangement  of  DNA  which  deviates  base  plane  normals 
from  the  optic  axis  by  —8°  in  posterior  and  anterior  regions  and  —12°  in  the  middle. 

The  observations  are  consistent  with  a  model  with  B-form  DNA  molecules  bundled  in 
helices  of  8°  pitch  from  their  main  axes,  and  arranged  longitudinally  during  spermiogenesis. 
In  the  model,  each  helix  represents  a  chromonema  and  is  paired,  forming  a  double-helical 
chromosome  with  chromonemata  separated  by  less  than  the  helix  diameter.  With  equal 
gyre  periods,  the  chromosomes  can  mesh  when  aligned,  becoming  unresolvable.  Placing 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         405 

shorter  chromosomes  posteriorly  and  anteriorly,  we  may  envision  a  twist  of  4°  in  the 
middle  region,  explaining  regional  variation  in  optic  axis  shifts.  This  model  agrees  with 
measured  birefringence  of  —2  X  10"2,  with  Wilkins'  x-ray  diffraction  studies  indicating  vertical 
B-form  DNA  in  squid  sperm,  and  with  visual  interpretations  of  phase,  polarization,  and 
electron  micrographs  during  spermiogenesis.  Other  models,  with  grounds  for  rejection, 
shall  be  discussed. 

Aided  by  grants  from  the  National  Cancer  Institute  CA  10171  and  the  National  Science 
Foundation,  GB-5120. 

The  effect  of  !).,(.)  on  the  uiitotic  spindle.  HIDEMI  SATO,  SHINYA  INOUE,  JOSEPH 
BRYAN,  NORA  E.  BARCLAY  AND  CHRISTOPHER  PLATT. 

The  effect  of  D2O  has  been  studied  in  the  first  meiotic  metaphase  spindles  of  Pectinaria 
i  oocytes  and  in  the  dividing  eggs  of  Arbacia  punctulata  and  Lyt echinus  varicgatus. 
Polarization  microscopy  reveals  that  the  volume  of  the  spindle  increases  8  times,  the  retarda- 
tion 2  times  while  the  coefficient  of  birefringence  remains  constant  at  3  X  10~*.  The  increase  in 
volume  and  retardation  is  a  function  of  D^O  concentration  and  the  stage  of  mitosis  at  which  it 
is  applied.  Maximum  increases  occur  with  45%  D2O  and  application  during  metaphase.  These 
changes  are  rapid,  being  80%  complete  within  40  seconds.  The  effect  of  D2O  is  completely 
reversible.  It  is  not  a  secondary  effect  brought  about  by  change  of  acidity,  even  though 
pD  *=»  pH  +  0.4.  The  medium  can  be  varied  from  pH  7.2  to  8.6  with  no  detectable  change  in 
spindle  size,  shape,  or  retardation. 

The  constant  coefficient  of  birefringence  suggests  that  there  is  an  increase  in  (micro- 
tubule  (?))  protein  in  deuterated  spindles.  To  measure  this  increase,  spindles  from  both  normal 
and  40%  deuterated  Arbacia  pnnctulata  eggs  were  isolated  in  1.0  M  hexylene  glycol.  Such 
isolated  spindles  show  volume  and  retardation  increases  equivalent  to  those  observed  in  vivo. 
The  isolated  spindles  were  dissolved  in  0.6  M  KC1  and  analyzed  in  the  ultracentrifuge  using 
Schlieren  optics  and  a  UV  scanner.  The  major  22S  protein  component  of  Kane  and 
Stephens  is  observed  in  both  preparations.  The  amount  of  this  22S  protein/spindle  increases 
2.6-10  times  in  the  deuterated,  isolated  spindles.  This  increase  agrees  with  the  predicted 
values  obtained  from  polarization  microscopy. 

Purified  22S  protein  was  obtained  from  normal  and  deuterated  unfertilized  Arbacia  eggs. 
The  amount  of  22S  protein  in  both  samples  is  essentially  equal,  suggesting  a  constant  pool  of 
22S  protein  in  the  whole  egg.  Thus  the  quantity  of  22S  protein  in  the  mitotic  apparatus  is 
enhanced  by  D2O. 

Aided  by  grants  from  the  National  Cancer  Institute,  CA  10171  CB,  and  the  National  Science 
Foundation,  GB-5120. 

Mercenene:  Preliminary  analysis  of  induced  focal  changes  in  the  Krcbs-2  carcinoma 
fine  structure.  SISTER  M.  ROSARII  SCHMEER,  O.P.,  AND  REV.  T.  D.  CASSIDY, 
O.P. 

Cytological  effects  of  Mercenene  on  the  Krebs-2  tumor  in  vivo  were  surveyed.  The  dearth 
of  published  research  on  the  ultrastructure  of  this  carcinoma  presented  a  major  difficulty. 
Mercenene  was  extracted  from  the  clam  Mcrccnaria,  purified,  and  administered  to  4-5-week-ol'd 
female  CF1  mice  implanted  with  the  Krebs-2  carcinoma.  The  7-day  treatment  was  followed 
by  autopsy  and  tumor  biopsy  for  one-half  of  the  treated  animals  and  for  all  the  untreated 
mice.  The  remaining  45  treated  animals  were  maintained  for  a  6-month  longevity  study. 
Carcinoma  tissue  from  treated  and  control  mice,  and  biopsied  tumors  from  the  longevity  group, 
were  then  prepared  for  ultrastructural  investigation.  Fine  structure  analyses  utilized  four 
parallel  experiments  with  complementary  methods  of  'cytological  preparation.  Three  gave 
excellent  and  reproducible  results:  (1)  Millonig's  sodium  phosphate-buffered  OsO4;  (2)  sodium 
phosphate-buffered  5%  glutaraldehyde,  and  OsO4;  (3)  s-collidine-buffered  5%  glutaraldehyde 
and  OsO4.  Thin  sections  of  an  Epon-Araldite  mixture  were  viewed  in  the  Siemen's  Elmiskop  I. 
Cytochemical  techniques  utilizing  azure  11-methylene  blue,  azure  B,  basic  fuchsin,  and  Nile 
blue  A  were  conducted  on  alternate  1 -micron  sections.  No  changes  were  detected  in  untreated 
cells  upon  examination  of  the  nuclei,  nucleolar  apparatus,  membranes,  Golgi  zone,  mitochondria, 


406          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

endoplasmic  reticulum,  and  RNP  particles.  In  Mercenene-treated  tumor  cells  changes  were 
observed  in  polysomes,  free  ribosomes,  the  nuclear  envelope  and  nucleolar  apparatus.  Quanti- 
tative alteration  at  these  sites  by  Mercenene  suggests  possible  antagonism  of  protein  metabolism 
in  the  cancer  cells  under  these  experimental  conditions.  Induced  oncolysis,  reflected  in  fine- 
structure  changes,  could  further  elucidate  the  antitumor  activity  of  Mercenene.  Correlated 
cytochemical  investigations  have  been  initiated  to  test  this  hypothesis. 

Supported  by  American  Cancer  Society  Grant  T-361,  NSF  Fellowship  73182,  USPHS 
Grant  GN  296,  Ashland  County  Health  Foundation,  Ashland  County  Cancer  Society. 

Bottom  temperature  and  faunal  provinces:  continental  shelf  from  Hudson  Canyon 
to  Nova  Scotia.     THOMAS  J.  M.  SCHOPF  AND  JOHN  B.  COLTON,  JR. 

In  open  marine  waters,  temperature  and  substrate  are  the  biologically  important  factors 
that  change  most  radically  and  thus  have  marked  influence  on  the  distribution  of  benthic 
organisms.  Bottom  temperature  regimes  reported  here  were  determined  from  approximately 
5000  measurements  collected  on  29  cruises  of  Bureau  of  Commercial  Fisheries  ships,  mostly  in 
1955,  1956,  1963,  1964  and  1965.  From  1  to  8  cruises  cover  each  month ;  all  but  two  months 
(January  and  June)  are  represented  by  data  from  three  or  more  cruises. 

Quarterly  (March,  June,  September  and  December)  average  bottom  temperatures  are  as 
follows:  Nantucket  Shoals,  (20-80  m.  depth),  3.6°,  7.5°,  11.3°,  9.5°  C. ;  Georges  Bank  (4-100 
m.  depth),  4.2°,  8.6°,  13.0°,  8.3°  C. ;  Gulf  of  Maine  (100-377  m.  depth),  4.8°,  5.9°,  6.7°,  6.1°  C. ; 
Browns  Bank  (40-100  m.  depth),  2.5°,  4.5°,  7.3°,  5.0°  C. ;  Nova  Scotia  Shelf  (40-140  m.  depth), 
2.2°,  4.6°,  7.0°,  4.6°  C.  The  subsidiary  influence  of  continental  slope  water,  which  sometimes 
rises  onto  the  continental  shelf,  is  not  considered  here. 

Most  striking  is  the  similarity  of  bottom  temperatures  on  Georges  Bank  and  Nantucket 
Shoals,  and  the  distinctly  lower  temperatures  on  nearby  Browns  Bank.  The  Gulf  of  Maine 
is  intermediate  in  its  temperature  characteristics.  This  temperature  distribution  is  apparently 
related  to  bottom  currents  that  circulate  water  over  the  Nova  Scotia  Shelf  and  Browns  Bank, 
through  the  Gulf  of  Maine,  and  then  onto  Georges  Bank.  The  Nantucket  Shoals  appear  to 
obtain  bottom  water  from  both  Georges  Bank  and  the  Gulf  of  Maine.  These  different 
temperature  regimes  suggest  inclusion  of  Georges  Bank  and  Nantucket  Shoals  in  the  same 
faunal  province  distinct  from  the  Gulf  of  Maine,  Browns  Bank  and  the  Nova  Scotia  Shelf. 

Supported  in  part  by  a  grant  from  the  Ford  Foundation  to  the  MBL  Systematics-Ecology 
Program. 

Evidence  against  the  presence  of  functional  pigment-dispersing  nerve  fibers  in  the 
sand  flounder  Scophthahnus  aquosus.     GEORGE  T.  SCOTT  AND  K.  K.  WONG. 

We  conclude  that  only  pigment-aggregating  nerve  fibers  are  active  in  the  sand  flounder  on 
the  basis  of  the  following  observations:  (1)  Nerve  sectioning  or  blocking  by  pressure  or  cold 
produces  only  pigment  dispersion.  Electrical  stimulation  of  sectioned  nerves  results  in 
blanching.  (2)  A  negative  "Parker  Effect"  is  observed.  A  branch  of  the  trigeminal  nerve 
ennervating  the  ventral  aspect  of  the  opercular  area  was  sectioned,  causing  a  dark  patch 
which  fades  within  6  to  23  hours.  Recutting  the  nerve  distal  to  the  first  cut  did  not  produce 
secondary  darkening.  (3)  The  most  active  melanophore  pigment-aggregating  agents,  when 
injected  subcutaneously,  were  epinephrine,  norepinephrine,  isopropylnorepinephrine,  dopamine, 
melatonin  and  serotonin,  with  effective  doses  ranging  from  0.02  to  0.10  micrograms.  Three 
energizers,  phenelzine,  pheniprazine  and  etryptamine,  were  active  at  0.3,  0.4  and  2.0  micrograms, 
respectively.  Iproniazid  and  isocarboxazid  were  inactive.  The  metabolic  products  of 
epinephrine,  metanephrine  and  mandelic  acid,  were  inactive.  Dihydroxyphenylalanine  (DOPA) 
was  also  inactive.  (4)  Pretreatment  with  pyrogallol  (5  mg.  per  kg.),  an  inhibitor  of  catechol- 
o-methyl  transferase,  resulted  in  marked  potentiation  of  certain  catechol  amines.  (5)  Of  the 
large  number  of  drugs  causing  localized  melanophore  dispersion,  the  phenothiazine  tranquilizers 
were  most  active,  in  an  effective  dose  range  of  0.08  to  5.0  micrograms.  The  most  active 
members  of  this  group  were  fluphenazine,  perphenazine,  fluorophenazine  and  thiopropazate. 
Pretreatment  with  pyrogallol  (5  mg.  per  kg.)  raised  the  effective  dose  of  these  agents  by  one 
or  two  orders  of  magnitude  but  had  no  influence  on  the  other  phenothiazines  examined 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         407 

(trifluoperazine,  prochlorperazine,  acetophenazine,  chlorpromazine,  mepazine,  proketazine).  A 
similar  response  following  pyrogallol  was  observed  with  Dibenamine  and  ergotamine.  The 
more  potent  phenothiazines,  therefore,  appear  to  act  as  adrenergic  blocking  agents. 

The  extent  of  pigment  aggregation  or  dispersion  would  presumably  depend  on  the  amount 
of  endogenous  catechol  amine  transmitter  available  to  active  sites  on  the  melanophores.  This 
would  depend  on  the  balance  between  neurosecretion  by  aggregating  nerve  fiber  endings  and 
physiological  inactivation  by  COMT. 

The  research  was  supported  by  Grant  MH  03903  from  the  National  Institute  of  Mental 
Health  to  Oberlin  College. 

Swelling  of  the  tubular  system  (TTS)  in  twitch  fibers  of  Carcinus  maenas.     A. 
SELVERSTON,  P.  W.  BRANDT  AND  J.  P.  REUBEN. 

The  closer  carpopodite  muscle  contains  fibers  which  in  response  to  direct  or  indirect 
stimulation  are  almost  exclusively  phasic.  At  approximately  15  mv  depolarization  all-or-none 
action  potentials  and  80-msec  twitches  are  produced.  Electron  microscopy  revealed  an  extensive 
network  of  sarcolemmal  invaginations.  Tubules  originating  from  these  invaginations  and  from 
the  surface  penetrate  into  the  fiber  and  form  extensive  diadic  contacts  with  the  sarcoplasmic 
reticulum  at  the  A-I  junctions.  The  crab  fibers  exhibit  finite  permeability  to  Cl,  depolarizing 
transiently  with  a  time  constant  of  about  20  minutes  when  Cl  is  removed  from  the  medium.  As 
in  gradedly  responsive  crayfish  muscle  fibers,  the  TTS  of  the  crab  twitch  fibers  can  be  caused 
to  swell  and  vesiculate.  Two  procedures  induced  these  changes.  In  one,  fibers  were  loaded 
with  KC1,  then  were  replaced  in  a  normal  or  a  Cl-free  Ringer.  In  the  second,  hyperpolarizing 
current  pulses  were  passed  through  a  KCl-filled  intracellular  microelectrode  for  15  minutes 
to  one  hour.  With  both  methods  the  swelling  and  subsequent  vesiculations  were  visible  with 
the  light  microscope.  Swelling  induced  with  currents  was  greatest  under  the  electrode. 
Electron  microscopy  revealed  that  the  swelling  of  the  TTS  was  mainly  in  the  tubules,  but  it 
could  also  be  seen  in  the  invaginations.  Only  the  tubular  components  of  the  diads  were  swollen. 
Since  Cl-efflux  was  the  common  condition  to  induce  the  morphological  changes,  the  tubular 
membrane  appears  to  be  anion  permselective.  Two  methods  were  used  to  estimate  changes 
in  membrane  capacity  when  the  fibers  were  vesiculated.  Estimates  based  on  the  foot  of  the 
spike  indicated  no  change  in  capacity,  but  cable  analysis  with  square  pulses  indicated  that  the 
capacity  was  more  than  doubled  when  the  tubules  were  swollen. 

This  work  was  supported  by  a  Grass  Foundation  Fellowship  to  A.  Selverston,  by  NIH 
grant  (NB  05910-01  Al)  to  P.  W.  Brandt,  NIH  grants  (NB  03270-05  and  NB  03728-04)  to 
Dr.  H.  Grundfest.  Dr.  J.  P.  Reuben  holds  a  career  development  award  from  NIH. 

Visualisation   of  radioactivity   in   Schistosoma   mansoni   by   means   of   an   image 
intensifier.     ALFRED  W.  SENFT  AND  GEORGE  T.  REYNOLDS. 

In  an  attempt  to  localize  radioactivity  in  schistosome  worms,  specimens  were  made 
highly  active  by  incubation  in  a  maintenance  medium  to  which  had  been  added  l-proline-U-Cu. 
After  6  hours'  incubation  the  worms  were  washed  and  placed  on  a  Millipore  filter  for  drying 
and  counting.  They  were  found  to  contain  about  25,000  decays/worm/minute.  The  disc 
containing  worms  was  then  placed  on  a  microscope  slide  and  covered  with  0.002"-thick  Nuclear 
Enterprise  scintillation  plastic. 

The  slide  was  mounted  at  the  focal  point  of  a  special  Elgeet  F  1.2  double  lens  focused 
so  as  to  produce  a  1:1  image  on  the  cathode  of  the  Image  Intensifier.  The  tube  used  was  an 
English  Electric  Valve  type  P829A,  with  tri-alkali  cathode  and  a  P-ll  phosphor  anode. 

Faint  incandescent  trans-illumination  was  employed  to  focus  the  worms  through  the  entire 
system,  using  the  intensifier  at  low  gain.  After  focusing,  the  specimen  was  viewed  by  means  of 
the  scintillation  of  the  plastic  cover  slip.  For  this  purpose  the  tube  was  driven  at  28-36  kilovolts 
which  provided  an  intensification  of  from  104  to  10°. 

In  some  trials  the  worms  were  dusted  with  ZnS  phosphor.  This  material  has  about  10 
times  the  photo-emission  per  scintillation  as  compared  to  the  plastic,  but  has  the  disadvantage 
of  impairing  ordinary  microscopy. 

Our  results  demonstrate  that  Cu-induced  scintillation  can  be  photographed  by  means  of 


408          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Polaroid  P-10,000  or  P-3000  film.  The  resolution  achieved  has  been  extremely  poor,  which 
is  a  reflection  of  deficient  optics  and  a  noisy  image  intensifier.  Improvement  in  optical  efficiency 
above  the  presently  calculated  1%  level  through  development  of  a  low-power  objective  having 
a  numerical  aperture  greater  than  0.30,  would  be  most  helpful.  Concurrently,  an  image  intensi- 
fier as  quiet  or  better  than  those  now  used  in  astronomy  will  be  required  for  optimal  resolution. 

However,  we  believe  that  it  is  technically  possible  to  develop  an  instrument  which  will 
allow  the  virtual  simultaneous  microscopic  observation  of  living  or  stained  material  and 
concomitant  estimation  of  weak  radioactivity  contained  therein. 

Supported  by  NSF  Grant  GB-2673  and  AEC  Contract  AT (30-1) -3406. 

Effects  of  dopamine  on  Mercenaries  mercenaries  heart.     MORRIS  A.  SPIRTES  AND 
DAVID  JACOBOWITZ. 

The  possibility  that  dopamine  is  a  neurohumor  in  the  heart  of  M.  mcrcenaria  has  been 
explored.  Treatment  of  the  heart  and  visceral  ganglia  with  paraformaldehyde,  according  to 
the  method  of  Falck,  resulted  in  the  appearance  of  green-fluorescent,  varicose  nerve  fibers. 
Because  of  a  reported  absence  of  other  catecholamines  in  the  nervous  system  of  M.  mcrcenaria 
by  Sweeney,  this  fluorescence  is  probably  due  to  dopamine.  The  latter  was  then  tested  on 
isolated  and  in  situ  heart  preparations.  The  minimal  effective  dose  of  dopamine  was  100  /x/^g. 
which  caused  a  negative  inotropic  and  a  positive  chronotropic  response.  With  large  doses, 
a  rapidly  appearing  increase  in  tonus  was  usually  observed,  as  well  as  an  occasional  post- 
inhibitory  positive  inotropy  and  negative  chronotropy.  These  effects  could  be  almost  com- 
pletely blocked  by  a  5  /ug./ml.  dose  of  propranolol,  a  beta-adrenergic  blocker.  Dibenamine,  an 
alpha-adrenergic  blocker  only  partially  inhibited  dopamine  effects.  Methysergide  (MLD), 
which  blocks  serotonin  responses,  had  no  effect  on  dopamine  phenomena.  These  reactions  were 
all  confirmed  using  the  in-situ  prepared  heart,  except  that  the  minimal  effective  dose  was 
usually  1  /ug.  of  dopamine.  A  rapidly  appearing  tachyphylaxis  was  noted,  often  within 
minutes,  to  all  doses  of  dopamine  in  both  types  of  preparations.  Although  this  phenomenon 
is  puzzling  for  a  neurohumoral  candidate,  because  of  the  probable  presence  of  dopamine  granules 
in  the  nerve  fibers  of  the  visceral  ganglion  and  the  heart  and  the  low  levels  of  the  minimal 
effective  doses,  it  is  concluded  that  dopamine  must  be  considered  for  such  a  role,  at  least 
for  the  heart. 

In  vivo  determination  of  sodium  turnover  of  tissues  in  the  smooth  dogfish,  Musielus 
canis.     JOHN  J.  STANGEL  AND  W.  T.  W.  POTTS. 

The  purpose  of  this  preliminary  investigation  was  to  measure  K,  the  fraction  of  total 
intracellular  sodium  exchanging  with  the  blood  per  hour  for  several  tissues  of  the  smooth 
dogfish. 

Thirty  to  50  microcuries  of  Na2'Cl  diluted  to  0.5  ml.  in  isotopic  NaCl  were  injected 
intravenously  into  the  caudal  vein  of  the  smooth  dogfish.  The  blood  of  several  fish  was 
sampled  at  various  intervals  and  a  log  plot  of  counts  per  minute,  cpm,  was  made.  During  the 
first  45-50  minutes  the  decline  of  activity  could  be  represented  by  a  single  exponential  term. 
The  rate  constant  of  decline  was  0.78/hour. 

Other  dogfish  were  injected  in  a  like  manner,  and  killed  at  five  minutes  and  at  30  minutes 
after  isotope  injection.  Tissue  samples  were  excised,  weighed,  and  dissolved  in  hot  nitric  acid. 
Activities  of  these  samples  were  measured  as  cpm/gram  wet  weight  of  tissue,  and  sodium 
was  measured  photometrically  as  /j,M  Na/gram  wet  weight  of  tissue.  Specific  activities  of 
tissues  and  blood  were  recorded  and  reported  as  the  ratio  of  tissue  specific  activity  to  blood 
specific  activity  from  the  same  animal.  It  was  assumed  that  after  five  minutes  the  extracellular 
compartment  was  fully  loaded,  but  the  intracellular  compartment  was  only  slightly  loaded. 
After  30  minutes  the  intracellular  compartment  was  substantially  loaded  as  well.  From  the 
specific  activity  of  the  tissues  after  five  minutes  and  after  30  minutes  both  the  size  of  the 
extracellular  compartment  and  the  rate  constant  for  the  intracellular  compartment  were 
computed  with  the  aid  of  Moser  and  Emerson's  equation  (/.  Clin.  Invest.  1955). 

The  following  are  the  preliminary  constants  found :  kidney,  15/hour ;  rectal  gland,  2.25/hour ; 
muscle,  0.42/hour.  These  values  seem  to  reflect  the  high  rate  of  sodium  extrusion  by  the 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         409 

kidney  and  the  rectal  gland  as  compared  to  muscle,  the  greatest  part  of  the  body  mass.     The 
high  activity  of  the  kidney  probably  represents  filtered  sodium  rather  than  intracellular  sodium. 

Sulfhydryl  balance  in   mitosis:    The   effect  of   mercaptoethanol   on   spindle   bire- 
fringence.    R.  E.  STEPHENS,  SHINYA  INOUE  AND  JOHN  I.  CLARK. 

Rapkine,  Sakai  and  others  have  shown  cyclic  changes  in  protein-bound  sulfhydryl  groups 
during  mitosis ;  Kawamura  and  Dan  found  such  changes  in  the  mitotic  apparatus.  Mazia 
and  co-workers  reported  that  mercaptoethanol  and  dithiodiglycol  disrupt  the  structure  of  the 
mitotic  apparatus  prior  to  isolation.  Experiments  were  carried  out  in  order  to  test  the 
hypothesis  of  Mazia  that  a  balance  between  sulfhydryl  and  disulfide  groups  is  involved  in  the 
maintenance  of  the  mitotic  apparatus. 

Application  of  mercaptoethanol  at  concentrations  greater  than  0.05  M  caused  complete 
disappearance  of  birefringence  within  two  minutes  in  meiotic  spindles  of  Pcctinaria  goitldi 
oocytes ;  concentrations  in  the  range  of  0.01  to  0.05  M  produced  a  rapid  decrease  in  birefringence 
which  thereafter  remained  constant.  The  birefringence  was  completely  restored  within  five 
minutes  upon  perfusion  with  aerated  sea  water.  Neither  ethylene  glycol  nor  ethanol  caused 
any  decrease  in  birefringence  at  similar  solute  concentrations.  On  the  contrary,  0.1  M  ethanol 
produced  a  marked  increase. 

Application  of  0.01  M  dithiodiglycol  (oxidized  mercaptoethanol)  also  caused  a  loss  of 
birefringence  which  was  irreversible  with  sea  water  perfusion.  If  the  eggs  were  perfused 
with  mercaptoethanol  before  the  asters  completely  disappeared  in  dithiodiglycol,  spindles 
re-formed  upon  sea  water  perfusion  but  were  generally  smaller  than  those  prior  to  treatment. 

Simultaneous  application  of  dithiodiglycol  and  mercaptoethanol  were  antagonistic,  causing 
a  decrease  in  the  rate  of  birefringence  loss.  Determination  of  exact  molar  ratios  involved  in 
such  action  was  difficult,  however,  due  to  the  questionable  purity  of  commercial  dithiodiglycol 
and  to  oxidation  of  dilute  mercaptoethanol  solutions. 

Thus,  formation  of  additional  sulfhydryl  groups  (through  the  action  of  mercaptoethanol) 
or  disulfide  linkages  (through  the  action  of  dithiodiglycol)  disrupt  the  ordered  structure  of  the 
spindle.  Dithiodiglycol  action  can  be  reversed  only  by  subsequent  reduction  through  mercapto- 
ethanol. Mercaptoethanol  and  its  oxidized  form  appear  to  act  antagonistically.  These 
findings  are  consistent  with  the  theory  that  a  change  in  balance  between  intramolecular 
sulfhydryl  and  disulfide  groups  prevents  association  of  spindle  precursors. 

Supported  in  part  by  National  Cancer  Institute  Grant  CA  10171,  National  Science 
Foundation  Grant  GB-5120,  and  National  Institutes  of  Health  Grant  GM  14363. 

Further  studies  on  dicjenetic  trematodes  of  the  family  Notocotylidae.     HORACE  W. 
STUNKARD. 

Hydrobia  salsa  is  a  somewhat  rare,  brackish-water  snail,  described  by  Pilsbry  (1905)  as 
Paludina  salsa  from  Cohasset,  Massachusetts.  During  the  summers  of  1963,  1964,  1965  and 
1966,  over  5000  individuals  have  been  examined  for  infection  by  larval  trematodes,  and  five 
species  of  notocotylid  cercariae  have  been  recognized.  These  larvae  emerge  chiefly  between 
10  AM  and  2  PM.  They  are  ocellate,  swim  actively  for  a  short  time,  the  tail  in  advance,  and 
accumulate  on  the  light  side  of  the  container.  In  the  course  of  one  to  three  or  four  hours  they 
encyst,  often  on  the  operculum  or  shell  of  the  snail  from  which  they  emerged,  but  on  any 
hard  surface,  including  the  wall  of  the  container.  The  cysts  are  firmly  attached  by  the 
hardening  of  the  cystogenous  material.  Miriam  Rothschild  (1938)  described  three  groups  of 
notocotylid  cercariae,  the  Yenchingensis  Group,  the  Monostomi  Group,  and  the  Imbricata 
Group,  based  on  differences  in  the  structure  of  the  excretory  system.  Feeding  of  cysts  from 
individual  snails  to  laboratory-reared  eider  and  domestic  ducklings  has  yielded  five  species  of 
adult  worms,  belonging  to  three  different  genera.  Two  of  the  cercariae  belong  to  the 
Yenchingensis  Group,  develop  to  maturity  in  the  digestive  ceca  of  the  ducks,  and  are  identified 
as  Notocotylus  tninutus  Stunkard,  1960  and  an  as  yet  undescribed  species  of  Notocotyhts.  Two 
cercariae  belong  to  the  Monostomi  Group,  develop  in  the  lumen  of  the  intestine,  and  are 
identified  as  Parainonostonniin  alveatum  (Mehlis  in  Creplin,  1846)  and  Paramonostomion  parvitm 
Stunkard  and  Dunihue,  1931.  The  fifth  cercarial  species  belongs  to  the  Imbricata  Group, 


410         PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

develops   in  the  bursa   Fabricius,   and  may  be   identical   with    Uniscrialis   gippyensis   Burton, 
1938,  from  mallard  ducks  taken  in  England. 

Chromatophore  response  in  Loligo  pealeii.  .  GEORGE  SZABO  AND  AARON  B.  LERNER. 

The  chromatophore  complex  of  the  squid  consists  of  a  central  pigment  cell  (chromatophore 
proper)  and  radial  myoepithelial  cells.  We  investigated  the  following  problems:  (1)  Are 
muscle  fibers  present  in  the  chromatophores  which  may  be  responsible  for  contraction  of  these 
cells?  (2)  Are  the  chromatophores  themselves  innervated?  (3)  How  can  the  autonomous 
pulsation  of  the  chromatophores  of  a  decapitated  animal  be  influenced  by  adrenergic  or 
cholinergic  drugs  or  by  other  compounds?  (4)  Has  melatonin  any  effect  on  the  chromatophores 
of  cephalopods  ? 

(1)  Electron  micrographs  showed  no  evidence  of  muscle  fibers  inside  the  chromatophores. 
There  are,  however,  abundant  collagen  fibers,  arranged  in  bundles,  around  the  chromatophores 
except  where  the  smooth  muscle  cell  or  nerves  are  attached  to  the  chromatophores.  (2)  There 
are  nerve  endings  in  close  proximity  to  the  cell  membrane  of  the  chromatophore,  inserting 
themselves  in  between  the  muscle  and  the  chromatophore.  (3)  Acetylcholine,  atropine, 
tyramine,  histamine,  alpha  and  beta  MSH,  ACTH,  melatonin,  adrenaline  and  noradrenaline 
were  injected  subcutaneously  into  decapitated  squids.  The  chromatophores  contract  immediately 
after  decapitation.  Several  doses  of  0.1  ml.  solutions,  in  concentrations  ranging  from  1000 
gamma  to  0.1  gamma  per  ml.  of  sea  water,  were  injected  at  one  side  of  the  animal  and  0.1  ml.  of 
sea  water  was  injected  on  the  contralateral  side.  In  both  control  and  experimental  sites, 
stretching  of  skin  due  to  the  blister  formation  resulted  in  eventual  expansion  of  chromatophores 
which  took  1-3  minutes  to  develop.  Only  acetylcholine  caused  an  immediate  chromatophore 
expansion  at  concentrations  1000-100  gamma.  It  took  progressively  longer  (10  seconds  to 
2  minutes)  for  the  expansion  to  develop  in  lower  concentrations.  Atropine  (1000  gamma  per 
ml.)  reversed  the  effect  of  acetylcholine,  whereas  the  expansion  of  chromatophores  caused  by 
sea  water  was  not  reversed  by  atropine.  Among  the  other  substances  tested  only  beta  MSH 
and  to  a  lesser  degree  alpha  MSH  showed  some  chromatophore-expansive  action,  which, 
however,  was  inconsistent.  (4)  When  a  subcutaneous  blister  is  caused  in  a  darkened,  intact 
animal  by  injecting  sea  water  under  the  skin,  there  is  a  slow  contraction  of  chromatophores. 
Melatonin  speeds  up  this  blanching ;  within  30  seconds  contraction  occurs,  whereas  it  takes 
60-90  seconds  for  the  sea  water  controls  to  develop  any  blanching. 

Supported  by  a  Research  Grant  CA  05401-07,  USPHS,  and  Career  Development  Award 
USPHS  Ke-GM-14.987  and  CA  04679-07,  USPHS. 

Behavior  and   settling   mechanism   of   planulae   of   Hydractinia   echinata.      MAE 
TEITELBAUM. 

Experimental  results  on  the  behavior  of  the  planulae  of  Hydractinia  echinata,  a  colonial 
hydrozoan  usually  occurring  on  shells  of  Pagurus  longicarpus,  demonstrate  that  specific  eco- 
logical factors  influence  the  settling  response.  Planulae  prefer  to  settle  in  pits  and  grooves 
and  select  the  sculptured  shell  fragments  of  Nassarhis  obsolcta  in  preference  to  the  smooth  ones 
of  Polyniccs  duplicates  and  Littorina  littorea.  Colonization  often  begins  in  the  anal  canal 
of  a  shell.  Mechanical  movement  of  shells  or  vibration  of  the  dish  increases  settlement.  The 
larvae  prefer  a  clean  glass  surface  to  one  covered  with  detrital  film.  Settlement  increases  as 
the  density  of  larvae  increases ;  2-10%  settlement  is  achieved  with  100  larvae  as  opposed  to 
42-94%  with  1000  larvae.  The  larvae  are  not  attracted  to  adult  colonies,  i.e.,  they  are  not 
gregarious.  Dishes  with  newly  settled  polyps  appear  to  be  favorable  for  metamorphosis,  despite 
the  fact  that  the  individual  polyps  do  not  fuse  to  form  one  colony  but  form  distinct 
colonies.  In  a  half-light  half-dark  dish,  the  planulae  prefer  to  settle  in  the  light.  Upon  shading 
the  light  source,  the  planula  straightens  out,  enabling  it  to  hit  an  oncoming  shell  perpen- 
dicuarly.  The  planulae  have  nematocysts  all  of  the  atrichous  isorhiza  type  which  are  most 
densely  distributed  at  the  posterior  end.  Their  function  is  believed  to  be  that  of  adhesion.  A 
larva  will  attach  to  a  moving  coverslip  with  its  nematocysts  or  will  hold  on  to  a  dish  bottom 
by  its  tail  despite  suction  of  a  pipette.  The  nematocysts  do  not  fire  upon  addition  of  hermit 
crab  extract,  Acartia  extract  or  electrical  stimulus.  They  do  fire  upon  application  of  dilute 
acid  or  a  mechanical  stimulus. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         411 

It  is  proposed  that  the  larva  attaches  to  a  moving  shell  or  crab  leg  by  firing  nematocysts 
and  settles  or  may  metamorphose  after  moving  to  a  more  protected  spot. 

Supported  in  part  by  Grant  GM  535-05  from  NIH  to  the  MBL  Marine  Ecology  Course, 
summer,  1965;  by  Grant  GB-4509  from  the  NSF  to  the  MBL  Systematics-Ecology  Program; 
and  an  NSF  Fellowship  for  Teaching  Assistants,  summer  1966.  The  author  gratefully 
acknowledges  the  helpful  suggestions  of  Dr.  D.  Crisp  during  the  summer  of  1965. 

The  effects  of  reserpine  and  guanethidine  suljate  on  serotonin  levels  in  Campanu- 
laria  colonies.  TAMARA  M.  THABES,  CHARLES  R.  WYTTENBACH  AND  SANDRA 
E.  COLLINS. 

The  capacity  of  reserpine  and  guanethidine  sulfate  to  inhibit  the  tentacle  closure  response 
to  tactile  stimulation  of  Campamilaria  hydranths  (Wyttenbach,  Thabes  and  Collins,  1966) 
suggested  that  we  investigate  the  serotonin  (5HT)  content  of  this  primitive  hydroid  and  its 
alterations  under  treatment  with  these  drugs. 

Serotonin  was  extracted  from  whole  colonies  by  the  technique  of  Bogdanski  ei  al.  (1956), 
then  complexed  with  ninhydrin  according  to  the  method  of  Snyder,  Axelrod  and  Zweig  (1965). 
Serotonin  so  extracted  shows  a  single  characteristic  excitation  peak  at  390  mp  and  a  correspond- 
ing emission  peak  at  about  480  m^.  Colonies  were  found  to  contain  on  the  average  1.70  ^g. 
of  serotonin  per  gram  wet  weight. 

Bioassay,  with  the  Venus  mercenaria  heart  preparation,  shows  serotonin  to  be  present  in 
all  parts  of  the  colony,  in  the  relative  concentration  (per  unit  protein)  of  gonangia)  hy- 
dranths )}  stems. 

Colonies  maintained  in  solutions  of  reserpine  (1  or  10  X  10~6  gm./ml.)  or  guanethidine 
(2.5  or  200  X  10~8  gm./ml.)  for  periods  of  30  minutes  or  36  hours  at  20°  C.  were  assayed 
for  serotonin  per  unit  protein.  In  5  cases  of  reserpine  treatment,  serotonin  showed  little 
change  relative  to  the  controls,  averaging  a  decrease  of  10%,  with  a  range  from  —21%  to 
+8%.  Guanethidine  treatment,  among  7  cases,  produced  an  average  elevation  of  serotonin 
level  to  20%,  with  a  range  from  —27%  to  +73%.  In  two  cases,  in  which  a  30-minute  recovery 
period  in  sea  water  had  been  allowed  after  36-hour  guanethidine  treatment,  a  drop  in  5HT 
back  toward  controls  was  noted. 

Thus,  reserpine  treatment,  of  a  dose  and  duration  which  produce  measurable  physiological 
effect,  causes  little  depletion  of  serotonin.  Physiologically  active  doses  of  guanethidine  cause, 
unexpectedly,  a  variable  but  in  most  cases  positive  change  in  serotonin  level.  A  clearcut 
relationship,  therefore,  betv/een  serotonin  depletion  and  impaired  neural  function  does  not  exist. 

Supported  by  NSF  grant  number  GB-2663. 

A  study  of  the  effects  of  divalent  cations  on  squid  giant  axons.  A.  WATANABE, 
I.  TASAKI  AND  L.  LERMAN. 

After  enzymatic  removal  of  axoplasm,  and  under  intracellular  perfusion  with  25  to  100  mM 
CsF,  the  squid  giant  axon  maintained  excitability  in  media  free  of  univalent  cation  salts. 
In  external  media  containing  Ca,  Ba,  or  Sr  as  the  sole  cation  species,  the  action  potential  was 
typically  of  long  duration  (0.1  to  20  seconds)  and  between  70  to  100  mV  in  amplitude.  Action 
potential  duration  decreased  as  the  calcium  concentration  was  increased  in  a  range  between 
50  and  400  mM.  With  excitation,  membrane  resistance  fell  sharply,  reaching  a  level  of 
approximately  one-sixth  its  resting  value  at  the  peak  of  the  action  potential.  Total  elimina- 
tion of  divalent  cation  with  EDTA  from  external  medium  containing  600  mM  NaCl  resulted 
in  a  rapid  loss  of  excitability.  Subsequently  recovery  occurred  with  addition  of  divalent  cation. 

"Abolition"  of  action  potentials  by  a  brief  inward  current  pulse  was  demonstrable.  When 
the  duration  of  an  action  potential  was  long,  an  inward  directed  current  pulse  applied  during 
the  plateau  of  the  action  potential  resulted  in  a  "all-or-none,"  premature  termination  of  the 
action  potential. 

In  external  media  containing  400  mM  NaCl  and  50  mM  Ca^CU,  the  calcium  influx  during 
resting  and  excited  states  was  measured.  Average  calcium  influx  increased  by  a  factor  of 
approximately  six  during  suprathreshold  biphasic  stimulation  at  50  shocks/second.  Rapid 
subthreshold  stimulation  showed  no  observable  effect  on  Ca  influx. 


412          PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Cytological  studies  on  the  inhibition  of  early  cleavage  by  estradiol  17  /?  in  Arbacia 
punctulata.  D.  G.  WHITTINGHAM  AND  C.  R.  AUSTIN. 

Early  cleavage  stages  in  Arbacia  were  inhibited  with  estradiol  17  ft  (Agrell,  1954)  at 
concentrations  of  10~4  M  and  10~5  M  in  sea  water. 

It  has  now  been  found  that  treatment  of  unfertilized  eggs  for  5,  10,  and  15  minutes  did  not 
prevent  cleavage  and  normal  development  if  they  were  returned  to  normal  sea  water  prior 
to  semination.  However,  if  eggs  remained  in  estradiol  solution  they  failed  to  divide  at  the  first 
cleavage  division  although  sperm  entry  and  elevation  of  the  fertilization  membrane  were 
unimpeded.  Pronuclear  fusion  failed  to  take  place  (60-70%  of  eggs)  if  unfertilized  eggs 
were  placed  in  estradiol  10  and  15  minutes  prior  to  semination;  the  chromosomes  appeared  at 
metaphase  in  two  separate  haploid  plates.  Pronuclear  fusion  may  have  been  prevented  by 
failure  of  aster  formation  normally  responsible  for  bringing  the  pronuclei  together. 

Unfertilized  eggs  placed  in  estradiol  5  minutes  before  semination  and  eggs  placed  in  it 
after  semination  revealed  no  impairment  of  pronuclear  fusion  and  aster  formation,  but  spindle 
formation  was  not  well  defined  and  chromosomes  appeared  scattered  at  the  metaphase  stage. 

Prolonged  treatment  beyond  the  metaphase  stage  (30-40  minutes)  led  to  the  development 
of  tripolar,  tetrapolar  and  more  complex  spindles  (40-50%  of  the  eggs  had  tetrapolar  spindles). 
Eventually  multinucleate  cells  were  formed  which  sometimes  fragmented,  yielding  "blastomeres" 
of  unequal  size  containing  one,  several  or  no  nuclei.  Formation  of  tetrapolar  spindles  at  the 
time  of  the  second  metaphase  suggests  that  centriolar  replication  takes  place,  with  the 
production  of  a  second  spindle.  Although  the  steroid  caused  a  delay  at  metaphase,  it 
apparently  did  not  prevent  further  development  of  the  nuclear  cycle  taking  place.  Essentially 
cytokinesis  was  delayed,  fragmentation  of  the  eggs  occurring  80-90  minutes  after  semination. 

The  primary  effect  of  estradiol  thus  appears  to  be  upon  spindle  structure,  interfering  with 
division  and  causing  detachment  of  chromosomes.  Centriolar  replication  and  the  nuclear 
cycle,  including  chromosome  replication,  evidently  proceed  unimpaired. 

Facilities  made  available  through  training  grant  5  Tl  HD  26-05  from  the  National 
Institutes  of  Health. 

The  physiological  effects  of  reserpine  and  guanethidine  snljate  on  Campannlaria 
hydranths.  CHARLES  R.  WYTTENBACH,  TAMARA  M.  THABES  AND  SANDRA 
E.  COLLINS. 

In  order  to  gain  insight  into  the  nature  of  nerve  transmission  in  coelenterates,  we  have 
studied  the  effects  of  two  serotonin-  and  catecholamine-depleting  drugs  upon  the  response  of 
Campannlaria  flcxuosa  hydranths  to  both  tactile  and  chemical  stimulation. 

Using  contraction  of  the  entire  tentacle  ring  upon  mechanical  stimulation  of  just  3  to  5 
tentacles  as  a  criterion  for  the  ability  to  receive  and  produce  an  integrated  response  to  tactile 
stimulation,  guanethidine  produced  a  very  clearcut  dose-effect  curve.  Average  non-responsive- 
ness at  the  extreme  concentrations  tested,  1.56  X  10"6  and  5  X  10'*  gm./ml.,  were  8.9%  and  97.5%, 
respectively,  among  nearly  200  hydranths  tested,  relative  to  a  control  value  of  6.7%.  At  all 
eight  doses  tested,  maximal  or  near  maximal  effect  was  noted  after  just  5  minutes'  exposure. 
Recovery  is  equally  rapid :  non-responsiveness  in  the  colony  held  at  5  X  10~4  gm./ml.  for  90 
hours  dropped,  after  return  to  normal  sea  water,  to  35%,  25%  and  15%  at  5  minutes,  90 
minutes  and  10  hours,  respectively. 

Dose-response  data  for  reserpine  are  incomplete  due  to  its  limited  solubility  in  sea  water 
and  because  considerable  fluctuation  in  response  throughout  the  observation  period  made  it 
unfeasible  to  determine  a  lower  limit  of  effective  concentration.  However,  at  saturation  (about 
1  X  10~5  gm./ml.)  and  at  1  X  10~8  gm./ml.,  reserpine  produced  an  average  non-responsiveness 
over  the  5-day  observation  period  of  respectively,  45%  and  35%  among  nearly  500  hydranths 
tested,  relative  to  a  control  figure  of  12%.  In  contrast  to  guanethidine,  the  reserpine  effect  is 
not  seen  until  after  three  hours  exposure,  and  recovery  after  prolonged  treatment  is  much  slower. 

Regardless  of  dose  or  time  in  either  drug,  all  hydranths  tested  reacted  positively  to  the 
chemical  stimulus  employed,  i.e.,  they  showed  a  normal  feeding  response  on  exposure  to  a  drop 
of  supernatant  from  a  centrifuged  Artcnria  homogenate. 

The  differential  effect  of  these  drugs  in  modifying  response  to  tactile  and  chemical  stimula- 
tion suggests  that  the  neural  pathways  involved  are  pharmacologically  distinct. 

Supported  by  NSF  grant  number  GB-2663. 


PAPERS  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY         413 
Protein  synthesis  in  dogfish  cornea  epithelial  cells.     S.  ZIGMAN,  S.  LERMAN,  S. 

ROCKFORD  AND  J.  TuTTLE. 

Dogfish  (Mustchis  canis)  cornea  epithelial  cells  are  able  to  incorporate  H3-uridine  into 
RNA  and  C14-amino  acids  into  protein  when  incubated  at  20°  C.  in  elasmobranch  Ringer's 
solution  with  95%  O3:5%  CO2.  The  amount  of  incorporation  into  insoluble  (nuclear), 
ribosomal,  and  soluble  RNA  and  protein  increases  from  a  low  level  in  15  minutes  to  a  plaetau 
at  6  hours  of  incubation. 

When  fresh  cells  are  homogenized  and  the  1400  g  residue  removed,  5%  to  20%  sucrose 
density  centrifugation  yielded  an  ultraviolet  (UV)  absorption  (260  rmt)  profile  with  a  peak 
due  to  heavy  particles  in  tube  7  and  lighter  particles  in  tube  27.  Electron  microscopy  of 
solutions  taken  from  these  tubes  showed  polyribosomes  (clusters  of  ribosomes  attached  by  RNA 
strands)  present  in  tube  7  and  single  ribosomes  present  in  tube  27.  Gentle  ribonuclease 
treatment  resulted  in  a  loss  of  the  polyribosome  peak  due  to  RNA  chain  breakage. 

When  actinomycin  D  (40  /xg./ml.)  was  added  to  3-hour  incubations,  a  marked  depression  of 
polyribosomal  RNA  and  protein  synthesis  was  found.  Differential  centrifugation  of  ceH 
homogenates  indicated  a  4-  to  5-fold  inhibition  of  ribosomal  RNA  and  protein  synthesis.  RNA 
and  protein  synthesis  in  the  insoluble  and  soluble  fractions  was  inhibited  2-  to  3-fold. 

Two  hours  of  UV  irradiation  led  to  a  less  marked  depression  of  polyribosomal  RNA 
and  protein  synthesis,  as  shown  by  gradient  centrifugation.  This  depression  was  limited  to 
ribosomal  and  soluble  fractions.  Control  to  irradiated  ratios  were  approximately  2. 

The  results  show  that  the  epithelial  cells  of  the  dogfish  cornea  can  synthesize  protein  on 
polyribosomes  during  incubation  in  simple  media,  and  that  protein  and  RNA  syntheses  are 
inhibited  by  actinomycin  D  and  UV  light. 

Supported  by  Fight  For  Sight  Grant  G325  and  Student  Fellowship  SF313  of  the 
National  Council  To  Combat  Blindness. 

A  new  method  for  the  extraction  of  living  Thalassopsammon  from  intertidal  and 
sub  tidal  marine  sands.     DONALD  J.  ZINN. 

A  gentle,  efficient  and  repeatable  method,  superior  to  manual  and  mechanical  stirring 
and  shaking  in  not  damaging  or  destroying  protozoa  and  contractile  metazoa,  has  been 
developed.  It  is  sturdier,  quicker  and  more  portable  than  the  Boisseau  Tubes,  and  it  is  faster 
and  more  readily  usable  for  sands  with  heavy  lacunar  detritus  than  the  Uhlig  Sea-ice  System. 
It  extracts  70%  to  80%  of  the  total  psammon  population  from  100-cc.  sand  samples. 

The  central  instrument  used  in  this  method  extracts  Annelida,  Mollusca,  Nematoda, 
Arthropoda,  Nemertinea  and  Foraminifera  best,  and  is  less  successful  in  varying  degree  in 
removing  Protozoa,  Tardigrada,  Gastrotricha,  Ostracoda  and  Turbellaria.  The  removal  of 
organisms  that  adhere  tenaciously  to  sand  grains  can  be  facilitated  by  treating  the  sample  for 
about  10  minutes  with  6%  magnesium  chloride  added  to  filtered  sea  water. 

In  practice,  a  100-cc.  sample  of  sand  is  placed  in  a  vertical,  transparent  plastic  tube  10" 
long  with  an  inside  diameter  li"  and  plugged  with  a  No.  8  rubber  stopper  at  the  bottom. 
Depending  on  the  dryness  of  the  sand,  50  to  100  cc.  of  filtered  sea  water  are  added  at  the  top. 
Air  is  then  bubbled  through  the  sand  from  a  small  piston-type  aquarium  pump  connected  to  a 
plastic  "Y"  tube  that  leads  to  two  12"  lengths  of  rigid  plastic  tubing  (i"  inside  diameter) 
cemented  on  opposite  sides  of  the  larger  tube  with  their  lower  ends  I"  from  the  rubber  stopper. 
After  5  minutes  bubbling,  and  with  the  pump  still  engaged,  the  tube  is  released  from  the  clamp 
holding  it  vertically  to  a  ring  stand,  and  the  water  with  the  Thalassopsammon  is  poured  into  a 
container.  It  is  gently  mixed,  poured  in  aliquots  into  small  plastic  petri  dishes,  permitted  to 
settle,  and  then  placed  on  the  dissecting  microscope  stage  for  counting  and  sorting. 

Supported  by  Grant  GB-4116  from  the  National  Science  Foundation,  and  Grant  GB-4509 
from  the  National  Science  Foundation  to  the  Marine  Biological  Laboratory,  Systematics- 
Ecology  Program,  Woods  Hole,  Massachusetts. 


Vol.  131,  No.  3  December,  1966 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE   MARINE   BIOLOGICAL  LABORATORY 


THE  ROLE  OF  SODIUM  CHLORIDE  IN  SEQUENTIAL  INDUCTION 

OF  THE  PRESUMPTIVE  EPIDERMIS  OF  RANA 

PIPIENS  GASTRULAE1 

LESTER  G.  EARTH 
Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts  02543 

Previous  investigations  have  shown  that  ions  will  induce  various  cell  types 
from  the  presumptive  epidermis  of  the  Rana  pipiens  gastrula  (Barth  and  Earth, 
1959,  1962,  1963,  1964,  1966).  When  it  was  found  that  sucrose  also  would  induce 
(Barth,  1965),  the  question  arose  as  to  whether  sucrose  acted  directly  as  an  in- 
ductor or  indirectly  by  facilitating  the  penetration  of  ions.  A  study  of  the  effects 
of  sucrose  in  relation  to  various  concentrations  of  sodium  chloride  was  undertaken. 

METHODS 

The  solutions  and  procedures  used  for  operation,  treatment,  and  culture  of 
small  aggregates  of  cells  from  the  Rana  pipiens  embryo  have  been  described  in 
detail  (Barth  and  Barth,  1959,  1962,  1963,  1964,  1966).  Essentially  the  procedure 
consists  of  the  following  steps :  ( 1 )  Presumptive  epidermis  regions,  for  example, 
are  dissected  out  in  standard  solution  and  treated  briefly  with  Versene  (EDTA) 
to  loosen  the  pigment  coat  layer  from  underlying  presumptive  epidermis  cells;  (2) 
aggregates  consisting  of  approximately  100  cells  each  are  teased  out  from  the 
presumptive  epidermis  and  allowed  to  heal  for  10-15  minutes  before  transfer  to 
treatment  or  culture  solutions;  (3)  cultures  of  such  aggregates  in  small  glass 
stender  dishes  prepared  and  maintained  under  sterile  conditions  are  able  to  be 
observed  daily  in  the  living  condition. 

RESULTS 

Table  I  records  the  data  obtained  with  sucrose  substituted  for  the  sodium 
chloride  in  the  standard  solution  used  for  culture  of  the  presumptive  epidermis. 
Low  concentrations  of  sucrose  (exps.  1  and  2)  induce  radial  nerve  and  slate  gray 
epithelium,  while  higher  concentrations  (exps.  7,  8  and  9)  induce  pigment  cells 
and  nerve.  The  higher  concentrations  applied  for  different  periods  of  time  induce 
first  nerve,  then  slate  gray  epithelium  and  finally  pigment  cells  (exp.  11).  Thus, 

1  This  work  was   supported  by  a  grant  from   the   Department   of  Health,   Education,   and 
Welfare,  HD  00701-02,  to  the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts. 

415 
Copyright  ©  1966,  by  the  Marine  Biological  Laboratory 


416 


LESTER  G.  EARTH 


TABLE  I 

Sequential  induction  by  solutions  in  which  had  is  replaced  by  sucrose 

NaCl  is  omitted  from  the  medium  and  varying  amounts  of  sucrose  added  in  its  place. 

In  this  and  succeeding  tables  the  headings  are  to  be  interpreted  as  follows: 

Stage  no.:  Shumway  (1940);  cone.:  concentration  of  substances  in  milligrams  per  milliliter  of 

solution;  hrs. :  time  in  hours  during  which  aggregates  are  exposed  to  the  substances  indicated; 

types  of  cellular  differentiation:  as  in  Earth  and  Earth  (1962,  1963,  1964);  UPS  =  unipolar 

spongioblasts. 


Exp. 
no. 

Stage 

no. 

Treatment 

No.  of 
aggregates 

Types  of  cellular  differentiation 

Cone. 

Hrs. 

1 

11 
11 

29.0 
29.0 

6.0 
8.5 

25 
42 

Radial  nerve 
Radial  nerve 

2 

11 
11 

43.0 
43.0 

3.3 
6.3 

75 
75 

Radial  nerve,  epithelium 
Nerve,  slate  gray  epithelium,  epithelium 

3 

11- 
11- 

43.0 
43.0 

4.0 
5.5 

40 

35 

Nerve 
Nerve,  pigment  cells 

4 

11  + 

58.0 

2.5 

35 

Nerve,  rare  pigment  cells 

5 

11 

58.0 

4.5 

75 

Nerve,  pigment  cells 

6 

11 

58.0 

5.0 

75 

Nerve,  pigment  cells 

7 

11- 

58.0 

5.0 

70 

Nerve,  pigment  cells 

8 

11- 

58.0 

6.0 

75 

Nerve,  pigment  cells 

9 

11 

58.0 

9.5 

50 

Pigment  cells,  slate  gray  epithelium,  nerve 

10 

11 
11 

58.0 
58.0 

5.0 
10.5 

75 
75 

Nerve,  slate  gray  epithelium,  UPS 
Pigment  cells,  nerve 

11 

11 
11 
11 

58.0 
58.0 
58.0 

3.0 
6.0 

8.5 

25 
50 

75 

Nerve,  UPS 

Nerve,  slate  gray  epithelium,  UPS 
Pigment  cells,  nerve 

12 

11 

65.0 

4.5 

75 

Nerve,  pigment  cells 

13 

11- 

65.0 

6.0 

75 

Nerve,  pigment  cells 

14 

11- 

87.0 

4.0 

60 

Nerve 

15 

11- 

11- 

116.0 
116.0 

3.0 
4.0 

28 
32 

Nerve 
Dead 

sucrose  appears  to  induce  sequentially  a  variety  of  cell  types  as  do  various  ions 
(Earth,  1965). 

Since  the  above  experiments  were  done  with  a  medium  containing  the  normal 
concentrations  of  K+,  Ca++,  Mg++,  HCO3~  and  phosphate  ions,  any  of  these  ions 


SODIUM  CHLORIDE  AND  INDUCTION 


417 


might  have  acted  as  the  inductor.     Therefore  sucrose  was  applied  to  the  cells  in 
the  complete  absence  of  ions. 

Table  II  records  the  results  of  experiments  in  which  various  concentrations  of 
sucrose  dissolved  in  glass-distilled  water  were  applied  to  presumptive  epidermis  for 
varying  lengths  of  time.  Experiment  1  shows  that  a  low  concentration  of  sucrose 
induces  nerve  and  slate  gray  epithelium.  Higher  concentrations  (exps.  3,  4  and  7) 
induce  pigment  cells  and  nerve.  Experiments  2  and  6  show  sequential  induction 
of  radial  nerve,  spreading  nerve  and  unipolar  spongioblasts,  while  in  experiment  3 
with  longer  times  of  exposure  pigment  cells  are  induced.  Thus,  sucrose  in  the 
complete  absence  of  ions  will  induce  the  various  cell  types  which  are  also  induced 
by  ions. 

TABLE  II 
Sequential  induction  by  sucrose  in  glass-distilled  water 


Treatment 

Exp. 

Stage 

No.  of 
aggre- 

Types of  cellular  differentiation 

Cone. 

Hrs. 

gates 

1 

11- 

43.0 

5.0 

28 

Nerve,  slate  gray  epithelium 

11 

58.0 

0.5 

20 

Ciliated  epithelium 

? 

11 

58.0 

0.75 

20 

Radial  nerve,  ciliated  epithelium 

11 

58.0 

1.0 

20 

Spreading  nerve 

11 

58.0 

1.5 

20 

Spreading  nerve,  UPS 

11 

58.0 

1.0 

25 

Nerve,  pigment  cells 

3 

11 

58.0 

2.0 

25 

Pigment  cells,  nerve 

11 

58.0 

2.5 

25 

Pigment  cells,  nerve,  dead  cells 

4 

11  + 

58.0 

2.5 

28 

Pigment  cells,  nerve 

5 

11- 

58.0 

0.75 

40 

Spreading  nerve,  UPS 

11- 

58.0 

0.1 

35 

Ciliated  masses 

11- 

58.0 

0.5 

40 

Nerve,  ciliated  epithelium 

6 

11- 

58.0 

1.25 

35 

Nerve,  ciliated  epithelium 

11- 

58.0 

1.75 

40 

Nerve,  UPS 

7 

12- 

58.0 

0.4 

40 

Spreading  nerve,  radial  nerve 

12- 

58.0 

1.5 

36 

Nerve,  slate  gray  epithelium,  pigment  cells 

8 

11 

68.0 

1.0 

40 

Spreading   nerve,    slate   gray   epithelium,    pig- 

ment cells 

11 

68.0 

1.7 

35 

Spreading  nerve,  pigment  cells 

q 

11 

90.0 

1.0 

Spreading  nerve,  slate  gray  epithelium 

11 

90.0 

1.7 

Spreading  nerve 

11 

137.0 

1.0 

Spreading  nerve,  radial  nerve 

10 

11 

137.0 

1.5 

Spreading  nerve,  UPS 

11 

137.0 

2.0 

Spreading  nerve,  UPS 

11 

137.0 

3.0 

Dead  cells 

418 


LESTER  G.  EARTH 


TABLE  III 

Induction  by  sugars  other  than  sucrose,  dissolved  in  glass-distilled  water 
gl  =  glucose;  lac  =  lactose;  xyl  =  xylose. 


Treatment 

Exp. 
no. 

Stage 
no. 

No.  of 
aggregates 

Types  of  cellular  differentiation 

Cone. 

Hrs. 

1 

11 

lac  58 

4.5 

35 

Pigment  cells 

11 

lac  58 

5.1 

40 

Pigment  cells 

9 

11- 

lac  58 

4.0 

28 

Pigment  cells 

11- 

lac  58 

4.5 

32 

Pigment  cells 

7 

11- 

g!31 

2.0 

75 

Spreading  nerve,  UPS 

11- 

g!31 

3.0 

75 

Spreading  nerve,  UPS,  slate  gray  epithelium 

A 

11- 

g!31 

4.0 

32 

Pigment  cells 

11- 

g!31 

4.5 

20 

Pigment  cells 

S 

11 

g!31 

4.5 

40 

Pigment  cells 

11 

g!31 

5.1 

25 

Pigment  cells 

11 

xyl  20 

0.5 

Nerve,  ciliated  masses 

6 

11 

xyl  20 

1.0 

Spreading  nerve 

11 

xyl  20 

2.0 

Pigment  cells,  slate  gray  epithelium 

The  next  question  asked  was :  Is  sucrose  peculiar  in  some  respect  as  regards 
induction,  or  will  other  sugars  induce?  Table  III  records  the  results  of  experi- 
ments with  other  sugars  when  these  compounds  are  dissolved  in  glass-distilled 
water.  Experiments  1  and  2  show  that  lactose  will  induce  pigment  cells,  while 
experiments  3,  4  and  5  demonstrate  the  sequential  action  of  glucose  when  applied 
for  different  intervals  of  time.  Experiment  6  shows  sequential  induction  by  xylose 
with  respect  to  time.  Thus  sucrose  is  not  unique  in  its  ability  to  induce  but  shares 
this  quality  with  lactose,  glucose  and  xylose. 

Speculations  on  the  nature  of  the  action  of  sugars  as  inductors  led  to  a  number 
of  possible  actions.  First  of  all  the  sugars  could  be  acting  directly  as  inductors 
by  some  unknown  mechanism  or  they  could  act  indirectly  by  altering  the  cells  in 
some  manner  so  that  subsequently  the  ions  of  the  standard  solution  would  induce. 
The  possibility  that  the  ions  of  the  standard  solution  might  induce  is  indicated  by 
the  fact  that  in  some  species  of  Amphibia,  namely  Ambystoma  maculatum  and 
Ambystoma  opacuin,  presumptive  epidermis  will  differentiate  into  brain  without 
the  presence  of  extraneous  inductors  (Barth,  1941  ;  Holtfreter,  1944).  Therefore 
the  standard  salt  solution  must  have  been  the  inductor.  We  also  had  some  evi- 
dence that  some  substances  would  induce  if  applied  for  a  short  time,  while  long 
periods  of  treatment  resulted  in  ciliated  epidermis.  Such  a  finding  would  be  con- 
sistent with  the  hypothesis  that  induction  occurred  in  the  standard  salt  solution  and 
not  in  the  substance  tested.  This  follows  from  the  known  fact  that  abilitv  of  cells 

j 

to  be  induced  by  an  inductor  disappears  with  time. 

Experiments  therefore  were  set  up  to  test  the  hypothesis  that  sugars  did  not 


SODIUM  CHLORIDE  AND  INDUCTION 


419 


actually  induce  but  that  the  induction  occurred  when  the  cells  were  transferred  to 
the  standard  salt  solution. 

Two  types  of  experiments  were  designed  to  test  the  idea.  In  one,  the  cells 
were  to  be  kept  in  sucrose  until  the  period  of  competence  was  concluded  and  then 
returned  to  the  standard  solution.  Under  these  conditions  no  induction  was  to 
be  expected.  The  other  type  of  experiment  consisted  in  reducing  the  ionic 
strength  of  the  standard  solution  in  an  attempt  to  reduce  its  inductive  capacity. 
The  first  type  of  experiment  proved  to  be  too  difficult  to  carry  out,  as  the  cells 
would  not  survive  sucrose  treatment  in  absence  of  ions  when  exposed  during  the 
entire  period  of  competence.  The  second  type  of  experiment  gave  definite  results. 

Table  IV  records  the  results  of  experiments  in  which  the  ionic  strength  of 
the  standard  salt  solution  was  reduced  by  varying  the  concentration  of  sodium 
chloride.  The  standard  solution  contains  5.15  mg.  NaCl/ml.  Experiment  1  shows 
that  a  concentration  of  1.28  mg./ml.  is  too  low  for  continuous  treatment  of  the 
cells,  although  ciliated  masses  will  develop  after  7.5  hours  tratment.  Experi- 
ments 2  and  3  show  that  2.0  mg./ml.  is  the  minimum  concentration  of  sodium 
chloride  which  may  be  used  for  continuous  culture  of  the  cells.  Experiments  4-8 
record  the  results  with  2.55,  2.57  and  3.0  mg./ml.  of  sodium  chloride. 

The  first  results  of  the  effect  of  a  reduction  in  ionic  strength  upon  induction 
are  shown  in  Table  V,  experiments  1,  2  and  3.  There  was  no  induction  by  sucrose 

TABLE  IV 

The  effect  of  various  concentrations  of  sodium  chloride 
Normal  concentration  is  5.15  mg./ml.  Other  ions  are  present  in  normal  concentration. 


Exp. 
no. 

Stage 
no. 

Treatment 

No.  of 
aggre- 
gates 

Types  of  cellular  differentiation 

Cone. 

Hrs. 

1 

11 
11 
11 

1.28 
1.28 
1.28 

1.5 

7.5 
cont. 

25 
25 
25 

Radial  nerve,  spreading  nerve,  little  ciliated  epithelium 
Ciliated  masses,  many  unattached  single  cells 
Dead  cells 

2 

11 
11 

2.00 
2.00 

7.5 
cont. 

30 
20 

Masses  with  voluminous  mucus,  some  cilia 
Ciliated  masses  with  voluminous  mucus 

3 

11- 
11- 

2.00 
2.00 

6.0 
cont. 

25 
20 

Ciliated  masses  with  some  mucus 
Ciliated  masses  with  voluminous  mucus 

4 

11 
11 
11 

2.55 
2.55 
2.55 

3.0 
6.0 
8.5 

25 
25 
25 

Ciliated  epithelium 
Ciliated  epithelium 
Ciliated  epithelium 

5 

11 
11 

2.57 
2.57 

6.0 

cont. 

25 

25 

Ciliated  masses  with  some  mucus 
Ciliated  masses,  ciliated  epithelium 

6 

11 

2.57 

cont. 

25 

Ciliated  masses 

7 

11 

2.57 

cont. 

25 

Ciliated  masses  with  a  little  mucus,  ciliated  epithelium 

8 

11- 

3.00 

cont. 

25 

Ciliated  epithelium 

420 


LESTER  G.  EARTH 


TABLE  V 

Lack  of  induction  by  sucrose  when  followed  by  culture  in  low  concentrations 

of  sodium  chloride 

Sucrose  dissolved  in  glass-distilled  water.  Standard  salt  solution  contains  5.15  mg.  NaCl/ml. 
Other  ions  are  present  in  same  concentrations  as  in  our  standard  solution. 


Sucrose 

Exp. 

Stage 

treatment 

No.  of 
aggre- 

NaCl 
cone,  in 

Types  of  cellular  differentiation 

gates 

culture 

Cone. 

Hrs. 

1 

11- 

58.0 

1.25 

35 

5.15 

Spreading  nerve,  UPS 

11- 

58.0 

1.25 

40 

2.00 

Ciliated  masses,  mucus 

2 

11- 

58.0 

2.0 

35 

5.15 

Spreading  nerve,  UPS 

11- 

58.0 

2.0 

40 

2.0 

Ciliated  masses 

11- 

58.0 

1.0 

38 

2.0 

Ciliated  masses 

11- 

58.0 

1.5 

40 

2.0 

Ciliated  masses 

11- 

58.0 

2.0 

35 

2.0 

Ciliated  masses 

11- 

58.0 

2.3 

35 

2.0 

Ciliated  masses 

11 

58.0 

2.0 

25 

2.57 

Ciliated  masses 

4 

11 

58.0 

2.5 

25 

2.57 

Ciliated  masses,  mucus 

11 

58.0 

3.0 

25 

2.57 

Ciliated  masses,  mucus 

5 

11 

58.0 

2.0 

40 

2.57 

Ciliated  masses 

6 

11 

58.0 

2.0 

40 

2.57 

Ciliated  masses,  little  mucus,  ciliated  epithelium 

7 

11- 

58.0 

2.0 

40 

2.57 

Ciliated  masses 

8 

11 

58.0 

2.0 

35 

2.57 

Ciliated  masses 

9 

11 

58.0 

2.3 

25 

2.57 

Ciliated  masses,  mucus,  ciliated  epithelium 

10 

11 

58.0 

2.2 

40 

2.57 

Ciliated  masses,  ciliated  epithelium 

11 

11 

58.0 

2.8 

25 

2.57 

Ciliated  masses,  mucus,  ciliated  epithelium 

12 

11 

58.0 

2.3 

25 

2.57 

Ciliated  masses,  mucus,  ciliated  epithelium 

13 

11 

58.0 

1.8 

40 

3.00 

Spreading  nerve,  radial  nerve 

11- 

59.0 

2.3 

40 

2.57 

Ciliated  epithelium 

11- 

59.0 

2.3 

40 

3.50 

Spreading  nerve 

11- 

59.0 

2.8 

40 

2.57 

Ciliated  epithelium,  rare  nerve 

11- 

59.0 

2.8 

40 

3.50 

Spreading  nerve,  UPS 

15 

11- 

58.0 

4.0 

25 

3.75 

Spreading  nerve 

16 

11 

58.0 

2.7 

35 

4.00 

Spreading  nerve,  pigment  cells 

when  the  cells  were  returned  to  2.0  mg./ml.,  but  good  induction  when  they  were 
returned  to  5.15  mg./ml.  Experiments  4  through  12  resulted  in  no  induction  with 
sucrose  when  the  cells  were  returned  to  sodium  chloride  at  a  concentration  of  2.57 


SODIUM  CHLORIDE  AND  INDUCTION 


421 


mg. /ml.  If,  after  sucrose  treatment,  the  cells  were  returned  to  a  solution  containing 
3.0  mg./ml.,  induction  of  radial  nerve  and  spreading  nerve  took  place.  Experiment 
14  shows  that  induction  occurs  in  a  solution  containing  3.50  mg.  of  sodium  chloride 
per  milliliter  but  not  at  a  concentration  of  2.57  mg./ml.  At  concentrations  of  3.75 
and  4.0  mg./ml.  induction  also  occurs  (exps.  15  and  16). 

It  is  clear  that  induction  with  sucrose  is  dependent  upon  the  concentration  of 
sodium  chloride  in  the  solution  into  which  the  cells  are  subsequently  transferred. 
Table  VI  records  the  data  from  experiments  in  which  after  treatment  with  sucrose 
the  cells  were  transferred  to  different  concentrations  of  sodium  chloride.  The 
extent  of  induction  is  proportional  to  the  concentration  of  sodium  chloride. 

Either  the  cells  are  not  induced  by  sucrose  or  the  cells  are  induced  by  sucrose 
but  cannot  differentiate  in  low  concentrations  of  sodium  chloride.  Table  VII 
demonstrates  that  induced  cells  do  differentiate  in  low  concentrations  of  sodium 
chloride.  In  these  experiments  the  cells  are  first  treated  with  sucrose,  then  trans- 
ferred to  high  concentrations  of  sodium  chloride  for  varying  periods  of  time,  and 
lastly  transferred  to  low  sodium  chloride  for  culture.  Experiment  1  shows  that 
while  a  two-hour  post-treatment  with  high  sodium  chloride  (4.25  mg./ml.)  results 
mostly  in  ciliated  cells  with  a  little  radial  nerve  present,  a  five-hour  post-treatment 

TABLE  VI 

After  treatment  with  sucrose,  induction  is  proportional  to  the  concentration  of 

NaCl  in  the  culture  medium 

Sucrose  dissolved  in  glass-distilled  water.  Concentration  of  NaCl  is  varied,  but 
other  ions  are  as  in  our  standard  solution. 


Sucrose 

Exp. 

Stage 

treatment 

No.  of 
aggre- 

NaCl 
cone,  in 

Types  of  cellular  differentiation 

no. 

ITO. 

gates 

culture 

Cone. 

Hrs. 

11- 

58.0 

2.0            35 

2.57 

Ciliated  masses 

11- 

58.0 

2.0            40 

3.00 

Ciliated  masses,  radial  nerve,  spreading 

i 

nerve 

11- 

58.0 

2.0 

35 

3.50 

Spreading  nerve,  radial  nerve 

11- 

58.0 

2.0 

40 

4.00 

Spreading  nerve,  UPS 

11- 

58.0 

2.1 

35 

2.57 

Ciliated  masses,  mucus 

2 

11- 

58.0 

2.1 

40 

3.00 

Ciliated  masses,  spreading  nerve 

11- 

58.0 

2.1 

40 

4.00 

Spreading  nerve,  UPS,  slate  gray  epi- 

thelium, pigment  cells 

11- 

58.0 

2.5 

30 

2.00 

All  ciliated  masses 

11- 

58.0 

2.5 

30 

2.57 

Ciliated  masses,  rare  nerve 

3 

11- 

58.0 

2.5 

30 

3.00 

Ciliated  masses,  ciliated  epithelium,  nerve 

11- 

58.0 

2.5 

30 

3.75 

Nerve,  no  ciliated  cells 

11- 

58.0 

2.5 

30 

4.50 

Spreading  nerve,  UPS 

11 

58.0 

2.1 

25 

2.25 

Ciliated  masses,  mucus 

11 

58.0 

2.1 

25 

2.57 

Ciliated  masses 

4 

11 

58.0 

2.1 

25 

3.00 

Nerve,  cilia  rare 

11 

58.0 

2.1 

25 

3.50 

Spreading  nerve,  UPS 

11 

58.0 

2.1 

25 

3.50 

UPS,  spreading  nerve 

422 


LESTER  G.  EARTH 


TABLE  VII 

After  pre-treatment  with  sucrose,  induction  is  proportional  to  the  time  of  exposure 

to  high  concentrations  of  NaCl 

Aggregates  are  pre-treated  with  sucrose  in  glass-distilled  water,  then  transferred  to  a  "high" 
concentration  of  NaCl  for  varying  lengths  of  time  (post-treatment),  and  finally  transferred  to  a 
culture  medium  of  "low"  NaCl  content.  "High"  concentration  is  from  4.25  to  5.15  mg.  of  NaCl 
per  ml.  of  solution  containing  the  other  ions  in  normal  concentrations. 


Sucrose 

NaCl 

Exp. 

Stage 

pre-treatment 

No.  of 
aggre- 

post-treatment 

NaCl 
cone. 

Types  of  cellular  differentiation 

gates 

Cone. 

Hrs. 

Cone. 

Hrs. 

11- 

58.0 

2.0 

35 

4.25 

2.0 

2.57 

Ciliated  masses,  ciliated  epithelium, 

radial  nerve 

i 

11- 

58.0 

2.0 

40 

4.25 

5.0 

2.57 

Spreading  nerve,  no  cilia 

11- 

58.0 

2.0 

40 

4.25 

7.0 

2.57 

Spreading  nerve,  no  cilia 

11- 

58.0 

2.0 

35 

4.25 

19.0 

2.57 

Spreading  nerve,  no  cilia 

11 

58.0 

2.3 

25 





2.25 

Ciliated  masses 

11 

58.0 

2.3 

25 

4.25 

1.0 

2.25 

Ciliated  masses 

2 

11 

58.0 

2.3 

25 

4.25 

2.0 

2.25 

Nerve,  ciliated  masses 

11 

58.0 

2.3 

25 

4.25 

5.0 

2.25 

Nerve,  no  cilia 

11 

58.0 

2.3 

25 

4.25 

19.0 

2.25 

Nerve,  no  cilia 

11- 

54.0 

2.0 

35 

5.15 

0.2 

2.57 

Ciliated  masses,  rare  nerve 

? 

11- 

54.0 

2.0 

40 

5.15 

2.0 

2.57 

Spreading  nerve,  no  cilia 

11  - 

54.0 

2.0 

40 

5.15 

4.5 

2.57 

Extensive  spreading  nerve,  no  cilia 

11- 

54.0 

2.0 

40 

5.15 

19.5 

2.57 

Extensive  spreading  nerve,  no  cilia 

4 

11- 

58.0 

2.0 

40 

5.15 

19.0 

2.57 

Nerve,  no  cilia 

5 

11- 

58.0 

2.0 

50 

5.15 

19.0 

2.57 

Nerve,  no  cilia 

induces  spreading  nerve.  In  experiment  3  where  post-treatment  consisted  of  5.15 
mg.  of  sodium  chloride  per  milliliter  of  solution,  induction  occurred  in  two  hours. 
The  extent  of  induction  is  proportional  to  the  time  of  post-treatment  with  high 
concentrations  of  sodium  chloride. 

It  is  clear  that  sucrose  will  not  induce  unless  followed  by  a  treatment  with  a 
solution  containing  from  3.0  to  5.15  mg.  of  sodium  chloride.  Do  sucrose  and  so- 
dium chloride  have  similar  effects  so  that  they  are  synergetic  or  does  sucrose  merely 
prepare  the  cells  for  induction  by  sodium  chloride?  Table  VIII  records  the  results 
of  experiments  designed  to  answer  this  question.  Experiment  1  shows  that  29.0 
mg.  of  sucrose  will  induce  nerve  while  2.57  mg.  of  sodium  chloride  has  no  inductive 
properties.  The  two  compounds  in  combination  have  no  inductive  properties. 
Sodium  chloride,  therefore,  applied  simultaneously  with  sucrose  antagonizes  the 
inductive  action  of  sucrose  and  the  cells  differentiate  into  ciliated  masses  instead 
of  nerve. 

Experiment  2  shows  that  29.0  mg.  of  sucrose  applied  for  8  hours  will  induce 
as  far  as  pigment  cells,  but  when  combined  with  sodium  chloride  the  sucrose  has 
no  inductive  ability.  Experiments  3,  4,  5  and  6  confirm  and  extend  the  results 
of  exps.  1  and  2. 


SODIUM  CHLORIDE  AND  INDUCTION 


423 


Therefore  sucrose  cannot  induce  by  itself  unless  followed  by  high  sodium  chlo- 
ride, nor  can  sucrose  in  combination  with  sodium  chloride  induce  regardless  of 
subsequent  treatment.  It  may  be  concluded,  therefore,  that  the  action  of  sucrose 
in  the  absence  of  sodium  chloride  is  to  alter  the  cell  surfaces  so  as  to  permit  sodium 
chloride  and  other  ions  to  penetrate. 

Actually  the  alteration  of  the  cell  surfaces  is  probably  due  to  lack  of  sodium 
chloride  in  the  solution  and  not  to  the  presence  of  sucrose.  Sucrose  probably 
merely  maintains  the  osmotic  pressure  necessary  for  survival  of  cells  while  lack 
of  sodium  chloride  produces  the  alteration  in  the  cell  surfaces.  The  fact  that 

TABLE  VIII 

The  antagonism  of  sucrose  and  NaCl  when  applied  together 

In  these  experiments  the  concentrations  of  all  ions  except  Na+  and  Cl~  are  kept  constant. 
Various  concentrations  of  NaCl  (Na)  and/or  sucrose  (S)  are  added  to  a  standard  solution  lacking 
NaCl.  Culture  of  the  aggregates  after  treatment  is  also  in  the  presence  of  the  normal  concentrations 
of  all  ions  except  Na+  and  Cl~. 


Exp. 
no. 

Stage 
no. 

Treatment 
concentrations 

Hrs. 

No.  of 
aggre- 
gates 

Culture 

Types  of  cellular  differentiation 

11 

29  S 

6.0 

25 

5.15  Na 

Nerve 

11 

29  S 

6.0 

25 

2.57  Na 

Nerve,  ciliated  masses 

1 

11 

2.57  Na 

6.0 

25 

2.57  Na 

Ciliated  masses,  some  mucus 

11 

2.57  Na 

6.0 

25 

5.15  Na 

Ciliated  masses,  ciliated  epithelium 

11 

29S  +  2.57Na 

6.0 

25 

2.57  Na 

Ciliated  masses,  some  mucus 

11 

29S  +  2.57Na 

6.0 

25 

5.15Na 

Ciliated  epithelium 

11- 

29  S 

3.0 

20 

5.15  Na 

Nerve 

11- 

29  S 

3.0 

20 

2.57  Na 

Nerve 

11- 

29S  +  2.57Na 

3.0 

20 

5.15  Na 

Ciliated  epithelium 

? 

11- 

29  S  +  2.57  Xa 

3.0 

20 

2.57  Na 

Ciliated  masses 

11- 

29  S 

8.0 

20 

5.15Na 

Nerve 

11- 

29  S 

8.0 

20 

2.57  Na 

Pigment  cells,  nerve 

11- 

29  S  +  2.57Xa 

8.0 

20 

5.15  Na 

Ciliated  epithelium,  radial  nerve 

11- 

29S  +  2.57Na 

8.0 

20 

2.57  Na 

Ciliated  masses 

3 

11 

29  S  +  2.57Na 

5.3 

25 

5.15Na 

Ciliated  masses 

11 

29  S  +  2.57Na 

5.3 

25 

29S  +  2.57Na 

Ciliated  masses 

11 

29  S 

6.0 

25 

5.15Na 

Radial  nerve,  spreading  nerve,  UPS 

11 

29  S 

8.0 

40 

5.15  Na 

Radial  nerve,  spreading  nerve,  UPS 

4 

11 

29  S 

3.0 

25 

5.15  Na 

Radial  nerve 

11 

2.55  Na 

3.0 

25 

5.15Na 

Ciliated  epithelium 

11 

2.55Na 

6.0 

25 

5.15  Na 

Ciliated  epithelium 

11 

2.55  Na 

8.0 

25 

5.15  Na 

Ciliated  epithelium 

11 

29S  +  2.55Na 

9.5 

50 

5.15  Na 

Ciliated  epithelium 

5 

11 

5.15Na 

9.5 

50 

5.15Na 

Ciliated  epithelium 

11 

58  S 

9.5 

50 

5.15  Na 

Pigment  cells,  nerve,  slate  gray 

epithelium 

11 

34S  +  5.15Na 

2.0 

35 

5.15  Na 

Ciliated  masses 

6 

11 

34S  +  5.15Na 

5.5 

40 

5.15Na 

Ciliated  masses 

11 

34S  +  5.15Na  18.0 

75 

5.15  Na 

Ciliated  masses 

424 


LESTER  G.  EARTH 


TABLE  IX 

Correlation  between  pre-treatment  with  solutions  lacking  sodium  chloride  and 
induction  by  standard  salt  solution 

Sodium  chloride  is  omitted  from  the  standard  salt  solution  and  other  substances  are  added 
with  or  without  sodium  chloride.  After  a  period  of  treatment  the  aggregates  are  transferred  to 
standard  solution  containing  5.15  mg.  NaCl/ml.  E.G.  =  ethylene  glycol;  gl  =  glycine;  sue  = 
sucrose. 


Exp. 
no. 

Stage 

Treatment 
concentrations 

hrs. 

No.  of 
aggre- 
gates 

Types  of  cellular  differentiation 

11 

13gl 

0.5 

30 

Spreading  nerve,  ciliated  masses 

1 

11 

13  gl 

1.0 

30 

Spreading  nerve 

1 

11 

13gl 

1.7 

30 

Dead  cells,  spreading  nerve 

11 

13  gl 

3.0 

10 

Dead  cells 

11 

31E.G.  +  2.0NaCl 

1.0 

25 

Radial  nerve,  spreading  nerve,  epithelium 

11 

31E.G.  +  2.0NaCl 

2.0 

25 

Epithelium,  ciliated  masses,  mucus 

11 

31  E.G.  +2.0  NaCl 

4.0 

25 

Ciliated  masses,  voluminous  mucus 

11 

31  E.G.+S.lSNaCl 

0.3 

25 

Ciliated  masses,  mucus,  epithelium,  nerve 

11 

31E.G.+5.15NaCI 

5.0 

25 

Ciliated  masses,  mucus 

2 

11 

31  E.G. 

1.0 

25 

Dead  cells 

11 

31  E.G. 

2.0 

25 

Dead  cells 

11 

6  E.G.  +5.15  NaCl 

1.5 

30 

Ciliated  masses,  mucus 

11 

6E.G.+5.15NaCl 

6.0 

35 

Ciliated  masses,  mucus 

11 

6E.G.  +  5.15NaCl 

16.0 

25 

Ciliated  masses,  mucus 

11 

O.ONaCl 

0.25 

35 

Ciliated  masses,  epithelium,  rare  nerve 

11 

0.0  NaCl 

0.5 

20 

Nerve,  epithelium,  rare  UPS 

11 

0.0  NaCl 

0.75 

12 

Spreading  nerve 

3 

11 

0.0  NaCl 

3.0 

50 

Cytolyzed 

11 

58  sue 

0.75 

40 

Spreading  nerve,  UPS 

11 

0.65  NaCl 

0.75 

25 

Spreading  nerve,  epithelium 

11 

0.65  NaCl 

1.0 

25 

Nerve,  epithelium 

sodium  chloride  added  to  sucrose  results  in  no  induction  suggests  strongly  that 
the  induction  is  by  means  of  a  lack  of  sodium  chloride. 

Additional  evidence  that  the  lack  of  sodium  chloride  so  alters  the  cell  surfaces 
that  subsequent  exposure  to  high  concentrations  of  sodium  chloride  results  in 
induction  comes  from  a  few  experiments  recorded  in  Table  IX.  Experiment  1 
shows  the  effect  of  substitution  of  glycine  for  sodium  chloride.  Short  exposures 
to  this  solution  result  in  the  induction  of  spreading  nerve  after  the  aggregates  are 
returned  to  high  concentrations  of  sodium  chloride.  When  ethylene  glycol  is 
substituted  for  sodium  chloride  the  cells  do  not  survive,  but  when  ethylene  glycol 
is  combined  with  a  low  concentration  of  sodium  chloride  induction  of  nerve  occurs 
when  the  cells  are  returned  to  a  high  concentration  of  sodium  chloride  (exp.  2). 
When  ethylene  glycol  is  added  to  a  high  concentration  of  sodium  chloride  there  is 
no  inductive  activity  of  the  solution.  Thus,  again  the  alteration  of  cell  surfaces 


SODIUM  CHLORIDE  AND  INDUCTION  425 

appears  to  be  the  result  of  low  sodium  chloride  content  rather  than  the  action  of 
ethylene  glycol  itself. 

Finally  in  experiment  3  the  cells  are  exposed  to  low  sodium  chloride  content 
and  lack  of  sodium  chloride  for  short  intervals  and  then  cultured  in  high  concen- 
trations of  sodium  chloride.  After  both  treatments  nerve  is  induced,  indicating 
that  the  very  low  concentrations  of  sodium  chloride  result  in  some  alteration  in 
the  cell  surfaces  so  that  induction  occurs  after  the  cells  are  returned  to  the  higher 
concentration  of  sodium  chloride.  It  is  interesting  that  the  treatment  with  lack 
of  sodium  chloride  results  in  the  same  type  of  induction  as  with  sucrose  (experi- 
ment 3). 

DISCUSSION 

Clearly  substances  such  as  sucrose  do  not  induce  by  themselves  but  rather 
prepare  the  cells  for  induction  by  the  salt  solution  in  which  they  are  cultured. 
The  induction  by  the  salt  solution  is  proportional  to  the  concentration  of  sodium 
chloride.  Do  all  or  most  of  the  so-called  inductors  act  in  the  same  manner  as 
sucrose?  Since  all  the  experiments  on  induction  have  been  carried  out  in  Holt- 
freter's  solution  containing  a  concentration  of  sodium  chloride  of  3.4  mg./ml., 
possibly  all  the  inductions  obtained  by  various  compounds  and  mixtures  may  be 
attributed  to  Holtfreter's  solution. 

The  above  possibility  is  reinforced  by  the  investigations  of  Earth  (1941)  and 
of  Holtfreter  (1944),  which  show  that  Holtfreter's  solution  will  induce  neural 
tissue  in  Ambystoma  tnaculatum  and  Ambystoina  opacum  presumptive  epidermis 
without  benefit  of  additives  of  any  sort.  This  is  a  clear-cut  demonstration  that 
Holtfreter's  solution  is  an  adequate  inductor  of  nervous  tissue.  Thus,  any  com- 
pound or  mixture  claimed  as  an  inductor  when  used  in  Holtfreter's  solution  or  its 
equivalent  may  merely  be  preparing  the  cells  for  induction  by  the  salt  solutions. 
In  order  to  prove  that  any  substance  is  an  inductor  it  will  be  necessary  to  show  that 
induction  occurs  during  the  time  of  treatment  and  not  after  the  cells  are  returned 
to  a  salt  solution. 

In  view  of  the  wide  variety  of  compounds  and  mixtures  which  have  been 
claimed  as  inductors,  it  does  seem  more  reasonable  to  suppose  that  all  induction  is 
brought  about  by  the  ions  in  the  salt  solutions  used  for  the  culture  of  presumptive 
epidermis.  If  so,  we  can  begin  to  think  more  clearly  about  the  mechanism  of 
induction  by  ions  instead  of  trying  to  make  sense  out  of  the  disorderly  array  of 
so-called  inductors. 

As  far  as  normal  induction  by  the  mesoderm  is  concerned,  it  may  now  be  sug- 
gested that  the  mechanism  of  normal  induction  is  by  ways  of  ions.  For  example, 
we  find  that  cultures  of  mesoderm  mixed  with  ectoderm  prepared  from  lateral 
blastoporal  lips  contain  functional  nerve  and  pigment  cells  as  well  as  muscle  and 
mesenchyme.  Thus,  mesoderm  induces  nerve  and  pigment  cells  under  the  condi- 
tions of  our  experiments.  However,  if  the  sodium  chloride  content  of  our  salt 
solution  is  reduced  to  2.57  mg./ml.  no  nerve  nor  pigment  cells  are  induced  but 
muscle  and  mesenchyme  differentiate  normally.  Thus,  the  normal  induction  of 
nerve  and  pigment  cells  by  mesoderm  is  dependent  upon  a  high  concentration  of 
sodium  chloride.  This  suggests  that  the  role  of  the  mesoderm  during  normal 
gastrulation  is  to  prepare  the  presumptive  neural  plate  for  induction  by  the  ions 


426  LESTER  G.  EARTH 

present  in  the  blastocoel  fluid.  This  preparation  may  simply  consist  in  an  increase 
in  permeability  so  that  the  ions  penetrate.  Experiments  designed  to  test  the  above 
suggestion  are  in  progress. 

If  we  accept  the  idea  that  basically  induction  is  brought  about  by  ions,  then 
we  have  first  the  problem  of  which  ions  in  the  salt  solution  are  necessary.  Pre- 
vious experiments  have  shown  that  Na+,  K+,  Ca++,  Mg++  and  HCO3~  can  induce 
(Earth  and  Earth,  1963,  1964  and  1965).  Secondly,  how  do  the  ions  act  inside 
the  cell  to  induce  cellular  differentiation?  A  previous  study  of  ion  induction 
(Earth,  1965)  showed  a  correlation  between  the  effects  of  ions  as  inductors  and 
their  effects  on  the  electrophoretic  mobility  of  DNA.  Possibly  the  ions  in  our  salt 
solution  act  directly  upon  DNA  complexes. 

SUMMARY 

1.  An  analysis  of  the  mode  of  action  of  sucrose  as  an  inductor  of  the  presump- 
tive epidermis  of  the  Rana  pipiens  gastrula  leads  to  the  conclusion  that  sodium 
chloride  is  the  actual  inductor. 

2.  After  treatment  with  sucrose,  induction  is  proportional  to  the  concentration 
of  sodium  chloride  in  the  culture  medium.     After  treatment  with  sucrose,  induc- 
tion is  proportional  to  the  length  of  exposure  to  a  solution  containing  3.4  to  5.15 
mg.  sodium  chloride  per  ml. 

3.  It  is  concluded  that  sodium  chloride  in  concentrations  of  from  3.4  to  5.15 
mg./ml.  is  an  adequate  inductor,  while  in  concentrations  from  2.00  to  2.57  mg./ml. 
sodium  chloride  does  not  induce  but  will  sustain  the  differentiation  of  various  cell 
types  after  induction. 

4.  It  is  suggested  that  normal  induction  by  the  mesoderm  during  gastrulation 
may  be  brought  about  by  the  ions  present  in  the  blastocoel.     The  hypothesis  that 
ions  act  directly  upon  DNA  complexes  has  been  advanced  in  a  previous  paper 
on  induction. 

LITERATURE  CITED 

EARTH,  L.  G.,  1941.     Neural  differentiation  without  organizer.     /.  Exp.  Zool.,  87:  371-384. 
EARTH,  L.  G.,  1965.     The  nature  of  the  action  of  ions  as  inductors.     Biol.  Bull,  129:  471-481. 
EARTH,  L.  G.,  AND  L.  J.  EARTH,  1959.     Differentiation  of  cells  of  the  Rana  pipiens  gastrula 

in  unconditioned  medium.     /.  Embryol.  Exp.  Morphol.,  7:  210-222. 
EARTH,  L.  G.,  AND  L.  J.  EARTH,  1962.     Further  investigations  of  the  differentiation  in  -vitro  of 

presumptive  epidermis  cells  of  the  Rana  pipiens  gastrula.    /.  Morphol.,  110:  347-373. 
EARTH,  L.  G.,  AND  L.  J.  EARTH,  1963.     The  relation  between  intensity  of  inductor  and  type  of 

cellular  differentiation  of  Rana  pipiens  presumptive  epidermis.     Biol.  Bull.,  124:   125- 

140. 
EARTH,  L.  G.,  AND  L.  J.  EARTH,  1964.     Sequential  induction  of  the  presumptive  epidermis  of 

the  Rana  pipiens  gastrula.     Biol.  Bull.,  127:  413-427. 
EARTH,  L.  G.,  AND  L.  J.  EARTH,  1966.     Competence  and  sequential  induction  in  presumptive 

epidermis  of  normal  and  hybrid  frog  gastrulae.     Physiol.  Zool.,  in  press. 
HOLTFRETER,  J.,  1944.     Neural  differentiation  of  ectoderm  through  exposure  to  saline  solution. 

/.  Exp.  Zool.,  95:  307-340. 
SHUMWAY,  W.,  1940.     Stages  in  the  normal  development  of  Rana  pipiens.    Anat.  Rec.,  78: 

139-147. 


THE  pH  TOLERANCE  OF  EMBRYOS  AND  LARVAE  OF  MERCE- 
NARIA  MERCENARIA  AND  CRASSOSTREA  VIRGINICA 

ANTHONY  CALABRESE  AND  HARRY  C  DAVIS 
U.  S.  Bureau  of  Commercial  Fisheries,  Biological  Laboratory,  Milford,  Connecticut  06460 

The  tidal  estuarine  waters  that  form  the  principal  habitat  of  most  commercial 
mollusks  are  some  of  the  most  complex  environments  in  nature.  Of  the  various 
interacting  biological,  physical,  and  chemical  factors  that  affect  commercial  mollusks 
in  these  waters,  pH  has  received  less  attention  than  any  other  major  factor.  Pry- 
therch  (1928)  measured  the  pH  at  several  stations  in  Milford  Harbor  and  the 
Milford  area  of  Long  Island  Sound.  He  found  a  pH  range  during  the  day  from 
7.2  to  8.4  and  observed  that  oysters  spawned  at  pH  7.8  to  8.2.  Prytherch  con- 
cluded that  low  pH  inhibited  oyster  spawning  and  that  oysters  in  Milford  Harbor 
spawned  at  high  tide  because  this  was  the  only  tidal  stage  at  which  the  pH  was 
between  7.8  and  8.2.  Korringa  (1940)  quoted  Gaarder  (1932)  and  Gaarder  and 
Sparck  (1932)  who  found  that  larvae  of  Ostrea  e dulls  died  when  the  pH  in  their 
oyster  polls  exceeded  9.0. 

In  laboratory  experiments,  Loosanoff  and  Tommers  (1947)  found  that  adult 
American  oysters,  Crassostrea  virglnica,  kept  in  pH  4.25  remained  open,  on  an 
average,  76%  of  the  time,  but  pumped  only  10%  as  much  water  as  did  the  controls. 
Oysters  kept  at  pH  6.75  and  7.00  initially  pumped  more  vigorously  than  the  con- 
trols but  the  rate  of  pumping  later  decreased  to  less  than  that  of  the  controls. 

Although  the  pH  of  the  open  ocean  usually  ranges  from  7.5  to  8.5  (the  higher 
values  are  at  the  surface  during  active  photosynthesis),  the  pH  in  tidepools,  bays, 
and  estuaries  may  decrease  to  7.0  or  lower  due  to  dilution  and  production  of  H2S 
(Sverdrup,  Johnson  and  Fleming,  1942).  These  inshore  areas  constitute  a  major 
portion  of  the  habitat  of  commercial  bivalves,  and  Davis  and  Calabrese  (1964)  sug- 
gested that  these  regions  may  be  exceedingly  important  also  as  the  nursery  grounds 
for  the  larval  stages.  Since  clam  and  oyster  larvae  must,  at  times,  encounter  a 
wide  range  of  pH  in  their  natural  habitat,  it  is  possible  that  success  or  failure  of 
recruitment  in  some  areas  may  be  determined  by  variations  in  pH.  The  present 
studies  were  designed  to  determine  the  pH  tolerance  of  the  embryonic  and  larval 
stages  of  hard  clams  (Mercenaria  tnercenaria)  and  American  oysters  (Crassostrea 
virginica)  under  laboratory  conditions. 

METHODS 

The  methods  at  this  laboratory  for  maintaining  spawners  and  obtaining  fer- 
tilized eggs  throughout  the  year  have  been  described  previously  (Loosanoff  and 
Davis,  1963).  The  effect  of  pH  on  the  percentage  of  eggs  of  clams  or  oysters  that 
develop  into  normal  straight-hinge  larvae  was  determined  by  placing  a  known 
number  of  fertilized  eggs  (usually  10,000  to  15,000)  into  each  of  a  series  of  1 -liter 
polypropylene  beakers  of  filtered,  ultraviolet-treated  sea  water  (salinity  27  ±  0.5/£c). 

427 


428 


ANTHONY  CALABRESE  AND  HARRY  C.  DAVIS 


The  pH  of  duplicate  cultures  was  adjusted  with  HC1  or  NaOH  to  each  of  the 
following  levels :  6.00,  6.25,  6.50,  6.75,  7.00,  7.50,  8.00,  8.25,  8.50,  8.75,  9.00,  9.25, 
and  9.50.  Finally,  one  pair  of  cultures  retained  at  the  pH  of  our  laboratory  sea 
water  (7.40-7.70)  served  as  controls.  All  cultures  were  kept  in  a  constant- 
temperature  bath  at  25°  ±  1°  C.  After  48  hours  at  the  experimental  conditions, 
the  larvae  from  each  culture  were  collected  on  a  stainless  steel  screen.  The  larvae 
were  resuspended  in  a  250-ml.  graduated  cylinder  and,  after  thorough  mixing,  a 


LJ 

o 

z 


9  5 


9.0 


85 


80 


I     7.5 

a 


70 


6.5 


60 


60 


6.5 


7.0 


75 


8.0 


85 


90 


95 


ADJUSTED    INITIAL     PH 


FIGURE  1.  Maximum  range  of  pH  (vertical  line)  and  average  pH  (horizontal  bar)  for 
each  initial  pH.  The  "adjusted  initial  pH"  was  established  at  the  beginning  of  each  experiment, 
and  readjusted  to  this  level  at  12-hour  intervals,  by  the  addition  of  HC1  or  NaOH. 

4-ml.  sample  was  withdrawn  and  preserved  in  5%  neutral  formalin.  The  larvae 
from  each  sample  were  then  counted  and  the  number  of  larvae  developing  normally 
at  each  pH  was  calculated  as  a  percentage  of  the  number  of  larvae  developing  nor- 
mally in  control  cultures. 

To  ascertain  the  effect  of  pH  on  survival  and  growth,  a  known  number  of  larvae 
(usually  8000  to  12,000),  which  had  been  reared  to  the  48-hour  straight-hinge 
stage  in  our  normal  sea  water  (pH  7.40-7.70),  was  placed  into  each  of  the  series 
of  cultures.  The  sea  water  in  these  beakers  was  changed  every  second  day  and 
supplemental  food,  consisting  of  a  mixture  of  Isochrysis  g  alb  ana,  Monochrysis 
lutherl,  and  Chlorella  sp.  580,1  was  added  to  each  beaker  daily.  The  pH  of  each 

1  Chlorella  sp.  (Indiana  U.  Collection  #580). 


pH  EFFECT  ON  OYSTERS  AND  CLAMS  429 

culture  was  adjusted  to  the  desired  level  immediately  after  each  change  of  sea  water 
by  the  addition  of  an  appropriate  amount  of  HC1  or  NaOH.  In  experiments  with 
clam  larvae  it  was  necessary  to  add  a  standard  dose  (50  ppm.)  of  Subnet 2  at  each 
change  of  sea  water  to  prevent  disease-induced  mortality  that  was  not  a  direct 
result  of  the  pH  being  tested.  Since  buffers  were  not  used,  it  was  necessary  to 
measure  and  readjust  the  pH  at  approximately  12-hour  intervals  using  a  line- 
operated,  solid-state  pH  meter  3  having  a  readability  of  0.02  pH  unit  and  a  repeat- 
ability of  0.01  pH  unit.  The  range  and  average  for  each  initial  pH  are  shown  in 
Figure  1. 

Experiments  with  clam  larvae  were  terminated  after  10  days  at  the  experi- 
mental pH  levels,  when  the  larvae  were  12  days  old,  because  at  favorable  pH  the 
majority  of  the  larvae  had  completed  larval  development  and  metamorphosis.  For 
similar  reasons  experiments  with  oyster  larvae  were  discontinued  after  12  days  at 
experimental  pH  levels,  when  the  larvae  were  14  days  old. 

Quantitative  samples  were  taken  from  each  culture  at  the  termination  of  an 
experiment  to  determine  the  percentage  of  larvae  surviving  and  their  increase  in 
mean  length.  In  each  of  these  samples,  all  survivors  were  counted  and  50  clam 
larvae  or  100  oyster  larvae  from  each  sample  were  measured  to  the  nearest  5  /*. 
The  increase  in  mean  length  of  larvae  during  the  test  period  was  calculated  for 
each  pH  as  a  percentage  of  the  increase  in  mean  length  of  larvae  in  the  control 
cultures. 

The  method  for  determining  the  number  of  larvae  surviving  or  the  percentage 
of  bivalve  eggs  developing  into  normal  straight-hinge  larvae  is  accurate  to  approxi- 
mately ±  10%  (Davis,  1958).  Differences  of  less  than  20%  in  the  percentage 
of  eggs  developing  normally  or  of  larvae  surviving  a  treatment  are,  therefore, 
considered  insignificant. 

Five  experiments  were  with  clam  larvae  and  four  with  oyster  larvae.  In  the 
first  three  experiments  with  clam  larvae  a  standard  technique  was  being  developed 
and  various  buffer  systems  were  being  tested.  In  these  initial  experiments  citric 
acid,  monobasic  potassium  phosphate,  dibasic  sodium  phosphate,  and  Tris  (hydroxy- 
methyl  aminomethane)  were  used  as  buffers  in  an  attempt  to  stabilize  the  pH  at 
desired  levels.  The  phosphates  and  citric  acid  were  not  effective  in  maintaining 
pH  levels  below  7.00;  these  buffers  also  appeared  somewhat  toxic  to  clam  larvae. 
When  concentrations  of  these  buffers  were  high,  a  white  flocculent  precipitate  was 
formed  in  the  cultures.  Tris  was  of  some  help  in  maintaining  relatively  stable  pH 
levels  above  7.00,  but  was  toxic  at  pH  8.50  or  higher,  even  though  apparently 
nontoxic  at  levels  below  8.50.  A  precipitate  which  adhered  to  the  sides  and  bottom 
of  the  beakers  was  formed  at  pH  9.50,  with  or  without  Tris.  The  results  of  these 
preliminary  experiments  are  not  included  in  our  graphs. 

Effect  of  pH  on  embryonic  development  of  clams  and  oysters 

The  number  of  clam  eggs  developing  normally  within  the  pH  range  from  7.00 
to  8.75  or  of  oyster  eggs  within  the  range  from  6.75  to  8.75  did  not  vary  sig- 
nificantly (Fig.  2).  The  number  of  both  clam  and  oyster  eggs  that  developed  nor- 
mally at  pH  9.00  was  greatly  reduced  and  at  9.25  to  9.50  almost  none  developed. 

2  Sulmet  (Sodium  sulfamethazine) — Trade  name  of  American  Cyanamid. 

3  Instrumentation  Laboratory's  Model  165  LAB-omatic. 


430 


ANTHONY  CALABRESE  AND  HARRY  C.  DAVIS 


100 


90 


5 

< 

2 

O 

Z 

o 

z 

£ 
o 


80 


70 


60 


u 
o 


50 


o 
o 
u 

u.    40 
O 

K 
Z 
kl 

U    30 

cc 
u 


20 


10 


6.0  6.5  7.0  7.5  8.0  8.5 

ADJUSTED    INITIAL     PH 


9.0 


9.5 


FIGURE  2.  Percentage  of  clam  and  oyster  eggs  that  developed  into  normal  straight-hinge 
larvae  at  different  pH  levels,  expressed  as  a  percentage  of  the  number  developing  into  normal 
larvae  in  control  cultures. 


Clam  eggs  apparently  were  not  able  to  tolerate  pH  values  as  low  as  did  oyster  eggs. 
At  pH  6.75  only  29.5%  of  the  clam  eggs  developed  but  92.4%  of  the  oyster  eggs 
developed  normally. 

Effect  of  pH  on  survival  of  clam  and  oyster  larvae 

Survival  of  both  clam  and  oyster  larvae  was  approximately  normal  throughout 
the  pH  range  from  6.25  to  8.75  (Fig.  3).  Oyster  larvae  were  somewhat  more 
tolerant,  however,  of  low  pH  than  clam  larvae.  At  pH  6.00,  for  example,  21.5% 
of  the  oyster  larvae  survived,  but  all  of  the  clam  larvae  died.  Survival  of  both 
clam  and  oyster  larvae  increased  sharply  from  20%  or  less  at  pH  6.00  to  approxi- 


pH  EFFECT  ON  OYSTERS  AND  CLAMS 


431 


mately  70%  at  6.25  and  decreased  sharply  from  70%  or  better  at  pH  8.75  to  approx- 
imately 40%  at  9.00.  Most  of  the  larvae  lived  a  few  days  at  pH  9.00  although 
eventually  more  than  50%  died.  No  larvae  of  either  species  survived  at  9.25 
and  higher. 

Effect  of  pH  on  growth  of  clam  and  oyster  larvae 

The  pH  range  for  normal  growth  was  6.75  to  8.50  for  clam  larvae  and  6.75  to 
8.75  for  oyster  larvae  (Fig.  4).  The  range  for  normal  growth  was,  therefore, 
slightly  narrower  than  the  range  for  normal  survival.  The  rate  of  growth  of  clam 
larvae  was  most  rapid  at  pH  7.50  to  8.00,  whereas  oyster  larvae  grew  most  rapidly 
at  8.25  to  8.50.  Although  oyster  eggs  and  larvae  survive  at  lower  pH  levels  than 
clam  eggs  and  larvae,  the  optimum  pH  for  growth  of  oyster  larvae  was  somewhat 


100 


10 


60 


70 


7.5  8.0  85 

ADJUSTED    INITIAL     PH 


90 


95 


FIGURE  3.     Percentage  of  clam  and  oyster  larvae  that  survived  at  different  pH  levels,  expressed 

as  a  percentage  of  survival  in  control  cultures. 


432 


ANTHONY  CALABRESE  AND  HARRY  C.  DAVIS 


120 


no 


100 


90 


80 


70 


60 


±    50 


z 

UJ 

o 

tf 
u 

0. 


40 


30 


20 


10 


6.0 


6.5 


7.0 


7.5 


8.0 


8.5 


9.0 


ADJUSTED   INITIAL     PH 


9.5 


FIGURE  4.  Increase  in  mean  length  of  clam  and  oyster  larvae  at  different  pH  levels  ex- 
pressed as  a  percentage  of  the  increase  in  mean  length  of  larvae  in  control  cultures.  Fifty  clam 
or  100  oyster  larvae  were  measured  from  each  of  duplicate  cultures  at  each  pH  in  each  of  two 
or  more  replicate  experiments. 

higher  than  the  optimum  for  clam  larvae.  The  rate  of  growth  for  both  clams  and 
oysters  varied  only  slightly  within  the  pH  range  6.75  to  8.50,  but  below  pH  6.75 
the  rate  of  growth  decreased  rapidly.  The  rate  of  growth  also  decreased  rapidly 
at  pH  values  above  8.75  for  oysters  and  above  8.50  for  clams.  Since  the  empty 
shells  of  dead  larvae  were  not  dissolved  at  the  higher  pH  levels,  it  was  possible  to 
measure  them.  At  pH  9.00  some  increase  in  length  had  taken  place  before  the 
larvae  died,  but  at  9.25  to  9.50  there  had  been  no  growth. 

Implications  for  distribution  and  survival  in  nature 

The  failure  of  bivalve  larvae  to  survive  and  grow  at  low  pH  levels  did  not  appear 
to  be  an  indirect  result  of  the  effect  of  pH  on  the  algal  cells  added  as  food.  That 


pH  EFFECT  ON  OYSTERS  AND  CLAMS 


433 


the  food  cells  were  not  destroyed  by  low  pH  and  that  they  remained  in  suspension 
was  shown  by  the  fact  that  they  were  ingested  by  the  larvae.  Since  the  larvae  were 
fed  supplemental  food  daily,  starvation  was  unlikely  even  if  some  algal  cells  were 
destroyed.  Even  those  larvae  that  survived  at  low  pH  and  had  food  in  their 
stomachs,  however,  did  not  grow  appreciably.  Gray  (1922)  found  that  movement 
of  gill  cilia  of  mussels  was  more  readily  inhibited  by  weak  acids  which  entered  the 
ciliary  cells  than  by  strong  acids  which  do  not  enter  the  cells  readily  and,  con- 
versely, that  weak  bases  were  more  efficient  restoratives  of  ciliary  movement  than 
strong  bases.  Because  some  food  was  ingested,  even  at  our  lowest  pH  levels,  it 
seems  unlikely  that  failure  of  these  larvae  to  grow  can  be  attributed  to  the  effect 
of  pH  on  ciliary  movement. 

It  should  be  emphasized  that  clam  larvae  can  survive  at  pH  levels  lower  than 
those  at  which  clam  eggs  can  develop  normally  (Fig.  5).     The  range  for  normal 


100 


90 


eo 


70 


60 

t- 
z 

III 

u 

5    50 


40 


30 


20 


10 


DEVELOPING     NORMALLY 


i •  SURVIVAL 

OF    LARVAE 


— •  INCREASE 
MEAN    LENGTH 


6.0 


6.5 


7.0 


9.0 


9.5 


7.5  8.0  8.5 

ADJUSTED    INITIAL     PH 

FIGURE  5.     The  pH  tolerance  of  clam  embryos  and  larvae  as  indicated  by  percentage  of  eggs 
that  developed  normally,  survival  of  larvae,  and  increase  in  mean  length  of  larvae. 


434 


ANTHONY  CALABRESE  AND  HARRY  C.  DAVIS 


120  - 


DEVELOPING    NORMALLY 


-•  SURVIVAL 

LARVAE 


75  8.0  8.5 

ADJUSTED   INITIAL    PH 

FIG.  6.     The  pH  tolerance  of  oyster  embryos  and  larvae  as  indicated  by  percentage  of  eggs 
that  developed  normally,  survival  of  larvae,  and  increase  in  mean  length  of  larvae. 

survival  of  larvae  was  6.25  to  8.75,  whereas  the  range  for  normal  embryonic  devel- 
opment was  only  7.00  to  8.75.  In  environments  with  a  pH  below  7.00,  failure  of 
clam  eggs  to  develop  normally  would  be  the  factor  that  would  limit  recruitment  of 
this  species.  At  pH  levels  9.00  and  above  the  percentage  of  clam  eggs  developing 
normally,  the  percentage  of  larvae  surviving,  and  the  percentage  increase  in  mean 
length  all  decrease  abruptly,  so  that  at  high  pH  levels  all  three  aspects  of  develop- 
ment limit  recruitment  of  the  species.  Variations  in  the  percentages  of  eggs  devel- 
oping normally  and  of  larvae  surviving  at  pH  levels  between  7.00  and  8.75  were 
erratic,  but  all  fell  within  the  ±  10%  confidence  limits  of  our  method.  Although 
the  pH  ranges  for  normal  survival  of  clam  larvae  were  6.25  to  8.75  and  those  for 
normal  rate  of  growth  were  6.75  to  8.50,  the  optimum  for  growth  was  7.50  to  8.00. 
The  differences  in  rates  of  growth  at  pH  6.75  to  8.50,  however,  were  slight  enough 


pH  EFFECT  ON  OYSTERS  AND  CLAMS  435 

to  be  negligible  in  recruitment  of  this  species  in  nature.  The  pH  of  our  laboratory 
sea  water  (7.40-7.70)  was  close  to  optimum  for  growth  of  hard  clam  larvae. 

Oyster  larvae,  like  clam  larvae,  survived  at  lower  pH  levels  than  those  at  which 
the  eggs  developed  (Fig.  6).  At  pH  6.00  none  of  the  oyster  eggs  developed  nor- 
mally, but  21.5%  of  the  larvae  survived.  At  pH  6.25  the  percentage  survival  of 
larvae  increased  sharply,  but  the  increase  in  the  percentage  of  eggs  developing 
normally  was  negligible.  Most  of  the  oyster  eggs  developed  normally  at  pH  6.75, 
whereas  a  pH  of  7.00  was  required  for  most  clam  eggs  to  develop.  Oysters, 
therefore,  should  be  able  to  penetrate  into  areas  of  lower  pH  than  clams  could 
tolerate.  The  range  for  normal  survival  of  oyster  larvae  was  pH  6.25  to  8.75,  and 
the  range  for  a  normal  rate  of  growth  of  the  larvae  was  6.75  to  8.75.  The  optimum 
pH  for  growth  of  oyster  larvae,  however,  was  8.25  to  8.50,  i.e.,  both  the  optimum 
and  the  upper  limit  for  normal  growth  were  somewhat  higher  than  for  clam  larvae. 
As  with  clams,  however,  the  percentage  of  eggs  developing  normally,  the  percentage 
survival  of  larvae,  and  the  rate  of  growth  all  decrease  rapidly  at  pH  9.00  and  above. 
Since  oyster  larvae  at  pH  8.00  to  8.50  outgrew  the  oyster  larvae  in  the  control 
cultures  (pH  7.40-7.70),  it  was  apparent  that  the  pH  of  our  normal  laboratory 
sea  water  was  somewhat  too  low  for  the  most  rapid  growth  of  these  larvae. 

It  can  be  concluded  that  for  successful  recruitment  of  clams  and  oysters  the 
pH  of  the  tidal  estuarine  waters  that  form  their  principal  habitat  must  not  fall 
below  7.00  for  clams  or  6.75  for  oysters  for  any  appreciable  time.  Moreover, 
neither  species  could  reproduce  successfully  in  waters  where  the  pH  remained 
appreciably  above  9.00. 

Laboratory  experiments  have  shown  that  high  concentrations  of  silt  can  lower 
the  pH  of  our  sea  water  to  6.40,  or  below  the  lower  limit  for  normal  development 
of  eggs  of  hard  clams  and  oysters.  It  is  apparent,  therefore,  that  heavy  siltation, 
or  any  pollution  that  can  change  the  pH  of  tidal  estuarine  waters,  could  cause  failure 
of  recruitment  of  hard  clams  and  oysters. 

SUMMARY 

1.  The  pH  range  for  normal  embryonic  development  of  oysters  was  6.75  to 
8.75,  and  for  clams,  7.00  to  8.75. 

2.  More  than  68%  of  the  larvae  of  both  clams  and  oysters  survived  at  pH  6.25 
to  8.75.     The  lower  pH  limit  for  survival  of  oyster  larvae  was  6.00  and  for  clam 
larvae,  6.25. 

3.  The  pH  range  for  normal  growth  was  6.75  to  8.50  for  clam  larvae  and 
6.75  to  8.75  for  oyster  larvae.     The  rate  of  growth  of  both  species  dropped  rapidly 
at  pH  levels  below  6.75. 

4.  The  optimum  pH  for  growth  was  7.50  to  8.00  for  clam  larvae  and  8.25  to 
8.50  for  oyster  larvae. 

5.  At  pH  9.00  to  9.50  the  percentage  of  eggs  that  developed  normally,  the 
percentage  of  larvae  that  survived,  and  the  percentage  increase  in  mean  length  of 
both  species  decreased  rapidly. 

LITERATURE  CITED 

DAVIS,  H.  C.,   1958.     Survival  and  growth  of  clam  and  oyster  larvae  at  different   salinities. 
Biol.Bull.,  114:296-307. 


436  ANTHONY  CALABRESE  AND  HARRY  C.  DAVIS 

DAVIS,   H.   C,  AND  A.   CALABRESE,    1964.     Combined   effects   of   temperature   and    salinity   on 

development  of  eggs  and  growth  of  larvae  of  M.  mercenaries  and  C.  virginica.    Fish. 

Bull.,  63:  643-655. 
*GAARDER,  T.,   1932.     Untersuchungen  iiber   Produktions-  und   Lebensbedingungen   in   Norwe- 

gischen  Austerpollen.    Bergens  Mus.  Arbok  1932  Naturv.  Rekke  No.  3. 
*GAARDER,  T.,  AND  R.  SPARCK,  1932.     Hydrographisch-Biochemische  Untersuchungen  in  Nor- 

wegischen  Austerpollen.     Bergens  Mus.  Arbok  1932  Naturv.  Rekke  No.  1. 
GRAY,  J.,  1922.     Ciliary  beat  in  Mytilus.     Influence  of  ions  on  ciliary  beat.     Proc.  Roy.  Soc. 

London,  Ser.  B,  93:  104-121. 
KORRINGA,  P.,   1940.     Experiments  and  observations  on  swarming,  pelagic  life  and  setting  in 

European  flat  oyster,  Ostrea  edulis  L.    Arch.  Neer.  Zool.,  5:   1-249. 
LOOSANOFF,  V.  L.,  AND  H.  C.  DAVIS,  1963.     Rearing  of  bivalve  mollusks.     In:   Advances  in 

Marine  Biology,  F.  S.  Russell,  Ed.,  Academic  Press,  Inc.,  London,  Vol.  I,  pp.  1-136. 
LOOSANOFF,  V.  L.,  AND  F.  D.  TOMMERS,  1947.    Effect  of  low  pH  upon  rate  of  water  pumping 

of  oysters,  Ostrea  virginica.    Anat.  Rec.,  99:  112-113. 
PRYTHERCH,   H.   F.,    1928.     Investigation   of  the   physical   conditions   controlling   spawning   of 

oysters  and  the  occurrence,  distribution,  and  setting  of  oyster  larvae  in  Milford  Harbor, 

Connecticut.     Bull.  U.  S.  Bur.  Fish.,  44:  429-503. 
SVERDRUP,  H.  U.,  M.  W.  JOHNSON  AND  R.  H.  FLEMING,  1942.     The  Oceans,  Their  Physics, 

Chemistry  and  General  Biology.     Prentice-Hall,  Inc.,  New  York,  pp.  1-1087. 

4  Reviewed  by  Korringa,  1940,  cited  above. 


DESCRIPTION  OF  A  ZOOCHLORELLA-BEARING  FORM  OF 
EUPLOTES,  E.  DAIDALEOS  N.  SP.  (CILIOPHORA, 

HYPOTRICHIDA) 

WILLIAM  F.  DILLER  AND  DEMETRIUS  KOUNARIS 

Department  of  Biology,  University  of  Pennsylvania,  Philadelphia,  Pa.  19104 

Marine  and  fresh-water  species  of  Euplotes  are  numerous  and  widely  distributed 
throughout  the  world.  Reviews  and  historical  accounts  have  been  given  by  Kahl 
(1932),  Pierson  (1943),  Bovee  (1957),  Tuffrau  (1960),  Wichterman  (1964)  and 
others.  In  spite  of  a  great  deal  of  morphological,  genetic  and  cultural  work,  the 
taxonomy  of  this  genus  is  still  in  an  unsettled  condition  due  to  variability  within 
species,  conjugation  between  recognized  different  species,  failure  of  morphologically 
similar  forms  to  mate  and  utilization  of  perhaps  unreliable  criteria  in  species  differ- 
entiation. Moreover,  it  seems  reasonable  to  believe,  as  Bovee  has  suggested,  that 
morphological  variation  and  speciation  have  followed  after  reproductive  and 
physiological  variation  in  this  genus ;  hence,  the  obvious  difficulty  of  dealing  with 
this  interesting  group  of  ciliates  and  the  need  for  the  employment  of  a  variety  of 
characters  in  the  recognition  of  species.  A  great  advance  in  the  analysis  of  the 
genus  Euplotes  was  made  by  Tuffrau  in  his  application  of  the  following  combination 
of  characters:  (1)  the  number  of  latero-dorsal  kinetics,  (2)  the  pattern  of  the 
argyrome  on  the  dorsal  surface,  (3)  the  number  of  frontoventral  cirri  and  (4)  the 
shape  of  the  meganucleus  in  the  vegetative  state.  He  based  his  own  extensive 
revision  of  the  genus  on  these  specific  traits.  Other  students  would  extend  diag- 
nostic criteria  to  further  features,  particularly  characters  relating  to  the  peristome 
(buccal  cavity).  Undue  reliance  on  argyrome  traits  alone  can  be  misleading.  For 
example,  the  number  of  kinetics  in  a  given  species  may  be  variable,  as  in  E.  tuffraiii 
(Berger,  1965),  E.  vannus  (Dusenberry,  1966)  and  one  stock  of  E.  crassits  (Dusen- 
berry,  1966).  In  addition,  there  may  be  several  species  possessing  the  same  num- 
ber of  kinetics  and  a  somewhat  similar  dorsal  argyrome  pattern  (Tuffrau,  1960). 
Recently  evolved  species  may  have  argyrome  patterns  very  similar  to,  if  not  iden- 
tical with,  the  ancestral  forms,  so  that  it  may  be  very  difficult  to  decide  whether  or 
not  a  given  variant  is  a  separate  species.  The  most  recently  described  species,  to 
the  writers'  knowledge,  are  E.  leticicnsis  Bovee  (1957),  E.  neapolitanus  Wichter- 
man  (1964)  and  E.  tuffraui  Berger  (1965).  Borror  (1962)  has  re-described 
Euplotes  minuta  Yocum.  Wichterman  reports  (1964,  pp.  368-369)  that  "Vacelot 
(1961)  described  what  he  believed  to  be  a  new  marine  species  which  he  named 
E.  psammophilus,  from  Amphioxus-sand  near  Marseilles;  but  his  brief  description 
and  poor  figure  are  inadequate  to  set  the  species  on  a  firm  foundation." 

MATERIALS  AND  METHODS 

Collections  of  samples  from  a  small,  permanent,  artificial  fresh-water  pond  in 
the  Biological  Gardens  on  the  campus  of  the  University  of  Pennsylvania  have  con- 

437 


438  WILLIAM  F.  DILLER  AND  DEMETRIUS  KOUNARIS 

sistently  yielded  over  the  past  seven  years  specimens  of  a  green  species  of  Euplotes 
which  is  here  named  and  described  as  E.  daidaleos  n.  sp.1  Not  every  collection 
was  positive  but  there  has  been  no  difficulty  in  securing  material  from  this  source. 
The  green  Euplotes  is  never  present  in  large  numbers  in  a  freshly  collected  sample 
and  it  is  often  found  together  with  one  or  more  colorless  species  of  Euplotes  from 
which  it  is  easily  distinguishable  because  of  its  different  size  and  its  characteristic 
bright  green  color.  However,  when  there  are  few  algae  in  E.  daidaleos  it  can  be 
confused  with  the  colorless  species  which  may  have  ingested  green  organic  food. 
The  green  species  is  not  difficult  to  maintain  in  the  laboratory  and  thrives  on  the 
usual  ciliate  culture  media — hay  infusion,  Cerophyl  infusion,  malted  milk,  powdered 
milk,  rice  grains  in  boiled  pond  water,  etc. 

Animals  were  studied  alive,  slowed  down  by  0.05  %  nickel  sulfate  or  by  methyl- 
cellulose,  as  well  as  in  fixed  and  stained  preparations.  Many  cytological  techniques 
were  employed,  the  most  useful  being  (1)  formalin-vapor  fixation  followed  by 
Bouin's  and  stained  by  the  Holmes  silver  technique  (Figs.  1-3),  (2)  the  Chatton- 
Lwoff  silver  impregnation  (Figs.  4,  5  and  8),  and  (3)  Perenyi  fixation  with 
carmine-picronigrosin  staining  (Figs.  6  and  7).  All  three  methods  have  been 
described  in  detail  in  a  previous  paper  (Diller,  1966). 

RESULTS 
Size  and  shape 

This  fresh-water  species  averages  92  //,  in  body  length  and  57  p.  in  width,  for 
vegetative  non-dividing  individuals.  A  range  of  77  ^  to  119//,  in  length  and  43 /A 
to  80  p.  in  breadth  is  encountered.  Dividing  animals  are  somewhat  larger,  as  might 
be  expected,  averaging  102  p.  in  length  and  59 /x  in  width.  Exconjugants  are 
shorter  and  more  rounded,  averaging  80  ^  long  and  55 /A  wide.  E.  daidaleos  is 
definitely  smaller  than  the  common  colorless  species  with  which  it  is  commonly 
found  in  nature.  As  is  true  of  all  species  of  Euplotes,  E.  daidaleos  has  a  very 
strongly  flattened  body  and  is  oval  to  rounded  in  face  view.  The  anterior  margin, 
bearing  a  low  collar,  is  truncated,  while  the  posterior  end  tends  to  be  bluntly 
pointed  (Figs.  1,  2  and  3).  The  right  anterior  margin  is  straighter  than  the  cor- 
responding corner  on  the  left  side  which  is  more  rounded.  The  dorsal  surface  is 
slightly  convex  and  bears  a  constant  number  of  ridges.  The  ventral  surface  is 
flatter  and  also  is  equipped  with  ridges  of  a  characteristic  nature.  The  collar  carries 
the  transverse  anterior  adoral  membranelles. 

Surface  organelles 

The  peristome  is  capacious  and  tapers  to  a  narrow  funnel  slightly  behind  the 
middle  of  the  body,  at  the  cytostome  (Figs.  1,  2  and  8).  It  is  bordered  on  its  left 
and  anterior  margins  by  the  adoral  zone  of  membranelles  (AZM).  These  are 
small  and  close-set  in  the  cytostomal  region,  becoming  wider  and  larger  anteriorly. 

1  The  much-studied  genus  Euplotes  with  its  numerous  species  and  many  synonyms  offered 
a  problem  in  the  selection  of  an  appropriate  name  for  a  new  species.  The  term  "daidaleos"  was 
finally  selected  after  reference  to  Brown  (1954).  He  (p.  742)  gives  the  meaning  of  the  Greek 
word  daidaleos  as  "dappled,  spotted."  It  has  an  additional  meaning  (daidalos,  p.  104)  :  "cun- 
ningly or  skilfully  made  in  the  manner  of  Daedalus,  the  Athenian  artificer."  The  species  name 
seemed  fitting,  especially  so  because  of  the  Greek  origin  of  the  word  Euplotes. 


EUPLOTES  DAIDALEOS  N.  SI'. 


439 


There  are  approximately  40-45  units  constituting  this  structure.  Posteriorly,  the 
AZM  is  opposed  by  the  prominent  hand  of  cilia  in  the  mouth  area  (Figs.  1  and  2) 
which  is  known  as  the  undulating  or  paroral  memhranelles.  The  exact  conforma- 
tion of  the  peristomal  cavity  is  a  little  difficult  to  determine  but  can  be  analyzed 
satisfactorily  from  silver  and  from  picronigrosin  preparations.  The  right  edge  is 


FIGURE  1.  Semi-diagrammatic  drawing  of  Euplotcs  daidalcos  n.  sp.  from  the  ventral  side, 
showing  structural  features.  Taken  from  a  fixed  specimen,  stained  with  Holmes  silver.  Note 
the  collar  at  the  anterior  margin  of  the  body  bearing  some  of  the  adoral  membranelles  (AZM) 
which  continue  along  the  left  wall  of  the  buccal  cavity  (peristome)  to  terminate  at  the  cyto- 
stome ;  the  paroral  membranelles  near  the  base  of  the  peristome  on  its  right  wall ;  the  peristomal 
plate  extending  along  the  left  wall  of  the  peristome ;  a  bulge  on  the  opposite  wall  of  the  peri- 
stome occupying  a  considerable  portion  of  the  roof  of  it ;  an  irregular  channel  between  these 
two  thickened  areas  of  the  peristomal  roof;  the  endoplasmic  sac  (corresponding  to  the  level 
of  the  paroral  membranelles)  ;  the  contractile  vacuole  just  posterior  to  the  sac  ;  the  meganucleus ; 
the  micronucleus  ;  the  eighteen  cirri  (six  frontals,  three  smaller  ventrals,  five  anals  and  four 
caudals)  ;  the  "neuromotor"  fibers  from  the  bases  of  the  anal  cirri  and  the  four  zoochlorellae 
in  the  cytoplasm. 


440 


WILLIAM  F.  DILLER  AND  DEMETRIUS  KOUNARIS 


FIGURES  2-8. 


EUPLOTES  DAIDALEOS  N.  SP.  441 

almost  straight,  being  formed  by  a  continuation  of  the  heavy  ridge  which  runs 
anteriorad  from  the  left  side  of  the  medial  anal  cirrus.  This  border  is  undercut 
by  a  shallow  furrow  so  as  to  form  a  narrow  longitudinal  lip  which  is  usually  a 
little  wider  at  its  anterior  and  posterior  limits  than  in  its  center.  The  lip  con- 
tinues posteriorly  over  the  paroral  membranelles  to  form  the  posterior  border  of 
the  peristome,  the  funnel  of  the  mouth.  Along  a  good  part  of  the  right  wall  of  the 
peristome  is  a  shallow  bulge  (Fig.  1,  stippled)  which  continues  over  part  of  the 
roof  of  the  peristomal  cavity  from  near  the  anterior  end  to  about  the  middle  of  the 
paroral  membranelles.  Opposite  this  bulge  is  another  thickening  of  the  left  wall 
and  roof  of  the  peristome,  the  so-called  peristomal  plate  (Fig.  1,  also  stippled).  It 
bears  a  very  small  lip.  A  trough  or  channel  is  formed  in  the  roof  of  the  peristome 
by  the  space  between  these  two  thickenings.  The  channel  is  expanded  anteriorly 
at  the  level  of  the  collar  and  also  posteriorly  in  the  region  where  actual  ingestion 
occurs.  In  other  species  of  Euplotes  we  have  seen  small  food  organisms  entrapped 
in  the  anterior  expansion  which  seems  to  serve  as  a  food-collecting  mechanism. 
However,  we  have  not  noticed  any  collection  of  food  material  in  this  structure  in 
E.  daidaleos. 

The  18  cirri  are  very  constant  in  number  and  position.  In  comparison  with 
colorless  species,  one  has  the  impression  that  those  of  E.  daidaleos  are  longer  and 
slenderer.  Very  rarely  five  caudal  cirri  (instead  of  four)  are  present.  These  may 
represent  reorganizations  from  division  of  the  cell.  Also,  in  exconjugants  (at 
certain  stages)  only  five  instead  of  six  frontal  cirri  are  present.  The  usual  "bio- 
logical variation"  can  account  for  the  infrequent  cirral  anomalies  which  affect  the 
caudals,  mainly.  The  number  and  position  of  the  cirri  are  as  follows :  six  frontals 
in  three  rows  of  two  each,  three  ventrals  in  an  oblique  row  on  the  right  ventral 


EXPLANATION  OF  FIGURES  2-8 

Euplotcs  daidaleos.  Photomicrographs.  Specimens  shown  in  Figures  2  and  3  were  fixed 
in  formalin  vapor  and  Bouin's,  followed  by  the  Holmes  silver  technique.  Figures  4,  5  and  8 
are  specimens  fixed  in  Champy,  DaFano  and  treated  with  the  Chatton-Lwoff  silver  impregna- 
tion. Figures  6  and  7  are  exconjugants  fixed  in  Perenyi's  fluid  and  stained  by  Grenadier's 
alcoholic  borax  carmine  and  picronigrosin. 

FIGURE  2.  Ventral  view.  Typical  individual  showing  the  locations  of  all  of  the  eighteen 
cirri,  the  shape  of  the  meganucleus,  the  fibrils  from  the  anal  cirri,  the  AZM,  the  paroral  mem- 
branelles and  some  of  the  zoochlorellae  (approx.  50).  X  700. 

FIGURE  3.  Ventral  view,  with  the  dorsal  argyrome  in  focus.  The  alternating  rows  of 
narrower  and  wider  polygons  or  "compartments"  are  easily  seen.  The  dorsal  bristles  are  not 
prominent.  X  615. 

FIGURE  4.  Dorsal  view.  Most  of  the  rows  of  dorsal  bristles  are  visible — located  in 
meridians  on  the  right  side  of  the  wider  "compartment"  rows.  X  650. 

FIGURE  5.  Dorsal  view.  All  nine  rows  of  latero-dorsal  bristles  are  visible.  The  zoo- 
chlorellae are  prominent,  as  are  the  elongated  basal  plates  of  the  four  caudal  cirri.  X  645. 

FIGURE  6.  Dorsal  view.  The  rows  of  narrow  polygons  contain  or  overlie  protoplasmic 
constituents  (probably  mitochondria)  which  are  thus  organized  differently  from  their  condition 
in  the  wider  "compartment"  rows  so  as  to  give  a  banded  organization  to  the  dorsal  cortex. 
X600. 

FIGURE  7.  Dorsal  view,  with  the  ventral  surface  in  focus.  The  stained  granules  noted  in 
Figure  6  are  differentially  distributed  in  the  ventral  cortex  also,  outlining  the  ventral  ridges. 
X62S. 

FIGURE  8.  Dorsal  view,  with  focus  on  the  ventral  surface  to  show  the  nature  of  the  ventral 
argyrome  pattern.  To  the  right  of  the  bases  of  the  anal  cirri  is  the  contractile  vacuole  pore. 
X690. 


442  WILLIAM  F.  DILLER  AND  DEMETRIUS  KOUNARIS 

surface,  five  large  anals  (four  in  line  on  the  right  side  and  the  medial  one  slightly 
anterior  to  the  last  of  this  line)  and  four  small  caudals.  The  two  right  caudals 
regularly  tend  to  be  fimbriated. 

Ridges  are  present  on  both  dorsal  and  ventral  surfaces.  On  the  latter  there 
are  six  ridges  supporting  the  grooves  in  which  the  anal  cirri  lie.  The  ridges  are 
of  varying  lengths  and  prominence  (Fig.  7).  Starting  from  the  right  side,  nos.  1, 
4  and  6  are  the  longest.  The  latter  participates  in  the  formation  of  the  major 
ridge  which  becomes  the  right  wall  of  the  buccal  cavity.  The  ridges  on  the  dorsal 
side  are  definite,  parallel,  equidistant  and  each  bears  a  row  of  dorsal  bristles. 
More  will  be  said  about  these  "latero-dorsal"  bristle  rows  in  connection  with  the 
discussion  of  the  argyrome. 

The  argyrome 

Various  silver  techniques  (Figs.  2,  3,  4,  5  and  8)  and,  under  certain  conditions, 
the  picronigrosin  stain  (Fig.  6)  demonstrate  the  "latero-dorsal  kinetics,"  the  dorsal 
bristles  and  associated  structures  which  may  be  interpreted  as  either  polygons, 
fibrils  or  a  meshwork.  Exactly  what  is  brought  out  is  somewhat  uncertain : 
kinetosomes,  pellicular  structures  or  actual  cortical  protoplasmic  elements.  Prob- 
ably a  variety  of  structures  is  indeed  impregnated  and  caution  is  required  in  inter- 
pretation. However,  with  the  use  of  the  standard  Chatton-Lwoff  technique,  e.g., 
in  Figures  4,  5  and  8,  striking,  reproducible  and  exceedingly  useful  preparations 
can  be  produced  so  as  to  compare  and  separate  different  forms  of  Euplotes.  Tuffrau 
has  made  extensive  use  of  this  silver  technique  in  his  admirable  study  of  the  genus. 
E.  daidaleos  possesses  nine  latero-dorsal  row^s  of  bristles  (Figs.  4  and  5  particu- 
larly). Eight  of  these  kineties  are  on  the  dorsal  surface,  the  ninth  definitely  on 
the  ventral  surface.  In  specimens  well  flattened  in  gelatine  all  nine  kineties  may 
be  visible  at  practically  the  same  focus.  The  number  of  kineties  is  remarkably  con- 
stant in  E.  daidaleos:  in  hundreds  of  specimens  carefully  studied  only  one  showed 
a  discrepancy  from  this  number,  nine.  The  number  of  kinetosomes  per  row  is 
also  fairly  constant.  Starting  with  the  ventral  row,  then  going  to  the  dorsal  side 
and  proceeding  to  the  right  edge  we  have  counted  the  following  numbers  of  bristles 
per  row:  13-16,  7-17,  15-18.  15-19,  17-20,  16-18,  16-18,  13-19,  10-19.  The 
total  number  of  bristles  per  animal  varied  from  123  to  158  in  the  specimens  counted. 
Between  the  kineties — kinetosomes  seem  to  lie  on  a  "fibril"-— are  stainable  lines 
(Figs.  4  and  5)  which  look  like  cross-connecting  fibrils.  With  the  picronigrosin 
technique  (Fig.  3)  one  gets  the  impression  that  this  configuration  may  be  shallow 
polygons  of  alternating  wider  and  narrower  rows  (Figs.  3  and  6)  of  pellicular  or 
of  superficial  cortical  material.  The  dorsal  bristles  always  lie  to  the  right  of  a 
wide  row  and  to  the  left  of  a  narrow  row  of  these  polygons.  It  would  appear  that 
the  wider  and  narrower  rows  may  represent  some  real  cytoplasmic  difference,  since, 
with  the  picronigrosin  stain  (Fig.  6),  there  is  a  differential  staining  reaction,  the 
narrow  rows  taking  up  the  blue  stain  much  more  intensely  than  the  wide  rows  do. 
Probably,  the  mitochondria  are  being  stained  and  this  banded  effect  represents  a 
differential  distribution  of  the  mitochondria.  One  can  think  of  no  logical  explana- 
tion for  this  condition.  On  the  ventral  surface  the  mitochondria  in  the  ridges 
take  up  the  picronigrosin  intensely  (Fig.  7).  The  argyrome  pattern  of  the  ventral 


EUPLOTES  DAIDALEOS  N.  SI'.  443 

surface  (Fig.  8)  is  more  irregular  and  finer  (including  the  roof  of  the  peristome) 
than  it  is  on  the  dorsal  surface.  As  is  characteristic  of  all  species  of  Euplotes,  the 
meshwork  immediately  surrounding  the  contractile  vacuole  pore,  to  the  right  of 
the  anal  cirri  (Fig.  8),  consists  of  much  smaller  polygons. 

Internal  structures 

The  shape  of  the  C-shaped  meganucleus  is  somewhat  variable  and  not  particu- 
larly distinctive  for  E.  daidaleos.  Typically,  the  anterior  arm  bends  backward  but 
the  posterior  arm  does  not  curve  forward  to  meet  it,  so  that  a  symmetrical  C  is  not 
formed  (Figs.  1  and  2).  Usually  the  anterior  arm  extends  forward  into  a  small 
hump  as  it  becomes  drawn  out  in  the  main  body  of  the  organelle.  The  position  of 
the  micronucleus  is  very  constant,  at  the  anterior  curvature  of  the  meganucleus 
slightly  dorsal  to  it  and  between  it  and  the  AZM.  It  is  a  small  vesicular  structure 
less  than  5  //,  in  diameter  enclosing  a  homogeneous  endosome. 

The  presence  of  symbiotic  algae  in  the  cytoplasm  of  E.  daidaleos  is  very  char- 
acteristic and  perhaps  a  constant  feature  of  this  species.  The  zoochlorellae  vary 
in  number  from  a  very  few  (Fig.  1  contains  four)  to  perhaps  100  (Figs.  4,  5  and 
8).  Their  size  ranges  from  3  to  5  //,  in  diameter.  When  a  culture  is  dividing 
rapidly  the  number  of  algae  is  less  per  host  than  when  the  population  is  static.  We 
do  not  know  whether  this  species  can  normally  exist  without  their  customary  algae ; 
if  so,  whether  they  can  reacquire  their  symbionts  from  the  medium.  In  clonal  cul- 
tures, all  of  the  individuals  possess  at  least  some  zoochlorellae  under  normal  con- 
ditions. When  colorless  Euplotes  and  E.  daidaleos  are  mixed  together,  both  color- 
less and  green  individuals  can  be  recognized  as  such  for  many  weeks.  Just  as 
Parainecium  bursaria  can  be  freed  of  its  algae,  so  it  might  be  expected  that  E. 
daidaleos  can  be  sterilized  of  its  zoochlorellae.  However,  critical  studies  have  not 
yet  been  made  on  these  matters  of  obligatory  symbiosis,  mode  of  "infection"  and 
survival  in  the  absence  of  algae.  It  should  be  noted  that  exconjugants  frequently 
contain  more  algae  than  their  non-conjugated  associates.  This  can  be  explained 
on  the  basis  of  a  long  reorganization  interval  between  conjugation  and  the  first 
postconjugant  fission  during  which  the  algae  divide  and  are  not  diluted  by  division 
of  their  host  cell. 

Many  of  our  Holmes  silver  preparations  bring  out  the  five  long  internal  fibers 
originating  at  the  bases  of  the  anal  cirri  and  converging  at  the  anterior  end  in  the 
region  of  the  medial  frontal  cirri  (Figs.  1  and  2),  as  well  as  other  fibers  associated 
with  other  cirri.  We  have  not  been  able  to  detect  any  structure  which  could  be 
interpreted  as  a  neuromotorium. 

The  food  material  appears  to  be  bacteria,  algae,  flagellates  and  small  ciliates  of 
the  pleuronematid  type.  Some  cultures  show  bacteria-like  bodies  in  the  cytoplasm 
which  resemble  Kappa  particles  in  Paramecium.  They  are  quite  distinct  from 
the  mitochondria  and  are  often  sharply  demonstrated  by  the  Holmes  technique.  A 
prominent  endoplasmic  sac  (Fig.  1)  is  present  on  the  right  side  of  the  body.  Its 
left  side  appears  to  be  very  intimately  associated  with  the  bases  of  the  paroral 
membranelles. 

Just  posterior  to  the  endoplasmic  sac  is  the  contractile  vacuole  (Fig.  1).  Its 
external  pore  is  a  fixed  spot  on  the  ventral  surface. 


444  WILLIAM  F.  DILLER  AND  DEMETRIUS  KOUNARIS 

Life  cycle 

Conjugation  has  been  observed  frequently  in  non-isolated  mass  cultures  of 
E.  daidaleos  and  in  cultures  started  from  a  few  (8-10)  individuals  from  wild  cul- 
tures. Studies  of  the  correlation  of  ciliary  and  nuclear  development  in  the  life  cycle 
of  this  species  have  been  published  elsewhere  (Kounaris,  1964;  Diller,  1966). 
These  report  binary  fission  and  conjugation.  Encystment  in  this  species  has 
never  been  seen  by  us. 

•/ 

DISCUSSION 

Colored  species  of  Euplotes  have  been  described  as  far  back  as  Ehrenberg 
(1840)  who  named  a  form  from  Berlin  E.  viridis.  Stein  (1859)  presented  three 
figures  of  forms  he  called  Euplotes  patella,  noting  that  very  frequently  their  cyto- 
plasm is  more  or  less  thickly  filled  with  bright  green  chlorophyll  bodies.  He  re- 
ferred to  one  of  these  as  "disc-forms."  Apparently  he  felt  that  there  were  several 
types  of  chlorophyll-bearing  Euplotes.  Stein  identified  Ehrenberg's  Euplotes  viri- 
dis from  Berlin  as  most  probably  nothing  more  than  the  chlorophyll-bearing  form 
of  E.  patella.  Pierson,  in  her  review  of  species  of  Euplotes  closely  related  to 
Euplotes  patella,  makes  no  reference  to  green  Euplotes.  However,  Kahl  (1932) 
listed  three  colored  types:  all  "formae"  of  E.  patella.  These  are  forma  typicus, 
80-100^1.  mostly  with  zoochlorellae  ;  forma  latns  90-120 /x,  often  with  zoochlorellae  ; 
forma  alatus,  broad  form  with  zoochlorellae.  Obviously,  he  recognized  different 
forms  as  capable  of  harboring  zoochlorellae.  Tuff  ran,  in  his  revision  of  the  genus 
Euplotes,  does  not  mention  the  occurrence  of  zoochlorella-bearing  forms  and  dis- 
misses Kahl's  alga-bearing  formae,  together  with  Ehrenberg's  E.  viridis,  as  syno- 
nyms of  E.  patella.  This  decision  was  made  largely  on  the  basis  of  culture  work 
by  Pierson  and  himself  which  indicated  intra-clonal  variation  of  E.  patella. 

Tuffrau  lays  particular  emphasis  on  the  distinctiveness  of  the  dorsal  argyrome 
pattern  of  E.  patella:  the  alternating  wide  and  narrow  polygonal  rows.  E.  daida- 
leos  has  a  dorsal  argyrome  pattern  similar  to  E.  patella — perhaps  not  quite  as 
regular  as  Tuffrau  has  shown.  His  size  of  E.  patella — 105-145  p. — is  greater  than 
we  have  found  for  E.  daidaleos.  However,  Pierson's  size  of  E.  patella — 90  by  52  ^ 
—is  practically  the  same  as  for  E.  daidaleos.  Kahl's  E.  patella  is  larger. 

Most  of  the  published  figures  and  descriptions  of  Euplotes  allow  for  some 
uncertainty  with  regard  to  the  detailed  structure  of  the  buccal  cavity :  its  wall, 
relative  length  and  curvature,  lips,  bulges,  channels,  plates  and  membranelles.  It 
is  uncertain  how  similar,  or  dissimilar,  E.  daidaleos  is  to  other  species  with  respect 
to  the  features  of  the  buccal  cavity  but  it  does  not  seem  to  fit  either  Pierson's  or 
Tuffrau's  description  of  E.  patella  exactly.  Pierson  has  emphasized  the  desirability 
of  including  these  features  in  the  diagnostic  determination  of  species  of  this  genus. 

Clearly,  E.  daidaleos  is  closely  related  to  E.  patella  but  on  the  basis  of  difference 
in  body  shape,  constant  possession  of  zoochlorellae,  smaller  size  and  details  of  the 
structure  of  the  peristome,  E.  daidaleos  is  considered  to  be  a  distinct  species, 
hitherto  undescribed. 

SUMMARY 

1.  Euplotes  daidaleos  n.  sp.  is  described  as  a  hitherto  unrecognized  species.  It 
is  a  fresh-water  form  found  in  Philadelphia,  Pa.,  and  appears  to  be  closely  related 


EUPLOTES  DAIDALEOS  N.  SP.  445 

to  E.  patella,  differing  from  it  in  shape  and  size,  in  the  possession  of  zoochlorellae 
and  possibly  in  the  structure  of  the  peristome. 

2.  Diagnostic    characteristics :    Fresh-water.     Contains    zoochlorellae    (few    to 
100).     Average  length  92 /A,  width  57 /A.     Body  flattened,  oval  in  face  view;  right 
anterior  margin  straighter  than  the  more  convex  shoulder  on  the  left  side ;  poste- 
rior end  bluntly  pointed.     Buccal  cavity    (peristome)    extending  slightly  beyond 
middle  of  body.     Adoral  membranelles  approximately  40,  arranged  in  a  smooth 
curve  terminating  on  the  right  edge  of  the  collar.     Right  edge  of  the  peristome  an 
almost  straight  wall  originating  as  an  extension  of  the  ventral  ridge  at  the  left  of 
the  anal  cirri ;  undercut  so  as  to  form  a  narrow  lip,  anterior  to  the  paroral  mem- 
branelles ;  attached  to  it  and  the  dorsal  wall  of  the  buccal  cavity  is  a  low  bulge 
partly  occluding   the   cavity.     Between   this   bulge   and   the   elongated    triangular 
peristomal  plate  on  the  left  anterior  side  of  the  buccal  cavity  is  a  trough  or  channel 
expanded  anteriorly  as  well  as  posteriorly  in  the  region  of  the  paroral  membranelles. 
The  latter  delimit  the  endoplasmic  sac  on  the  right.     Eighteen  cirri :  six  frontals, 
three  ventrals,  five  anals  and  four  caudals,  two  of  the  latter  on  the  right  side  tending 
to  be  fimbriated.  Meganucleus  C-shaped.     Nine  latero-dorsal  rows  of  bristles,  cor- 
responding in  position  to  the  dorsal  ridges  (the  left  one  distinctly  ventral).     Be- 
tween each  dorsal  "kinety"  are  two  rows  of  alternating  wider  and  narrower  poly- 
gons, revealed  by  silver  impregnation  techniques.     The  dorsal  bristles  lie  on  the 
right  border  of  the  wide  rows  and  to  the  left  of  the  narrow'  rows. 

3.  Conjugation  is  common  in  the  stocks  of  E.  daidaleos  examined.     Encyst- 
ment  has  not  been  found. 

LITERATURE  CITED 

BERGER,  J.,  1965.     The  infraciliary  morphology  of  Enplotcs  tuff  rani,  n.   sp.     Protistologica,  1: 

17-32. 
BORROR,  A.   C.,   1962.     Euplotcs  ininuta  Yocum    (Ciliophora,   Hypotrichida).     /.  Protozool.,  9: 

271-273. 
BOVEE,  E.  C.,  1957.     Euplotes  leticiensis,  n.  sp.,  from  the  Letician  Drainage  into  the  Amazon 

River.     /.  Protozool.,  4:  124-128. 
BROWN,  ROLAXD  W.,   1954.     Composition  of  Scientific  Words.     Pub.  by  the  author ;    George 

W.  King  Printing  Co.,  Baltimore,  Md. 
DILLER,   W.    F.,    1966.     Correlation   of   ciliary   and   nuclear    development   in   the    life    cycle    of 

Enplotcs.    J.  Protozool.,  13:  43-54. 
DUSENBERRY,  P.  A.,  1966.     Genetics  of  Euplotes.     M.S.  Thesis  in  Biology,   Library,   LTniv.  of 

Pennsylvania,  Philadelphia,  Pa. 

EHRENBERG,  C.  G.,  1840.     Monatsber.  der  Berl.  Acad.  von  1840.     S.  200. 
KAHL,   A.,   1932.     Urtiere   oder   Protozoa.     I.   Wimpertiere   oder    Ciliata    (Infusoria').     In:    F. 

Dahl's  Die  Tierwelt  Deutschlands,  Gustav  Fischer,  Jena,  Teil  25,  399-650. 
KOUNARIS,  D.,   1964.     Conjugation   in  a  green  Euplotes  with   special   reference   to  the  kineto- 

somal  activity.     M.S.  Thesis  in  Biology,  Library,  Univ.  of  Pennsylvania,  Philadelphia, 

Pa. 
PIERSON,  B.  E.,  1943.     A  comparative  morphological  study  of  several  species  of  Euplotes  closely 

related  to  Euplotes  patella.    J.  Morph.,  72:  125-165. 
STEIN,   F.,    1859.     Der   Organismus   der    Infusionsthiere.     I.    Allgemeine    Theil    und    Naturge- 

schichte  der  Hypotrichen  Infusionsthiere.     Leipzig. 
TUFFRAU,  M.,   1960.     Revision  du  genre  Euplotcs,  fondee   sur  la  comparaison   des   structures 

superficielles.     Hydrobiologia,  15:  1-77. 
WICHTERMAN,    RALPH,    1964.     Description    and    life    cycle    of   Euplotcs    iicapolitainis    sp.    nov. 

(Protozoa,  Ciliophora,  Hypotrichida)  from  the  Gulf  of  Naples.     Trans.  Aincr.  Micros. 

Soc.,  83 :  362-370. 


THE  INFLUENCE  OF  LIGHT  ON  THE  SIZE  OF  AGGREGATIONS 
IN  DICTYOSTELIUM  DISCOIDEUM 

THEO  M.  KONIJN  1  AND  KENNETH  B.  RAPER 

Departments  of  liactcriolnf/y  and  Botany,  University  of  Wisconsin,  Madison,   Wisconsin  53706 

Fruiting  structures  of  the  Dictyosteliaceae  formed  in  light  are  smaller  and  more 
numerous  than  those  produced  in  comparahle  cultures  incubated  in  darkness  (Potts, 
1902;  Harper,  1932;  Raper,  1940;  Heller  and  Miles,  1961;  Shaffer,  1961;  Kahn. 
1964).  Since  the  size  of  a  fructification,  or  sorocarp,  is  dependent  to  a  large  extent 
upon  the  number  of  cells  that  enter  an  aggregation,  it  is  logical  to  seek  the  bases 
for  this  behavior  in  the  aggregative  process  per  sc.  Bonner  and  co-workers 
(Bonner  and  Dodd,  1962;  Bonner  and  Hoffman,  1963)  have  reported  that  for 
certain  species  of  Dictyosteliwm  the  size  of  the  aggregation  territory  remains  the 
same  in  cultures  grown  under  constant  environmental  conditions.  They  believe  it 
possible,  as  does  Shaffer  for  Polysphondyliitin  violaceutn  (Shaffer,  1961),  that  an 
inhibitory  substance  diffuses  outward  from  the  first-formed  centers  and  prevents 
the  formation  of  additional  ones,  thus  determining  the  disposition  of  developing 
aggregations.  To  this  putative  factor  they  have  applied  the  term  "spacing  sub- 
stance." More  recently,  Kahn  (1964)  has  suggested  that  cell  aggregation  in 
PolyspJwndylhnn  paUidinn  may  be  inhibited  in  darkness  by  a  center-suppressing 
factor,  the  effect  of  which  is  erased  by  illumination.  For  a  summary  of  published 
information  and  opinion  prior  to  1962  regarding  the  process  of  cell  aggregation  in 
the  Dictyosteliaceae,  the  reader  is  referred  to  Shaffer's  comprehensive  review 
(1962),  "The  Acrasina." 

The  effect  of  different  light  conditions  on  the  time  of  aggregation  in  Dictyo- 
steliiini  discoidenm  has  been  investigated  (Konijn  and  Raper,  1965).  But  no 
studies  have  been  made  to  determine  at  what  time  during  the  preaggregative  stage 
the  existing  light  conditions  influence  the  number  of  aggregations  formed  (and  by 
inference  the  size  of  aggregation  territories)  or  the  number  of  fruiting  bodies  that 
subsequently  develop.  The  present  study  attempts  to  assess  the  effect  of  light 
during  the  preaggregative  stage  on  the  number  of  aggregations  that  subsequently 
arise,  and  to  correlate  differences  in  the  size  of  such  aggregations  in  light  and  in 
darkness  with  possible  changes  in  the  activity  of,  or  the  cellular  responses  to,  the 
chemotactic  substance (s)  secreted  by  the  converging  myxamoebae. 

MATERIALS  AND  METHODS 

Dictyostelium  discoideum  Raper,  NC-4(H),  a  haploid  strain  derived  from 
the  diploid  stock,  NC-4,  was  the  culture  most  used  during  this  research.  The 
myxamoebae  were  grown  in  either  light  or  darkness  and  on  either  a  solid  medium 

1  Present  address :  Hubrecht  Laboratory,  Universiteitscentrum  de  Uithof,  Utrecht,  the 
Netherlands. 

446 


LIGHT  AND  AGGREGATION  SIZE  IN  DICTYOSTELIUM  447 

(Bonner,  1947)  in  association  with  Escherichia  coll  #281,  E.  coli  B/r  or  ^icro- 
bactcr  acrogcnes  #900,  or  in  shaken  tube  cultures  with  pregrown  E.  coli  B/r  ac- 
cording to  the  technique  of  Gerisch  (1959).  The  details  of  harvesting  the  myx- 
amoebae  were  given  in  our  previous  report  (Konijn  and  Raper,  1965). 

Populations  of  two  sizes  and  densities  were  employed  in  the  present  study: 

(1)  For  investigating  the  number  and  size  of  aggregations  that  would  develop 
under  different  light  conditions,  washed  myxamoebae  were  restispended   in   dis- 
tilled water,  or  in  Bonner's  salt  solution  (Bonner,  1947),  at  a  dilution  of  4  X  105 
cells/ml,  and  deposited  on  non-nutrient  agar  (1.5%)  as  0.1-ml.  aliquants.     Three 
such  drops  were  implanted  per  Petri  dish  and,  after  having  spread  on  the  agar 
surface,  covered  areas  1.5  to  1.9  cm.  in  diameter.     The  resultant  cell  density  was 
usually  about  200  to  250  myxamoebae  per/mm.2,  there  being  some  increase  in  the 
cell  populations  after  deposition  (see  Konijn  and  Raper,  1961). 

(2)  For  investigating  the  influence  of  light  on  cell  attraction,  minute  droplets 
of  a  much  denser  suspension   (1.5  X  107  cells/ml.)   were  deposited  on  a  soft  gel 
made  with  highly  purified  agar  and  Bonner's  salt  solution    (Konijn  and  Raper, 
1961).     The  number  of  cells  per  population  was  800  to  1600,  and  the  areas  covered 
by  the  droplets  were  approximately  0.5  mm.  in  diameter.     Depending  upon  the 
time  of  deposition,  populations  were  designated  as  either  "attracting"  or  "respond- 
ing."    From  150  to  200  droplets  containing  attracting  populations  were  first  de- 
posited on  the  surface  of  an  agar  plate  with  hand-drawn  micropipettes,  while  an 
equal  number  of  similar  droplets  containing  responding  populations  were  deposited 
later  at  distances  of  400  to  1200  /*  from  the  former.     The  concentration  of  the  agar 
used  to  form  the  gel  was  ca.  0.5%,  or  sufficient  to  yield  a  rigidity  of  35  to  40  grams 
expressed  as  the  weight  required  to  cause  the  end  of  a  microscope  slide  to  break 
the  agar  surface  (Konijn  and  Raper,  1961).     For  a  particular  experiment,  a  single 
cell  suspension  was  used  as  the  source  of  both  attracting  and  responding  popula- 
tions, the  suspension  being  held  in  a  refrigerator  until  the  latter  were  placed  on  agar. 

The  light  source  employed  was  "cool  white"  fluorescent  tubes,  and  the  light 
intensity  was  ca.  60  foot  candles  at  the  level  of  the  agar  surface  on  which  the 
myxamoebae  were  deposited.  The  incubation  temperature  was  23  ±  1°  C. 

RESULTS 

1.  The  influence  of  light  on  the  number  and  she  of  developing  aggregations  and 
sorocarps 

Myxamoebae  of  Dictyostelium  discoidcwn,  NC-4(H),  were  grown  on  agar  in 
darkness  and  harvested  in  the  preaggregative  stage.  After  removal  of  excess  bac- 
teria by  centrifugation,  the  cells  were  restispended,  deposited  on  non-nutrient  agar 
and  incubated  under  different  light  conditions.  The  total  number  of  fruiting  or- 
ganizations formed  within  the  populations  was  recorded  after  25  hours.  As  shown 
in  Figure  1,  more  and  smaller  aggregations  and  fruiting  structures  were  formed  in 
constant  light  than  in  constant  darkness.  Since  only  a  small  minority  of  the 
myxamoebae  stayed  outside  the  developing  aggregations  in  either  case,  such  cells 
could  not  account  for  the  lower  number  produced  in  the  dark.  Of  special  interest 
was  the  influence  of  an  initial  period  of  dark  incubation  of  varying  duration.  The 
number  of  sorocarps  formed  in  light  gradually  decreased  as  an  initial  dark  period 


448 


THEO  M.  KONIJN  AND  KENNETH  B.  RAPER 


was  increased,  and  the  lowest  number  developed  if  a  dark  period  optimal  for  early 
aggregation  (e.g.,  8  to  9  hours  darkness)  preceded  exposure  to  light.  Myxamoebae 
that  were  transferred  from  darkness  to  light  shortly  before  aggregation  began,  or 
in  the  early  aggregative  stage,  usually  produced  a  near  maximum  to  maximum 
number  of  fruiting  structures,  as  for  example  in  plates  incubated  in  darkness  for 
12  or  13  hours.  The  number  of  fruiting  structures  formed  under  similar  light  con- 
ditions varied  considerably  in  different  experiments.  After  an  initial  dark  period 
of  14  hours,  aggregations  in  some  experiments  were  already  well  advanced,  and  the 


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Time     of    Aggregation     in      Hours 

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Number    of    Aggregations 


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FIGURE  1.  The  effect  of  an  increasing  initial  dark  period  on  the  number  of  aggregations 
and  sorocarps  in  Dictyostelium  discoideum,  NC-4(H).  The  myxamoebae  were  grown  in  the 

dark  on  a  solid  medium.     The   results   of  five  experiments   are   included,    n^n  Light.  •• 

Darkness.      |    Number   of  aggregations  per  population   in   individual   experiments.     Each  mark 

represents  the  average  of  6  drops.  |  Average  number  of  aggregations  per  population  in  all 
experiments.  |  [  Average  time  of  aggregation. 

final  count  of  fructifications  in  such  plates  was  close  to  the  total  number  formed  in 
complete  darkness.  In  other  experiments,  the  myxamoebae  were  in  a  less  ad- 
vanced stage  and  in  these  the  number  of  aggregations  was  increased  substantially 
by  exposure  to  light.  Variation  in  the  diameters  of  the  drop  areas  due  to  minor 
differences  in  the  non-nutrient  agar  may  have  contributed  somewhat  to  the  incon- 
stant behavior.  However,  such  variation  should  not  have  been  greater  in  plates 
incubated  for  14  hours  in  darkness  than  in  the  other  series. 

The  same  general  approach  was  used  in  a  second  set  of  experiments  except  that 
the  myxamoebae  were  grown  in  shaken  cultures  in  the  dark.  In  these  tests,  varia- 
tion caused  by  differences  in  the  areas  covered  by  the  drops,  or  by  a  possible  "edge 


LIGHT  AND  AGGREGATION  SIZE  IN  DICTYOSTELIUM 


449 


effect"  at  the  boundaries  of  the  drops,  was  minimized  by  counting  only  the  number 
of  fruiting  structures  in  an  area  1.0  cm.  square  at  the  center  of  each  population. 
Counts  obtained  in  this  way  were  roughly  proportional  to  those  obtained  in  the 
earlier  experiments  when  all  the  sorocarps  that  developed  within  a  drop  were 
counted  (Fig.  2).  The  onset  of  aggregation  occurred  somewhat  earlier  than  with 


Periods  of  Dark  Incubation(hours)  followed  by  Light 
^>  ro  o  oo  a)  ^  ro 

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Time     of     Aggregation     in      Hours 
32                   4                   6                  8                  10                 12                 14 

1                       i                          n 

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1                          §                               n 

•               B                                n 

1                     §                         n 

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•                           a                            f] 

•                        a                               g 

•                                     a                                        n 

•            s                                                                     n 

50                     80                      100                     120                     140                     160 

Number    of    Aggregations 

FIGURE  2.  A  comparison  of  the  numbers  of  aggregations  in  entire  populations  of  Dictyo- 
stcliniii  discoidcum,  NC-4(H),  and  from  areas  1  cm.  square  at  centers  of  populations  of  com- 
parable size  and  density.  The  myxamoebae  for  the  latter  tests  were  grown  in  the  dark  in  a 
liquid  medium.  The  results  of  four  experiments  are  averaged.  I^HL  Light.  ••  Darkness. 
|  Average  number  of  aggregations  in  centrally  placed  areas  1  cm.  square.  Each  mark  repre- 
sents the  average  of  24  determinations,  j  \  Average  time  of  aggregation  in  the  four  experiments. 
I!  Average  numbers  of  aggregations  in  entire  populations  of  comparable  size  and  density. 
(Data  from  Fig.  1.) 


myxamoebae  dark-grown  on  the  solid  medium,  this  difference  being  as  much  as 
two  hours  in  cultures  incubated  in  the  dark  for  5  or  6  hours  prior  to  being  trans- 
ferred to  light. 

Other  species  and  strains  were  examined  in  a  third  set  of  experiments.  In 
Dictyostelium  purpurenui,  \YS  321,  and  in  Polysphondylium  pallidnni.  WS  320r 
the  number  of  aggregations  was  much  reduced  when  the  populations  of  myx- 
amoebae were  transferred  from  light  to  darkness  at  an  early  aggregative  stage.. 
If  the  cells  were  first  kept  in  the  dark  for  a  few  hours  and  later  exposed  to  light,, 
the  number  of  aggregations  increased  if  the  transfer  took  place  at  or  near  the 
beginning  of  aggregation.  Also,  in  D.  discoideum,  strain  Acr  12,  aggregations 


450 


THEO  M.  KONIJN  AND  KENNETH  B.  RAPER 


'occurred  earlier  and  in  greater  numbers  in  light  than  in  darkness.  Thus  inter-  and 
intraspecific  differences  in  response  to  light  must  occur  since  cell  populations  of 
low  density  (200-250  myxamoebae/mm.2)  were  employed,  as  for  D.  discoideitin, 
KC-4(H). 

2.   The  influence  of  lit/lit  during  the  preaggregatvue  stage  on  the  rate  of  increase  in 
the  number  of  aggregations 

Myxamoebae  of  Dictyostelium  discoideuui,  NC-4(H),  were  grown  in  shaken 
cultures  in  the  dark,  harvested,  washed  and  deposited  as  0.1 -ml.  drops  on  non- 
nutrient  agar.  Surveys  at  half-hour  or  hourly  intervals  after  the  cells  started  to 
aggregate  indicated  that  the  number  of  aggregations  increased  slowly  if  an  initial 


8 


10  12          14          16          18 

Time      in      Hours 


20         22 


24         26 


FIGURE  3.  The  effect  of  an  increasing  initial  dark  period  on  the  rate  of  increase  in  number 
of  aggregations.  L :  Light ;  D  :  Darkness ;  D-L :  the  drops  were  in  darkness  for  the  number 
of  hours  indicated  before  exposure  to  the  light. 

dark  period  was  lacking,  or  if  this  was  insufficient  to  induce  early  aggregation 
(Fig.  3).  After  a  dark  period  of  8  hours  or  longer,  however,  the  number  of 
aggregations  increased  rapidly,  and  within  a  period  of  one  to  three  hours  the  final 
number  was  reached.  Primary  aggregations  in  populations  exposed  to  light  after 
a  dark  incubation  of  10-13  hours  were  relatively  large  and  the  increase  in  number 
of  sorocarps  formed  in  such  plates  resulted,  in  substantial  part,  from  a  breaking 
up  of  the  streams  of  pre-existing  aggregations  into  smaller  pseudoplasmodia.  The 
sample  curves  presented  in  Figure  3  are  taken  from  a  much  more  extensive  series 
twice  confirmed  in  which  periods  of  light  and  dark  incubation  were  varied  at  incre- 
ments of  less  than  four  hours. 

3.  The  Influence  of  light  on  chemotaxis 

Information  relating  to  the  phenomenon  of  chemotaxis  was  obtained  by  observ- 
ing the  behavior  of  small  populations  of  myxamoebae  when  deposited  on  the  suface 


LIGHT  AND  AGGREGATION  SIZE  IN  DICTYOSTELIUM 


451 


FIGURE  4.  A  small  population  of  myxamoebae  deposited  on  washed  non-nutrient  agar  of 
low  rigidity.  Without  attracting  forces  from  the  outside  the  myxamoebae  stay  inside  the 
boundary  of  the  drop.  X  140. 

FIGURE  5.  Myxamoebae  in  a  responding  population  attracted  outside  the  boundary  of  the 
original  drop  by  an  aggregation  (a)  in  the  neighboring  drop.  The  myxamoebae  move  toward 
the  attracting  aggregation  by  wriggling  through  the  agar.  The  rigidity  of  the  agar  surface 
was  the  same  as  in  Figure  4.  X  100. 


452 


THEO  M.  KONIJN  AND  KENNETH  B.  RAPER 


of  washed,  non-nutrient  agar  of  low  rigidity  (Konijn  and  Raper,  1961).  The  soft 
agar  gel  provided  a  means  of  measuring  interpopulational  responses  since  it  was 
rigid  enough  to  keep  all  the  cells  within  the  confines  of  the  drop  in  the  absence 
of  an  extraneous  stimulus  (Fig.  4),  but  was,  at  the  same  time,  sufficiently  soft  to 
allow  the  myxamoebae  to  move  outside  the  drop  boundary  if  attracted  by  a  chemo- 
tactic  stimulus  secreted  by  a  neighboring  population.  Fortunately  for  our  pur- 
poses, the  myxamoebae  that  were  attracted  beyond  the  edge  of  such  a  drop  moved 
into  the  agar  (Fig.  5)  and  did  not  return  to  their  "home"  drop  until  its  residual 
cells  formed  their  own  aggregates.  Thus  data  relating  to  the  effect  of  chemotactic 
stimuli  could  be  obtained  by  employing  droplets  of  cells  of  one  preaggregative 
"age"  to  act  as  attractors  and  cells  that  were  less  mature  in  point  of  time  to  serve 
as  responders.  Drops  containing  attracting  and  responding  myxamoebae  were 


500  700  900  1100 

Distance  of  Attraction  in  Microns 


1300 


FIGURE  6.  The  percentages  of  myxamoeba  populations  that  showed  a  response  to  develop- 
ing aggregations  under  different  light  conditions,  and  the  distances  between  the  nearest  margins 
of  these  populations  and  the  aggregations  that  attracted  them.  Graphic  representation  of  data 
contained  in  Table  I.  O  :  Constant  light.  w  :  Constant  light,  but  with  plates  sealed  after  the 
responding  drops  were  deposited  on  the  agar.  •  :  Constant  darkness. 


deposited  on  the  agar  surface  at  different  distances  from  each  other  and  the  plates 
were  then  incubated  in  either  light  or  darkness.  The  distance  over  which  attraction 
could  take  place  was  taken  as  a  measure  of  the  strength  of  the  stimulus  produced 
by  the  population  of  aggregating  myxamoebae.  This  distance  wras  measured  not 
between  the  proximal  edges  of  the  two  drops  but  from  the  center  of  the  developing 
aggregation  in  the  attracting  drop  to  the  nearest  margin  of  the  responding  drop. 
The  response  was  considered  as  positive  when  two  or  more  cells  moved  outside  the 
edge  of  the  responding  drop  toward  the  attracting  population.  Responding  myx- 
amoebae rarely  moved  more  than  500 /A  outside  their  "home"  drops. 

For  the  actual  tests,  minute  droplets  of  a  dense  suspension  of  cells  pregrown 
on  a  solid  medium  were  deposited  on  low-rigidity  \vashed  agar  and  occupied  areas 
approximately  0.5  mm.  in  diameter.  In  preliminary  experiments,  responding 
populations  plated  three  hours  after  the  attracting  populations  showed  less  response 


LIGHT  AND  AGGREGATION  SIZE  IN  DICTYOSTELIUM 


453 


when  incubated  in  the  light  than  in  darkness.  Fifty  per  cent  of  the  attracting 
small  populations  incubated  in  the  light  aggregated  after  about  8-10  hours,  which 
was  on  average  one  hour  earlier  than  those  in  the  dark,  and  it  was  thought  that 
responding  cells  that  were  themselves  nearer  in  time  to  aggregation  might  have  an 
increased  sensitivity  to  chemotactic  substances.  In  order  that  the  "physiological 
age"  of  the  responding  drops  in  light  and  in  dark  might  be  more  nearly  identical 
at  the  time  aggregation  began  in  their  counterpart  attracting  drops,  the  responding 
cells  to  be  incubated  in  the  dark  were  deposited  one  hour  later  than  those  to  be 
incubated  in  the  light.  The  influence  of  light  on  attraction  by  aggregating  myx- 
umoebae  is  graphically  presented  in  Figure  6.  Attraction  in  the  dark  was  ob- 
served to  occur,  on  the  average,  over  a  longer  distance  than  in  the  light  (Table  I). 
For  example,  50%  of  the  illuminated  populations  showed  a  response  over  a  distance 
of  940  fj.,  while  an  equal  percentage  of  those  incubated  in  the  dark  responded  over 
a  distance  of  1090  \*.. 

TABLE  I 

Attraction  of  myxamoebae  in  populations  of  responding  celh  by  developing  aggregations  in 

plates  incubated  in  light,  in  darkness,  and  in  light  with  plates  sealed.      The  2056 

populations  represented  in  this  table  were  tested  in  three  different  experiments 


Distance  between 

Light 

Light,  sealed 

Darkness 

aggregations  and  > 

the  responding 

populations 

+ 

— 

% 

+ 

— 

% 

+ 

— 

% 

600-  700  n 

17 

0 

100 

700-  800  n 

47 

2 

96 

30 

0 

100 

17 

0 

100 

800-  900  p 

126 

22 

85 

137 

23 

86 

153 

0 

100 

900-1000  n 

98 

111 

47 

125            77 

62 

233 

25 

90 

1  000-  11  00  ft 

45 

118 

28 

49 

84 

37 

159 

76 

68 

1  100-1200  n 

2 

61 

3 

2 

57 

3 

20 

58 

26 

1200-1  300  M 

0 

27 

0 

0 

22 

0 

0 

33 

0 

Code:  +  Test  populations  responding  to  an  aggregation  in  the  "attracting  drop";  -  Test 
populations  not  responding  to  an  aggregation  in  the  "attracting  drop";  %  Percentage  of  test 
populations  responding. 

A  possible  decrease  of  humidity  in  plates  incubated  in  the  light  could  have 
affected  the  agar  surface  and  consequently  limited  the  movement  of  the  responding 
cells  outside  the  edge  of  the  drop.  For  this  reason,  control  plates  in  the  light  were 
sealed  with  masking  tape  to  prevent  evaporation  and  to  reduce  any  possible  gaseous 
exchange  which  might  influence  aggregation,  as  has  been  reported  by  Bonner  and 
Hoffman  (1963).  The  distance  over  which  attraction  occurred  in  sealed  plates 
in  three  different  experiments  was  equal  to  or  slightly  greater  than  that  observed 
in  the  unsealed,  light-incubated  plates,  but  it  was  never  equivalent  to  that  recorded 
in  the  dark  (Fig.  6). 

DISCUSSION 

When  an  initial  dark  period  optimal  for  early  aggregation  in  Dictyostelium 
dlscoideum,  NC-4(H),  was  employed  (see  also  Konijn  and  Raper,  1965),  the 
number  of  sorocarps  was  less  than  in  constant  light  and  greater  than  in  constant 


454  THEO  M.  KONIJN  AND  KENNETH  B.  RAPER 

darkness.  A  further  increase  in  the  period  of  dark  incubation  resulted  in  a  delay 
of  aggregation  and  an  increase  in  the  number  of  sorocarps,  particularly,  if  the 
transfer  to  light  occurred  near  or  at  the  onset  of  aggregation.  The  number  of 
sorocarps,  however,  was  not  necessarily  related  to  the  time  of  aggregation.  Myx- 
amoebae  of  D.  disco  id  cum  incubated  in  continuous  light  aggregated  slightly  later 
than  cells  kept  in  darkness,  but  formed  sorocarps  more  abundantly.  Light  could 
exert  its  effect  by  a  stimulation  of  center  formation,  or  by  an  inhibition  of  the 
spacing  substance  (Bonner  and  Hoffman,  1963),  e.g.,  by  reducing  the  sensitivity 
of  cells  to  it.  It  is  questionable  whether  a  gaseous  spacing  substance  determines 
the  number  of  aggregations  and  consequently  influences  the  number  of  fruiting 
structures  in  this  species,  for  Bonner  and  Hoffman  (1963)  noticed  that  the  gaseous 
spacing  substance  that  has  such  a  pronounced  effect  on  D.  muc  oroides  does  not 
affect  the  myxamoebae  of  D.  discoideum,  although  the  latter  are  able  to  produce 
a  gaseous  substance  that  influences  the  spacing  in  other  species. 

The  increase  in  the  number  of  sorocarps  produced  when  myxamoebae  are 
exposed  to  light  at  an  early  aggregative  stage  is  dependent,  at  least  in  part,  on 
a  delicate  balance  within  the  aggregations.  For  example,  if  populations  in  the 
process  of  aggregating  were  placed  under  fluorescent  light,  the  aggregates  already 
formed  would  sometimes  break  up  into  several  smaller  pseudoplasmodia.  At  other 
times  the  binding  forces  within  the  aggregations  were  strong  enough  to  prevent 
severance  of  streams  when  exposed  to  light,  and  no  significant  increase  in  the 
number  of  sorocarps  occurred. 

When  a  very  few  aggregations  are  formed  early,  as  after  a  short  initial  dark 
period  (Konijn  and  Raper,  1965),  one  would  expect  these  centers  to  extend  their 
spheres  of  attraction  over  large  areas  and  subsequently  to  produce  only  a  few  and 
large  sorocarps.  However,  this  does  not  occur ;  instead  many  aggregations  are 
formed  although  the  increase  in  number  occurs  slowly.  When  all  aggregations 
appear  at  about  the  same  time,  as  after  a  long  initial  dark  period,  their  size  is  gen- 
erally larger  and  their  number  less. 

The  limited  size  of  aggregations  in  the  former  populations  may  be  due  to  a 
reduced  acrasin  secretion  per  cell  as  the  size  of  the  aggregate  increases,  and,  after 
a  certain  acrasin  concentration  has  been  reached,  additional  cells  entering  the 
aggregate  may  not  further  increase  the  level  of  acrasin.  This  concentration  may 
be  sufficient  to  attract  all  cells  within  the  aggregate's  territory.  This  would  con- 
form with  Shaffer's  observation  that  acrasin  sources  of  various  sizes  seem  to  secrete 
at  the  same  concentration  (Shaffer,  1957),  and  would  support  his  assumption  that 
the  acrasin  secretion  per  cell  is  inversely  related  to  the  size  of  the  aggregate 
(Shaffer,  1962). 

It  is  a  common  observation  that  streams  of  myxamoebae  flowing  into  centers 
in  darkness  are  longer  than  those  in  light.  This  may  result  in  part  from  an  in- 
creased stickiness  of  the  cells  in  darkness  that  is  reflected  in  somewhat  longer 
streams,  which  in  turn,  since  these  also  secrete  acrasin,  attract  additional  sensitive 
myxamoebae  to  further  enlarge  the  aggregations.  But  this  is  only  one  of  many 
ways  in  which  light  may  act  on  aggregating  myxamoebae. 

Light  may  depress  the  formation  of  acrasin,  or  its  precursors,  or  have  a  regu- 
lating action  on  the  secretion  of  acrasin,  e.g.,  by  changing  the  permeability  of  the 
cell  membranes.  If  light  affects  the  attraction  of  the  cells  it  may  do  so  by  an 


LIGHT  AND  AGGREGATION  SIZE  IN  DICTYOSTELIUM  455 

inactivation  of  acrasin,  or  by  altering  the  sensitivity  of  the  myxamoebae  to  it. 
That  inactivation  of  acrasin.  presumably  enzymatic,  does  occur  has  been  shown 
by  Shaffer  (1956).  Another  effect  of  increased  inactivation  of  acrasin  could  be 
the  occurrence  of  a  steeper  gradient,  which  would  favor  earlier  aggregation.  If 
inactivation  of  acrasin  is  enhanced  by  light,  attraction  should  occur  over  a  shorter 
distance  in  light,  hence  results  in  the  formation  of  smaller  aggregations  and  soro- 
carps.  Smaller  sorocarps  were  actually  observed  in  plates  incubated  in  the  light, 
and  a  reduced  attraction  of  responder  cells  in  populations  exposed  to  light  was 
observed  in  an  assay  system  in  which  attracting  and  responding  cells  were  separated 
from  each  other  by  different  distances  at  the  time  attraction  occurred. 

This  work  was  made  possible  by  research  grants  from  the  National  Institutes 
of  Health  (CA  02119-09),  U.  S.  Public  Health  Service,  and  the  National  Science 
Foundation  (G-24953). 

SUMMARY 

1.  Myxamoebae  of  Dictyostelium   dlscoidcuin,   NC-4(H),   were   pregrown   in 
the  dark  on  Eschcricliia  coll  or  Aerobacter  aerogcncs,  washed  and  deposited  on 
non-nutrient  agar.     Populations  incubated  in  constant  light  produced  more  and 
smaller  aggregations  and  sorocarps  than  similar  populations  incubated  in  the  dark. 
If  populations  were  incubated  in  darkness  for  several  hours  and  then  transferred 
to  light,  the  number  of  aggregations  was  reduced  and  the  dimensions  of  these  and 
the  resulting  sorocarps  were  correspondingly  greater.     The  rate  of  increase  in  the 
number  of  aggregations  was  most  rapid  if  the  myxamoebae  were  exposed  to  a  long 
initial  dark  period  followed  by  light. 

2.  The  chemotactic  response  of  myxamoebae  incubated  in  light  or  darkness  was 
studied  by  depositing  "attracting"  and  "responding"  cells  in  separate  small  popula- 
tions at  predetermined  distances  from  each  other.     The  sphere  of  attraction  by 
myxamoebae  aggregating  in  light  was  found  to  extend  over  a  shorter  distance  than 
that  of  cells  aggregating  in  darkness.     Among  other  possibilities,  inactivation  of 
the  attracting  substance  (s)  in  the  light  may  account  for  reduced  attraction,  hence 
result  in  smaller  aggregations. 

LITERATURE  CITED 

BONNER,  J.  T.,  1947.  Evidence  for  the  formation  of  cell  aggregates  by  chemotaxis  in  the 
development  of  the  slime  mold  Dictyostelium  discoideitm.  J.  E.rp.  Zool.,  106:  1-26. 

BONNER,  J.  T.,  AND  M.  R.  DODD,  1962.  Aggregation  territories  in  the  cellular  slime  molds. 
Biol.BulL,  122:  13-24. 

BONNER,  J.  T.,  AND  M.  E.  HOFFMAN,  1963.  Evidence  for  a  substance  responsible  for  the  spac- 
ing pattern  of  aggregation  and  fruiting  in  the  cellular  slime  molds.  /.  Embryol.  E.vp. 
Morph.,2:  571-579. 

GERISCH,  G.,  1959.  Ein  Submerskulturverfahren  fur  entwicklungsphysiologische  Untersuch- 
ungen  an  Dictyostelium  discoideitm.  Naturwiss,,  23:  654-656. 

HARPER,  R.  A.,  1932.  Organization  and  light  relations  in  Polysphondylium.  Bull.  Torrey  Bot. 
Club,  59:  49-84. 

HELLER,  S.  A.,  AND  M.  G.  MILES,  1961.  The  effect  of  humidity  and  light  on  the  concentration 
and  distribution  of  sorocarps  of  Dictyostelium  pnrpitrcitin,  str.  2.  Senior  thesis, 
Princeton. 


456  THEO  M.  KONIJN  AND  KENNETH  B.  RAPER 

KAHN,  A.  J.,  1964.  The  influence  of  light  on  cell  aggregation  in  Polysphondylium  pallidum. 
Biol.Bitll.,  127:  85-96. 

KONIJN,  T.  M.,  AND  K.  B.  RAPER,  1961.  Cell  aggregation  in  Dictyostelium  discoideum. 
Develop.  Bio!.,  3:  725-756. 

KONIJN,  T.  M.,  AND  K.  B.  RAPER,  1965.  The  influence  of  light  on  the  time  of  cell  aggregation 
in  the  Dictyosteliaceae.  Biol.  Bull.,  128:  392-400. 

POTTS,  G.,  1902.     Zur  Physiologic  des  Dictyostelium  mucoroides.    Flora,  91:  281-347. 

RAPER,  K.  B.,  1940.  Pseudoplasmodium  formation  and  organization  in  Dictyostelium  discoideum. 
J.  Elisha  Mitchell  Sci.  Soc.,  56:  241-282. 

SHAFFER,  B.  M.,  1956.  Acrasin,  the  chemotactic  agent  in  the  cellular  slime  moulds.  /.  Exp. 
Biol.,  33:  645-657. 

SHAFFER,  B.  M.,  1957.  Properties  of  slime-mould  amoebae  of  significance  for  aggregation. 
Quart.  J.  Micr.  Sci.,  98:  377-392. 

SHAFFER,  B.  M.,  1961.  The  cell  founding  aggregation  centres  in  the  slime  mould  Polysphon- 
dylium violaccum.  J.  Exp.  Biol.,  38:  833-849. 

SHAFFER,  B.  M.,  1962.  The  Acrasina.  Adv.  in  Morphogenesis.  Vol.  II.  pp.  109-182.  Aca- 
demic Press  Inc.  New  York  and  London. 


PREDICTING  DEVELOPMENT  RATE  OF  COPEPOD  EGGS  l 

IAN  A.  MCLAREN  2 

Marine  Sciences  Centre,  McGill  University,  Mnutrcul,  Quebec 

This  paper  is  part  of  a  continuing  study  of  intrinsic  controls  of  growth,  develop- 
ment and,  implicitly,  productivity  of  marine  zooplankton.  Copepods  are  particularly 
suitable  for  comparative  studies  of  embryonic  development  rate  because  most  spe- 
cies hatch  at  a  morphologically  equivalent  first  naupliar  stage.  The  effect  of  tem- 
perature on  size  varies  markedly  among  different  geographical  populations  of  the 
copepod  Pseudocalanus  in  nut  t  us  Krjziyer,  and  it  was  assumed  that  embryonic  devel- 
opment rate  would  vary  likewise  (McLaren,  1965a).  However,  after  trips  in  the 
spring  of  1965  to  Woods  Hole,  Mass.,  Halifax,  N.  S.,  and  Millport,  Scotland,  it 
was  clear  that  embryonic  development  rate  varied  only  slightly,  and  attention  was 
directed  to  other  species.  A  summer  trip  to  Frobisher,  N.  W.  T.,  was  made  par- 
ticularly to  secure  data  on  a  large-egged  form  of  Pseudocalanus  living  in  Ogac 
Lake,  a  warm,  landlocked  fiord  off  Frobisher  Bay  (McLaren,  1965a).  Successful 
experiments  at  Frobisher  on  the  large  Calanus  glacialis  Jaschnov  (see  Grainger, 
1961)  were  doubly  useful  because  of  published  data  on  C.  fininarcliicits  (Gunnerus  ) 
from  Scotland  and  Norway  (Marshall  and  Orr,  1953).  A  brief  return  to  Halifax 
in  April,  1966,  added  two  more  species,  Acartia  claiisi  Giesbrecht  and  Tor  tan  us 
discandatits  (Thompson  and  Scott). 

Although  the  number  of  forms  studied  is  rather  small,  the  results  are  published 
at  this  time  because  they  seem  to  have  some  general  and  theoretical  interest.  More 
work  will  be  done  to  confirm  the  results  and  hopefully  to  extend  their  predictive 
value  to  other  developmental  stages. 

Use  will  be  made  of  Belehradek's  (1935,  1957)  equation,  in  which  rate  of  a 
metabolic  function  (here,  development  time  D  in  days)  is  given  by 

D==a(T    -«)ft 

where  a,  b  and  a  are  constants  and  T  is  the  temperature.  The  empirical  superiority 
of  this  equation  and  the  conceptual  meaning  of  its  parameters  have  been  discussed 
by  McLaren  (1963,  1965b).  Briefly,  the  formula  is  the  simplest  of  several  equa- 
tions describing  the  three  ways  in  which  montonic  responses  to  temperature  may 
differ :  a  accounts  for  differences  in  mean  slope,  a  for  shifts  on  the  temperature 
scale,  and  b  depicts  the  degree  of  curvilinearity  of  the  response  quite  adequately 
over  the  vital  temperature  range.  This  paper  will  show  that  the  three  parameters 
are  related  to  separate  biological  properties  as  well.  The  equation  is  fitted  by 
conversion  to  logarithms  and  successive  approximation  to  that  value  of  a  having 
smallest  sums  of  squares  of  deviations  of  observed  from  calculated  development 
times. 

1  Supported  by  a  grant  from  the  National  Research  Council  of  Canada  and  a  fellowship 
from  the  Canada  Council. 

2  Present  address  :  Biology  Department,  Dalhousie  University,  Halifax,  Nova  Scotia. 

457 


458  IAN  A.  MCLAREN 

The  author  is  grateful  to  the  Woods  Hole  Oceanographic  Institution,  the  Insti- 
tute of  Oceanography  at  Dalhousie  University  in  Halifax,  the  Marine  Station  at 
Millport  in  Scotland,  and  the  Department  of  Northern  Affairs  and  National  Re- 
sources at  Frobisher  in  N.W.T.  for  working  facilities  and  assistance.  Drs.  Carl 
M.  Boyd,  J.  C.  Carter,  R.  C.  Conover,  and  Sheina  M.  Marshall,  F.R.S.,  were 
especially  helpful  during  the  experimental  programs.  Mr.  R.  E.  Banks,  Naval 
Research  Establishment,  supplied  temperature  data  from  Halifax. 

MATERIALS  AND  METHODS 

The  methods  used  were  simple  but  at  times  onerous.  Animals  were  captured 
by  fine-mesh  nets  and  samples  kept  cool  during  the  return  to  the  laboratory.  Indi- 
vidual females  were  removed  from  the  samples  within  an  hour  or  two  of  capture 
with  the  aid  of  dissecting  microscopes  and  eyedroppers.  The  females  were  kept 
in  small  groups  in  bottles  at  temperatures  about  as  cool  as  the  waters  from  which 
they  were  captured.  During  earlier  series  of  experiments  on  P.  uiinittus,  concen- 
trations of  diatoms  from  net  samples  were  added  to  the  bottles  in  the  hope  of 
stimulating  egg-laying.  It  was  later  found  that  eggs  would  be  produced  without 
the  addition  of  food,  provided  the  females  came  from  phytoplankton-rich  water. 
The  bottles  were  observed  regularly  and  free  eggs  (most  species)  or  females  carry- 
ing egg  sacs  (Pseudocalamts^  were  removed  by  eyedroppers  as  they  appeared. 
These  were  placed  in  small  vials  with  about  10  cc.  of  filtered  sea  water.  Only  a 
small  proportion  of  females  produced  eggs,  usually  within  a  day  or  so  of  capture. 
Pertinent  collection  and  experimental  data  are  given  on  Table  I. 

The  vials  were  kept  at  controlled  temperatures  until  the  eggs  hatched.  Accu- 
rate constant-temperature  baths  were  available  at  Woods  Hole,  Halifax,  and  Mill- 
port.  Baths  in  less  well-controlled  ambient  temperatures  of  cold  rooms  were  also 
available,  and  somewhat  variable  temperatures  of  around  2-3°  C.  were  obtained 
in  domestic-type  refrigerators.  Only  a  domestic  refrigerator  was  available  at 
Frobisher,  and  higher  temperatures  \vere  maintained  there  by  periodic  additions 
of  ice  or  warm  water  to  large,  covered,  styrene-foam  containers  kept  in  the  cool 
out-of-doors.  These  portable  containers  were  also  useful  in  completing  experi- 
ments begun  at  Ogac  Lake  and  finished  at  the  townsite  at  Frobisher,  a  day's  boat- 
trip  from  Ogac  Lake.  A  temperature  of  0°  was  easily  obtained  in  ice-water  baths 
kept  just  under  the  freezing  compartments  of  domestic  refrigerators.  Because  of 
the  long  time  required  for  development,  most  of  the  experiments  at  0°  and  some  of 
those  at  higher  temperatures  were  carried  out  on  eggs  produced  by  females  brought 
by  air  to  Montreal  in  vacuum  bottles.  A  few  experiments  begun  elsewhere  were 
finished  in  Montreal — the  vials  kept  at  the  appropriate  temperatures  in  vacuum 
bottles  during  air  transport.  Clearly  it  was  impossible  to  maintain  rigidly  con- 
stant temperatures  under  some  of  the  above  experimental  conditions.  However, 
temperatures  were  kept  within  narrow  limits  and  observed  frequently  (at  least 
every  3-4  hours),  24  hours  a  day.  Since  the  effect  of  temperature  is  virtually 
linear  over  a  reasonably  narrow  range,  the  graphically  estimated  mean  tempera- 
tures may  be  taken  as  extremely  accurate  measures  of  the  effective  temperatures 
during  development. 

Experimental  salinities  were  not  quite  the  same  in  all  localities,  but  differed 
markedly  only  in  a  second  series  of  experiments  on  Pscudocalaniis  from  Ogac  Lake. 


DEVELOPMENT  RAT?:  OF  COPEPOI)  E(,(iS 


459 


TABLE  I 

Mean  development  times  of  copepod  eggs  at  different  temperatures.     Each  experiment  is  a  single  egg 
sac  (Pseudocalanus)  or  a  batch  of  eggs  produced  more  or  less  synchronously  by  one  or  more  females 

(other  spp.) 


Mean 

Species 

Locality 

Time-  oi 
capture 

Kxperi- 
mental 
salinity 

(  '".,  ) 

No.  of 
experi- 
ments 

experi- 
mental 
tem- 
pera- 

Time ot  50%  hatch  of 
viable  eggs  (days) 

\       f  (    i 

ture 

Mean  +95% 

range 

(°  C.) 

f.l. 

Pseudocalanus  mi  nut  us 

Woods  Hole,  Mass. 

Jan.  27- 

31.8 

7 

0 

10.  89  ±0.20 

10.54-11.14 

Feb.  Id, 

31.8 

3 

3.18 

6.61 

6.49-6.75 

'65 

31.8 

9 

4.60 

5.91  ±0.09 

4.43-4.70 

31.8 

1 

11.38 

3.07 

— 

31.8 

3 

13.13 

2.78 

2.69-  2.83 

I'ti-uitofiilanus  minutus 

Halifax,  N.S. 

Mar.  19- 

30.4 

9 

0 

10.71  ±0.22 

10.56-11.14 

29,  '65 

30.4 

1  1 

2.73 

6.92  ±0.09 

6.71-   7.16 

30.4 

4 

5.35 

5.05.J-0.27 

4.90-  5.31 

30.4 

1  1 

9.27 

3.  48  ±0.04 

3.38-  3.58 

30.4 

14 

12.01 

2.  90  ±0.05 

2.75-  3.00 

Pseudocalanus  mi  nut  us 

Millport,  Scotland 

Mav  17-31, 

30.4 

4 

0 

11.15±0.19 

11.03-11.24 

'65 

30.4 

2 

2.65 

7.50 

7.42-   7.58 

30.4 

1 

3.14 

7.15 

— 

31.3 

5 

4.53 

6.05  ±0.14 

5.92-  6.19 

31.3 

5 

6.64 

4.84  ±0.20 

4.64-   4.94 

31.3 

7 

10.05 

3.35  ±0.13 

3.20-  3.49 

31.3 

5 

12.95 

2.  75  ±0.10 

2.63-  2.79 

31.3 

2 

14.90 

2.61 

2.57-  2.65 

I'tfudnfaliinus  in  in  itt  us 

Frobisher.  \.\V.T. 

I  une  24- 

32.3 

7 

0 

10.44±0.19 

10.18-10.71 

July  8.  '65 

32.3 

9 

2.49 

7.50±0.23 

7.13-  8.00 

32.3 

8 

5.42 

5.32  ±0.13 

5.19-  5.67 

32.3 

5 

7.90 

4.14±0.20 

3.98-  4.40 

f'si'ititm'tiliinits  ininitlHS 

Ogac  Lake,  N.W.T. 

July  18 

32.3 

1 

0 

11.22 

_ 

& 

25.7 

3 

0 

10.86 

10.61-11.21 

Aug.  3.  '65       25.7 

3 

2.62 

7.43 

7.33-  7.62 

32.3                2               3.30 

6.94 

6.87-   7.00 

25.7 

3 

5.34 

5.55 

5.46-  5.67 

32.3 

1 

7.10 

4.35 

— 

Pseudocalanus.  large  form 

Ogac  Lake,  N.W.T. 

July  18 

32.3 

2 

0 

17.91 

17.08-18.75 

& 

25.7 

1 

0 

17.27 



Aug.  3. 

32.3 

1 

2.67 

12.23 

— 

'65 

25.7 

1 

3.31 

10.46 

— 

25.7 

3 

5.19 

9.06 

8.92-  9.30 

Calanus  glacialis 

Frobisher,  N.W.T. 

July  8  '65 

32.3 

1 

0 

6.35 



32.3 

1 

2.60 

4.30 



32.3 

1 

5.23 

3.23 



32.3 

1 

7.92 

2.61 

— 

Aini'tia  clausi 

Halifax,  N.S. 

April  5  & 

30.5 

1 

0 

15.33 

_ 

7,  '66 

30.5 

3 

4.88 

5.98 

5.88-  6.05 

30.5 

3 

10.80 

2.78 

2.69-  2.87 

Tint  anus  di  scandal  us 

Halifax,  N.S. 

April  5  & 

30.5 

1 

0 

19.71 

_ 

7.  '66                 30.5 

3 

4.85 

8.15 

8.00-  8.25 

30.5 

3 

10.80 

3.93 

3.82-  3.98 

1 

Much  mortality  had  occurred  during  the  first  series  of  experiments  at  higher  salini- 
ties. Since  Ogac  Lake  is  somewhat  brackish,  it  was  thought  that  filtered  lake 
water  would  be  more  suitable,  but  mortality  was  still  quite  high.  The  possibility 
that  salinity  had  an  effect  on  development  rate  of  eggs  from  Ogac  Lake  will  be 
considered. 

Not  all  eggs  hatched,  but  those  that  did  produced  nauplii  within  a  period  of  a 
few  hours  at  most  in  a  given  vial.  After  eggs  began  hatching,  experimental  vials 
-were  observed  frequently  so  that  time  of  50%  hatch  of  viable  eggs  could  be  deter- 


460 


IAN  A.  MCLAREN 


mined  accurately.  Above  certain  critical  temperatures,  which  varied  with  locality,, 
no  eggs  hatched.  At  lower  temperatures,  some  hatches  of  eggs  failed  partly  or 
completely  to  hatch,  and  this  sometimes  appeared  to  be  associated  with  bacterial 
or  protozoan  infestation.  No  antibiotics  were  used,  but  the  sea  water  in  some 
vials  was  changed  when  it  appeared  dirty.  Arbitrarily,  all  experiments  in  which 
fewer  than  half  the  eggs  proved  viable  are  excluded  from  the  following  analyses, 
in  view  of  the  possibility  that  high  mortality  is  accompanied  by  pathological  or 
"unphysiological"  retardation  of  development  among  the  survivors. 


TABLE  II 
Parameters  of  Belehrddek's  temperature  function  fitted  to  development  time  of  cope  pod  embryos 


b  taken  as 

Mean  egg 

Relative  optical 

Three  constants  fitted 

-1.68 

Species 

Locality 

diameter 

density  (ratio 

0*±95%f.I.) 

±95%  f.l.) 

a 

a. 

b 

a 

a 

Pseudocalanus 

Woods  Hole 

127.4±3.7 

— 

325 

-   9.2 

-1.53 

552 

-10.52 

minutus 

P.  minutus 

Halifax 

121.6±1.8 

1.04±0.10 

159 

-   6.3 

-1.23 

516 

-10.20 

p.  minutus 

Millport 

123.6±1.8 

— 

425 

-   9.4 

-1.62 

536 

-   9.99 

P.  minutus 

Frobisher 

130.4±3.3 

(taken  as  1.00) 

8433 

-16.6 

-2.39 

572 

-10.77 

P.  minutus 

Ogac  Lake 

108.5±2.3 

0.95  ±0.11 

3296 

-14.0 

-2.16 

543 

-10.27 

Pseiidocalanus, 

Ogac  Lake 

155.3±2.6 

1.04±0.14 

82 

-   5.0 

-0.95 

908 

-10.47 

large  form 

Calanus  glacialis 

Frobisher 

178.6±2.5 

0.39±0.03 

44 

-   6.1 

-1.07 

308 

-11.23 

C.finmarchicus 

Troms0 

ca.  145 

•  — 

155 

-   8.7 

-1.57 

231 

-   9.72 

C.  finmarchicus 

Millport 

ca.  145 

— 

6 

1.2 

-0.65 

204 

-   9.43 

Acartia  chnisi 

Halifax 

79.4±3.4 

2.21±0.35 

1679 

-  8.9 

-2.15 

322 

-   6.02 

Tortanus 

Halifax 

102.4±4.7 

1.29±0.18 

2307 

-   9.4 

-2.12 

477 

-   6.55 

discatidatus 

Eggs  were  measured  with  optical  micrometers  at  X  40  or  greater.  Diameters 
given  are  of  unpreserved  eggs.  Maximum  and  minimum  diameters  were  averaged 
for  near-spherical  eggs,  but  the  three  appropriate  diameters  were  taken  of  the 
flattened  spheroid  eggs  of  T.  discaudatus. 

A  simple  and  perhaps  rather  crude  method  was  used  to  determine  optical 
density  of  formalin-preserved  eggs,  using  a  photomicrographic  exposure  meter 
(Photovolt  Corp.,  New  York,  model  514-M).  Eggs  were  placed  on  depression 
slides  in  clean  formalin-sea  water.  Each  egg  was  measured,  then  centered  alone 
in  the  microscopic  field  at  X  250,  in  low,  unfiltered  illumination  from  a  6-volt  wet- 
battery.  The  light  cell  was  then  applied  to  the  photographic  ocular  and  the  meter 
deflection  caused  by  moving  the  egg  in  and  out  of  the  center  of  the  field  was  noted. 
Since  the  eggs  are  rather  uniformly  granular,  it  is  assumed  that  no  defraction 
problems  were  involved  and  that  the  meter  deflections  were  a  valid  measure  of 
the  optical  extinction  caused  by  the  volume  of  matter  in  the  egg.  Deflections  were 
of  the  range  of  5-20  units  per  egg ;  calibration  is  not  exact,  but  one  unit  is  of  the 
order  of  10~5  foot  candles. 


DEVELOPMENT  RATE  OF  COPEPOD  EGGS  461 

RESULTS 
Effects  of  temperature 

The  experimental  results  are  summarized  on  Table  I.  Clearly  there  are  dif- 
ferences in  temperature  response  among  the  various  species.  Development  rate 
of  P.  miinitits  varies  only  slightly  in  different  parts  of  its  range.  The  differences 
are  in  some  cases  significant  (compare,  for  example,  the  rate  at  0°  at  Frobisher 
and  Millport),  but  not  nearly  as  great  as  expected  from  preliminary  experiments 
on  relative  rate  of  development  (McLaren,  1965a). 

Belehradek's  temperature  functions  have  been  fitted  to  all  the  data  on  Table  I, 
and  the  resulting  parameters  are  on  Table  II.  For  simplicity,  mean  development 
times  were  used,  weighted  by  number  of  experiments  at  each  temperature;  the 
error  in  this  procedure  is  probably  small  and  unsystematic.  Published  data 
(Marshall  and  Orr,  1953)  on  development  times  of  C.  finntarchicus  are  also 
analyzed  on  Table  II.  The  data  were  published  as  ranges  in  hours  and  were 
averaged  for  purposes  of  calculation. 

Since  the  calculated  values  of  a  on  Table  II  are  generally  well  below  the  mini- 
mal experimental  temperature  (0°),  the  resolution  of  the  function  is  very  low,  and 
the  wide  ranges  of  the  three  fitted  parameters  may  be  largely  spurious.  McLaren 
(  1965b)  suggested  that  the  degree  of  curvilinearity  of  response  to  temperature  (b) 
might  be  the  same  among  related  groups.  The  "real"  value  of  b  may  be  taken  as 
the  mean  of  estimates  on  Table  II,  each  estimate  weighted  by  the  square  root  of 
number  of  determining  experiments  (excluding  C.  finmarchicits  from  Millport,  for 
which  number  of  experiments  was  not  given).  This  mean  may  differ  slightly  from 
the  true  statistical  mean,  but  the  arguments  that  follow  would  not  differ  for  any 
chosen  value  of  b  within  the  range  on  Table  II,  since  a,  log  a,  and  b  are  all  linearly 
related.  The  new  values  of  a  and  a  are  listed  on  Table  II,  and  the  empirical 
adequacy  of  the  resulting  curves  is  clear  on  Figure  1.  Only  two  points  deviate 
much  from  the  curves;  these  points,  at  2.7°  and  3.3°  for  the  large  form  of  Pseudo- 
calanns,  represent  individual  experiments,  whereas  almost  all  other  points  are 
means.  Assuming  that  b  is  in  fact  constant  greatly  increases  the  resolution  of  the 
function,  even  with  inaccurate  or  biased  data. 

Taking  b  as  1.68  for  all  localities  and  species  reduces  the  great  range  in  values 
of  a  and  regularizes  them  in  a  more  logical  way.  Differences  within  species  from 
various  localities  are  then  found  to  be  very  slight  (range  of  0.8°  in  P.  niinntns  and 
0.3°  in  C.  finmarchicus) ,  but  differences  between  species  are  more  marked.  The 
most  strictly  arctic  species,  C.  glacialis,  has  the  lowest  value  of  a.  Unlike  the 
other  species,  A.  clausi  and  T.  discaudatus  do  not  extend  to  cold,  northern  regions, 
and  this  seems  to  be  reflected  in  their  higher  values  of  «.  At  temperatures  of  -  -  1°, 
which  may  be  expected  during  the  spring  in  arctic  wraters,  A.  clausi  and  T.  dis- 
candatus would  take  about  three  and  four  weeks  simply  to  hatch  their  eggs. 

Different  values  of  a  for  embryonic  development  among  thermal  races  of  frogs 
(McLaren,  1956b)  are  significantly  correlated  with  latitude  or  altitude,  and  there- 
fore with  environmental  temperature.  The  C.  fimnarchicus  studied  by  Marshall 
and  Orr  (1953)  were  said  by  these  authors  to  be  living  at  2-3°  and  6°,  respec- 
tively, at  Tromsp'  and  Millport.  The  appropriate  temperatures  experienced  by 
females  of  P.  uihuitns  can  only  be  approximated.  Temperatures  for  Halifax, 


462 


IAN  A.  MCLAREN 


Woods  Hole,  and  Millport  are  available  as  daily  surface  temperatures  recorded  by 
institutions  at  these  localities.  These  are  averaged  for  the  periods  during  which 
females  were  captured.  Temperatures  were  not  taken  at  Frobisher  or  Ogac  Lake 
during  1965.  The  waters  below  a  few  meters  at  Frobisher  in  late  June  and  early 
July  may  be  assumed  to  be  at  the  winter  minimum  of  ca.  -  1.7°.  A  published 
estimate  (McLaren,  1965a,  his  Figure  2)  of  mean  temperature  in  Ogac  Lake  ex- 


22 

doys_ 

20  - 


16   - 


12   - 


X 

o 


< 

X 


8  - 


4  - 


A  TORTANUS    DISCAUDATUS 

D  LARGE  FORM,    PSEUDOCALANUS 

A  ACARTIA    CLAUSI 

•  PSEUDOCALANUS    MINUTUS,     MILLPORT 

o  PSEUDOCALANUS  MINUTUS,  FROBISHER 

•  CALANUS    GLACIALIS 

o  CALANUS    FINMARCHICUS,    TROMS0 


-2    °C 


8 

TEMPERATURE 


12 


16 


FIGURE  1.  Belehradek's  temperature  functions  fitted  to  development  times  of  copepod  em- 
bryos. The  parameter  b  is  taken  as  —  1.68  for  all  curves,  and  the  fitted  values  of  a  and  a  are 
given  on  Table  II. 


DEVELOPMENT  RATE  OF  COPEPOD  EGGS 


463 


•II. 0 

°c 


-10.6  -H 


,-10.2  - 


LL 

o 

LU 

I 


-9.8  - 


-9.4  - 


-9.0 


I  i  I  I  I  I  I  I  I 


P  minutus 


C.  finmarchicus 


-2  °C 


1^        I  \^        l^       r^        \^        \^         I  I 

02468 

TEMPERATURE  AT  TIME   OF  COLLECTION 


10 


FIGURE  2.  Relationship  between  the  scale  correction  or  "biological  zero"  a  of  Belehradek's 
function  for  development  time  of  copepod  embryos  and  estimated  temperatures  at  the  times  egg- 
producing  females  were  collected. 

perienced  by  P.  minutus  maturing  in  early  summer  of  1957  may  be  used  as  a  rough 
indication  of  temperatures  at  the  same  season  in  1965.  Values  of  a  are  plotted 
against  these  temperature  estimates  on  Figure  2.  The  correlation  for  P.  minutus, 
for  which  there  are  sufficient  points  to  test,  falls  short  of  significant  (P~  0.10). 
But  the  relationship  is,  as  expected,  positive  for  both  species.  The  amount  of  intra- 
specific  "adaptation"  is  very  small,  and  is  of  about  the  same  order  as  that  found 
among  thermal  races  of  frogs  (McLaren,  1965b). 


•  Effects  of  egg  size 

Berrill  (1935)  found  that  development  time  of  ascidian  eggs  was  linearly  related 
to  egg  diameter,  provided  the  eggs  were  comparable  in  yolkiness.  McLaren 
(1965b)  found  that  among  thermal  races  of  the  frog  Rana  pipiens,  a  of  Belehradek's 
function  for  embryonic  development  time  was  significantly  correlated  with  egg 
diameter,  at  least  within  the  United  States.  The  same  seems  to  be  true  among 
closely  related  forms  of  copepods  (Fig.  3a).  The  correlation  between  a  and  egg 
diameter  is  significant  (0.05  >  P  >  0.01)  for  P.  minutus,  excluding  those  from 
Ogac  Lake.  This  is  remarkable  enough,  considering  the  small  number  and  re- 


464 


IAN  A.  MCLAREN 


stricted  range  of  values,  and  again  seems  to  justify  the  assumption  that  b  is  the 
same  for  all  populations. 

In  spite  of  their  smaller  size,  the  eggs  of  P.  minutus  from  Ogac  Lake  developed 
at  much  the  same  rate  as  those  from  other  areas.  Differences  in  egg  and  body  size 
of  this  species  in  the  cold  waters  of  Frobisher  Bay  and  the  warm  waters  of  Ogac 
Lake  are  phenotypic  (McLaren,  1965a).  It  may  be  that  differences  in  egg  size 
and  its  effects  on  a  are  not  comparable  to  those  occurring  among  the  more  widely 


10004 


800- 


0    eoo- 


UJ 

U 

t     400- 

UJ 
O 


200- 


0 


A 
A 


Large  form  Pseudocalanus 
Pseudocalanus  minutus 
Tort  anus  discaudatus 
Acartia  clausi 
Calanus  glacialis 
Co  I  anus  finmarchicus 


D 


8 


a 


1000 


800  - 


I    6°°" 

UJ 
Q 


9     400- 

H 
CL 

o 


200  - 


0 


— i 1 1 1 1 1 1 1 1 r~ 

60  80  100  120  140  160 

EGG       DIAMETER      (JU  ) 


180 


200 


FIGURE  3.  (a)  Relationship  between  the  proportionality  coefficient  a  of  Belehradek's  tem- 
perature function  for  development  time  of  copepod  embryos  and  egg  diameter,  (b)  The  same 
after  correction  for  yolk  concentration  (optical  density).  See  text. 


niCYKLOI'MKNT  RATE  OF  COPEPOD  EGGS 


465 


C.  glacial  is 


,r,  - 


T.  discaudatus 


A,  clausi 


P.  minutus 
(Halifax) 


P,  minutus 
(Frobisher) 


FIGURE  4.     Representative  copepod  eggs  to  show  differing  optical  density  and  size. 


separated  populations  of  the  species.  It  is  also  possible  that  experimental  salinities 
(see  Materials  and  Methods)  had  an  effect.  Development  was  slower  at  32.3%e 
(Table  I),  which  is  abnormally  high  for  the  brackish  population  of  Ogac  Lake. 
There  is  evidence  (Kinne,  1964)  that  growth  rates,  development  rates,  and  sizes 
have  optimal  salinities.  Whatever  the  explanation  of  the  results  from  Ogac  Lake, 
it  seems  possible  to  conclude  that  P.  minutus  and  the  large  form  of  Pseudocalanus 
would  have  produced  considerably  larger  eggs  which  would  have  developed  only 
slightly  more  slowly  than  indicated,  if  they  had  been  captured  from  the  colder,  more 
saline  waters  of  Frobisher  Bay. 

The  parameter  a  is  not  proportional  to  egg  diameter.  Assuming  linearity, 
a  --  0  at  an  egg  size  of  about  35  ^  for  marine  P.  minutus.  For  the  large  form  of 
Pseudocalanus  and  P.  niinntiis  from  Ogac  Lake,  the  intercept  is  at  about  54  p,  and 
for  C.  finmarchicus  and  glacialis,  at  about  64  //..  Since  it  is  impossible  that  real 
eggs  of  these  intercept  sizes  would  develop  infinitely  rapidly,  the  assumed  linear 
relationships  are  probably  roughly  tangential  to  shallow,  concave  functions,  with 
origins  at  O/*. 

It  is  clear  from  Figure  3a  that  development  times  within  species  or  among 
closely  related  forms  may  be  partly  predicted  from  egg  size,  but  the  same  rule  does 
not  apply  between  distantly  related  species.  For  example,  the  eggs  of  Calami s, 
although  much  larger  than  those  of  Pseudocalanus,  develop  much  more  rapidly. 

Effects  of  yolk  concentration 

Berrill  (1935)  concluded  that  development  rate  of  ascidian  eggs  was  retarded 
in  proportion  to  the  ratio  of  yolk  to  cytoplasm,  although  he  made  no  quantitative 
measurements  of  yolk.  All  the  copepod  eggs  studied  here  seem  to  be  unpigmented, 
and  are  white  to  pale  yellow  in  reflected  light.  Under  transmitted  light  they  differ 


466  IAN  A.  MCLAREN 

markedly  in  transparency  (Fig.  4).  It  seems  probable  that  transparency  is  related 
to  concentration  of  yolk  substances. 

Newly  produced  eggs  may  be  somewhat  darker,  and  advanced  embryos  are 
more  transparent,  except  for  dark  centers,  which  appear  to  be  fat  globules.  Inter- 
mediate stages  do  not  appear  to  vary  systematically,  and  embryos  ranging  from 
few-celled  to  probable  gastrula  stages  were  chosen  at  random  for  measurements. 
Eggs  of  Pseudocalanus,  Calanus,  and  A.  clausi  presented  no  special  difficulties, 
although  only  a  total  of  11  eggs  of  the  last  species  were  available.  Eggs  of  T. 
discaudatus  are  surrounded  by  a  thick  membrane,  which  is  not  completely  trans- 
parent, and  which  often  had  a  faint,  reddish  tinge.  Thus,  some  of  the  extinction 
of  transmitted  light  by  these  eggs  is  caused  by  the  membrane.  Fortunately,  the 
complete  membranes  (Fig.  4)  are  cast  by  the  hatching  nauplii.  A  number  were 
preserved  for  light  measurements,  and  the  results  subtracted  from  measurements 
of  intact  eggs.  Unfortunately,  no  corrections  can  be  made  for  correlations  of 
transparency  of  membranes  and  intact  eggs. 

The  results  are  listed  on  Table  II  as  mean  extinctions  per  unit  volumes  of  eggs, 
relative  to  the  values  for  P.  minutus  from  Frobisher  Bay.  Although  they  repre- 
sent a  3-fold  range  in  egg  volumes,  the  eggs  of  the  various  populations  and  forms 
of  P.  minutus  do  not  differ  in  optical  density.  This  suggests  that  yolk  concentra- 
tion is  the  same  among  closely  related  forms,  which  also  seems  to  be  true  of  ascid- 
ians  (Berrill,  1935).  It  also  indicates  the  validity  of  the  optical  methods.  In 
spite  of  large  fiducial  limits,  due  to  small  samples  in  most  cases,  there  are  marked 
and  significant  differences  between  species. 

The  exact  relationship  between  yolk  concentration  and  optical  density  cannot 
be  proportional,  but  it  may  be  nearly  so,  since  cytoplasm  is  very  transparent.  The 
effect  of  yolk  concentration  on  development  time  may  be  proportionate,  but  the 
effect  of  egg  size  is  not  (see  above).  Therefore  the  best  way  to  express  the  possible 
effect  of  yolk  is  by  dividing  development  time  at  a  given  temperature  (or  a  among 
forms  which  differ  in  temperature  characteristics)  by  yolk  concentration  (relative 
optical  density).  The  results  on  Figure  3b  clearly  represent  a  marked  regulariza- 
tion  of  the  data  on  Figure  3a.  Figure  3b  also  assumes,  from  arguments  given 
above,  that  P.  minutus  is  equivalent  in  size  and  development  rate  to  the  species  in 
nearby  Frobisher  Bay,  and  that  the  large  form  of  Pseudocalanus  would  be  propor- 
tionately larger  and  slower  in  development  if  it  occurred  in  Frobisher  Bay. 

The  relationship  on  Figure  3b  seems  adequately  described  by 

o=  Y  (6.51  £-317) 

where  a  is  the  proportionality  coefficient  of  Belehradek's  function  in  days,  Y  is 
the  optical  density  relative  to  eggs  of  Pseudocalanus,  and  D  is  egg  diameter  in  /*. 
Again,  the  real  effect  of  D  is  probably  not  linear,  and  the  relationship  may  take  its 
origin  from  0  p. 

DISCUSSION 

Belehradek's  temperature  function  clarifies  analysis  of  the  data.  From  the 
results,  it  should  be  possible  to  predict  development  rate  of  eggs  of  other  species 
of  copepods  with  a  minimum  of  experimental  data.  Of  perhaps  more  general 
interest  is  the  further  support  for  the  conclusion  of  McLaren  (1965b)  that  the 


DEVELOPMENT  RATE  OF  COPEPOD  EGCS  467 

three  conceptually  separable  parameters  of  Belehradek's  function  have  separable 
biological  meaning. 

Temperature  adaptation  per  sc  can  be  considered  in  relation  to  a  single  parame- 
ter, the  scale  correction  a.  This  seems  much  simpler  than  discussion  of  "Q10 
shifts,"  "translation,"  "rotation,"  and  like  terms,  some  of  which  are  artifacts  of 
the  semilogarithmic  plot  and  combine  differences  in  slope  (a),  curvilinearity  (b ) , 
and  position  on  the  Celsius  scale  (a). 

Among  closely  related  forms  differences  in  a  may  be  predicted  from  size  alone. 
The  large  form  of  Pseitdocalanus,  with  eggs  and  bodies  about  three  times  the  vol- 
ume of  those  of  co-existing  P.  111  i  nut  us,  has  the  same  chromosome  number,  but 
the  chromosomes  are  much  larger  and  contain  several  times  as  much  DNA  (Mc- 
Laren, Woods,  and  Shea,  1966).  A  similar  mechanism  may  account  for  the  larger 
size  of  C.  glacialis,  which  has  the  same  chromosome  number  but  larger  eggs  than 
C.  fimnarchiciis.  The  inherent  differences  in  size  and  development  rate  are  per- 
haps related  to  DNA  content  in  the  manner  suggested  by  Commoner  (1964). 
However,  a  is  not  proportional  to  volume  or  DXA  content,  so  that  another  form 
of  control  must  be  superimposed. 

It  is  of  interest  to  note  that  Berrill  (1935)  found  a  similar  pattern  of  develop- 
ment among  ascidian  eggs.  Among  eggs  larger  than  about  250  p.,  development  time 
was  linearly  related  to  egg  diameter,  with  an  intercept  at  about  125  /*.  For  smaller 
eggs  between  100  and  170^  the  relationship  was  slightly  curvilinear,  with  an 
apparent  intercept  at  about  60  p.  This  is  comparable  with  copepod  egg  develop- 
ment, with  an  apparent  intercept  at  about  50  p.  (Fig.  3b).  Berrill  argued  from 
proportionality  (although  this  is  not  strictly  true)  of  development  time  and  egg 
diameter  that  control  is  imposed  by  surface-to-volume  restrictions  on  CO2  exchange 
of  the  whole  embryo,  and  offered  some  experimental  evidence  for  this.  Recently 
Daykin  (1965)  applied  mass  transfer  theory  to  the  uptake  of  oxygen  by  fish  eggs. 
It  is  not  possible  to  define  conditions  applying  to  ascidian  and  copepod  eggs,  but 
representative  solutions  of  Daykin's  equations  imply  that  the  mass  transfer  coeffi- 
cient is  itself  a  positive  function  of  egg  diameter,  with  a  negative  second  derivative. 
If  the  same  form  of  coefficient  applies  to  the  outward  transfer  of  CO,  (which  is 
much  more  likely  than  O2  to  be  limiting),  then  the  control  suggested  by  Berrill 
would  not  result  in  strict  proportionality  between  development  time  and  egg  diam- 
eter. Instead  development  time  would  increase  curvilinearly  with  diameter  among 
small  eggs  and  more  nearly  linearly  among  larger  eggs,  more  or  less  in  the  manner 
suggested  for  ascidian  and  copepod  eggs.  This  suggests  that  Berrill's  general  ideas 
are  sound,  and  that  detailed  analysis  and  prediction  might  be  possible. 

The  influence  of  yolk  does  not  seem  explicable  in  energetic  or  biochemical  terms. 
Although  more  yolky  eggs  take  longer  to  hatch,  many  analyses  of  fat,  protein,  and 
energy  have  shown  that  very  little  is  used  by  most  developing  embryos  before  they 
hatch  (e.g.,  Hayes,  1949).  There  may  well  be  qualitative  differences  in  the  yolk 
of  the  several  kinds  of  copepods,  but  its  effect  seems  proportional  to  its  crudely 
defined  concentration.  This  seems  to  support  Berrill's  conclusion  that  yolk  simply 
"dilutes"  the  metabolically  active  cytoplasm  of  the  egg  and  embryo. 

Belehradek  (1935,  1957)  believed  he  had  a  theoretical  basis  for  his  temperature 
function  in  observations  that  diffusion  and  viscosity,  but  not  chemical  reaction  rates 
in  vitro,  are  affected  in  a  comparable,  double  logarithmic  way.  McLaren  (1965b) 


468  IAN  A.  MCLAREN 

suggested  that  yolk,  by  affecting  diffusion  characteristics,  might  be  involved  in 
temperature  adaptations.  There  is  no  evidence  that  a  of  copepod  eggs  is  affected 
by  concentration  of  yolk;  the  relationship  (Table  II)  is  positive  but  far  from  sig- 
nificant. If  Belehradek's  general  ideas  are  correct,  it  may  be  that  qualitative 
differences  of  yolk  are  involved,  or  that  control  resides  in  biophysical  properties  of 
the  cytoplasm,  rather  than  yolk. 

SUMMARY 

1.  The  development  times  to  hatching  of  eggs  of  several  kinds  of  copepods  were 
studied  at  controlled  temperatures.     Data  are  analyzed  from  several  geographical 
populations  of  Pscudocalanus  minutus,  a  large  form  of  Pseiidocalanus,   Calanus 
ftnmarchicus,  C.  glacialis,  Acartia  clausi,  and  Tortanus  discaudatiis. 

2.  Belehradek's  temperature  function,  expressed  as  D  --  a(T  —  a)6,  where  D 
is  development  time,  T  the  temperature,  and  a,  a  and  b  are  constants,  was  fitted 
to  the  results.     Assuming  that  b  is  the  same  (--  1.68,  the  mean  of  fitted  values) 
for  all  species  results  in  several  regularities. 

3.  The  scale  correction  or  "biological  zero"  a  varies  little  within  species,  but 
seems  positively  related  to  environmental  temperature.     C.  glacialis,  with  the  most 
northerly  range,  has  the  lowest  value  of  a,  and  A.  clausi  and  T.  discaudatus,  which 
are  the  most  southerly,  have  highest  values  of  a.     Temperature  adaptation  per  se 
may  be  considered  in  relation  to  this  parameter  alone. 

4.  The  proportionality  coefficient  a  varies  significantly  with  egg  diameter  within 
species  or  between  closely  related  species.     Differences  in  a  and  egg  size  are  related 
to  differences  in  DNA  content  between  P.  minutus  and  the  large  form  of  Pseudo- 
calanus, and  the  same  may  be  true  between  other  closely  related  forms. 

5.  The  coefficient  a  is  not  exactly  proportional  to  egg  diameter  or  DNA  con- 
tent, but  the  relationship  resembles  predictions  from  mass   transfer   theory,   and 
supports  Berrill's   (1935)   belief  that  control  is  superimposed  by  surface/volume 
restrictions  on  CO2  exchange  by  the  whole  embryo. 

6.  Differences  in  optical  density  of  eggs  are  attributed  to  yolk  concentration. 
The  parameter  a  is  proportionate  to  relative  optical  density,  which  supports  Berrill's 
(1935)    conclusion    that    yolk    simply    "dilutes"    metabolically    active    cytoplasm. 
Yolkiness  does  not  appear  to  affect  other  parameters,  which  it  might  do  if  it  im- 
posed restrictions   on  diffusion,   as   implied   by  the  possible  biophysical   basis   of 
Belehradek's  temperature  function. 

LITERATURE  CITED 

BELEHRADEK,    J.,    1935.     Temperature    and    living    matter.     Protoplasma    Monograph,    No.    8. 

Borntraeger,  Berlin,  277  pp. 

BELEHRADEK,  J.,  1957.  Physiological  aspects  of  heat  and  cold.  Ann.  Rev.  PhysioL,  19:  59-82. 
BERRILL,  N.  J.,  1935.  Studies  in  tunicate  development.  Part  III — differential  retardation  and 

acceleration.     Phil.  Trans.  Roy.  Soc.   (London),  Ser.  B,  225:  255-326. 
COMMONER,  B.,  1964.     DNA  and  the  chemistry  of  inheritance.     Amer.  Sci.,  52:  365-388. 
DAYKIN,  P.  N.,  1965.     Application  of  mass  transfer  theory  to  the  problem  of  respiration  of  fish 

eggs.     /.  Fish.  Res.  Bd.  Canada,  22:  159-171. 
GRAINGER,  E.  H.,   1961.     The  copepods  Calanus  glacialis  Jaschnov  and   Calanus  finmarchiciis 

(Gunnerus)   in  Canadian  arctic-subarctic  waters.     /.  Fish.  Res.  Bd.  Canada,  18:  663- 

678. 


DEVELOPMENT  RATE  OF  COPEPOD  EGGS  469 

HAYES,  F.  R.,  1949.     The  growth,  general  chemistry,  and  temperature  relations  of  salmonid 

eggs.     Quart.  Rev.  Biol,  24:  281-308. 
KINNE,  O.,  1964.     The  effects  of  temperature  and  salinity  on  marine  and  brackish  water  animals. 

II.  Salinity  and  temperature  salinity  combinations.     Oceanogr.  Mar.  Biol.  Ann.  Ret'., 

2:  281-339. 

MARSHALL,  S.  M.,  AND  A.  P.  ORR,  1953.     Calatnts  finmarchicus:  egg  production  and  egg  devel- 
opment in  Troms0  Sound  in  spring.     Acta  Borealis,  A.  Scientia,  No.  5,  21  pp. 
McLAREN,   I.   A.,   1963.     Effects   of  temperature  on  growth   of  zooplankton,   and   the   adaptive 

value  of  vertical  migration.     /.  Fish.  Res.  Bd.  Canada,  20:  685-727. 
MCLAREN,   I.   A.,    1965a.     Some   relationships   between   temperature   and   egg   size,   body   size, 

development  rate,  and  fecundity,   of  the   copepod  Pscndocalamis.     Limnol.    Oceanogr., 

10:  528-538. 
MCLAREN,  I.  A.,  1965b.     Temperature  and  frog  eggs.     A  reconsideration  of  metabolic  control. 

/.  Gen.  Physiol..  48:  1071-1079. 
MCLAREN,  I.  A.,  S.  M.  WOODS  AND  J.  SHEA,  JR.,  1966.     Polyteny :  a  source  of  cryptic  specia- 

tion  among  copepods.     Science,  153  :  1641-1642. 


FACTORS  INFLUENCING  THE  RESPONSE  OF  ISOLATED 

DOGFISH  SKIN  MELANOPHORES  TO  MELANOCYTE- 

STIMULATING  HORMONE1 

RONALD  R.  NOVALES  AND  BARBARA  J.  NOVALES 

Department  of  Biological  Sciences,  Northwestern  University,  Evanston,  Illinois  60201,  and 
the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts  02543 

Although  a  good  deal  is  known  about  the  endocrinology  of  melanophores  in 
elasmobranch  fishes  (Parker,  1948;  Waring,  1963),  little  has  been  published  re- 
garding the  hormonal  responses  of  elasmobranch  melanophores  in  vitro.  The  iso- 
lated skin  of  several  species  of  dogfish  pales  in  isotonic  media  and  darkens  upon 
the  addition  of  pituitary  extracts  containing  melanocyte-stimulating  hormone 
(MSH),  according  to  Lundstrom  and  Bard  (1932)  and  Waring  (1936,  1960). 
This  fact  correlates  well  with  the  established  role  of  MSH  in  producing  the  dark- 
ening of  elasmobranchs  upon  an  illuminated  black  background.  It  also  shows  that 
dogfish  melanophores  are  similar  to  frog  melanophores  in  their  in  vitro  behavior 
(Hogben  and  Winton,  1922).  Although  the  molecular  and  cellular  mechanism 
of  the  melanin-dispersing  action  of  MSH  is  still  unknown,  MSH  may  produce 
melanin  dispersion  in  amphibian  melanophores  by  a  sodium-dependent  mechanism 
involving  the  uptake  of  water  (Novales,  1959,  1962).  Osmotic  and  ionic  factors 
were  also  found  to  be  of  importance  for  the  action  of  eye  stalk  hormone  on  the 
melanophores  of  the  fiddler  crab,  Uca  pugnax,  by  Fingerman,  Miyawaki  and  Oguro 
(1963).  In  view  of  the  relatively  primitive  nature  of  elasmobranch  fishes,  a  study 
has  been  conducted  of  osmotic  and  ionic  effects  on  the  response  of  dogfish  melano- 
phores to  MSH  (Novales  and  Novales,  1966).  It  was  hoped  to  learn  more  about 
the  endocrine  cellular  physiology  of  elasmobranch  melanophores  and  possibly  to 
shed  light  on  the  evolution  of  melanophore  control  mechanisms.  Although  simi- 
larities exist  between  frog  and  dogfish  melanophores,  there  are  also  differences, 
which  could  be  of  theoretical  importance. 

MATERIALS  AND  METHODS 

The  majority  of  experiments  were  performed  with  the  isolated  skin  from  a 
total  of  12  female  spiny  dogfishes  (Sqnaliis  acanthias).  Some  work  was  also  done 
with  three  smooth  dogfishes  (Mustelus  canis}.  Fishes  were  caught  with  hook 
and  line  and  stored  in  sea  water  pens  until  use.  They  were  kept  overnight  in  an 
illuminated  tank  with  a  light  background,  resulting  in  some  paling  of  their  skin. 
The  following  day  the  fishes  were  decapitated.  Skin,  removed  from  the  dorso- 
lateral  trunk  region,  was  placed  in  elasmobranch  Ringer's  solution  (Cavanaugh, 
1956),  which  had  the  following  composition  in  g.  per  1.  of  distilled  water:  NaCl, 

1  This  study  was  aided  by  grants  from  the  National  Science  Foundation  (G-24017)  and  the 
Office  of  Research  Coordination,  Northwestern  University. 

470 


DOGFISH  MELANOPHORE  RESPONSE  TO  MSH  471 

16.38;  KCL  0.89;  CaCl2-2H2O,  1.47;  NaHCO3,  0.38;  dextrose,  1.00;  urea,  21.6; 
NaH.,PO4<H.,O,  0.07.  Pieces  of  skin  were  mounted  on  aluminum  frames  of  the 
type  described  by  Shizume,  Lerner  and  Fitzpatrick  (1954)  and  immersed  in  20  ml. 
of  Ringer's  solution  in  a  50-ml.  beaker.  The  inside  diameter  of  the  upper  elements 
of  these  frames  had  to  be  about  one  mm.  larger  than  originally  described,  due  to 
the  greater  thickness  of  dogfish  skin.  One  fish  furnished  sufficient  skin  for  24 
such  frames,  enough  for  a  typical  experiment  of  6  experimental  groups.  The  bath 
fluid  was  changed  three  times  in  a  period  of  several  hours,  in  order  to  produce 
complete  blanching  of  the  skin.  At  the  end  of  this  time  the  experiment  was  begun, 
utilizing  the  paled  skin,  with  highly  aggregated  melanin  in  the  melanophores.  An 
initial  reflectance  reading  was  taken  with  a  Photovolt  photoelectric  reflection  meter, 
model  No.  610,  standardized  to  read  100  with  the  red  filter  and  porcelain  plate  pro- 
vided with  the  meter.  The  proper  amount  of  MSH  was  then  added  to  20  ml.  of 
Ringer  or  to  the  experimental  medium.  Preliminary  time  curves  showed  that  full 
darkening  requires  90  minutes.  However,  since  virtually  complete  darkening  had 
occurred  within  60  minutes,  the  decrease  in  reflectance  in  galvanometer  units 
(G.U.)  occurring  in  one  hr.  (A)  was  used  as  the  standard  measure  of  darkening, 
as  in  previous  work  with  frog  skin  (Novales,  1959).  A  standard  MSH  (S-MSH  ), 
a  highly  purified  bovine  /?-MSH  (/3b-MSH)  and  a  synthetic  «-MSH  (syn.  a-MSH) 
were  used  in  the  experiments.  The  S-MSH  (Shizume,  Lerner  and  Fitzpatrick, 
1954)  and  /?b-MSH  (Geschwind,  Li  and  Barnafi,  1957)  were  obtained  from  C.  H. 
Li;  the  syn.  a-MSH  (Hofmann  and  Yajima,  1962)  was  provided  by  K.  Hofmann. 
All  three  hormones  were  prepared  in  lactose  by  the  method  of  Novales,  Novales, 
Zinner  and  Stoner  (1962),  to  facilitate  transporting,  weighing  and  storage.  The 
S-MSH  had  an  activity  of  109  units/g.,  when  bioassayed  by  the  method  of  Shi- 
zume, Lerner  and  Fitzpatrick  (1954).  The  other  MSH's  showed  comparable 
activity  after  bioassay  at  the  sea  shore.  In  view  of  the  uniformity  of  the  results 
obtained  in  the  various  experiments  and  with  the  three  MSH's,  the  results  from 
separate  experiments  were  pooled.  However,  the  majority  of  experiments  were 
performed  with  the  syn.  a-MSH,  which  had  a  formyl  group  on  the  lysine  at 
position  10. 

Microscopic  examination  of  melanophores  showed  that  decrease  in  reflectance 
is  accompanied  by  melanin  dispersion  in  the  dogfish  melanophores.  Cleared  whole 
mounts  were  also  prepared  in  some  cases.  Sodium  analyses  of  the  skin  were  per- 
formed by  a  similar  method  to  that  used  for  frog  skin  by  Novales,  Novales,  Zinner 
and  Stoner  (1962).  Skin  was  either  removed  directly  from  the  fish  or  cut  from 
the  experimental  frames  after  treatment.  Wet  weights  were  taken  and  dry  weights 
obtained  after  drying  overnight  at  105°  C.  Samples  were  digested  in  one  ml.  of 
concentrated  nitric  acid  in  a  boiling  water  bath  and  analyzed  for  sodium  after 
dilution  with  distilled  water,  using  a  Coleman  model  21  flame  photometer. 

RESULTS 

The  isolated  skin  of  dogfish  darkens  when  MSH  is  added  to  the  medium  bath- 
ing the  skin.  The  log  dose-response  curves  for  the  three  MSH's  acting  on  Sqitalus 
skin  are  given  in  Figure  1.  The  response  to  one  unit/ml,  of  syn.  a-MSH  was 
significantly  greater  than  the  response  to  the  same  concentration  of  S-MSH  (p 
<  0.05  >  0.01),  but  not  significantly  more  than  that  to  /?b-MSH.  Since  100 


472 


RONALD  R.  NOVALES  AND  BARBARA  J.  NOVALES 


units/ml,  produced  marked  darkening,  this  concentration  was  chosen  for  further 
experiments  on  factors  influencing  the  response.  Urea  was  not  required  for  the 
response.  Thus,  S-MSH  at  10  units/ml,  gave  a  A  of  28  ±  2  G.U.  (2)  in  urea- 
free  Ringer.  The  numbers  of  skins  are  in  parentheses. 

The  effects  of  a  number  of  osmotic  and   ionic  variables  on   the   response  of 
Sqnaliis  skin  are  given  in  Table  I.     There  was  a  very  uniform  response  in  Ringer. 


ID 


E 
o 

c 
o 

_> 

D 
O 


g 

LU 

— I 
LJ_ 
LU 
CXL 

z 

LU 
CO 

LU 

U 

LU 

O 


1.0 

MSH 


100 


(U/ml.,Log  Scale) 


FIGURE  1.  Log  dose-response  curves  of  paled  Squalus  acanthias  skin  melanophores  to 
various  types  of  MSH.  Exposure  was  one  hour.  Points  are  means.  (•)  standard  MSH,  24 
skins;  (O)  syn.  a-MSH,  36  skins;  (£>)  /VMSH,  14  skins. 

The  sodium  content  of  skin  freshly  removed  from  the  fish  was  753  ±  49  (4) 
mM/kg.  dry  wt,  which  is  not  significantly  different  from  that  found  after  main- 
tenance in  Ringer  for  hours,  as  given  in  Table  I.  This  testifies  to  the  good  physio- 
logical qualities  of  the  Ringer's  solution  used  here. 

Bathing  in  hypotonic  Ringer  containing  £  of  normal  Ringer  sodium  produced 
slight  darkening,  as  seen  in  Table  I.  MSH  produced  a  normal  total  darkening 
in  the  hypotonic  Ringer  (Table  I).  Hypotonic  Ringer  of  ^  and  f  normal  Ringer 
sodium  produced  no  significant  darkening  alone,  showing  the  resistance  of  dogfish 


DOGFISH  MELANOPHORE  RESPONSE  TO  MSH 


473 


melanophores  to  the  darkening  effect  of  hypotonicity.  Very  dilute  Ringer  with 
•^  normal  sodium  produced  a  marked  darkening  of  18  ±  1  (2)  G.U.  However, 
MSH  failed  to  produce  any  more  darkening  than  this,  showing  the  unphysiological 
nature  of  this  medium.  Melanin  was  dispersed  in  melanophores  treated  with  the 
hypotonic  Ringer.  However,  distilled  water  produced  a  "cloudy"  partially  dis- 
persed condition,  suggesting  that  possibly  osmotic  cytolysis  of  the  melanophores 
had  occurred.  The  sodium  content  of  skin  treated  with  ^  X  Ringer  was  223  ±  16 
(5)  rml//kg.,  a  marked  reduction  below  that  of  fresh  skin  or  skin  treated  with 
normal  Ringer's  solution. 

Although  hypertonicity  by  itself  has  no  effect  on  the  melanophores,  Ringer's 
solution  made  hypertonic  with  sucrose  (Table  I)  blocks  the  action  of  MSH.  MSH 
at  the  lower  concentration  of  10  units/ml,  was  also  inhibited  in  the  hypertonic 

TABLE  I 

Factors  influencing  the  response  of  Squalus  melanophores  to  MSH 


1-Hr,  darkening  (i,  in  G.U.)* 

Skin  Na  after  treatment 

-Medium 

(m.I/  /kg.  dry  wt.) 

MSH  Absent 

MSH  Presentf 

Elasmobranch  Ringer 

1   ±  0.4  (9)** 

29  ±  1       (25) 

713  ±  50  (5) 

Hypotonic  Ringer  (i  X) 

8±2       (8) 

29  ±  2      (8) 

•  — 

Hypertonic  Ringer  (2  X) 

1  ±  1     (7) 

11  ±  1      (8) 

— 

Na-free  Ringer  (lithium) 

1  ±  1      (6) 

27  ±  3      (4) 

130  ±  10  (4) 

Na-free  Ringer  (choline) 

4  ±  1      (8) 

26  ±  3      (12) 

— 

Isotonicff  XaCl  (313  m.I/) 

+  1  ±  1      (3) 

23  ±  3      (4) 

— 

Isotonic  sucrose  (625  m.V) 

6  ±  1      (5) 

9  ±  1      (8) 

— 

Isotonic  KC1  (313  m.I/) 

8  ±  0.3  (4) 

27  ±  2      (4) 

146  ±     1  (4) 

Isotonic  MgCl2  (208  ml/) 

10  ±2      (4) 

21  ±  2      (4) 

146  ±  14  (4) 

Isotonic  CaCl2  (208  ml/) 

0  ±  1      (5) 

0  ±  0.3  (8) 



*  Decrease  in  reflectance  in  galvanometer  units. 
T  MSH  concentration  of  100  units/ml. 

*  Figures  are  means  ±  standard  errors;  numbers  of  skins  in  parentheses. 
ft  Isotonic  solutions  contained  urea  and  dextrose  as  in  Ringer. 

medium.  Ringer  made  hypertonic  with  NaCl  to  give  a  final  NaCl  concentration 
of  0.56  M  completely  inhibited  MSH  action,  indicating  that  a  high  sodium  content 
is  toxic  to  the  melanophores.  Furthermore,  MSH  was  markedly  inhibited  in  sea 
water,  which  was  calculated  to  be  about  1.6  X  isotonicity.  The  hypertonic  inhibi- 
tion was  reversible,  for  skins  darkened  with  MSH  upon  transfer  to  normal  Ringer 
after  having  been  in  a  hypertonic  medium. 

The  effect  of  replacement  of  sodium  by  a  variety  of  other  substances  is  also 
shown  in  Table  I.  Sodium-free  Ringer  was  prepared  with  LiCl  replacing  the 
NaCl,  and  potassium  salts  the  other  sodium  salts.  MSH  was  fully  active  in  the 
lithium  Ringer,  even  though  this  medium  produced  an  82%  reduction  in  the 
sodium  content  of  the  skin.  MSH  was  also  active  in  sodium-free  choline  Ringer 
(Table  I).  After  these  experiments  showing  that  sodium  can  be  replaced  in 
normal  Ringer,  a  simplified  type  of  medium  was  used,  consisting  of  a  single  chlo- 
ride as  well  as  urea  and  dextrose  as  in  normal  Ringer.  An  isotonic  NaCl  solution 


474 


RONALD  R.  NOVALES  AND  BARBARA  J.  NOVALES 


TABLE  II 

Factors  influencing  the  response  of  Mustelus  melanophores  to  MSH 


Medium 

1-Hr,  darkening  (A.  in  G.U.)* 

MSH  absent 

MSH  presentf 

Elasmobranch  Ringer 
Hypotonic  Ringer  (j  X) 
Hypertonic  Ringer  (2  X) 
Na-free  Ringer  (choline) 

2  ±  1   (4)** 
+  1   ±  1   (4) 
6  ±  2  (4) 
2  ±  2  (4) 

41  ±  4  (8) 
27  ±  3  (5) 
17  ±2  (7) 
32  ±  2  (8) 

*  Decrease  in  reflectance  in  galvanometer  units. 

f  MSH  concentration  of  100  units/ml. 

**  Figures  are  means  ±  standard  errors;  numbers  of  skins  in  parentheses. 

of  this  type  fully  supported  the  response  to  MSH  (Table  I).  However,  there  was 
a  two-thirds  reduction  in  the  amount  of  response  in  an  isotonic  sucrose  medium, 
showing  the  need  for  cations  in  the  response.  MSH  action  was  also  inhibited  in 
sodium-free  Ringer  prepared  with  sucrose,  for  10  units/ml,  gave  a  darkening  of 
only  11  ±2  (4),  much  lower  than  the  response  in  normal  Ringer,  which  was  about 
twice  as  great  (Fig.  1).  Surprisingly,  a  strong  response  was  obtained  in  the  iso- 
tonic KC1  and  MgCL  media  (Table  I).  Either  of  these  solutions  alone  produced 
a  substantial  darkening,  probably  as  a  result  of  their  acidity,  for  their  pH  values 
were  6.3  and  6.0,  respectively.  Shizume,  Lerner  and  Fitzpatrick  (1954)  found 
that  solutions  below  a  pH  of  6.5  will  darken  frog  skin.  Both  media  were  effective 
in  reducing  the  sodium  content  of  dogfish  skin.  Finally,  the  CaCU  medium  com- 
pletely failed  to  support  darkening.  This  failure  was  not  reversible,  indicating  that 
a  high  calcium  concentration  is  probably  toxic  to  the  melanophores. 

Similar  results  were  obtained  with  the  skin  of  the  smooth  dogfish,  M.  canis,  as 
shown  in  Table  II.  Mustelus  skin  gave  a  greater  reflectometric  change  with  MSH 
than  did  S 'qttal 'us  skin.  However,  (^  X )  hypotonic  Ringer  had  no  significant 
darkening  alone  and  the  response  to  MSH  was  significantly  lower  in  this  medium 
than  in  normal  Ringer  (P  --  0.01).  Thus,  Mustelus  is  slightly  more  resistant  to 

TABLE  III 
The  effect  of  caffeine*  on  dogfish  skin 

1-Hr,  darkening!  (A,  in  G.U.) 


ivieuuim 

S.  acanlhias 

M.  canis 

Elasmobranch  Ringer 
Na-free  Ringer  (choline) 
Isotonicft  sucrose  (625  mM) 
Isotonic  CaCb  (208  mM) 

19  ±  2  (6)** 
26  ±  3  (4) 
15  db  1  (4) 
0(4) 

16  ±  6  (2) 

28  ±  1  (2) 

*  Caffeine  concentration  of  0.01  M. 

t  Decrease  in  reflectance  in  galvanometer  units. 

*  Figures  are  means  ±  standard  errors;  numbers  of  skins  in  parentheses. 
ft  Isotonic  solutions  contained  urea  and  dextrose  as  in  Ringer. 


DOGFISH  MELANOPHORE  RESPONSE  TO  MSH  475 

hypotonic  darkening  than  Sqiialns.  In  hypertonic  Ringer  there  was  about  a  60% 
reduction  in  the  response  to  MSH.  However,  in  sodium-free  choline  Ringer  the 
response  to  MSH  was  not  significantly  different  than  in  normal  Ringer;  thus 
sodium  can  be  replaced  in  the  case  of  Mustelus  also. 

Experiments  with  caffeine  shed  light  on  the  specificity  of  the  above  effects  on 
MSH  darkening.  This  agent  darkened  the  skin  of  both  species  at  0.01  M,  as  seen 
in  Table  III.  Moreover,  in  sodium-free  Ringer,  there  was  a  greater  darkening, 
although  the  difference  is  not  statistically  significant.  This  difference  was  not  seen 
in  the  isotonic  sucrose,  however.  No  response  was  obtained  in  the  isotonic  Cad2, 
further  demonstrating  the  toxic  effect  of  this  solution  on  the  melanophores. 

DISCUSSION 

The  present  study  extends  earlier  results  with  the  isolated  skin  of  Mustelus 
canis  and  ScyUhun  canicula  to  Squalus  acanthias.  Lundstrom  and  Bard  (1932) 
first  showed  that  hypophysectomy  causes  paling  of  the  dogfish.  Melanophores  in 
isolated  skin  of  M.  canis  contracted  their  pigment  in  dilute  sea  water.  Melanin 
dispersion  occurred  with  mammalian  or  Mustelus  posterior  lobe  extract.  Waring 
(1936)  obtained  similar  results  with  vS".  canicula  skin.  Because  he  found  that  mela- 
nin aggregation  occurs  slowly  in  dilute  sea  water,  we  allowed  at  least  two  hours 
of  Ringer  rinses  to  produce  maximum  paling.  Waring  (1960)  also  obtained  a 
graded  response  to  increasing  amounts  of  hormone,  as  we  did  in  Figure  1.  He 
obtained  a  good  response  in  Young's  elasmobranch  saline  (dilute  sea  water  and 
urea)  and  we  did  also  in  our  earliest  experiments.  However,  we  adopted  the 
elasmobranch  Ringer,  because  of  the  ease  of  modifying  its  composition. 

The  present  results  also  support  the  conclusion  of  Parker  (1936)  that  darken- 
ing is  brought  about  in  6".  acanthias  by  a  pituitary  hormone  and  paling  by  the 
absence  of  this  hormone,  since  isolated  skin  pales  maximally  when  rinsed  and 
darkens  maximally  upon  addition  of  MSH.  Highly  purified  mammalian  hor- 
mones act  on  dogfish  melanophores,  just  as  they  do  on  frog  melanophores,  in  con- 
firmation of  earlier  work  showing  that  a  variety  of  MSH-containing  preparations 
disperse  melanin  in  elasmobranch  melanophores  (Pickford  and  Atz,  1957). 

The  dispensability  of  urea  in  the  response  of  Squahis  melanophores  is  another 
indication  that  the  high  blood  urea  of  elasmobranchs  probably  functions  solely  to 
maintain  osmotic  balance  (Smith,  1936).  Fredericq  (1922)  found  that  the  heart 
of  the  dogfish  Scyllmm  catulus  continued  to  beat  in  a  urea-free  salt  solution. 
Squahis  melanophores  are  also  capable  of  responding  to  MSH  in  the  absence  of  urea. 

Waring  (1936)  observed  that  dogfish  color  changes  are  largely  due  to  changes 
in  the  dermal  melanophores.  The  gross  and  microscopic  aspects  of  color  change 
in  5\  acanthias  are  illustrated  in  Waring  and  Landgrebe  (1950).  Dogfish  color 
changes  require  days  to  occur  in  vivo  (Parker,  1948),  but  only  a  few  hours  in  vitro. 
Thus,  the  greater  length  of  time  required  in  vivo  must  be  due  to  the  slowness  of 
the  control  of  MSH  secretion,  rather  than  to  any  slowness  in  the  response  of 
melanophores  to  MSH. 

The  darkening  effect  of  hypotonic  media  shows  that  water  entry  is  capable  of 
producing  melanin  dispersion  in  Squalus  melanophores.  Furthermore.  Squahis 
melanophores  are  less  sensitive  than  frog  melanophores  to  the  dispersing  effect  of 


476         RONALD  R.  NOVALES  AND  BARBARA  J.  NOVALES 

hypotonic  media.  Thus,  whereas  $  X  Ringer  darkens  frog  skin  about  50%  as 
much  as  MSH  (Novales,  1959),  it  only  darkens  Squahis  skin  28%  as  much  as 
MSH  (Table  I).  Hypotonic  media  also  disperse  melanin  in  the  melanophores  of 
frog  (Shizume,  Lerner  and  Fitzpatrick,  1954),  bony  fish  (Spaeth,  1913),  fiddler 
crabs  (Fingerman,  Miyawaki  and  Oguro,  1963),  and  salamanders  in  tissue  culture 
(Novales  and  Novales,  1965).  This  effect  has  been  used  to  support  the  view  that 
MSH  action  may  involve  the  uptake  of  water  by  the  melanophore  (Novales,  1959, 
1962).  Another  way  of  demonstrating  the  role  of  water  movement  is  to  show  an 
inhibitory  effect  of  hypertonicity  on  the  response  to  MSH.  This  was  done  in  both 
dogfish  species  studied  (Tables  I,  II).  Hypertonicity  inhibits  melanin  dispersion 
in  frog,  fiddler  crab  and  salamander  melanophores  (loc.  cit.).  However,  since 
dispersion  produced  by  drugs  as  well  as  aggregation  are  inhibited  (Novales,  1959), 
this  effect  does  not  establish  a  role  of  water  entry  in  MSH  action,  since  the  inhibi- 
tion is  not  specific  to  the  effect  of  MSH. 

The  present  study  has  also  shown  that  dogfish  melanophores  are  able  to  respond 
to  MSH  in  sodium-free  media.  Sodium  can  be  replaced  by  lithium,  choline,  potas- 
sium or  magnesium  ions  in  the  response  of  Squalus  melanophores,  but  sucrose  or 
calcium  fail  to  replace  sodium  (Table  I).  Thus,  a  cation  must  be  present  for  the 
response  to  occur.  The  toxicity  of  calcium  is  shown  by  the  failure  of  caffeine  to 
act  in  a  sodium-free  calcium  medium,  whereas  it  is  effective  in  a  sucrose  medium, 
in  contrast  to  MSH  (Table  III).  The  cation  requirements  for  dogfish  melano- 
phore responses  are  thus  clearly  different  from  those  for  frog  melanophores. 
Whereas  sodium  can  be  replaced  by  a  variety  of  other  cations  in  the  dogfish 
response,  the  requirement  for  sodium  is  absolute  in  the  case  of  the  frog  response 
to  MSH  (Novales,  Novales,  Zinner  and  Stoner,  1962).  It  is  unlikely  that  suf- 
ficient sodium  was  present  in  the  dogfish  skin  to  permit  a  response  to  MSH,  if 
sodium  were  required.  About  80%  of  skin  sodium  was  removed  by  the  sodium- 
free  media  (Table  I).  Since  frog  skin  fails  to  respond  when  90%  of  its  skin 
sodium  is  removed  {loc.  cit.),  it  is  unlikely  that  dogfish  skin  would  respond  with 
80%  of  its  sodium  gone,  if  sodium  were  required  for  the  response.  Of  the  ions 
capable  of  replacing  sodium  in  the  response  of  Squalus  skin  to  MSH,  neither 
lithium,  choline  or  potassium  is  capable  of  replacing  sodium  in  the  response  of  frog 
skin  melanophores.  The  present  results  also  recall  those  obtained  with  the  fiddler 
crab  by  Fingerman,  Miyawaki  and  Oguro  (1963).  They  found  that  sodium  can 
be  replaced  by  other  monovalent  cations  such  as  potassium  or  lithium  in  the  response 
of  melanophores  to  eyestalk  hormone,  but  divalent  cations  such  as  magnesium  and 
calcium  fail  to  replace  sodium.  Thus,  this  system  differs  in  that  magnesium  is  able 
to  replace  sodium  in  the  dogfish  response. 

Sodium  can  be  replaced  by  other  cations  in  a  variety  of  other  excitable  systems, 
such  as  nerve  and  muscle  (Spyropoulos  and  Tasaki,  1960).  However,  this  does 
not  necessarily  mean  that  sodium  ions  are  not  involved  in  the  responses  in  vivo. 
Thus,  sodium  has  so  far  been  irreplaceable  in  the  response  of  Rana  pipiens  melano- 
phores to  MSH,  for  virtually  all  the  cations  capable  of  replacing  sodium  in  nerve 
and  muscle  excitation  have  been  tried  and  failed  (Novales,  1959;  Wright  and 
Lerner,  1960;  Novales,  Novales,  Zinner  and  Stoner,  1962).  On  the  other  hand, 
the  present  study  has  shown  that  Squalus  melanophores  will  respond  to  MSH 
when  sodium  is  replaced  by  a  variety  of  cations.  However,  these  results  do  not 


DOGFISH  MELANOPHORE  RESPONSE  TO  MSH  477 

mean  that  sodium  is  not  involved  in  the  Sqitaltis  response  in  vivo.  Sodium  prob- 
ably is  involved.  It  merely  means  that  the  specificity  of  the  cation-requiring  system 
is  broad  in  Squalus  but  extremely  narrow  in  Rana.  This  difference  in  specificity 
could  be  a  reflection  of  the  more  primitive  nature  of  the  elasmobranch  fish  when 
compared  with  the  anuran  amphibian.  The  sodium-requiring  process  involved  in 
MSH  action  has  apparently  increased  in  its  specificity  to  sodium  during  evolution. 
Another  possibility  is  that  modern  elasmobranchs  have  lost  the  high  specificity  of 
the  sodium  requirement  possessed  by  their  ancestors.  However,  this  is  an  unlikely 
explanation  which  would  be  difficult  to  prove.  In  view  of  the  phylogenetic  position 
of  bony  fishes  between  the  cartilaginous  fishes  and  amphibians,  information  is 
needed  regarding  the  cation  requirements  for  MSH  action  in  teleost  fishes.  Infor- 
mation about  the  requirements  in  cyclostome  fishes  would  also  be  of  interest,  since 
they  are  more  primitive  than  elasmobranchs. 

The  authors  are  indebted  to  Dr.  C.  H.  Li  of  the  University  of  California, 
Berkeley,  for  providing  the  natural  MSH.  Dr.  Klaus  Hofmann  of  the  University 
of  Pittsburgh  kindly  provided  the  synthetic  MSH.  Dr.  Lois  TeWinkel  of  Smith 
College  aided  us  greatly  in  our  early  fishing  expeditions  and  the  Staff  of  the  Marine 
Biological  Laboratory  provided  facilities  and  numerous  essential  services. 

SUMMARY 

1.  The  log  dose-response  curves  to  standard  MSH,  bovine  /3-MSH,  and  syn- 
thetic a-MSH  were  obtained  for  the  melanophores  of  isolated  Squalus  acanthias 
skin,  using  a  reflectometric  technique. 

2.  Urea  is  not  required  for  the  response  of  Squalus  melanophores  to  MSH, 
further    supporting   the   view    that   urea    is    required    solely   for    maintaining    the 
osmotic  balance  of  elasmobranchs. 

3.  Hypotonic   Ringer    (ro  X )    produces   marked   darkening   of   Squalus   skin, 
indicating  that  water  entry  can  cause  melanin  dispersion. 

4.  Hypertonic  Ringer   (2  X  )   inhibits  MSH  action  on  Squalus  melanophores, 
indicating  that  water  entry  may  occur  during  MSH  action. 

5.  MSH  can  act  on  Squains  or  Mustelus  melanophores  in  the  absence  of  sodium 
and  lithium ;  choline,  potassium  and  magnesium  are  all  capable  of  replacing  sodium 
in  the  response  of  Squalus  melanophores. 

6.  MSH  action  is  reduced  in  a  sodium-free  sucrose  medium ;  thus  there  is  a 
cation  requirement  for  MSH  action  on  Squalus  melanophores. 

7.  Either  there  is  no  sodium  requirement  for  MSH  action  on  the  dogfishes 
studied,  or  the  specificity  of  the  sodium  requirement  for  sodium  in  the  dogfish  is 
much  lower  than  in  the  frog. 

LITERATURE  CITED 

CAVANAUGH,  G.  M.,  1956.  Formulae  and  Methods  IV  of  the  Marine  Biological  Laboratory 
Chemical  Room,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts,  61  pp. 

FINGERMAIST,  M.,  M.  MiYAWAKi  AND  C.  OcuRO,  1963.  Effects  of  osmotic  pressure  and  cations 
on  the  response  of  the  melanophores  in  the  fiddler  crab,  Uca  pugnax,  to  the  melanin- 
dispersing  principle  from  the  sinus  gland.  Gen.  Comp.  Endocr.,  3:  495-504. 

FREDERICQ,  L.,  1922.  Pulsations  de  coeur  de  Scvlliitin  catitlus  en  1'absence  d'uree.  Arch.  int. 
Physiol,  19:  253-256. 


478         RONALD  R.  NOVALES  AND  BARBARA  J.  NOVALES 

GESCHWIND,  I.  I.,  C.  H.  Li  AND  L.  BARNAFI,  1957.  The  isolation  and  structure  of  a  melanocyte- 
stimulating  hormone  from  bovine  pituitary  glands.  /.  Anier.  Chan.  Soc.,  79:  1003-1004. 

HOFMANN,  K.,  AND  H.  YAJIMA,  1962.  Synthetic  pituitary  hormones.  Recent  Prog.  Harm. 
Res.,  18:41-83. 

HOGBEN,  L.,  AND  F.  R.  WiNTON,  1922.  The  pigmentary  effector  system.  I-Reaction  of  frog's 
melanophores  to  pituitary  extracts.  Proc.  Roy.  Soc.  London,  Scr.  B,  93:  318-329. 

LUNDSTROM,  H.  M.,  AND  P.  BARD,  1932.  Hypophysial  control  of  cutaneous  pigmentation  in  an 
elasmobranch  fish.  Biol.  Bull.,  62:  1-9. 

NOVALES,  R.  R.,  1959.  The  effects  of  osmotic  pressure  and  sodium  concentration  on  the  response 
of  melanophores  to  intermedin.  Pliysiol.  Zool.,  32:  15-28. 

NOVALES,  R.  R.,  1962.  The  role  of  ionic  factors  in  hormone  action  on  the  vertebrate  melano- 
phore.  Atner.  Zool.,  2:  337-352. 

NOVALES,  R.  R.,  AND  B.  J.  NOVALES,  1965.  The  effects  of  osmotic  pressure  and  calcium  de- 
ficiency on  the  response  of  tissue-cultured  melanophores  to  melanocyte-stimulating 
hormone.  Gen.  Comp.  Endocr.,  5 :  658-676. 

NOVALES,  R.  R.,  AND  B.  J.  NOVALES,  1966.  Factors  influencing  the  response  of  dogfish  melano- 
phores to  MSH.  Amer.  Zool.,  6:  311-312. 

NOVALES,  R.  R.,  B.  J.  NOVALES,  S.  H.  ZINNER  AND  J.  A.  STONER,  1962.  The  effects  of  sodium, 
chloride,  and  calcium  concentration  on  the  response  of  melanophores  to  melanocyte-stimu- 
lating hormone  (MSH).  Gen.  Comp.  Endocr.,  2:  286-295. 

PARKER,  G.  H.,  1936.     Color  changes  in  elasmobranchs.     Proc.  Nat.  Acad.  Set.,  22:  55-60. 

PARKER,  G.  H.,  1948.  Animal  Colour  Changes  and  their  Neurohumors.  Cambridge  Univ. 
Press,  Cambridge,  Eng.,  377  pp. 

PICKFORD,  G.  E.,  AND  J.  W.  ATZ,  1957.  The  Physiology  of  the  Pituitary  Gland  of  Fishes. 
X.  Y.  Zool.  Soc.,  N.  Y.,  613  pp. 

SHIZUME,  K.,  A.  B.  LERNER  AND  T.  B.  FITZPATRICK,  1954.  In  -vitro  bioassay  for  the  melano- 
cyte-stimulating hormone.  Endocrinology,  54 :  553-560. 

SMITH,  H.  W.,  1936.  The  retention  and  physiological  role  of  urea  in  the  Elasmobranchii. 
Biol.  Rev.,  11:49-82. 

SPAETH,  R.  A.,  1913.  The  physiology  of  the  chromatophores  of  fishes.  /.  Exp.  Zool.,  15: 
527-585. 

SPYROPOULOS,  C.  S.,  AND  J.  TASAKI,  1960.  Nerve  excitation  and  synaptic  transmission.  Ann. 
Rev.  Physiol,  22 :  407-432. 

WARING,  H.,  1936.  Colour  changes  in  the  dogfish  (Scyllium  canicula).  Proc.  Trans.  Liver- 
pool Biol.  Soc.,  49:  17-64. 

WARING,  H.,  1960.  The  effect  of  pituitary  extracts  on  melanophores  in  isolated  elasmobranch 
skin.  Aust.  J.  Exp.  Biol.  Med.  Sci..  38:  187-194. 

WARING,  H.,  1963.  Color  Change  Mechanisms  of  Cold-blooded  Vertebrates.  Academic  Press 
Inc.,  N.  Y.,  266  pp. 

WARING,  H.,  AND  F.  W.  LANDGREBE,  1950.  Hormones  of  the  posterior  pituitary-  In:  The 
Hormones,  pp.  427-514.  Ed.  by  G.  Pincus  and  K.  V.  Thimann,  Academic  Press  Inc., 
N.  Y. 

WRIGHT,  M.  R.,  AND  A.  B.  LERNER,  1960.  On  the  movement  of  pigment  granules  in  frog 
melanocytes.  Endocrinology,  66:  599-609. 


DURATION  AND  FREQUENCY  OF  WING  BEAT  IN  THE  AGING 
HOUSE  FLY,  MUSCA  DOMESTICA  L.1 

MORRIS  ROCKSTEIN  AND  PREM  LATA  BHATNAGAR 

Department  of  Physiology,  University  of  Miami  School  of  Medicine, 
Coral  Gables,  Florida  33134 

In  a  long-range  study  of  aging  in  the  house  fly,  Rockstein  and  his  co-workers 
(Rockstein,  1956,  1957;  Rockstein  and  Brandt,  1963;  Rockstein  and  Gutfreund, 
1961)  have  previously  noted  that  abrading  and  ultimate  loss  of  wings,  especially 
in  the  male  house  fly,  is  preceded  and  then  accompanied  by  the  failure  or  decline- 
of  specific  intracellular  biochemical  components  (enzymes,  coenzymes,  etc.)  of  the 
thoracic  flight  muscle,  which  are  directly  or  indirectly  concerned  with  the  energizing 
of  the  contraction  of  flight  muscle.  Corresponding  microanatomical  changes  were 
similarly  reported  by  Rockstein  and  Bhatnagar  (1965)  in  describing  the  age-related 
distribution  of  number  and  size  of  giant  mitochondria  of  maturing  and  senescent 
male  and  female  house  flies. 

The  study  on  which  this  report  is  based  was  undertaken  in  order  to  establish 
more  precisely  the  age-related,  quantitative  changes  in  flight  ability,  i.e.,  the  wing 
beat  frequency  and  duration  of  flight,  for  male  and  female  house  flies  from  emer- 
gence to  senility. 

MATERIALS  AND  METHODS 

A  long-inbred  (NAIDM)  strain  of  Musca  doinestica  L.,  maintained  in  our 
laboratory  at  80°  F.  and  45%  R.H.,  was  used  in  this  study.  The  experimental 
population  was  obtained  by  allowing  4-  to  5-day-old  females  to  lay  eggs  on  a 
standardized  artificial  medium  described  earlier  (Rockstein,  1957).  From  the  time 
of  emergence  and  during  the  course  of  the  entire  experiment,  all  flies  were  allowed 
to  feed  ad  libitum  on  sucrose,  twice  daily,  for  a  period  of  one  hour  each  feeding; 
such  flies  were  considered  to  be  fully  satiated  as  far  as  their  carbohydrate  require- 
ments were  concerned.  A  continuous  supply  of  water  was  provided  throughout 
the  period  of  study. 

For  the  study  of  wing  beat  frequency  (WBF),  flies  of  known  age,  immediately- 
after  having  been  previously  fed  on  sucrose  for  an  hour,  were  anesthetized  under 
carbon  dioxide,  sexed,  and  mounted  ( attached  individually  in  the  dorsal  midthoracic 
region  with  Duco®  cement)  onto  thin,  inverted  "J"-shaped  copper  wire  supports, 
which  have  been  set  in  fine  holes  drilled  in  a  wooden  block  6"  X  1.5".  Removal; 
of  tarsi  or  of  any  substratum,  essential  for  the  initiation  and  sustenance  of  flight  in 
the  case  of  Phonnia  (Friedman,  1959;  Clegg  and  Evans,  1961)  or  of  Drosophila 
(Williams  et  al.,  1943),  was  found  to  be  quite  unnecessary  for  initiating  or  main- 
taining flight  in  the  house  fly,  which  normally  flies  spontaneously  when  so  sup- 

1  Supported  in  part  by  funds  from  the  United  States  Public  Health  Service,  Research  Grant 
No.  HD  00571  from  the  National  Institute  of  Child  Health  and  Human  Development.. 

479 


480 


MORRIS  ROCKSTEIN  AND  PREM  LATA  BHATNAGAR 


ported.  This  total  process  of  anesthetizing  and  mounting  takes  less  than  5  minutes 
and,  if  normal,  the  flies  begin  to  fly  spontaneously  within  two  to  three  minutes 
after  having  been  mounted  and  after  recovery  from  CO2  anesthesia.  In  fact,  those 
flies  which  did  not  fly  spontaneously  would  not  do  so  even  upon  stimulation  of  their 
tarsi  or  of  the  ventral  surfaces  of  the  abdomen.  All  phases  of  the  experiments 
were  conducted  under  constant  conditions  of  temperature,  humidity  and  light  as 
previously  described  by  Rockstein  (1956). 

Wing  beat  frequency  was  measured  by  means  of  a  Xenon  Stroboscope  (Cenco), 
with  the  mounted  flies  placed  about  6  inches  from  the  emission  tube  and  observa- 
tions made  at  5 -minute  intervals  and  expressed  as  a  percentage  of  the  initial  rate 
(Clegg  and  Evans,  1961).  In  each  case,  such  observations  were  continued  until 
90%  or  more  of  the  flies  could  no  longer  fly.  Such  experiments  were  repeated 
over  a  period  of  four  generations. 

TABLE  I 

Wing  bent  frequency  and  duration  of  flight  of  the  fetuale  house  fly  as  a  function  of  age 


Age  (days) 

No.  of  specimens 

Initial  WBFiS.K. 

Flight  duration  (in  minutes) 

1 

16 

8463  ±109 

500 

2 

17 

9584  ±151 

475 

4 

22 

9598  ±  129 

455 

5 

10 

9838  ±  189 

465 

6 

25 

9852  ±  136 

470 

7 

29 

9918  ±111 

440 

10 

28 

9965  ±  164 

398 

15 

33 

9869  ±  166 

225 

19 

19 

9857  ±  207 

105 

22 

22 

9855  ±  150 

110 

RESULTS  AND  DISCUSSION 


Females 


Table  I  shows  that  the  average  WBF  of  a  one-day-old  female  fly  is  8500  beats 
per  minute  (bpm)  and  that  this  increases  to  9600  by  the  second  day  and  reaches 
a  maximum  of  approximately  10,000  bpm  by  the  seventh  day;  thereafter,  there  is 
very  little  change  in  WBF  up  to  the  third  week,  with  perhaps  a  slight  (if  at  all 
significant)  diminution  in  WBF  at  the  beginning  of  the  third  week  and  into  the 
last  day  of  adult  life  on  which  such  observations  could  be  made. 

The  time  course  of  WBF  of  a  one-day-old  female  is  represented  in  Figure  1. 
For  the  first  155  minutes,  the  flies  fly  at  a  maximum  WBF  and  thereafter  they 
show  a  gradual  decline.  Even  after  500  minutes  of  continuous  flight,  however, 
the  flies  show  speeds  as  high  as  90%  of  the  initial  WBF.  Two-day-old  female 
flies,  writh  a  WBF  of  9600  beats  per  minute,  show  a  constant  WBF  flight  pattern 
tip  to  about  250  minutes  (Fig.  2),  when  all  of  the  flies  fly  at  about  95%  of  the 
initial  WBF.  However,  during  the  last  200  minutes  of  flight,  the  WBF  of  such 
two-day-old  females  declines  gradually  to  a  low  of  less  than  80%  of  the  original. 
For  female  flies  up  to  10  days  of  age,  the  WBF  and  the  time  course  of  flight  pat- 
terns are  quite  similar  to  those  of  younger,  two-day-old  females.  However,  only 


WING  BEAT  OF  AGING  HOUSE  FLIKS 


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100      150      200      250      300     350     400      450     500      550 
FLIGHT    DURATION     IN    MINUTES 


FIGURE  1.     Time  course  of  wing  beat  frequency  in  one-day-old  female  house  flies. 

after  12  days,  when  the  WBF  is  still  at  its  peak  level,  does  the  time  course  of  flight 
change  appreciably  (Fig.  3),  with  a  more  rapid  decline  within  310  minutes  to  a 
WBF  minimum.  Finally,  in  19-day-old  females  (Fig.  4),  there  is  a  rapid  decline 
to  a  minimum  in  WBF  in  a  little  over  100  minutes. 

Thus,  the  most  conspicuous  manifestation  of  senescence  in  flight  function  is 
the  inability  of  very  old  flies  to  sustain  flight  for  any  extended  periods  of  time.  To 
recapitulate  (as  can  be  seen  from  Table  I),  there  is  a  slow,  steady,  day-to-day 
decline  in  the  duration  of  flight,  so  that  by  the  third  week,  the  ability  of  the  female 
house  fly  to  maintain  flight  for  long  periods  of  time  is  reduced  markedly  from  500 
minutes  in  one-day-old  to  110  minutes  in  22-day-old  female  flies.  This  represents 
a  decline  in  the  ability  of  aging  females  to  maintain  flight  to  about  50%  of  the 


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50         100      150      200      250      300      350     400     450     500      550 
FLIGHT    DURATION    IN    MINUTES 

FIGURE  2.     Time  course  of  wing  beat  frequency  in  two-day-old  female  house  flies. 


482 


MORRIS  ROCKSTEIN  AND  PREM  LATA  BHATNAGAR 


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maximum  level  of  flight  duration  at  two  weeks  of  age  and  to  20%  of  the  initial 
maximum  by  the  third  week  of  adult  life. 

Males 

As  regards  age-related  failure  in  flight  ability  of  the  senescent  male  house  fly, 
it  should  be  emphasized  that  this  particular  study  was  carried  out  under  special 
conditions  of  maintenance  of  all  adult  flies  (i.e.,  feeding  them  sucrose  alone). 
Under  such  conditions,  the  rate  of  dying  is  much  accelerated,  i.e.,  60%  of  the  males 
were  dead  by  the  7th  day,  and  of  the  remaining  males  at  this  age,  only  a  few 
retained  their  wings  (see  Rockstein,  1956). 

TIME    COURSE   OF   WING    BEAT   FREQUENCY 
19-DAY  OLD  FEMALES 


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WING  BEAT  OF  AGING  HOUSE  FLIES 


483 


TABLE  II 
Wing  beat  frequency  and  duration  of  flight  of  the  male  house  fly  as  a  function  of  age 


Age  (days) 

No.  of  specimens 

Initial  WBFiS.K. 

Flight  duration  (in  minutes) 

1 

19 

8639  ±141 

420 

2 

21 

9328  ±  109 

365 

4 

28 

9710  ±  193 

265 

5 

13 

9698  ±  183 

220 

6 

26 

9733  ±  123 

180 

7 

17 

9801  ±  138 

135 

8 

9 

9821  db  163 

125 

9 

8 

9700  d=  183 

63 

Moreover,  under  the  above-mentioned  conditions,  60%  to  70%  of  the  male 
population  in  the  fly  colony  died  within  five  to  six  days  of  eclosion  and,  out  of  the 
30%  to  40%  remaining  survivors  which  could  he  studied  for  flight  ability,  only  a 
few  retained  intact  wings,  even  as  early  as  the  end  of  the  first  week  of  imaginal  life. 

From  Table  II,  it  can  be  seen  that  one-day-old  males  fly  at  about  8600  wing 
beats  per  minute,  which  compares  favorably  with  that  for  females  of  the  same  age 
(see  Table  I.  above).  This  increases  to  about  9300  wing  beats  per  minute  during 
the  following  24  hours.  Maximum  WBF  of  about  9700  to  9800  bpm  is  reached 
within  four  days  after  emergence  and  this  remains  unchanged  through  the  ninth 
day  of  adult  life,  following  which  time  no  winged  flies  were  available. 

Figure  5  shows  WBF  as  a  function  of  flight  duration  in  the  one-day-old  male 
house  fly.  The  time  pattern  of  the  WBF  of  such  young  flies  appears  to  be  very 
similar  to  that  of  two-day-old  female  house  flies  (Fig.  2),  i.e.,  the  WBF  declines 
by  about  18%  by  the  end  of  420  minutes,  at  which  time  the  majority  of  the  flies 
have  stopped  flying. 

By  the  fifth  day  after  emergence,  male  flies  show  a  sharp  decline  in  their  WBF 
(Fig.  6)  with  time,  i.e.,  from  the  onset  to  the  termination  of  flight,  with  the  majority 


TIME  COURSE  OF  WING  BEAT  FREQUENCY 
1-DAY  OLD  MALES 


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FIGURE  5.     Time  course  of  wing  beat  frequency  in  one-day-old  male  house  flies. 


484 


MORRIS  ROCKSTEIN  AND  PREM  LATA  BHATNAGAR 


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of  flies  showing  a  decline  of  about  30%  within  215  minutes.  The  rapidity  of  such 
decline  becomes  more  pronounced  with  age,  so  that  seven-day-old  males  (Fig.  7) 
also  show  a  decline  by  about  30%  of  WBF,  but  at  130  minutes  after  the  initiation 
of  flight. 

It  is  quite  apparent  that  the  detailed  data  from  these  experiments  confirm  the 
more  gross  manifestations  of  senescence  in  flight  ability,  i.e.,  the  gradually  increas- 
ing rate  of  failure  of  wing  retention  (male  flies  especially)  previously  observed  by 
Rockstein  (1956)  and  by  Rockstein  and  Brandt  (1963). 

It  would  therefore  appear  from  these  data  that  the  onset  of  decline  in  the  motor 
function  of  flight  in  this  holometabolous  species  clearly  begins,  as  might  be  expected, 


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FLIGHT     DURATION    IN     MINUTES 


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FIGURE  7.     Time  course  of  wing  beat  frequency  in  7-day-old  male  house  flies. 


WING  BEAT  OF  AGING  HOUSE  FLIES  485 

shortly  after  emergence,  when  the  insect  is  fully  mature  and  all  of  its  tissues  are 
essentially  post-mitotic   (with  the  exception  of  sexual  maturation). 

However,  Williams  et  al.  (1943)  found  this  not  to  be  true  in  Drosophlla  junc- 
bris,  where  both  flight  duration  and  wing  beat  frequency  reach  a  peak  by  about 
7  days  and  then  drop  off  rapidly  to  a  minimum  by  35  days  of  adult  life.  However, 
no  distinction  as  to  relative  flight  ability  of  male  and  female  Drosophila  (either  as 
regards  WBF  or  duration  of  flight)  was  made  by  these  authors,  a  distinction  which 
both  our  present  study  and  others  have  indicated  may  be  a  significant  factor  in 
all  species  studied. 

In  the  two  other  important  reports  in  which  flight  ability  was  studied  (both  in 
PJiorniia  regitia),  unfortunately  only  WBF,  with  no  distinction  as  to  sex,  was 
determined  for  aging  adult  blow  flies  by  Levenbook  and  Williams  (1956)  and 
without  regard  to  sex  or  age  by  Clegg  and  Evans  (1961).  It  is  therefore  difficult 
to  attempt  comparisons  between  data  for  this  present  study  and  those  in  the  rela- 
tively few  past  studies  of  this  kind. 

As  for  the  trends  observed  in  Tables  I  and  II  for  wing  beat  frequency,  values 
of  about  10,000  beats  per  minute  in  mature  males  and  female  house  flies  resemble 
closely  those  reported  both  by  Levenbook  and  Williams  (1956),  and  by  Clegg  and 
Evans  (1961)  for  Phonnia  regina.  It  is  clear,  however,  that  the  parameter  of 
duration  of  flight  is  much  more  significant,  from  the  standpoint  of  aging,  and  that 
this  parameter  must  also  be  measured  separately  for  male  and  female  adults,  at 
least  as  far  as  the  common  house  fly,  Musca  domestica,  is  concerned. 

Finally,  from  the  standpoint  of  senescence  of  flight  ability,  the  steady  diminu- 
tion in  the  capacity  of  male  house  flies  to  sustain  the  original  high  levels  of  flight 
intensity  (WBF)  for  extended  periods  of  time  confirms  previously  obtained  quanti- 
tative, time-related  data  for  wTing  loss  as  such,  and  decline  in  ATP-ase,  alpha- 
glycerophosphate  dehydrogenase  and  acid  phosphatase  activity,  both  in  the  sarco- 
somes  and  in  the  extrasarcosomal  elements  of  the  flight  muscle  (Rockstein,  1956; 
Rockstein  and  Brandt,  1963). 

SUMMARY 

1.  The  age-related  changes  in  wing  beat  frequency  and  duration  of  flight  were 
studied  in  senescent  male  and  female  NAIDM  house  flies. 

2.  The  average  wing  beat  frequency  increases  to  a  maximum  by  the  fifth  day 
in  female  and  by  the  fourth  day  in  male  house  flies. 

3.  Duration  of  flight  shows  a  steady,  day-to-day  decline  with  age.     In  females, 
this  falls  from  500  minutes  for  one-day-old  to   110  minutes  for  22-day-old  flies. 
For  males,  this  drop  is  more  striking,  with  duration  of  flight  falling  from  420 
minutes  in  one-day-old  to  63  minutes  for  nine-day-old  males. 

4.  These  findings  correspond  to  and  confirm  quantitatively  previously  reported 
data  for  wing  loss  and  similar  age-related  changes  in  enzyme  and  coenzyme  content 
in  the  flight  muscle  of  senescent  house  flies. 

LITERATURE  CITED 

CLEGG,  J.  S.,  AXD  D.  R.  EVANS,  1961.  The  physiology  of  blood  trehalose  and  its  function  dur- 
ing flight  in  the  blowfly.  /.  £.v/>.  Biol.,  38:  771-792. 

FRIEDMAN",  S.,  1959.  Sustained  flight  in  Phonnia  (by  a  new  method)  and  its  effect  on  blood 
pH.  /.  Ins.  Physiol.,  3 :  118-1 19. 


486  MORRIS  ROCKSTEIN  AND  PREM  LATA  BHATNAGAR 

LEVENBOOK,  L.,  AND  C.   M.  WILLIAMS,   1956.     Mitochondria  in  the  flight  muscles   of  insects. 

III.  Mitochondrial  cytochrome  c  in  relation  to  the  aging  and  wing  beat  frequency  of  flies. 

/.  Gen.  Physiol,  39:  497-512. 

ROCKSTEIN,  M.,  1956.     Some  biochemical  aspects  of  aging  in  insects.     /.  Gerontol.,  11:  282-285. 
ROCKSTEIN,  M.,  1957.     Longevity  of  male  and  female  house  flies.     /.  Gerontol.,  12:  253-256. 
ROCKSTEIN,  M.,  AND  P.  L.  BHATNAGAR,  1965.     Age  changes  in  size  and  number  of  the  giant 

mitochondria  in  the  flight  muscle  of  the  common  house  fly    (Musca  domestica  L.). 

/.  Ins.  Physiol.,  11:  481-491. 
ROCKSTEIN,  M.,  AND  K.  BRANDT,  1963.     Enzyme  changes  in  flight  muscle  correlated  with  aging 

and  flight  ability  in  the  male  house  fly.     Science,  139:   1049-1051. 
ROCKSTEIN,  M.,  AND  D.  E.  GVTFREUND,   1961.     Age  changes  in  adenine  nucleotides  in  flight 

muscle  of  male  house  fly.     Science,  133:  1476-1477. 
WILLIAMS,  C.  M.,  L.  A.  BARNESS  AND  W.  H.  SAWYER,  1943.     Utilization  of  glycogen  by  flies 

during  flight  and  some  aspects  of  the  physiological  ageing  of  Drosophila.     Biol.  Bull.. 

84:263-272. 


ARTIFICIAL  CULTURE  OF  MARINE  SEA  WEEDS  IX 
RECIRCULATION  AQUARIUM  SYSTEMS 

JOHN  A.  STRAND,  JOSEPH  T.  CUMMINS  AND  BURTON  E.  VAUGHAN 

Biological  and  Medical  Sciences  Division,   U.  S.  Naval  Radiological  Defense  Laboratory, 

San  Francisco,  California  94135 

Purely  artificial  sources  of  sea  water  at  present  are  still  totally  inadequate  to 
facilitate  normal  and  prolonged  growth  of  any  of  the  more  exacting  marine  sea 
weeds  (Provasoli,  1963,  p.  9).  Enriched  sea  water  techniques,  however,  have  pro- 
vided a  suitable  means  to  establish  speciation  and  life-cycle  determinations  of  sev- 
eral highly  organized  forms.  In  1934  Foyn  successfully  cultured  Ulva  lactuca 
through  a  complete  life-cycle  in  enriched  media,  using  "Erdschreiber"  sea  water 
with  X,  P,  and  soil  extract.  Subsequent  studies  on  artificial  culture  of  Ulva  and 
related  genera  have  developed  through  empirical  refinement  of  this  basic  technique. 

Provasoli  (1963,  p.  11)  was  able  to  obtain  nearly  natural  morphological  develop- 
ment, i.e.,  a  flat  blade-like  thallus  2-3  cm.  long,  in  sporelings  of  Ulva  lactuca  only 
with  initial  samples  of  enriched  sea  water.  Subsequent  samples  enriched  in  the 
same  manner  yielded  varying  results.  The  inability  to  provide  for  sustained  growth 
and  development  in  a  reproducible,  synthetic  medium  has  thus  greatly  restricted  our 
understanding  of  salient  ecological  and  physiological  processes  involved  in  growing 
marine  sea  weeds.  Specific  disadvantages  of  older  enrichment  methods  are  obvious, 
and  they  include :  significant  variability  in  micro-element  composition  of  different 
sea  water  samples ;  reliance  upon  heat  sterilization  to  provide  contaminant-free 
media,  often  resulting  in  precipitation  of  essential  additives ;  and  variability  in  soil 
extract  samples  utilized. 

It  is  evident  that  current  methods  for  simulation  of  natural  conditions  are  far 
from  adequate.  Information  available  provides  exhaustive  description  of  the  uses 
of  trace  elements,  growth  regulators,  and  vitamin  sources,  but  specifically  fails  to 
provide  fresh  insight  into  the  complex  of  physical,  chemical,  and  biological  factors 
and  the  way  in  which  they  alter  the  bathing  medium.  In  many  respects  oceans  are 
relatively  constant  biological  environments,  for  example,  with  respect  to  dissolved 
gases,  salinity,  pH,  temperature,  and  illumination  (Provasoli  et  al.,  1957).  Wider 
variations  are  evident  in  littoral  or  estuarine  waters,  but  again,  transitions  are 
probably  gradual  and  seasonal  under  most  natural  conditions.  In  a  laboratory 
situation  these  properties  can  be  controlled  readily.  Biotic  interaction  and  micro- 
element composition  present  additional  complications,  but  in  principle  at  least  can 
be  brought  under  laboratory  control.  For  the  wTork  reported  here,  a  closed-system 
approach  was  implemented  to  facilitate  systematic  study  of  essential  parameters. 

MATERIALS  AND  METHODS 

Marine  sea  weeds  utilized  in  present  investigations  were  gathered  from  Mon- 
terey Bay,  Moss  Beach,  Point  Reyes,  and  San  Francisco  Bay.  Most  species  col- 

487 


488 


J.  A  STRAND,  J.  T.  CUMMINS  AND  B.  E.  VAUGHAN 


lected  were  accurately  identified  in  the  field.  Verification  of  closely  related  forms 
was  achieved  through  standard  histological  technique.  A  fixing  fluid  consisting 
of  1  g.  chromic  acid,  1  ml.  propionic  acid  in  90  cc.  sea  water  proved  to  be  satis- 
factory for  most  marine  algae.  Delafield's  or  Harris'  hematoxylin  was  a  suitable 
stain  for  most  preparations ;  an  appropriate  counter  stain  of  erythrosin  B,  orange  G 
(0.8%)  or  fast-green  (Johansen,  1951,  pp.  359,  361)  was  also  used. 

Transplants,  prior  to  placement  into  aquaria,  were  rinsed  in  fresh  sea  water 
and  pesticide,  using  Lindane    (hexachloro-cyclohexane),   5  ppm.  for   15   minutes, 


NRDL  389-66 


SUPPLY   TO 
OTHER  AQUARIA 


OTHER 
AQUARIA 


FIGURE  1.  Flow  diagram  for  recirculation  aquarium  system:  temperature  16  ±  1°  C. ; 
salinity  25  ±  3%c ;  total  filtration  from  4  to  10/x;  pH  7.9  ±  0.3 ;  recirculation  190  L./min. ;  sea 
water  replacement  4%  per  day.  Details  described  in  text. 

to  kill  resident  herbivorous  gastropods  and  polychaetes.  Polarographic  measure- 
ments at  the  surface  of  Ulva  after  24  hours  exposure  indicate  that  Lindane  at 
concentrations  of  650  ppm.  did  not  appreciably  inhibit  oxygen  evolution  (report 
in  preparation). 

Of  eight  requirements  to  obtain  optimal  growth,  five  were  adjusted  empirically: 
(1)  micronutrient  enrichment,  (2)  CO2  as  a  carbon  source,  (3)  salinity  and  pH 
stabilization,  (4)  light  source  of  suitable  intensity  and  spectral  quality,  and  (5) 
agitation  and  aeration.  Additional  requirements  were  necessitated  due  to  the  re- 
circulation  of  sea  water  within  the  system :  (6)  nonmetallic  construction,  (7)  filtra- 
tion and  sterilization,  and  (8)  sea  water  replenishment.  A  schematic  diagram  of 


CULTURE  OF  MARINE  SEA  WEEDS  489 

the  closed-recirculating  aquarium  system  is  shown  in  Figure  1  ;  its  components  will 
be  described  separately  below  : 

Fresh,  sand-filtered  sea  water  from  off-shore  was  provided  by  Steinhart  Aqua- 
rium, California  Academy  of  Sciences,  and  this  was  enriched  with  phosphate, 
nitrate,  and  EDTA  and  other  micronutrients.  A  mixture  like  that  of  Haxo  and 
Sweeney  (Provasoli  et  al,  1957)  provided  a  medium  favorable  to  growth  of  Viva 
lobata  and  related  species,  except  that  thiamin,  biotin,  and  B12  were  substituted  for 
soil  extract  (Table  I).  Tris  (hydroxymethyl)  aminomethane  buffer,  pH  range 
7.5-8.5,  also  was  added  at  0.3  part  per  thousand.  The  salinity  of  fresh  sea  water 
with  its  micronutrients  then  was  adjusted  to  25  ±  3.0  parts  per  thousand.  Daily 
replacement  was  maintained  at  the  rate  of  4%  of  the  total  volume  of  the  aquarium 
system. 

TABLE  I 
Sea  water  enrichment  mixture 

KNO3  20.0  mg. 

K,HPO4  3.5  mg. 

FeCh  0.097  mg. 

MnCl2  0.0075  mg. 

Glycerophosphate  di-sodium  pentahydrate  1.0  mg. 

EDTA  1.0  mg. 

B13  1.0  Mg- 

Thiamin  HC1  0.2  mg. 

Biotin  1.0  jug. 

Tris  (hydroxymethyl)  aminomethane  30.0  mg. 

Fresh  off-shore  sea  water  75.0  ml. 

Distilled  water  25.0  ml. 

Vitamins,  organic  phosphate,  and  Tris  buffer  not  included  in  Haxo  and  Sweeney  formulation 
(1955). 

The  recirculating  aquarium  system  was  constructed  of  fiberglass,  polyvinyl 
chloride  (PVC),  hard  rubber,  plastic,  and  glass,  in  order  to  reduce  to  a  negligible 
degree  contamination  by  undesirable  metallic  ions. 

Water  entering  the  closed  system  passed  initially  through  a  20 /u,  mesh  pre- 
filter  within  the  addition  tank.  Water  leaving  the  addition  tank  passed  through 
the  pump  (Duriron  Company,  Inc.)  into  a  filter  column  containing  a  non-impreg- 
nated cellulose  cartridge.  The  cartridge  was  designed  to  remove  particles  within 
a  4  to  10 /A  range  (Hilliard-Hilco  Corp.).  Efficient  filtration  was  achieved  at  flow 
rates  in  excess  of  190  liters  per  minute,  and  at  pressures  not  exceeding  60  psi. 
Temperature  control  at  16  ±  1°  C.  was  maintained  by  a  two-stage  system.  The 
initial  level  was  a  heat  exchanger  in  which  10-20%  ethylene  glycol  in  water  was 
cooled  with  freon  refrigerant  by  a  5-ton  capacity  compressor  (Dunham-Bush,  Inc.). 
The  ethylene  glycol  solution  in  turn  exchanged  heat  through  an  impervious  graphite 
shell  and  tube  heat  exchanger,  the  latter  of  which  circulated  aquarium  water  (Na- 
tional Carbon  Co.). 

Alkalinity  was  monitored  continually,  and  it  was  automatically  stabilized  at 
pH  7.9  ±  0.3  by  continuous  metering  of  CO,  gas.  For  this  purpose,  standard  pH 
electrodes  were  placed  just  below  surface  water.  A  pH  meter  (Beckman,  Model 


490 


J.  A  STRAND,  J.  T.  CUMMINS  AND  B.  E.  VAUGHAN 


2.5 


NRDL  389-66 


2.0    - 


ir 

UJ 


1.5 


o 
^ 

t- 
< 

5. 

LJ 
O 

O 

cr 

_i 
o: 

i 

CL 


1.0 


0.5 


W 
I 


U/ 
/ 

/ 
/_ 


\ 


\ 


V 


400 


500  600 

WAVELENGTH  (NANOMETER) 


700 


800 


FIGURE  2.  Spectral  energy  distribution  (S.E.D.)  at  water  surface  (dotted  line)  ;  energy 
detected  through  wide  and  narrow  band-pass  interference  filters  (horizontal  bars).  S.E.D. 
curve  was  interpolated,  amounting  to  approximately  3.7  kilolux  as  the  integrated  area  from  375 
to  725  nanometers.  For  combined  source,  using  40  watts  Gro-lux  and  40  watts  warm  white 
(fluorescent).  Details  in  text. 


CULTURE  OF  MARINE  SEA  WEEDS  491 

H-2)  was  modified  by  replacing  the  indicating  meter  with  a  meter  relay  switch. 
This  switch  activated  a  solenoid  valve  on  the  CO2  cylinder  when  pH  increased. 
Gaseous  CCX  entering  the  system  in  this  manner  not  only  enhanced  pH  stabiliza- 
tion hut  also  provided  an  additional  carbon  source  for  photosynthesis. 

Contaminating  bacteria,  protozoa,  and  diatoms  were  controlled  through  use 
of  an  ultraviolet  sterilization  source.  This  unit  was  constructed  entirely  of  PVC 
and  quartz  glass  tubing,  discharging  radiations  in  the  2537  A  range  (Aqua-Fine 
Corp.).  The  unit  efficiently  killed  biotic  contaminants  after  24  hours  of  recircula- 
tion.  at  flow  rates  in  excess  of  190  liters  per  minute.  Elimination  of  biotic  con- 
taminants was  confirmed  bacteriologically. 

Overhead  lighting  was  provided  for  each  aquarium.  Two  40-watt,  fluorescent 
lamps,  1  Gro-lux  (Sylvania),  and  1  Warm-white  (General  Electric),  were  placed 
in  a  standard  white,  painted  reflector  at  the  spacing  recommended  by  the  manu- 
facturer ( Mpelkas,  1964a).  This,  when  located  50  cm.  above  aquarium  top, 
yielded  an  irradiance  almost  entirely  in  the  visible  region,  amounting  to  3.7  kilolux 
at  the  water  surface  (Fig.  2).  Energy  emission  of  the  Gro-lux  lamps  follows 
closely  the  absorption  spectrum  of  chlorophyll  pigments  with  peak  energy  output 
in  the  440-460  and  660-680  nanometer  range  (Mpelkas,  1964b).  Peak  emission 
of  the  \Yarm-white  lamp  occurs  within  the  mid-region  of  the  action  spectrum,  490- 
590  nanometers  (Mpelkas,  1964b)  fitting  the  absorption  spectra  of  accessory  pig- 
ments. Spectral  energy  distribution  for  the  combined  sources  at  50  cm.  is  reported 
in  Figure  2.  This  was  determined  using  narrow  band  pass  interference  filters  (Set 
60;  Optics  Technology,  Inc.)  in  line  with  an  optical  power  meter  (Model  610; 
Optics  Technology,  Inc.).  Response  characteristics  of  the  meter-filter  system  were 
standardized  against  a  known  source  for  spectral  irradiance.  The  photoperiod  was 
maintained  automatically,  and  was  varied  according  to  conditions  to  be  described 
at  a  later  point.  Culture  tanks  were  installed  in  a  windowless  room,  devoid  of 
natural  sunlight. 

RESULTS 
Ta.i'onoinv  and  natural  liistor\  of  Ulva  lobata 

Thalli,  like  those  described  by  Setchell  and  Gardner  (1920)  and  Smith  (1944) 
were  found  along  the  California  Coast ;  they  attained  moderate  size,  nearly  50  cm. 
tall,  20-30  cm.  broad,  and  wrere  usually  rich  green  in  color.  The  plants  observed 
were  saxicolous  and  occasionally  epiphytic,  and  they  were  found  in  the  mid-littoral 
zone  between  2.0-  and  •  -  2.0-ft.  tide  levels.  Blades  were  membranaceous,  broadly 
expanded,  deeply  divided,  and  slightly  ruffled  at  margins.  The  thalli  gradually 
narrowed  to  a  stipe-like  holdfast.  Holdfast  structures  were  perennial ;  blade  por- 
tions, annual.  This  species  differed  from  the  closely  related  Uk'a  c.rpansa,  pri- 
marily in  extent  of  division  and  size  of  blade.  The  latter  was  not  deeply  divided 
and  was  observed  to  attain  a  length  in  excess  of  150  cm.  Like  those  of  all  species 
of  the  genera,  the  blade  of  Ulva  lobata  was  distromatic.  It  varied  from  40  ^  thick- 
ness at  the  margin  to  90  /j.  in  the  more  central  portion.  Cells  as  examined  micro- 
scopically are  shown  in  Figure  3. 

The  literature  indicates  that:  reproduction  follows  an  alternation  of  identical 
asexual  and  sexual  generations ;  each  fertile  cell  of  the  diploid  sporophyte  is  capable 


492 


J.  A  STRAND,  J.  T.  CUMMINS  AND  B.  E.  VAUGHAN 


of  producing  8  or  16  quadri-flagellate  zoospores;  meiosis  occurs  during  the  first 
and  second  divisions,  the  zoospores  developing  into  haploid  gametophytes ;  and 
that  the  gametophyte  generation  is  heterothallic  and  anisogamous  (Smith,  1944). 
Gametes  released  by  mature  plants  were  measured  in  the  present  study  by  stage 


CHLOROPLAST 


NUCLEUS 


FIGURE  3.  Ulva  lobata,  cross-section,  H&E  stain.  Cells  appear  tightly  packed  with  walls 
of  adjacent  cells  confluent  to  form  a  rigid  matrix.  Note  distromatic  organization.  (A)  X  760, 
(B)  X  1900. 


micrometer,  verifying  reported  descriptions  of  anisogamy.  Male  gametes  here 
measured  2.0-3.5  X  5. 5-7.0  ^  in  size;  female  gametes  measured  2.5-4.0  X  6.0-8.0/z. 
Each  was  ovate  or  pear-shaped,  showing  a  conspicuous  eyespot  and  chloroplast, 
and  each  was  bi-flagellate. 


CULTURE  OF  MARINE  SEA  WEEDS  493 

Gametes  and  zoospores  were  observed  to  lie  discharged  through  a  small  pore 
on  the  external  border  of  marginal  cells.  The  exit  pore  was  not  observed  until 
the  onset  of  sporulation.  Instantaneous  with  release,  gametes  or  zoospores  col- 
lected into  clumps,  containing  often  as  many  as  50-100  cells.  This  clumping  was 
somewhat  larger  than  that  reported  by  others  (Smith,  1947).  Gametic  union 
occurred  during  clumping,  pairs  fusing  side-by-side  or  anteriorly  end-on-end. 
Clumps  disintegrated  within  2  to  3  minutes,  and  the  quadri-flagellate  zygotes  re- 
mained motile  for  several  hours.  Zoospores  were  observed  to  remain  motile  longer, 
e.g.,  4  to  5  hours. 

Available  literature  (Smith,  1947)  shows  that  fruiting  or  sporulation  of  both 
gametophyte  and  sporophyte  generations  occurs  at  predictable  28-day  intervals, 
but  only  during  the  spring  tides  of  the  lunar  month,  as  observed  during  summer 
months.  Gametophytic  plants  sporulate  early  during  the  series  of  spring  tides, 
while  sporophytic  plants  liberate  zoospores  late  during  the  series.  Thalli  of  both 
generations  are  usually  found  closely  associated  and  in  approximately  equal  num- 
bers. Gametes  have  been  reported  to  germinate  parthenogenetically  (Smith,  1947; 
Moewus,  1938;  and  Yamada  and  Saito,  1938). 

Aquarium  development  and  differentiation  of  Ulz>a  hbata 

Fertile  thalli  of  Ulra  lobata  were  placed  into  plastic  bags  containing  fresh  sea 
water  and  transported  to  the  laboratory  in  ice  chests  maintained  at  10-15°  C. 
Mature  plants  transported  in  this  manner  often  discharged  swarmers  (gametes  or 
zoospores)  within  a  few  hours,  as  they  also  did  when  placed  into  fresh  sea  water 
of  normal  seasonal  temperature.  The  slightest  change  in  environmental  conditions 
often  stimulated  sporulation,  for  example  decreasing  temperature,  desiccation  or 
stimulation  by  intense  light.  Contents  of  bags  showing  spore  liberation  were 
mixed  and  gently  agitated  for  one  hour  to  insure  fertilization.  The  resulting 
zygospores  were  poured  into  a  225-liter  culture  tank  and  water  turnover  was 
reduced  to  allow  their  implantation  on  the  aquarium  bottom. 

Infertile  thalli  usually  developed  to  maturity  within  a  few  days  after  placement 
into  laboratory  aquaria.  Occasionally  blades  would  sporulate  spontaneously,  usu- 
ally 2-3  weeks  after  transplant.  Even  as  late  as  December  or  January  infertile 
thalli  were  seen  to  develop  mature  reproductive  cells  and  could  be  stimulated  to 
release  swarmers. 

Development  of  sporelings  was  followed  on  a  daily  basis  and  compared  to 
existing  accounts  of  similar  species.  Photomicrographs  were  prepared  to  record 
the  significant  stages  of  differentiation  not  previously  described  for  Ulva  lobata 
(Fig.  4).  Development  of  the  sporophyte  generation  will  be  described  below. 

One  to  two  days  after  fertilization  and  implantation,  zygotes  became  more 
spherical,  approximately  6-8  /*  in  size.  Two  eye-spots  and  a  single  pyrenoid  were 
still  recognizable  (Fig.  4A,  B).  In  from  2  to  4  days,  zygotes  increased  their 
size  to  8-10  /JL  and  showed  a  thin,  clear  membrane  which  gradually  thickened  as 
the  onset  of  germination  approached.  Germination  occurred  in  from  6  to  7  days 
and  the  eye-spots  at  this  stage  were  no  longer  visible.  At  9  to  10  days,  the  spore- 
ling  appeared  as  a  slender  filament  of  8-10  cells,  40-45  p,  in  length,  since  division 
only  occurred  along  the  transverse  axis  (Fig.  4C).  At  this  time,  the  basal  cell 
sometimes  elongated  to  form  a  primary  rhizoid.  At  14  days,  and  at  nearly  100  ^  in 


J.  A  STRAND,  J.  T.  CUMMINS  AND  B.  E.  VAUGHAN 


length,  the  first  cellular  divisions  along  the  longitudinal  plane  of  the  filament  oc- 
curred (Fig.  4D)  ;  secondary  rhizoids  were  then  observed  to  develop.  Through 
continued  longitudinal  divisions,  at  40  days,  the  sporeling  represented  the  flattened 
memhranaceous  structure  of  the  mature  thallus.  At  60  days,  the  young  plant  was 
4.5-5.0  cm.  in  length  and  1.5-2.0  cm  in  breadth  (Fig.  4E).  Although  the 
young  sporophytes  were  of  sufficient  size  and  maturity  to  liberate  zoospores,  sub- 
sequent development  of  the  gametophyte  generation  was  not  followed.  In  these 


NRDL   798-65 


A 


o 


, 

' 


RESTING 
ZYGOTE 


PYRENOID 


H 

10/A 


D 


LATERAL 
DIVISION 


H 
10/i 


A  Resting  phase   zygotes 
6-8/j.  ,  3  days 

B  Germination  of  zygotes, 
7  days 

C  Elongation  into  simple 
filament,  10  days 

D  Cell  division,  longitudinal 
plane,  14  days 

E   Membraneous  sporeling, 
40  days 


FIGURE   4.     Photomicrographs    showing   development    and    differentiation    of    Ult'a    lobata 
sporelings  in  recirculation  aquarium  system. 

studies  it  was  not  possible  to  distinguish  morphological  differences  in  the  develop- 
ment of  Ulva  lobata,  when  compared  to  the  closely  related  species,  Viva  (lactuca) 
(Miyake  and  Kunieda,  1931)  or  Ulva  pertusa  (Yamada  and  Saito,  1938).  Nor- 
mal elongation  and  subsequent  differentiation  were  clearly  demonstrated  in  Figure 
4.  Deformities  such  as  the  acute  twisting  and  bulging  of  Ulva  lactuca  filaments, 
described  by  Rea  (1964),  were  not  evident. 

Longitudinal  division  of  filaments  of   Ulva  lobata  occurred  in   approximately 
10-14  days  here,  whereas  in  a  study  of  Ulva  pertusa,  it  occurred  no  earlier  than 


CULTURE  OF  MARINE  SEA  WEEDS  495 

30  clays  after  germination  ( Yainacla  and  Saito,  1938).  Also,  in  the  present  study 
sporelings  were  nearly  ten-fold  larger  at  45  days,  compared  to  those  of  Ulva  pert  lisa 
as  measured  at  60  days.  These  differences  probably  do  not  indicate  different  spe- 
cies characteristics,  but  rather  attest  to  the  adequacy  of  the  present  culture  condi- 
tions. It  seems  likely  that  earlier  accounts  of  sporeling  development  were  hampered 
by  less  adequate  growth  conditions. 

Laboratory-maintained  Ulra  transplants 

The  genus  Ulva  demonstrates  a  marked  seasonal  periodicity  with  respect  to 
both  vegetative  and  reproductive  growth,  and  this  is  mediated  undoubtedly  through 
both  environmental  and  less  understood  internal  factors.  Among  the  former,  dura- 
tion of  photoperiod  and  water  temperature  are  of  particular  interest. 

In  general,  vegetative  growth  in  Ulva  is  observed  to  begin  slowly  in  early 
Spring  with  the  known  lengthening  of  photoperiod  and  rise  in  water  temperature. 
Maximum  attainment  of  both  vegetative  and  reproductive  processes  is  reached  by 
mid-summer.  The  growth  response  gradually  diminishes  by  late  summer,  the 
thallus  dying  back  to  where  only  the  holdfast  and  a  small  portion  of  the  leafy  blade 
survive  the  winter  months.  This  attrition  seems  to  be  determined  by  changes  in 
duration  of  photoperiod  and  environmental  temperature.  It  affects  both  gameto- 
phyte  and  sporophyte  generations  alike. 

Care  was  exercised  during  collection  of  specimens  not  to  damage  or  disrupt 
holdfast  structures.  Usually  small  plants,  less  than  20  cm.  tall  and  firmly  anchored 
to  small  rocks  or  pebbles,  were  selected.  Thalli  in  which  holdfasts  were  carefully 
cleaved  from  attachment  occasionally  would  continue  differentiation  of  this  struc- 
ture and  subsequently  reattach  on  aquarium  bottoms.  Usually  samples  of  Ulva 
lobata,  Ulva  lactuca,  and  Ulva  linza,  with  holdfasts  intact,  transplanted  better; 
however,  this  was  not  true  of  Ulva  e.vpansa.  In  the  latter  species,  free-living  thalli 
frequently  break  away  from  the  holdfast  and  continue  to  grow,  if  free-floating  in 
more  quiet  backwaters  (Smith,  1944). 

Seasonal  rhythm  was  preserved  during  laboratory  culture  and  was  paramount 
in  determining  transplant  longevity  (Table  II).  If  viable  plants  were  removed 
for  transplant  early  during  the  growth  season,  that  is,  during  May  or  June,  they 
were  usually  adequately  maintained  for  three  months  or  more,  comparable  to  the 
natural  seasonal  growth.  Thalli  introduced  to  laboratory  culture  late  during  the 
growth  season  exhibited  a  less  extensive  vegetative  development  or  none  at  all. 
Exposure  to  those  environmental  conditions  correlated  with  maximal  vegetative 
and  reproductive  development  did  not  alter  the  onset  of  senescence;  e.g.,  water 
temperature  (surface)  16-18°  C.  and  photoperiod  13-15  hours,  which  are  mean 
summer  conditions  in  Monterey  Bay.  Long-day  illumination,  13-15  hours,  under 
present  methods  of  culture  favored  longevity  of  Ulva  transplants  removed  from  the 
sea  during  the  months  of  May,  June  and  July  (Table  II).  Thalli  removed  during 
late  summer,  August  and  September,  were  maintained  only  if  the  photoperiod  was 
decreased  to  10-12  hours.  Exposure  to  photoperiods  above  16  hours  or  below 
10  hours  caused  a  gradual  degeneration  of  tissues  in  7-10  days.  Exposure  to 
continuous  light  produces  a  rapid  shriveling  and  hardening  of  the  thallus,  evident 
in  48  hours.  Microscopic  examination  of  tissues  illuminated  continually  for  96 
hours  revealed  a  marked  dissolution  of  mucilage  material  associated  \vith  the  free 


496 


J.  A  STRAND,  J.  T.  CUMMINS  AND  B.  E.  VAUGHAN 


TABLE  1 1 

Effect  of  photoperiod  and  other  lighting  conditions  on  longevity ;  summarized  data 
for  Ulva  transplants  maintained  in  recirculation  aquaria* 


Species 

Transplants 
or  sporel. 

(N) 

Natural 
photoper. 
(hr.) 

Artificial 
photoper. 
(hr.) 

Source  of 
ilium. 

Irrad.  level 
(kilolux) 

Longevity! 

May-Ju.  collections: 
U.  lobata 

12 

14.0 

13-15 

GL+WW 

3.7 

3  mo. 

U.  lobata 

12 

14.5 

16-18 

GL+WW 

3.7 

1  wk. 

U.  expansa 

12 

14.5 

16-18 

GL  +  WW 

3.7 

1  wk. 

U.  lobalu 

4 

14.5 

continuous 

GL  +  WW 

3.7 

48  hr. 

U.  expansa 

4 

14.5 

continuous 

GL  +  WW 

3.7 

48  hr. 

U.  lobata 

12 

14.5 

13-15 

GL  +  WW 

7.4 

1-2  wk. 

Oct.  collections  : 

U.  lobata 

12 

11.5 

10-12 

GL  +  WW 

3.7 

2  mo. 

U.  ex  pa  HSU 

12 

11.5 

10-12 

GL  +  WW 

3.7 

2  mo. 

Sporelings,  U.  lob. 
Sporelings,  U.  lob. 

300* 

11.5 
11.5 

13-15 
13-15 

GL  +  WW 

GL+WW 

3.7 
7.4 

3  mo. 
3  mo. 

£7.  lobata 

12 

11.5               13-15 

GL  +  WW 

3.7 

1-2  wk. 

U.  expansa 

12 

11.5 

13-15 

GL  +  WW 

3.7 

U.  lobata 

12 

11.5 

8 

GL  +  WW 

3.7 

1  wk. 

U.  expansa 

12 

11.5 

8 

GL+WW 

3.7 

1  wk. 

U.  lobata 

24 

11.5 

10-12 

DL  +  DL 

3.7 

1-2  wk. 

U.  expansa 

24 

11.5               10-12 

DL  +  DL 

3.7 

1-2  wk. 

U.  lobata 

24 

11.5 

10-12 

cw+cw 

3.7 

1-2  wk. 

U.  expansa 

24 

11.5 

10-12 

cw+cw 

3.7 

1-2  wk. 

Dec.—  Jan.  collections: 
U.  lobata 

24 

10.0 

10-12 

GL+WW 

3.7 

2  mo. 

U.  expansa 

24 

10.0 

10-12 

GL  +  WW 

3.7 

2  mo. 

*  Nutrient  factors,  salinity,  pH  and  other  conditions  are  constant;  see  text. 

f  Forty-watt  fluorescent  tubes:  GL  =  Gro-Lux;  WW  =  Warm-white;  DL  =  Daylight; 
C\V  =  Cool  white. 

J  Bleaching  and  autolysis  first  evidenced;  for  continuous  ilium.,  dissolution  of  mucilaginous 
envelope  and  shriveling  of  thallus  were  evidenced  at  48  hours. 

A  Number  estimated  from  area  and  number  in  microscopic  field. 


cell  border  surfaces  of  the  blade.  Comparable  tissue  breakdown  occurred  if  light 
intensities  exceeded  3.7  kilolux,  although  sporelings  of  Ulva  lobata  tolerated  inten- 
sities at  a  level  of  7.4  kilolux,  as  well  as  a  longer  photoperiod. 

Unbuffered  aquarium  waters  showed  the  well  known  rapid  decrease  in  hydrogen 
ion  concentration  when  transplants  were  illuminated  (Shelbourne,  1964,  p.  29). 
During  dark  periods  pH  values  fell,  but  not  to  initial  levels :  thus,  total  alkalinity 
gradually  increased  within  5-7  days  to  pH  values  in  excess  of  9.0.  Sustained 


CULTURE  OF  MARINE  SEA  WEEDS  497 

alkalinity  of  this  magnitude  usually  resulted  in  a  progressive  degeneration  of  trans- 
planted thalli.  Comparable  effects  resulted  if  pH  values  were  maintained  below  7.5. 

DISCUSSION 

Possible  seasonal  change  in  the  water  occurring  along  the  mid-California  coast 
did  not  affect  the  success  of  present  cultures.  Sterilization  of  media  by  autoclaving 
was  not  necessary  to  avoid  gross  biotic  infection,  as  effective  control  was  achieved 
by  ultraviolet  irradiation.  This  also  permitted  use  of  a  wider  salinity  range,  as 
well  as  greater  concentrations  of  both  inorganic  and  organic  constituents  precipit- 
able  by  heat  sterilization.  Vitamins,  chelators,  and  other  organics  could  be  added 
to  media  precisely  with  results  comparable  to  those  using  soil  extracts.  While  the 
B  vitamins,  thiamin,  B]2,  and  biotin,  have  been  shown  to  stimulate  growth  of  many 
unicellular  algae  including  diatoms  their  role  is  less  understood  for  marine  sea 
weeds.  Of  the  Rhodophyceae,  Goniotrichum,  N  emotion,  Antithamnion,  and  Bangia. 
all  are  found  to  utilize  one  or  more  of  the  B  vitamins  in  laboratory  culture  (Prova- 
soli,  1963,  p.  13).  Kylin  (1942)  reported  enhanced  growth  of  both  Ulva  and 
Enteroinorpha  with  thiamin  at  an  optimal  concentration  of  10  mg./liter.  None 
of  the  Phaeophyceae  thus  far  studied  are  known  to  require  these  additives,  but  since 
insufficient  information  is  available,  it  seemed  inadvisable  to  omit  these  constituents. 

Tris  ( hydroxymethyl )  aminomethane  at  0.03%  concentration  in  the  system 
buffers  adequately  between  pH  7.5  and  8.5.  Thus,  pH  levels  in  the  aquaria  were 
stabilized  at  7.9  ±  0.3,  with  gaseous  CO2  (1-10%)  bubbled  through  the  medium 
intermittently.  Provasoli  (1957)  indicated  that  Tris  (0.1%)  was  not  inhibitory 
to  the  most  sensitive  of  marine  algae,  and  in  the  present  work,  comparable  concen- 
trations were  not  inhibitory  to  either  transplants  or  developing  sporelings  of  Ulva. 
EDTA  at  a  final  concentration  of  3  X  10~5  M  is  sufficient  to  bind  the  trace  elements 
in  sea  water  (Johnston,  1964).  EDTA  has  the  added  advantage  of  being  meta- 
bolically  inactive  for  most  organisms,  and  it  evidently  does  not  promote  growth  of 
contaminating  biota  within  non-sterile  media  (Hutner  ct  a!.,  1950). 

In  maintaining  proper  alkalinity  levels,  ultraviolet  sterilization  and  microfiltration 
supplanted  chemical  buffering  by  eliminating  bacterial  and  other  organic  growth 
which  would  otherwise  acidifv  the  medium.  Many  sea  weeds  are  found  to  be 

•/  -- 

tolerant  of  wide  fluctuations  in  pH,  due  primarily  to  tidal  influence.  Blinks  (1963) 
demonstrated  that  photosynthetic  rate  decreased  by  50%  only,  when  fronds  of  Ulva 
and  Enteroinorplia  were  maintained  for  about  6  hours  at  pH  9.8  or  above.  Kylin 
(1927)  indicated  that  certain  intertidal  sea  weeds  survived  for  1-3  days  within  the 
pH  range  of  6.8  to  9.6.  From  the  present  studies,  it  would  appear  that  Ulva  and 
related  genera  are  more  closely  restricted  in  pH  requirements  than  might  first  be 
suspected,  and  that  even  high  ranges  as  encountered  in  isolated  tide  pools  would 
be  inhibitory  if  sustained  longer  than  normal  tidal  influence  allows. 

Typically  Ulva-  lobata  and  related  species  are  long-day,  short-night  plants.  At 
an  irradiance  of  3.7  kilolux,  illumination  for  13-15  hours  and  uninterrupted  dark- 
ness for  9-11  hours  favor  longevity  of  transplants.  In  nature,  both  vegetative  and 
reproductive  development  reach  maturity  during  similar  long-day  seasons.  Also 
it  is  noted  that  lunar  periodicity  governs  the  release  of  gametes  and  zoospores, 
undoubtedly  correlated  to  the  24-hour  light-dark  cycle  (Smith,  1944). 


498  J.  A  STRAND,  J.  T.  CUMMINS  AND  B.  E.  VAUGHAN 

From  our  field  observations,  water  temperature  seemed  to  have  a  greater  effect 
on  the  rapid  maturation  of  gametes  and  zoospores  than  duration  of  photoperiod. 
Although  approaching  maximum  vegetative  growth  in  late  spring,  fertile  thalli  were 
not  evident  until  water  temperatures  reached  summer  levels,  16-18°  C.  It  fol- 
lowed that  incidence  of  fertile  plants  decreased  abruptly  during  early  September  as 
water  temperatures  within  the  vicinity  of  collection  sites  fell  below  16°  C.  That 
the  response  was  a  consequence  of  temperature  change  and  not  a  consequence  of 
the  shortened  photoperiod  was  shown  by  the  following  observation.  Placing  infer- 
tile plants  collected  as  late  as  December  or  January  into  aquaria  maintained  at 
16-18°  C.,  with  an  artificial  photoperiod  of  9-10  hours,  did  induce  subsequent 
maturation  and  sporulation  (data  not  reported). 

The  influence  of  temperature  on  reproductive  growth  of  many  plants  is  closely 
interrelated  with  photoperiodism.  Depending  upon  the  species  of  plant  and  other 
conditions,  temperature  may  enhance  or  oppose  the  effect  of  photoperiod  on  repro- 
ductive maturity  (Meyer  and  Anderson,  1952,  p.  681).  Temperature  changes  may 
initiate  synthesis  or  breakdown  of  hormonal  compounds,  rates  of  translocation,  and 
relative  effectiveness  of  specific  morphogenic  change  (Meyer  and  Anderson,  1952, 
p.  681).  Aquatic  plants  such  as  Ulva  perhaps  have  biochemical  similarity  to  ter- 
restrial plants,  since  comparable  phyto-hormone  responses  are  present  (Provasoli, 
1957,  1958),  and  since  metabolic  effects  can  be  triggered  by  photoperiod  and  tem- 
perature changes  like  those  known  to  depend  on  phyto-hormone  systems. 

Scant  information  is  available  concerning  either  the  intensity  levels  or  the 
spectral  quality  essential  to  artificial  maintenance  of  marine  sea  weeds.  An  in- 
tensity range  of  1.1  to  5.4  kilolux  has  been  reported  for  Rhodophyceae  (Provasoli, 
1963, "p.  10;  Iwasaki,  1961).  Foyn  (1934,  1960).  however,  with  Ulva  did  not 
report  intensity  ranges.  Other  investigators  have  simply  exposed  cultures  to  nat- 
ural sunlight.  Investigations  by  Dello\v  and  Cassie  (1955)  on  the  littoral  zonation 
in  caves  demonstrated  the  adaptability  of  such  forms  as  Cladofora  to  grow  nor- 
mally at  low  intensity  levels,  in  the  range  of  0.005  to  0.250  kilolux.  In  similar 
experiments,  the  intertidal  alga,  Ulva  lobata,  could  survive  5  hours  exposure  to 
direct  sunlight  (104  kilolux)  (Biebl,  1952).  However,  in  the  natural  environ- 
ment, constantly  changing  intensities,  attenuation  of  red,  and  scattering  of  blue 
wave-lengths,  are  all  complicating  factors. 

While  the  energy  distribution  of  the  Sylvania  Gro-lux  fluorescent  lamp  approxi- 
mated a  photosynthetic  action  spectrum  (Mpelkas,  1964b),  the  combined  sources 
here  used  provided  a  better  spectral  balance  for  growth  and  differentiation.  Haxo 
and  Blinks  (1950),  in  comparing  light  absorption  and  photosynthetic  action  spectra 
in  Ulva  and  Monostroma,  demonstrated  that  higher  rates  occurred  in  spectral  bands 
corresponding  to  absorption  by  chlorophyll  A,  at  435  and  675  nanometers.  In  our 
studies,  when  Ulva  transplants  were  illuminated  at  comparable  intensities  but  with 
Gro-lux  lamps  alone  or  with  Cool-white  or  Daylight  lamps,  the  transplants  gradu- 
ally bleached  and  decayed.  These  adverse  changes  presumably  were  due  to  inade- 
quate energy  emission  in  the  red  spectral  regions  when  the  sources  indicated  were 
used  individually  (Mpelkas,  1964;  Haxo  and  Blinks,  1950).  For  the  combined 
light  sources  finally  adopted,  abrupt  drop  in  energy  distribution  below  400  and 
above  725  nanometers  (Mpelkas,  1964b)  lessens  concern  about  possibly  unfavor- 
able emissions,  e.g.,  mercury  lines,  ultraviolet  and  infrared  wave-lengths. 


CULTURE  OF  MARINE  SEA  WEEDS  499 

With  little  modification,  the  hasic  system  has  facilitated  cultures  of  other  marine 
forms,  c.(/.,  sea  weeds  such  as  Porpliyra  pcrforata,  Polyncura  latissima  and  Schizy- 
iiteiiia  pacifica,  and  diatoms  such  as  Ditylnm  brightwelli,  Nitzchia  anyularls  and 
Nai'icnla  (sps.  und.).  It  has  also  permitted  successful  culture  of  such  animal 
forms  as  protozoa,  annelids,  molluscs  and  crustaceans. 

The  authors  are  grateful  for  the  kind  assistance  of  Mr.  Clay  P.  Butler  of  this 
Laboratory,  who  standardized  their  tungsten  source  against  NBS  calibrated  lamp 
QL-50  (tungsten). 

SUMMARY 

Ulva  as  either  sporeling  or  transplant  could  be  cultured  for  periods  of  three 
months  in  closed  recirculating  aquarium  systems.  Early  development  of  Ulva 
lobata  sporelings  proceeded  normally  and  rapidly  under  conditions  imposed  and 
compared  to  closely  related  species,  i.e.,  Ulva  pcrtusa  and  Ulva  lactuca.  A  modified 
Haxo-Sweeney  enrichment  was  used,  substituting  B  vitamins  and  organic  phos- 
phate for  soil  extract.  Continuous  flow  ultraviolet  sterilization  and  microfiltration 
were  provided.  The  pH  was  maintained  automatically  at  7.9  ±  0.3,  using  Tris 
buffer  and  gaseous  CO2.  Improved  fluorescent  illumination  for  13-15  hours 
favored  culture  of  sporelings  and  summer  transplants.  Irradiance  was  confined 
to  the  spectrum  lying  between  approximately  380-725  in//,,  and  amounted  to  3.7 
kilolux.  From  field  observations,  photoperiod  appeared  closely  correlated  to  initia- 
tion of  vegetative  growth  during  early  spring.  Water  temperature  seemed  to  have 
a  greater  effect  on  the  rapid  maturation  of  gametes  and  zoospores. 

LITERATURE  CITED 

BIEBL,   R.,    1952.     Resistenz  der   Merresalgen   gegen   sichtbares   Licht   und   gegen   kurzwellige 

UV-Strahlen.     Protoplasma,  44:  353-377. 
BLINKS,   L.   R.,    1963.     The  effect   of  pH   upon   the  photosynthesis   of  littoral    marine   algae. 

Protoplasma,  57:  126-136. 
DELLOW,  V.,  AND  R.  M.  CASSIE,  1955.     Littoral  zonation  in  two  caves  in  the  Aukland  district. 

Trans.  Roy.  Soc.  New  Zealand,  83:  321-331. 
FOYN,  B.,  1934.     Lebenszyklus  und  Sexualitat  der  Chlorophycee  Ulva.     Arch.  Protistenk.,  83: 

154-177. 

FOYN,  B.,  1960.     Sex-linked  inheritance  in  Ulva.     Biol.  Bull.,  118:  407-411. 
FRIES,  L.,  1960.     The  influence  of  different  B]2  analogues  on  the  growth  of  Goniotriclutin  ele- 

gans  (Chauv.).     Physiol.  Plant.,  13:  264-275. 
HAXO,  F.  T.,  AND  L.  R.  BLINKS,  1950.     Photosynthetic  action  spectra  of  marine  algae.     /.  Gen. 

Physiol.,  33:  389-442. 
HUTNER,  S.  H.,  L.  PROVASOLI,  A.  SCHATZ  AND  C.  P.  HASKINS,  1950.     Some  approaches  to  the 

role   of  metals   in   the   metabolism   of   microorganisms.     Proc.   Amcr.   Phil.    Soc..   94: 

152-170. 

IWASAKI,  H.,  1961.     The  life-cycle  of  Porphyra  fencra  in  vitro.     Biol.  Bull.,  121:  173-187. 
JOHANSEN,   D.  A.,   1951.     Microtechnique.     In:    Manual   of   Phycology,   Ed.   by   G.   M.    Smith. 

Chronica  Botanica  Co.,  Waltham,  Mass.,  pp.  359-363. 
JOHNSTON,  R.,  1964.     Sea  water,  the  natural  medium  of  phytoplankton.     /.  Mar.  Biol.  Assoc., 

44:  87-109. 
KYLIN,   H.,    1927.     Uber   den   Einfluss   der    Wasserstoffionenkonzentration   auf   einige    Meere- 

salgen.     Botan.  Notiser,  pp.  243-254. 
KYLIN,  H.,  1942.     Influence  of  glucose,  ascorbic  acid,  and  heteroauxin  on  the  seedlings  of  Ulva 

and  Enter omorpha.     Kgl.  Fysiograf.  Sallskap.  i.  Lund  Fork.,  12:  135-148. 


500  J.  A  STRAND,  J.  T.  CUMMINS  AND  B.  E.  VAUGHAN 

MEYER.   B.    S.,   AND   D.   B.   ANDERSON,    1952.     Environmental   factors    influencing   reproductive 

growth.     In:    Plant   Physiology.     Chapter   32:   667-688.     D.   Van   Nostrand   Company, 

Inc.,  Princeton. 
MIYAKE,  K.,  AND  H.  KUNIEDA,  1931.     On  the  conjugation  of  the  gametes  and  the  development 

of  the  zoospores  in  Ulvaceae.     /.  Coll.  Aiiriculture,  Imp.  Univ.  Tokyo,  11:  341-357. 
MOEWUS,    F.,    1938.     Die    Sexualitat   und   der    Generations-wechsel    der    Ulvaceae    und    Unter- 

suchungen  iiber  die  Pathenogense  der  Gameten.     Arch.  Protistcnk.,  91:  357-441. 
MPELKAS,  C.  C.,   1964a.     Guide  to  better  plant  growth  with   Sylvania   Gro-lux.     In:    Sylvania 

Lighting  Products  Bull. 

MPELKAS,  C.  C.,  1964b.     The  Gro-lux  fluorescent  lamp.     In:  Sylvania  Lighting  Products  Bull. 
PROVASOLI,  L.,  1957.     Effect  of  plant  hormones  on  sea  weeds.     Biol.  Bull.,  113:  321. 
PROVASOLI,  L.,  1958.     Effect  of  plant  hormones  on   Ulra.     Biol.  Bull..  114:   375-384. 
PROVASOLI,  L.,  1963.     Growing  marine  seaweeds.     /;;:  IV.  Intern.  Seaweed  Symp.,  Ed.  by  A.  D. 

DeVirville  and  J.  Feldman. 
PROVASOLI,  L.,  J.  J.  A.  MCLAUGLIN  AND  M.  R.  DROOP,   1957.     The  development  of  artificial 

media  for  marine  algae.     Arch.  f.  Mikrobiologie,  25:   392-428. 
REA,  I.  K.,  1964.     Some  effects  of  salinity,  temperature  and  photoperiodism  on  the  growth  and 

morphogenesis  of  Ulz'a  lactuca.     Biol.  Bull..  127:  386. 
SETCHELL,  W.  A.,  AND  N.  L.  GARDNER,  1920.     Chlorophyceae.     In:  The  Marine  Algae  of  the 

Pacific  Coast  of  North  America.     Univ.  Calif.  Publ.  Botany,  8:   139-374. 
SHELBOURNE,  J.  E.,  1964.     The  artificial  propagation  of  marine  fish.     In:  Advances  in  Marine 

Biology,  Ed.  by  F.  S.  Russell.     Academic  Press,  London,  pp.   1-83. 
SMITH,  G.  M.,   1944.     Marine  Algae  of  the   Monterey  Peninsula.     Stanford  University   Press, 

Stanford,  California,  622  pp. 
SMITH,  G.  M.,   1947.     On  the  reproduction  of  some  Pacific  Coast  species  of   Ulva.     Ainer.  J. 

Botany,  34:  80-87. 
YAM  ADA,  Y.,  AND  E.  SAITO,  1938.     On  some  culture  experiments  with  the  swarmers  of  certain 

species  belonging  to  the  Ulvaceae.     Sci.  Papers  Inst.  Algological  Research  Hokkaido 

Imp.  Univ.,  2:  35-51. 


THE  .MORPHOLOGY  AND  LIFE-HISTORY  OF  NOTOCOTYLUS 
ATLANTICUS  N.  SP.,  A  DIGENETIC  TREMATODE  OF  EIDER 
DUCKS,  SOMATERIA  MOLLISSIMA,  AND  THE  DESIG- 
NATION, NOTOCOTYLUS  DUBOISI  NOM.  NOV., 
FOR  NOTOCOTYLUS  IMBRICATUS   (LOOSS, 
1893)   SZIDAT,  1935  l 

HORACE  W.  STUNKARD 

The  American  Museum  of  Natural  History,  Central  Park  JTcst  at  79th  Street,  New  York,  N.  Y. 

The  genus  Notocotylus  was  erected  by  Diesing  (1839)  with  Notocotylus  tri- 
serialis Diesing,  1839,  from  wild  and  domestic  European  ducks,  as  type  species. 
Diesing  confused  dorsal  and  ventral  aspects  and,  as  the  generic  name  implies, 
regarded  the  characteristic  pits  as  dorsal.  The  genus  is  worldwide  in  distribution 
and  contains  species  from  various  birds  and  mammals.  On  the  presumption  by 
Creplin  (1846)  that  N.  triserialis  is  identical  with  Monostomuw  attenuatnm  Ru- 
dolphi,  1809,  from  Scolopa.v  gallinago,  Kossack  (1911)  designated  Notocotylus 
attenuatus  (Rudolphi,  1809)  as  type  of  the  genus  and  this  determination  has  been 
accepted  by  most  subsequent  authors.  However,  Dubois  (1951)  questioned  the 
identity  of  the  two  species  and  presented  strong  evidence  to  support  his  contention 
that  N.  attenuatus  is  not  identifiable  and  that  it  should  be  considered  a  "species 
inquirende."  Accordingly,  he  restored  N.  triserialis  as  type  of  the  genus. 

'  The  generic  concept  of  Notocotylus  is  vague,  uncertain,  and  the  diagnostic  cri- 
teria are  disputed.  Many  species,  named  and  described  as  new,  have  been  sup- 
pressed as  synonyms  and  the  number  of  valid  species  is  problematical.  Yamaguti 
(1958)  listed  29  species  from  birds  and  five  species  from  mammals,  including 
Notocotylus  urbanensis  (Cort,  1914)  Harrah,  1922,  which  has  been  reported  from 
experimental  infections  of  both  birds  and  mammals  (Luttermoser,  1935  ;  Herber, 
1939,  1942;  Acholonu,  1964).  Dubois  (1951)  recognized  19  species  and  Odening 
(1964)  included  26  species  in  a  differential  key.  Certain  species  have  been  desig- 
nated as  types  of  new  genera,  but  these  proposals  have  not  been  adopted.  As 
synonyms  of  Notocotylus,  Yamaguti  (1958)  listed  H India  Lai,  1935;  Naviformia 
Lai,  1935 ;  Kossackia  Szidat,  1936 ;  and  Hindolana  Strand,  1942.  Baer  and 
Joyeux  (1961)  suppressed  Hippocrepis  Travassos,  1922;  Hindolana  Strand,  1942 
(-- Hindia  Lai  nee  Duncan);  Quinqucscrialis  Skwortzow,  1933  (-- Barkeria 
Szidat,  1936)  and  Uniserialis  Burton,  1958  as  synonyms  of  Notocotylus.  Revi- 
sions of  the  genus  have  been  made  by  Kossack  (1911),  Harrah  (1922),  Szidat  and 
Szidat  (1933),  Harwood  (1939),  Dubois  (1951),  and  Odening  (1964)  ;  the  last 
two  contain  extensive  bibliographies. 

Several  life-history  studies  have  been  reported  but  the  data  are  confusing, 
since  the  specific  identity  of  the  adults  is  uncertain  and  the  correlation  of  larval 

1  Investigation  supported  by  Grant  NSF-GB-3606,  continuation  of  G-23561. 

501 


502  HORACE  W.  STUNKARD 

and  adult  stages  with  the  type  of  snail  intermediate  host  is  obscure.  Some  of  the 
snail  hosts  are  pulmonates,  some  are  prosobranchs ;  some  are  fresh-water  and 
others  are  marine.  Sewell  (1922)  erected  the  "Ephemera  Group"  of  monostome 
cercariae,  based  on  Cercaria  ephemera  Nitzsch,  1807  from  Planorbis  corneus  Linn., 
taken  near  Halle,  Germany.  Marie  Lebour  (1907)  described  cercariae  from  the 
marine  prosobranch  snail,  Peringia  ulvae,  as  Cercaria  ephemera.  Sewell  predi- 
cated that  C.  ephemera  Lebour  could  not  be  identical  with  C.  ephemera  Nitzsch 
and  stated  (p.  40),  "Although  nothing  is  definitely  known  regarding  the  ultimate 
development  of  any  of  these  monostome  forms,  several  suggestions  have  been  put 
forward  by  different  authors."  After  the  description  of  more  than  50  species 
belonging  to  various  monostome,  amphistome  and  distome  groups,  he  declared  (p. 
309),  "The  classification  of  larval  trematodes  cannot  be  based  on  any  one  morpho- 
logical system  or  groups  of  characters,  but  must  include  a  study  of  their  develop- 
ment, as  well  as  of  the  morphology  of  both  parent  and  offspring."  Similarity 
between  the  "Ephemera"  cercariae  and  notocotylid  trematodes  had  long  been  noted, 
but  correlation  between  particular  larvae  and  their  adult  stages  remained  uncertain. 
Joyeux  (1922)  reported  that  cercariae  from  Planorbis  rotundatus  Poiret,  taken  in 
a  lake  at  the  Bois  de  Verrieres  near  Paris,  developed  in  domestic  ducklings  to 
adult  worms  that  were  identified  as  Notocotylus  attenuatus  (Rudolphi,  1809). 
Eggs  from  these  worms  provided  miracidia  which  infected  laboratory-reared  snails 
and  afforded  the  first  experimentally  demonstrated  life-cycle  in  the  group.  Harper 
(1929)  reported  that  trioculate  monostome  cercariae  from  Lymnaea  peregra  and 
Physa  jontinalis  developed  in  ducklings  to  adults  identified  as  Notocotylus  seineti 
Fuhrmann,  1919.  Mathias  (1930)  found  that  cercariae  from  Lymnaea  limosa 
developed  to  adults  in  ducklings ;  these  were  determined  as  N.  attenuatus.  Fur- 
ther, he  believed  that  the  specimens  of  Joyeux,  which  were  only  about  one-half  as 
large,  represented  a  related  but  different  species.  Lothar  and  LTrsula  Szidat  (1933) 
reported  that  Cercaria  ephemera  Nitzsch,  1807  from  Planorbis  corneus  developed 
into  adults,  which,  in  violation  of  the  rules  of  nomenclature,  they  designated  as  a 
new  species,  Notocotylus  thienemanni.  A  cercaria  from  Lymnaea  palustris  was 
described  by  them  as  a  new  species,  Cercaria  vaga.  Ursula  Szidat  (1935)  fed 
larvae  from  Bithynia  tentaculaia,  identified  as  Cercaria  imbricata  Looss,  1893.  to 
chicks  and  ducklings  and  recovered  adults,  strikingly  like  N.  attenuatus,  but 
designated  erroneously  as  a  new  species,  Notocotylus  imbricatus.  She  remarked  on 
the  difficulty  of  distinguishing  between  these  closely  related  cercariae  on  purely 
morphological  grounds  and  (1936)  insisted  on  the  marked  specificity  of  larval 
stages  and  snail  hosts. 

The  first  comprehensive  study  of  the  "Ephemera  Group"  was  done  by  Miriam 
Rothschild  when  in  1932  she  began  an  investigation  of  the  larval  trematodes  of 
Peringia  ulvae  (Pennant,  1777)  at  the  suggestion  of  Dr.  Marie  Lebour.  Miss 
Rothschild  (1935)  published  a  note  on  the  development  and  structure  of  the  excre- 
tory system  of  C.  ephemera  Lebour,  1907  (nee  Nitzsch,  1807).  She  (1936a) 
reported  three  species :  Cercaria  E,  Cercaria  F,  and  Cercaria  G,  in  addition  to 
C.  ephemera  and  (1936b),  discussed  the  development  of  normal  pigmentation  and 
its  variations  in  C.  ephemera  and  allied  species.  After  studying  infections  in 
P.  ulvae  and  Hydrobia  ventrosa  (Montagu,  1803),  Rothschild  (1938)  arranged 


LIFE-CYCLE  OE  NOTOCOTYLUS  503 

the  notocotylid  cercariae  in  three  groups,  based  on  the  form  of  the  anterior  trans- 
verse portion  of  the  excretory  vesicle.  In  the  Monostomi  group,  the  anterior 
transverse  portion  of  the  vesicle  is  a  closed  tubular  circuit  situated  posterior  to 
the  cerebral  ganglion  and  median  eye-spot ;  it  contained  1 2  described  species, 
including  two  from  P.  uh'ae.  In  the  imbricata  group,  the  anterior  portion  of  the 
vesicle  forms  a  loop,  between  the  lateral  eye-spots,  which  passes  anterior  to  the 
cerebral  ganglion  and  median  ocellus ;  four  described  and  one  undescribed  species 
from  Hydrobia  ventrosa  were  assigned  to  this  group.  In  the  yenchingensis  group, 
there  is  an  unpaired,  finger-like  diverticulum  which  extends  anteriad  from  the 
transverse  portion  of  the  vesicle ;  three  described  species  were  included  in  this 
group,  together  with  two  undescribed  species  from  P.  nlvac  and  another  from 
Littorina  neritoidcs  (Linn.).  Rothschild  (1940)  explained  that  the  imbricata  group 
was  based  on  Cere  aria  imbricata  Looss,  1896  and  that  C.  imbricata  Looss,  1893 
from  Palitdina  imp nra  (  =  Bithynia  tentaculata)  in  Germany  is  a  nomen  nudum, 
since  the  species  was  not  described  or  figured,  and  that  C.  imbricata  Looss,  1896 
from  Mclania  tubcrculata  Bourg  in  Egypt  is  distinct  and  different  from  C.  imbricata 
in  Germany.  She  stated  that  the  cercaria  from  B.  tentaculata  which  U.  Szidat 
found  to  mature  in  ducks,  and  which  she  named  Notocotylus  imbricatus,  was  the 
one  studied  by  Looss  and  his  surmise  that  it  is  the  larva  of  N.  attenuatus  was 
probably  correct.  Attempts  to  solve  the  life-cycles  of  these  species,  carried  on  for 
five  years  by  Miss  Rothschild,  were  only  partially  successful.  She  reported  (1941  ) 
that  cysts  of  the  Monostomi  and  Yenchingensis  groups  were  fed  to  laboratory- 
reared  ducklings  and  two  unidentified  species  of  Paramonostomiim  were  recovered 
from  birds  fed  Yenchingensis  metacercarial  cysts. 

In  his  revision  of  the  genus  Notocotylus,  Dubois  (1951)  recognized  two  sub- 
genera:  Hindia  Lai,  1935  ;  parasites  of  Ralliformes,  with  4-6  glands  in  the  median 
row,  cuticula  unarmed  or  provided  with  minute  spines,  type,  Notocotylus  gibbus 
(Mehlis  in  Creplin,  1846),  syn.  Kossackia  U.  Szidat,  1936;  and  Notocotylus 
Dubois,  1951,  parasites  of  Anseriformes,  with  10-25  glands  in  the  median  row, 
cuticula  armed,  type,  //.  triserialis  Diesing,  1839.  According  to  him,  the  subgenus 
Notocotylus  is  composed  of  two  distinct  groups,  in  one  of  which  the  cercariae 
develop  in  pulmonate  snails,  in  rediae  which  lack  locomotor  appendages  (apophyses 
of  the  German  authors )  ;  whereas  the  asexual  generations  of  the  other  group  develop 
in  prosobranch  snails  in  rediae  that  are  provided  with  locomotor  appendages.  The 
first  group  was  based  on  N.  triserialis  with  Notocotylus  urbanensis  (Cort,  1914)  ; 
Notocotylus  intestinalis  Tubangui,  1932;  Notocotylus  dcfilac  Harwood,  1939;  and 
Notocotylus  stagnicolac  Herber,  1942.  listed  as  synonyms.  The  second  group  was 
based  on  Notocotylus  imbricatus  (Looss,  1893)  U.  Szidat.  1935,  with  Notocotylus 
lucknowensis  (Lai,  1935)  and  Notocotylus  anatis  Ku,  1937,  listed  as  synonyms, 
while  Notocotylus  magnioi'atus  Yamaguti,  1934  and  Notocot\lus  babai  Bhalero. 
1935  (syn.  Notocotylus  indicus  Lai,  1935)  were  distinguished  only  by  relative 
extent  of  vitellaria  and  uterine  loops.  Referring  to  the  subgenus  Notocotvlus. 
Dubois  observed  (p.  64),  "Les  deux  groupes  biologiques  'triserialis'  et  'imbricatus' 
constituent,  en  realite.  deux  especes  polymorphes  au  sein  des  Notocotylides  que 
U.  Szidat  (1935,  p.  270)  envisageait  comme  tin  ensemble  cle  Trematodes  phylo- 
genetiquement  jeune  et  susceptible,  par  consequent,  d'une  difrerenciation  progressive 


504  HORACE  W.  STUNKARD 

a  partir  des  'races  physiologiques'  actuelles  (1936,  p.  234).  L'examen  du  tableau 
comparatif  des  stades  larvaires  confirm  cette  maniere  de  voir  et  ne  leve  aucune  des 
difficultes  recontree  dans  la  systematique  des  vers  adultes."  Dubois  examined 
the  specimens  of  Joyeux  (1922),  developed  in  ducklings  from  cercariae  of  Planorbis 
rotundatus,  and  identified  them  as  Catatropis  verrucosa  (Frohlich,  1789). 

Rothschild  (1940)  noted  that  Cercaria  imbricata  Looss,  1893,  was  from  Bithynia 
tentaculata  in  Germany  and  that  C.  imbricata  Looss,  1896,  was  from  Melania 
tuberculata  in  Egypt.  Apparently  Dubois  (1951)  accepted  the  allocation  by  U. 
Szidat  (1935)  of  C.  imbricata  Looss,  1893,  to  the  genus  Notocotylus  as  a  valida- 
tion of  the  specific  name  since  he  employed  the  combination,  Notocotylus  imbricatus 
(Looss,  1893).  But  C.  imbricata  Looss,  1893,  from  Bithynia  tentaculata  was 
named  provisionally  in  a  footnote,  without  description  or  figure  and,  accordingly, 
has  no  validity.  The  specific  name,  imbricatus,  is  valid  only  for  the  Egyptian 
species,  and  if  the  opinion  of  Dubois  that  C.  imbricata  is  the  larval  stage  of  Noto- 
cotylus aegyptiacus,  Odhner,  1905,  is  correct,  the  name  of  that  species  is  Notoco- 
tylus imbricatus  (Looss,  1896),  not  N.  aegyptiacus,  a  name  proposed  by  Odhner 
(1905)  to  replace  Monostomum  verrucosum  Looss,  1896,  nee  Frohlich,  1789.  It 
is  interesting  to  note  that  Lithe  (1909)  listed  C.  imbricata  from  B.  tentaculata, 
but  based  the  diagnosis  on  Looss'  description  of  the  African  species.  A  genus  can 
not  have  two  species  with  identical  names  and  the  species  whose  larvae  occur  in 
B.  tentaculata  and  which  has  been  known  as  N.  imbricatus  (Looss,  1893)  has  no 
valid  name.  For  it  I  propose  the  name,  Notocotylus  duboisi.  According  to 
Dubois  (1929),  it  is  distinct  from  the  species  from  B.  tentaculata  described  by 
him  as  Cercaria  Helvetica  I.  Odening  (1963)  compared  the  cercariae  from  B. 
tentaculata  described  as  C.  imbricata  Looss,  1893,  by  Wesenberg-Lund  (1934)  and 
by  Emmel  (1943)  with  Cercaria  Helvetica  I,  but  was  uncertain  of  their  identity. 

Recent  studies  have  only  partially  clarified  the  situation.  Herber  (1955) 
studied  the  life-cycle  of  N.  urbanensis  (Cort).  He  noted  errors  in  the  accounts 
of  Barker  (1915)  and  Harrah  (1922)  ;  stated  that  the  species  described  by  Barker 
in  the  text  as  Catatropis  fimbriata  and  figured  as  Catatropis  filamentis  and  the  spe- 
cies described  by  Harrah  as  Paramonostomum  echinatum  were  based  on  poorly 
preserved  specimens  of  N.  urbanensis  and  that  both  names  should  be  suppressed  as 
synonyms.  According  to  Herber,  the  specimens  described  by  Harrah  (1922)  as 
N.  urbanensis  are  not  conspecific  with  Cercaria  urbanensis  Cort,  1914,  but  com- 
prise a  heterogeneous  collection  of  immature  specimens  and  the  only  ones  identified 
were  referred  to  the  genus  Quinqueserialis. 

Stunkard  and  Dunihue  (1931)  described  specimens  from  an  unidentified  duck 
as  Notocotylus  gibbus  (Mehlis  in  Creplin,  1846).  Determination  was  based  on 
Kossack's  description  of  the  original  specimens.  The  species  was  found  again  and 
redescribed  by  L.  and  U.  Szidat  (1933).  Harwood  (1939)  then  assigned  the 
specimens  of  Stunkard  and  Dunihue  to  Notocotylus  imbricatus  (Looss).  Stunkard 
(1960)  reported  notocotylid  cercariae  from  Hydrobia  minuta  taken  near  Boothbay 
Harbor,  Maine,  that  encysted  on  the  opercula  of  the  snails  and  on  empty  shells  of 
Gemma  gemma.  Encysted  metacercariae  were  fed  to  laboratory-reared  birds: 
chicks,  eider  ducks,  herring  gulls,  and  common  terns,  as  well  as  white  mice  and 
hamsters.  Infection  was  obtained  only  in  eider  ducks,  Somateria  mollissima,  and 


LIFE-CYCLE  OF  NOTOCOTYLUS  505 

adult  worms  taken  from  the  caeca  proved  identical  with  those  described  by  Stunkard 
and  Dunihue  (1931).  With  the  more  complete  description  of  N.  gibbus  by  L.  and 
U.  Szidat,  it  was  apparent  that  the  specimens  described  by  Stunkard  and  Dunihue 
differ  from  that  species  in  host  and  geographical  distribution,  in  size  of  worms,  size 
and  shape  of  gonads,  and  in  the  number  and  arrangement  of  ventral  glands.  The 
worms  could  not  be  assigned  to  N.  imbricatus  or  any  other  known  species  and  were 
described  by  Stunkard  (1960)  as  a  new  species,  Notocotylus  minutus. 

Determination  of  species  in  the  genus  Notocotylus  is  difficult.  The  worms  are 
very  similar  (witness  the  keys  by  Harwood,  1939;  Dubois,  1951;  and  Odening, 
1964)  ;  specific  differences  in  morphology  are  small,  and  the  extent  of  individual 
variation  is  unknown.  Host  specificity  of  the  adult  stage  is  not  definite,  because 
the  same  metacercariae  can  mature  in  different  host  species.  Acholonu  (1964)  re- 
ported the  development  of  metacercariae  of  N.  urbanensis  and  N.  stagnicolae  (pos- 
sibly identical  forms)  in  ducklings,  goslings,  chicks,  white  rats  and  muskrats. 
Apparently  the  snail  hosts  are  more  specific,  and  U.  Szidat  (1935)  predicated 
precise  specificity  in  C.  ephemera,  C.  attenuatus,  and  C.  imbricata.  But  there  is 
so  much  confusion  in  the  identification  of  larvae  that  final  conclusions  are  impos- 
sible. No  less  than  five  different  cercariae  have  been  described  as  larvae  of  N. 
attenuatus  by  Joyeux  (1922),  Mathias  (1930),  L.  and  U.  Szidat  (1933),  Yama- 
guti  (1938)  and  Wright  and  Bennett  (1964),  and  the  identity  of  N.  attenuatus 
is  precarious. 

Other  more  recent  studies  on  the  life-history  of  notocotylid  species  include  the 
report  by  Wu  (1953)  on  the  development  of  cercariae  from  Stagnicola  palustris  to 
adults  of  N.  stagnicolae  in  chicks;  and  by  Erkina  (1954)  and  Bychovskaja- 
Pavlovskaja  (1962)  on  the  development  of  Notocotylus  chionis  of  the  Soviet 
authors  (Erkina,  1954  and  Bychovskaja-Pavlovskaja,  1962)  nee  Baylis,  1928.  from 
cercariae  of  Bithynia  tentaculata  to  adults  in  ducks  and  Fulica  atra.  According 
to  Odening  (1964),  the  N.  chionis  of  the  Russian  investigators  is  actually  Noto- 
cotylus parvlovatus  Yamaguti,  1934.  Hsu  (1957)  reported  the  development  of 
Notocotylus  maniii  Hsu,  1954,  from  cercariae  of  Melania  (Melanoides}  tuberculata 
chinensis  to  adults  in  ducks  and  guinea  pigs.  Kupriyanova-Shakhmatova  (1959) 
recalled  that  L.  and  U.  Szidat  (1933)  reported  that  C.  ephemera  Nitzsch,  1807, 
from  Coretus  corneus  developed  to  adults  designated  as  Notocotylus  thienemanni 
and  that  cercariae  from  Galba  palustris,  named  C.  vaga,  developed  into  adults  desig- 
nated as  N.  attenuatus.  The  Russian  author  fed  metacercariae  from  infections  in 
the  two  snails  and  obtained  adults  that  were  regarded  as  specifically  identical. 
Accordingly,  C.  vaga  was  considered  a  synonym  of  C.  ephemera  and  N.  thienemanni 
as  synonym  of  N.  attenuatus.  If  this  observation  is  correct,  both  N.  thienemanni 
and  TV.  attenuatus  (Rudolphi,  1809)  are  synonyms  of  Notocotylus  ephemera 
(Nitzsch,  1807)  Harwood,  1939.  Donges  (1962)  reported  that  cercariae  from 
Tropidiscus  carinatus  are  larvae  of  Notocotylus  ralli  Baylis,  1936.  Odening 
(1963)  suggested  that  Cercaria  monostomi  von  Linstow,  1896,  is  the  larval  stage 
of  Notocotylus  seineti  Fuhrmann,  1919.  Wright  and  Bennett  (1964)  found  that 
cercariae  from  Lymnaea  peregra  develop  into  N.  attenuatus  when  the  metacer- 
cariae are  fed  to  ducklings,  Anas  platyrhyncha.  Zdarska  (1964)  reported  that 
C.  ephemera  from  planorbid  snails  is  the  larva  of  N.  ephemera  and  that  C.  raga 


506  HORACE  W.  STUNKARD 

from  lymnaeid  snails  is  the  larva  of  N.  attenuatus.  According  to  this  author,  the 
adults  differ  in  number  and  arrangement  of  ventral  glands  and  in  relative  length 
of  metraterm  and  cirrus  sac.  Odening  (1964)  reported  that  cercariae  from  Physa 
fontalis  (probably  Cercaria  monostomi  v.  Linstow)  developed  in  chicks  to  Noto- 
cotylus pacifera  (Noble,  1933)  Harwood,  1939.  This  species,  according  to  Oden- 
ing, is  identical  with  N.  gibbus  Szidat  and  Szidat,  1933,  nee  Mehlis  in  Creplin,  1846. 
Also,  Odening  and  Bockhardt  (1965)  found  that  cercariae  from  the  planorbid  snail, 
Bathyomphalus  contortus  (Linn.),  developed  in  the  guinea  pig  to  adults  of  Noto- 
cotylus noyeri  Joyeux,  1922  (syn.  Notocotylus  neyrai  Gonzales  Castro,  1945),  a 
parasite  of  the  caecum  of  European  rodents,  Arvicola  spp.  Odening  (1966)  re- 
ported the  life-cycles  of  Notocotylus  pacijcr,  Notocotylus  ephemera,  Notocotylus 
noyeri,  Notocotylus  regis,  Notocotylus  ralli,  and  Catatropis  verrucosa.  All  employ 
pulmonate  snails  as  intermediate  hosts  and  all  the  species  of  Notocotylus  have  cer- 
cariae of  the  Monostomi  type.  The  cercaria  of  C.  verrucosa  has  an  excretory 
system  of  the  Imbricata  type. 

These  accounts,  while  somewhat  discordant,  add  materially  to  knowledge  of 
notocotylid  species  and  provide  data  that  will  aid  in  clarifying  the  taxonomic  prob- 
lems encountered  in  the  genus  Notocotylus.  The  morphology  and  life-cycle  of  an 
additional  species,  Notocotylus  atlanticus  n.  sp.,  whose  asexual  generations  occur 
in  the  brackish-water  prosobranch  snail,  Hydrobia  salsa,  are  described  in  the  present 
paper.  Type  and  paratype  specimens  deposited  in  the  Helminthological  Collection 
of  the  U.  S.  National  Museum  under  the  numbers  61,184  and  61,185. 

MATERIAL  AND  METHODS 

During  the  summers  of  1963,  1964,  1965  and  1966,  over  5000  specimens  of 
Hydrobia  salsa  have  been  examined  for  infection  by  larval  trematodes.  The  snails 
were  identified  by  Dr.  W.  K.  Emerson  of  the  American  Museum  of  Natural  His- 
tory. This  is  a  somewhat  rare,  brackish-water  species,  described  by  Pilsbry 
(1905)  as  Paludestrina  salsa  from  Cohasset,  Massachusetts.  During  the  summers 
of  1963  and  1964,  the  species  was  common  in  Nobska  Pond,  near  Woods  Hole, 
Massachusetts,  and  most  of  the  snails  were  taken  from  an  area  near  the  connection 
of  the  pond  with  Vineyard  Sound.  Infection  with  larval  trematodes  was  heavy  and 
five  species  of  notocotylid  cercariae  were  recognized.  In  the  fall  of  1964  the  pond 
was  "treated"  and  most  of  the  invertebrates,  including  snails,  were  killed.  Hy- 
drobia salsa  is  still  absent  from  Nobska  Pond,  but  in  the  summer  of  1965  the  spe- 
cies \vas  found  in  Oyster  Pond,  near  Falmouth,  Massachusetts,  and  the  study  of 
the  notocotylid  cercariae  has  been  continued.  The  incidence  of  infection  varied 
from  0.05%  to  more  than  4%,  dependent  on  the  size  and  age  of  the  snails,  time 
of  year  and  field  location.  Large  snails,  in  autumn,  and  from  locations  where 
ducks  congregate,  yielded  the  highest  rate  of  infection.  Incidence  was  determined 
by  isolation  of  the  snails  in  groups  of  ten  and,  when  an  infection  was  observed,  by 
further  isolation  of  these  snails.  To  discover  latent  infections  one  snail  of  each 
group  was  crushed,  and  young  cercariae  released  from  rediae  were  used  for  study 
of  the  development  of  the  excretory  system.  Cercariae  were  studied  alive  and  were 
killed  under  light  coverglass  pressure,  but  the  best  method  for  obtaining  relaxed 
specimens  of  uniform  shape  is  by  the  addition  of  hot  fixative,  AFAG  (alcohol, 


LIFE-CYCLE  OF  NOTOCOTYLUS  507 

formalin,  acetic  acid,  glycerine)  or  the  solution  of  Duboscq-Brasil  to  a  small  beaker 
in  which  the  larvae  are  being  whirled  rapidly.  The  use  of  vital  dyes,  Nile  blue 
sulphate  and  neutral  red,  facilitated  the  examination  of  living  larvae. 

These  notocotylid  cercariae  are  similar  in  size,  shape,  and  swimming  behavior. 
They  swim  with  the  tail  in  advance,  are  photosensitive,  and  accumulate  on  the 
light  side  of  the  container.  As  a  rule,  they  emerge  between  10  AM  and  2  PM  and 
encyst  soon  thereafter,  often  within  an  hour,  on  the  operculum  of  the  snail  from 
which  they  emerged  or  other  hard  surface.  Initial  feeding  of  cysts  to  experimental 
birds  yielded  different  kinds  of  adult  worms  and  showed  that  more  than  one 
species  was  involved.  Cysts  attached  to  the  shell  of  a  snail  may  have  come  from 
that  individual  or  from  other  snails. 

Specific  determination  of  the  infection  carried  by  individual  snails  is  a  tedious 
task,  since  the  snails  are  small,  the  cercariae  are  relatively  large  and  the  number 
liberated  is  small,  two  to  ten  in  24  hours,  although  an  infected  snail  often  will  not 
shed  cercariae  for  several  days.  The  cerceriae  leave  the  rediae  before  they  are 
mature  and  complete  their  development  in  the  haemocoele  of  the  snail.  The  water 
in  the  laboratory  is  warmer  than  in  the  ponds ;  accordingly,  in  the  first  days  after 
collection  the  snails  usually  shed  the  cercariae  that  are  mature  and  some  time  may 
elapse  before  additional  cercariae  are  liberated.  Study  of  the  cercariae  after  emer- 
gence provides  information  on  morphology  and  behavior;  sacrifice  of  the  snail 
ensures  that  only  one  species  of  parasite  is  present  and  affords  successive  stages 
in  the  development  of  the  rediae  and  cercariae.  Since  identification  of  encysted 
metacercariae  is  difficult  if  not  impossible,  specific  determination  and  correlation 
of  larval  and  adult  states  were  accomplished  by  feeding  metacercariae  from  indi- 
vidual snails  with  known  cercariae  to  laboratory-reared  hosts.  Of  the  five  noto- 
cotylid species,  two  belong  to  the  Yenchingensis  Group,  two  to  the  Monostomi 
Group,  and  the  other  to  the  Imbricata  Group  of  cercariae.  The  present  report  is 
concerned  with  the  Yenchingensis  cercariae.  One  species  proved  identical  with 
Notocotylus  minutus  Stunkard,  1960,  and  the  other  is  described  as  a  new  species, 
Notocotylus  atlanticus.  Both  species  develop  to  maturity  in  the  caeca  of  eider 
ducks  and  N.  minutus  (Fig.  8)  has  been  recovered  from  experimental  infections 
of  domestic  ducklings  and  natural  infections  of  hybrid  wild  ducks  taken  off  Cutty- 
hunk  Island.  The  eider  chicks  were  provided  by  Walter  R.  Welch  and  his  asso- 
ciates on  the  staff  of  the  U.  S.  Fish  and  Wildlife  Service  at  Boothbay  Harbor, 
Maine,  who  collected  the  eggs  and  hatched  the  chicks.  Their  kindness  is  grate- 
fully acknowledged.  Domestic  ducklings  were  purchased  from  a  local  hatchery. 

DESCRIPTIONS 
Notocotylus  minutus  Stunkard,  1960 

Yenchingensis-type  cercariae  from  H.  salsa,  encysted  as  metacercariae,  were  fed 
to  domestic  ducklings  and  worms  recovered  from  the  caeca  agree  with  those  from 
eider  ducks  described  by  Stunkard  (1960).  Other  specimens  were  found  in  the 
caeca  of  wild  hybrid  ducks  dead  at  Cuttyhunk  Island,  frozen  and  sent  to  Woods 
Hole  for  autopsy.  The  worms  were  in  poor  condition,  the  ventral  glands  were 
indistinct  and  their  number  could  not  be  determined.  A  specimen,  shown  in  Figure 
8,  is  referred  to  N.  minutus. 


508 


HORACE  W.  STUNKARD 


PLATE  I 

FIGURE  1.  A',  atlanticus,  holotype,  3.44  mm.  long,  from  the  caecum  of  an  eider  duck,  ex- 
perimental infection,  ventral  view. 

FIGURE  2.  N.  atlanticus,  paratype,  2.35  mm.  long,  flattened,  from  caecum  of  eider  duck,  ex- 
perimental infection,  ventral  view. 

These  figures  show  variation  in  arrangement  of  ventral  pits  and  loops  of  the  uterus ;  also 
differences  in  size  of  organs  in  a  pressed  specimen. 

Notocotylus  atlanticus  n.  sp. 

(Figs.  1-7) 
Adult   (Figs.  1,  2) 

The  description  is  based  on  ten  gravid  specimens  mounted  in  toto  and  six  cut 
in  serial  sections,  in  sagittal,  transverse  and  frontal  planes.  The  worms  are  ovate 
to  elongate,  flattened  dorsoventrally,  convex  dorsally  and  concave  ventrally.  They 
measure  2.20  to  3.65  mm.  in  length  and  0.80  to  1.10  mm.  in  width.  The  cuticula 
is  unarmed,  except  for  the  ventral  surface,  which  bears  very  fine  spines,  observed 


LIFE-CYCLE  OF  NOTOCOTYLUS  509 

only  on  living  specimens.  The  anterior  portion  of  the  body  may  bear  flecks  of 
pigment  from  dispersal  of  the  ocelli  of  the  larva,  but  the  pigment  disappears  in 
older  worms.  The  body  wall  is  delicate,  with  the  usual  circular,  longitudinal  and 
oblique  muscle  layers ;  the  longitudinal  fibers  are  most  conspicuous.  There  are 
three  rows  of  ventral  glands,  16  in  each  row ;  those  in  the  median  row  may  be 
one-half  interval  anterior  or  one-half  interval  posterior  to  those  in  the  lateral  rows. 
In  the  specimen  shown  in  Figure  1,  the  median  glands  are  anterior  to  the  lateral 
ones  whereas  in  Figure  2,  from  a  much  flattened  specimen,  the  lateral  glands  are 
anterior.  Possibly  the  arrangement  may  vary  with  different  states  or  degrees  of 
contraction  of  different  sets  of  muscles.  The  most  anterior  glands  in  each  row 
are  very  small  and  often  hardly  recognizable.  The  oral  sucker  is  circular  to  oval 
and  measures  from  0.15  by  0.12  to  0.18  by  0.15  mm.  The  esophagus  is  short  and 
the  caeca  have  irregular  lumina.  They  extend  posteriad,  turn  mediad  at  the  ante- 
rior ends  of  the  testes  and  pass  between  the  testes  and  ovary,  ending  blindly  behind 
the  level  of  the  gonads. 

The  testes  are  lateral,  lobed  to  branched,  longer  than  broad,  situated  near  the 
posterior  end  of  the  body,  and  measure  0.28  by  0.16  to  0.48  by  0.26.  Sperm  ducts 
arise  at  the  anterior  ends  of  the  testes,  pass  anteriad  and  mediad,  uniting  just 
anterior  and  medial  to  Mehlis'  gland  to  form  the  vas  deferens  which  passes  for- 
ward, dorsal  to  the  uterus.  It  forms  two  to  four  distended  irregular  coils  com- 
prising the  external  seminal  vesicle,  which  is  situated  dorsal  to  the  anterior  uterine 
loops,  and  then  enters  the  cirrus  sac  where  it  forms  a  coiled  internal  seminal 
vesicle,  0.25  to  0.375  mm.  in  extent.  The  vesicle  is  followed  by  a  prostatic  portion 
of  the  male  canal  and  a  long  ejaculatory  duct.  The  common  genital  pore  is  poste- 
rior or  immediately  anterior  to  the  bifurcation  of  the  digestive  tract.  The  cirrus 
is  protrusible  and  in  one  specimen  measures  0.19  by  0.028.  The  cirrus  sac  is  0.60 
to  0.84  mm.  in  length  and  the  posterior  end  is  about  three-eighths  of  the  body 
length  from  the  anterior  end.  The  posterior  end  of  the  cirrus  sac  is  situated  in 
the  interval  between  median  glands  five  and  six. 

The  ovary  is  lobed  and  measures  0.19  by  0.15  to  0.25  by  0.20  mm.  The  oviduct 
arises  at  the  anterior  median  face  and  passes  anteriad  where  it  receives  the  common 
vitelline  duct  and  enters  Mehlis'  gland.  In  one  series  of  sections  there  is  a  lateral 
outpocketing  of  the  oviduct,  filled  with  spermatozoa,  but  a  seminal  receptacle  or 
Laurer's  canal  were  not  observed.  The  initial  part  of  the  uterus  is  filled  with 
spermatozoa ;  it  coils  ventrad  and  there  is  a  loop  of  the  uterus,  with  thin-shelled 
eggs,  ventral  to  Mehlis'  gland.  There  are  12  to  16  transverse  loops  of  the  uterus 
to  the  level  of  the  anterior  lobes  of  the  vitellaria  and  an  additional  two  to  eight 
loops  in  front  of  the  vitellaria.  The  disposition  of  the  uterine  loops  is  dependent 
on  the  extension  or  retractions  of  the  body.  The  metraterm  is  less  than  one-half 
the  length  of  the  cirrus  sac ;  it  measures  0.28  to  0.37  mm.  in  length.  The  vitellaria 
are  situated  in  the  extracaecal  fields  and  extend  from  the  testes  to  a  level  slightly 
anterior  to  the  middle  of  the  body.  Collecting  ducts  course  posteriad  along  the 
median  faces  of  the  vitellaria,  pass  mediad  around  the  anterior  ends  of  the  testes 
and  unite  dorsal  to  Mehlis'  gland  to  form  the  common  duct  that  opens  into  the 
oviduct.  The  eggs  measure  0.017  to  0.019  by  0.011  mm.,  and  bear  long  polar  fila- 
ments. They  are  embryonated  when  passed.  The  excretory  system  becomes  very 


510 


HORACE  W.  STUNKARD 


FIGURES  3-8. 


LIFE-CYCLE  OF  NOTOCOTYLUS  511 

complex,  with  many  dendritic  branches  arising  from  both  the  lateral  and  medial 
faces  of  the  longitudinal  trunks  of  the  collection  ducts. 

Redia  (Figs.  5,  6) 

The  haemocoele  of  the  snail  contains  many  rediae  of  varying  sizes,  from  very 
small  active  individuals,  much  smaller  than  cercariae,  to  large  sluggish  specimens 
filled  with  progeny.  A  small  redia,  0.19  mm.  long  and  0.048  mm.  wide,  had  a 
pharynx  which  measured  0.036  by  0.040  mm.,  and  contained  eight  germ  balls,  the 
largest  of  which  was  0.043  by  0.020  mm.  Large  rediae  may  be  1.2  mm.  long  by 
0.14  mm.  wide,  with  a  pharynx  that  measures  0.045  to  0.052  mm.  in  diameter,, 
and  contain  four  or  five  cercariae  together  with  a  number  of  germ  balls  of  varying 
dimensions.  Small  rediae  are  colorless,  but  large  ones  are  orange-yellow  in  color, 
with  yellow  spherules,  some  10  microns  in  diameter,  in  the  body  wall.  Other  yel- 
low droplets  and  blackish  amorphous  material  are  present  in  the  lumen  of  the 
intestine.  The  anterior  end  bears  prepharyngeal  lips  and  short  setae.  There  is  a 
short  esophagus,  about  as  long  as  the  pharynx,  and  a  sac-like  intestine  extends 
about  one-half  the  length  of  the  body.  There  are  no  locomotor  appendages.  The 
birth  pore  is  ventral,  at  the  level  of  the  posterior  margin  of  the  pharynx.  The  ex- 
cretory system  is  double.  The  pores  are  lateral,  situated  posterior  to  the  middle 
of  the  body.  From  each  pore  a  common  duct  extends  anteriad  for  a  short  distance 
and  then  divides  into  an  anterior  and  a  posterior  branch,  each  of  which  terminates 
in  a  flame-cell.  The  anterior  cell  is  situated  near  the  posterior  end  of  the  esophagus 
and  the  posterior  cell  is  located  about  halfway  between  the  excretory  pore  and  the 
posterior  end  of  the  body.  The  cilia  of  the  flame-cells  are  10  to  12  microns  long. 
Each  collecting  duct  is  a  coiled  tube,  enclosed  in  a  straight  sinus. 

Cercaria  (Fig.  4) 

Living  specimens  shift  from  0.18  to  0.35  mm.  in  length  and  0.11  to  0.19  mm. 
in  width.  Fixed  in  hot  killing  fluid,  they  vary  from  0.29  by  0.12  mm.  to  0.24  by 
0.15  mm.  The  tail  is  simple,  slender;  it  may  be  shorter  than  the  body  or  extend 
to  a  length  of  0.43  mm.,  in  which  condition  it  is  only  0.025  mm.  wide  at  the  base. 
In  swimming,  the  body  is  contracted,  bent  ventrally,  while  the  tail  is  extended  and 
beats  violently.  Under  a  coverglass,  when  the  body  is  extended  the  tail  is  con- 
tracted and  vice  versa.  The  tail  is  subterminal  in  attachment  and  in  fixed  speci- 
mens tends  to  extend  at  a  right  angle  from  the  body.  The  wall  of  the  tail  is  com- 

PLATE  II 

FIGURE  3.  N.  atlanticus,  juvenile  specimen,  1.36  mm.  long,  from  caecum  of  eider  duck,  8 
days  development,  ventral  view. 

FIGURE  4.    N.  atlanticus,  cercaria. 

FIGURE  5.     N.  atlanticus,  redia,  1.00  mm.  long,  a  fixed  and  stained  specimen. 

FIGURE  6.  N.  atlanticus,  redia  from  sketches,  showing  the  excretory  system  of  one  side  ; 
alive  it  was  0.3  to  0.7  mm.  long. 

FIGURE  7.  N.  atlanticus,  parasagittal  section  showing  relative  positions  of  oral  sucker, 
esophagus,  digestive  caecum,  cirrus  sac  and  metraterm  at  the  genital  pore,  and  most  anterior 
median  gland. 

FIGURE  8.  N.  minutus,  specimen  1.21  mm.  long,  natural  infection,  from  caecum  of  hybrid 
wild  duck  from  Cuttyhunk  Island. 


512  HORACE  W.  STUNKARD 

posed  of  external  circular  and  internal  longitudinal  muscles,  disposed  around  a 
loose  parenchymal  matrix.  The  posterolateral  ends  of  the  body  bear  eversible 
and  retractile  locomotor  appendages  which  function  in  creeping  movements.  When 
the  body  is  extended  these  appendages  are  close  together  and  they  separate  as  the 
body  contracts.  While  in  the  redia,  the  cercaria  has  only  two  ocelli,  but  after 
emergence  there  may  be  a  small,  ring-like  medial  fleck  of  pigment.  The  ocelli 
measure  0.013  to  0.016  mm.  in  diameter  and  have  lenses  which  are  often  conspicuous 
and  give  the  larva  a  "spectacled"  appearance.  The  anterior  one-third  of  the  body 
has  more  or  less  diffuse,  dendritic  brown  pigment,  accumulated  around  the  ocelli, 
with  strands  that  extend  posteriad  along  the  digestive  caeca.  The  oral  sucker 
measures  0.033  to  0.038  mm.  in  diameter;  the  esophagus  is  about  as  long  as  the 
oral  sucker  and  is  dorsal  to  the  excretory  ring  and  its  anterior  projection.  The 
digestive  caeca  extend  posteriad,  just  mediad  of  the  excretory  ring  and  near  the 
posterior  end  of  the  body  they  turn  laterad  to  cross  the  ring  and  end  blindly,  ante- 
rior to  the  level  of  the  excretory  vesicle.  In  emerged  cercariae,  the  parenchyma  is 
filled  with  cystogenous  cells  containing  short,  bacilliform  rods,  2  to  3  microns  long 
and  about  one-half  as  wide. 

The  development  of  the  excretory  system  of  Cercaria  ephemera  Lebour,  1907, 
was  described  by  Rothschild  (1935),  with  three  tufts  of  actively  beating  cilia  in 
each  recurrent  tubule.  Miss  Rothschild  accepted  the  idea  of  Faust  (1919),  that 
flame-cells  increase  in  number  by  division,  but  this  postulate  has  never  been  con- 
firmed. I  have  never  seen  a  dividing  flame-cell  or  one  with  two  nuclei,  and  do 
not  believe  flame-cells  divide ;  new  cells  probably  arise  from  undifferentiated  cells 
in  the  parenchyma.  The  system  in  the  cercaria  of  N.  atlanticus  is  virtually  iden- 
tical with  that  of  C.  ephemera.  The  primary  ducts  of  the  very  young  cercaria, 
which  extend  from  the  flame-cells  to  the  excretory  pores,  fuse  posteriorly  and 
anteriorly  to  form  the  excretory  ring ;  from  the  locus  of  anterior  fusion  a  finger-like 
diverticulum  extends  forward  below  the  esophagus,  and  posteriorly  from  the  fused 
common  stem,  a  tubule  leads  to  the  excretory  pores  on  the  sides  of  the  developing 
tail.  From  the  lateral  ends  of  the  anterior  transverse  portion  of  the  ring,  recurrent 
tubules  pass  posteriad  to  the  middle  of  the  body  where  they  divide  into  anterior  and 
posterior  branches.  In  young  cercariae,  before  the  cystogenous  cells  become  filled 
with  secretion,  three  flame-cells  were  observed  on  each  branch  and  at  that  stage 
the  system  has  the  formula  2  [(1  +  1  +  1)  +  (1  +  1  +  1)].  Odening  (1963) 
reported  that  the  definitive  flame-cell  formula  of  notocotylid  cercariae  is  [(3  +  3 
+  3)  +  (3  +  3  +  3)],  but  his  figure  of  the  origin  of  the  secondary  tubules  from 
the  lateral  walls  of  the  ring  is  not  in  agreement  with  present  observations.  In 
young  cercariae,  the  constriction  that  cuts  off  the  tail,  delimits  a  primary  excretory 
vesicle  in  the  anterior  end  of  the  common  stem ;  it  is  immediately  posterior  to  the 
posterior  transverse  portion  of  the  ring  and  in  communication  with  it.  As  the 
cercaria  matures,  a  secondary  excretory  pore  is  formed  from  the  excretory  bladder 
to  the  dorsal  wall  of  the  body  and  the  portion  in  the  tail  atrophies. 

Metacercaria 

The  cercariae  emerge  principally  between  10  AM  and  2  PM.  They  encyst 
promptly  when  irritated,  otherwise  after  swimming  for  two  to  three  hours.  When 
encysting  the  larva  attaches  by  the  oral  sucker,  contracts  until  the  body  is  circular, 


LIFE-CYCLE  OF  NOTOCOTYLUS  513 

and  rhythmic  contractions  of  the  body  wall  cause  the  extrusion  of  the  cystogenous 
material.  The  tail  remains  outside  the  cyst  and  eventually  lashes  itself  free.  As 
the  cyst  forms,  the  external  layer  remains  hyaline,  amorphous  and  jelly-like;  as  it 
condenses,  a  second  layer,  which  has  the  appearance  of  flattened,  hemispherical  disks 
or  droplets,  may  be  recognized,  and  then  a  concentric  layer  of  fibrous  material 
encloses  the  larva.  The  outside  diameter  of  a  newly  formed  cyst  is  0.18  mm.;  the 
inside  diameter  is  0.16  mm.  but  the  cyst  wall  becomes  thinner,  darker  and  denser 
with  age.  The  larvae  do  not  develop  in  the  cysts  and  are  infective  soon  after 
encystment. 

DISCUSSION 

The  present  specimens  can  be  distinguished  from  species  with  described  life- 
cycles  since,  with  the  exception  of  Notocotylus  minutus,  all  others  use  fresh- water 
snails  as  intermediate  hosts.  In  size,  they  agree  with  N.  aegyptiaais,  N.  babai, 
N.  filamentis,  N.  noyeri,  N.  seineti,  and  N.  dafilae.  But  N.  aegyptiaciis  and  N. 
seine ti  have  fewer  ventral  glands,  N.  babai  has  a  smaller  oral  sucker,  longer  cirrus 
sac,  larger  gonads,  larger  eggs,  and  a  different  disposition  of  uterine  loops ;  N. 
dafilae  has  a  longer  cirrus  sac  and  different  arrangement  of  ventral  glands  and 
uterine  loops,  while  N.  filamentis  and  N.  noyeri  are  parasites  of  mammals  with 
distinct  morphological  differences.  These  specimens  can  not  be  assigned  to  any 
known  species  and  are  described  as  a  new  species,  Notocotylus  atlanticus. 

LITERATURE  CITED 

ACHOLONU,  A.  D.,  1964.  Life  history  of  two  Notocotylidae  (Trematoda).  /.  Parasitol.,  50 
(Suppl.)  :  28-29. 

BAER,  J.  G.,  AND  CH.  JOYEUX,  1961.  Classe  des  Trematodes,  in  Traite  de  Zoologie,  P.  P. 
Grasse,  4:  561-694. 

BARKER,  F.  D.,  1915.  Parasites  of  the  American  muskrat,  Fiber  sibethicus.  J.  Parasitol.,  1: 
184-197. 

BYCHOVSKAJA-PAVLOVSKAJA,  I.  E.,  1962.  Tremarody  ptic  fauny  SSSR.  Ekologo-geografices- 
kij  obzor.  Moskau-Leningrad ;  408  pp.  Cited  after  Odening  (1964). 

CREPLIN,  F.  C.  H.,  1846.  Nachtrage  zur  Gurlt's  Verzeichnis  der  Thiere,  bei  welchen  Entozoen 
gefunden  worden  sind.  Arch.  Naturg.,  1:  129-160. 

DIESING,  C.  M.,  1839.  Neue  Gattungen  von  Binnenwiirmern  nebst  einem  Nachtrage  zur  Mono- 
graphic der  Amphistomen.  Ann.  Wiener  Museums  der  Naturgesch.,  2:  219-242. 

DONGES,  JOHANNES,  1962.  Entwicklungsgeschichtliche  und  morphologische  Untersuchungen  an 
Notocotyliden  (Trematoda).  Zeitschr.  Parasitenk.,  22:  43-67. 

DUBOIS,  G.,  1929.  Les  cercaires  de  la  region  de  Neuchatel.  Bull.  Soc.  Neuchatel  Sci.  Nat., 
53:  1-177. 

DUBOIS,  G.,  1951.  fitude  des  trematodes  nord-americains  de  la  collection  E.  L.  Schiller  et 
revision  du  genre  Notocotylus  Diesing,  1839.  Bull.  Soc.  Neuchatel  Sci.  Nat.,  74:  41-76. 

EMMEL,  L.,  1943.  Die  Cercarien  von  Bithynia  tentaculata  L.  und  B.  Icachi  Leach  aus  einem 
Berliner  Standort,  ihre  jahreszeitliche  Verteilung  und  die  Specifitat  ihrer  Anpassung 
an  den  Zwischenwirt.  Zentrbl.  Bakt.,  1  Abt.,  Orig.,  149:  81-98. 

ERKINA,  N.  G.,  1954.  Der  Entwicklungscyclus  des  Trematoden  Notocotylus  chionis,  eines 
Parasiten  von  Wasservogeln.  C.  R.  Acad.  Sci.  URSS,  n.  s.,  97:  559-560. 

FAUST,  E.  C.,  1919.  The  excretory  system  in  Digenea,  III.  Notes  on  the  excretory  system 
in  a  monostome  larva,  Cercaria  spatula  nov.  spec.  Biol.  Bull.,  36:  340-344. 

HARPER,  W.  F.,  1929.  On  the  structure  and  life  histories  of  British  freshwater  larval  trema- 
todes. Parasitol.,  21 :  189-219. 

HARRAH,  E.  C.,  1922.  North  American  monostomes  primarily  from  freshwater  hosts.  Illinois 
Biol.  Monogr.,  7 :  225-324. 


514  HORACE  W.  STUNKARD 

HARWOOD,  P.  D.,  1939.     Notes  on  Tennessee  helminths.     IV.  North  American  trematodes  of 

the  subfamily  Notocotylinae.    /.  Tenn.  Acad.  Sci.,  14:  332-341,  421-437. 
HERBER,  E.  C,  1939.     Life  history  studies  on  monostomes  of  the  genus  Notocotylus   (Trema- 

toda).     /.  Parasitol,  25  (Suppl.)  :  18-19. 
HERBER,  E.  C.,  1942.     Life  history  studies  on  two  trematodes  of  the  subfamily  Notocotylinae. 

/.  Parasitol,  28:  179-196. 
HERBER,  E.  C.,  1955.     Life  history  studies  on  Notocotylus  urbancnsis   (Trematoda:  Notocoty- 

lidae) .     Proc.  Perm.  Acad.  Sci.,  29 :  267-275. 
Hsu,  P.  K.,  1957.     On  the  life  history  of  Notocotylus  mainii  Hsu,  1954  (Trematoda :  Notocoty- 

Hdae).    Ada  Zool.  Sinl,  9:  121-130.     Cited  after  Odening   (1964). 

JOYEUX,  CH.,  1922.     Recherches  sur  les  Notocotyles.    Bull.  Soc.  Path.  Exot.,  15:  331-343. 
KOSSACK,  W.,  1911.     Uber  Monostomiden.    Zool.  Jahrb.,  Syst.,  31:  491-590. 
KUPRIYANOVA-SHAKHMATOVA,  R.  A.,  1959.     Experimental  evidence  of  the  specific  identity  of 

Notocotylus  attenuatus  (Rud.,  1809)   and  Notocotylus  thienemanni  L.  und  U.  Szidat, 

1933.     Rabot.  Gel'mintol.  80  let.  Skrjabin,  pp.  185-187.     Referat.  Zhur.  Biol.  1960,  No. 

20864. 
LEBOUR,  MARIE  V.,  1907.     Larval  trematodes  of  the  Northumberland  coast.     Trans.  Nat.  Hist. 

Soc.  Northumberland,  n.  s.,  1 :  437-454. 
LUHE,  M.,   1909.     Parasitische   Plattwiirmer   I.     Trematoden.     Siissivasserjauna  Deutschlands, 

17:  215  pp. 

LUTTERMOSER,  G.  W.,  1935.     A  note  on  the  life  history  of  the  monostome,  Notocotylus  urba- 
ncnsis.   J.  Parasitol.,  21:  456. 
MATH  IAS,    P.,   1930.     Sur  le  cycle   evolutif  d'un   trematode   de   la   famille   des    Notocotylidae 

Liihe  (Notocotylus  attenuatus  Rud.).     C.  R.  Acad.  Sci.  Paris,  191:  75-77. 
ODENING,  K.,  1963.     Das  Exkretionsystem  monostomer  Cercarien  (Trematoda:  Notocotylidae) 

aus  Gewassrn  von  Berlin  und  Umgebung.    Limnologia   (Berlin),  1:  356-373. 
ODENING,  G.,  1964.     Zur  Trematodenfauna  von  Nettapus  c.  coromandeliamis  in  Indien.    Ange- 

wandte  Parasitol.,  5:  228-241. 
ODENING,  K.,  1966.     Physidae  und   Planorbidae  als  Wirte  in  den   Lebenszyklen  einheimisher 

Notocotylidae   (Trematoda:  Paramphistomida).    Zeitschr.  Parasitenk.,  27:  210-239. 
ODENING,  K.,  AND  I.  BOCKHARDT,  1965.     Der  Entwicklunszyklus  des  Trematoden  Notocotylus 

noyeri  Joyeux,  1922,  im  Raum  Berlin.     Monatsber.  Dent.  Akad.   Wissens.,  Berlin,  7: 

51-52.  " 

ODHNER,  T.,  1905.     Die  Trematoden  des  arktischen  Gebietes.     Fauna  Arctica,  4:  291-372. 
PILSBRY,  H.  A.,  1905.     A  new  brackish-water  snail  from  New  England.     The  Nautilus,   19: 

90-91. 
ROTHSCHILD,  MIRIAM,  1935.     Note  on  the  excretory  system  of  Cercaria  ephemera  Lebour,  1907 

(nee  Nitzsch).     Parasitol.,  27:  171-174. 
ROTHSCHILD,  MIRIAM,  1936a.     Preliminary  note  on  the  trematode  parasites  of  Peringia  uh'ae 

( Pennant,  1777) .     Novit.  Zool.,  39 :  268-269. 
ROTHSCHILD,  MIRIAM,  1936b.     A  note  on  the  variation  of  certain  cercariae.     Novit.  Zool.,  40: 

170-175. 
ROTHSCHILD,  MIRIAM,  1938.     Notes  on  the  classification  of  cercariae  of  the  super-family  Noto- 

cotyloidea  (Trematoda),  with  special  reference  to  the  excretory  system.    Novit.  Zool., 

42:75-83. 

ROTHSCHILD,   MIRIAM,   1940.     Cercaria  imbricata  Looss,   1896  nee   1893.     A   note   on   nomen- 
clature.   Novit,  Zool,  42 :  215-216. 
ROTHSCHILD,  MIRIAM,  1941.     Note  on  life  histories  of  the  genus  Paramonostomum  Liihe,  1909 

( Trematoda:    Notocotylidae)     with    special    reference    to    the    excretory    vesicle.    /. 

Parasitol,  27 :  363-365. 

SEWELL,  R.  B.  S.,  1922.     Cercariae  Indicae.     Ind.  Jour.  Med.  Res.,  10  (Suppl.  Number)  :  1-370. 
STUNKARD,  H.  W.,  1960.     Studies  on  the  morphology  and  life-history  of  Notocotylus  minutus 

n.  sp.,  a  digenetic  trematode  from  ducks.    /.  Parasitol,  46:  803-809. 
STUNKARD,  H.  W.,  AND  F.  W.  DUNIHUE,  1931.     Notes  on  trematodes  from  a  Long  Island  duck 

with  description  of  a  new  species.     Biol  Bull,  60:  179-186. 
SZIDAT,  L.,  AND  URSULA  SZIDAT,  1933.     Beitrage  zur  Kenntnis  der  Trematoden  der  Monosto- 

midengattung  Notocotylus  Diesing.     Zentrbl  Bakt.,  1  Abt.,  Orig.,  129:  411-422. 


LIFE-CYCLE  OF  NOTOCOTYLUS  515 

SZIDAT,  URSULA,  1935.  Weitere  Beitrage  zur  Kenntnis  der  Trematoden  der  Monostomiden- 
gattung  Notocotylus  Diesing.  Zentralbl.  Bakt.,  1  Abt.,  Orig.,  133:  265-270. 

SZIDAT,  URSULA,  1936.  Beitrage  zur  Kenntnis  der  Trematodengattung  Notocotylus  Diesing. 
III.  Notocotylus  lincaris  (Rud.  1819?)  n.  sp.,  aus  den  Blinddarmen  des  Kiebitz  (Vanel- 
lus  vancllus  L.).  Zcntrbl.  Bakt.,  1  Abt.,  Orig.,  136:  231-235. 

WESENBERG-LUND,  C.,  1934.  Contributions  to  the  development  of  the  Trematoda  Digenea. 
Part  II.  The  biology  of  the  freshwater  cercariae  in  Danish  freshwaters.  Mem.  Acad. 
Roy.  Sci.  ct  Lett.  Dancmark;  Sect.  Sci.,  5:  1-223. 

WRIGHT,  C.  A.,  AND  M.  S.  BENNETT,  1964.  The  life  cycle  of  Notocotylus  attenuatus.  Para- 
sitol,  54  (4)  :  14. 

Wu,  L.  Y.,  1953.  On  the  life  history  and  biology  of  Notocotylus  stagnicolae  Herber,  1942 
(Family  Notocotylidae).  Canad.  J.  Zool.,  31:  522-527. 

YAMAGUTI,  S.,  1938.  Zur  Entwicklungsgeschichte  von  Notocotylus  attenuatus  (Rud.,  1809) 
und  N.  magniovatus  Yamaguti,  1934.  Zeitschr.  Parasitenk.,  10:  288-292. 

YAMAGUTI,  S.,  1958.  Systema  Helminthum.  I.  The  Digenetic  Trematodes.  Interscience  Publ., 
Inc.,  New  York. 

ZDARSKA,  ZDENKA,  1964.  The  development  and  specific  independence  of  the  trematode,  Noto- 
cotylus ephemera  (Nitzsch,  1807).  Cesk  Parasitol.,  11:  309-318.  Cited  from  Biol. 
Abstr.  Feb.  1966;  No.  14690. 


OOCYTE  DEVELOPMENT  AND  INCORPORATION  OF  H3- 

THYMIDINE  AND  HMJRIDINE  IN  PECTINARIA 

(CISTENIDES)  GOULDII 

KENYON  S.  TWEEDELL 

Department  of  Biology,  University  of  Notre  Dame,  Notre  Dame,  Indiana  46556,  and 
the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts  02543 

Transformation  from  the  oogonium  and  development  of  the  primary  oocyte 
involves  intricate  morphological  and  metabolic  changes  in  the  cell.  Among  these 
are  enlargement  of  the  germinal  vesicle,  development  of  the  nucleolus  and  cyto- 
plasmic  growth  (Raven,  1961).  It  is  generally  believed  that  the  necessary  pre- 
meiotic  changes  in  the  nucleus  precede  these  phenomena. 

Most  evidence  indicates  that  H3-thymidine  is  not  incorporated  into  the  germinal 
vesicle  of  the  mature  oocyte  (Ficq,  1961a;  Favard-Sereno  and  Durand,  1963b; 
Ficq,  Aiello  and  Scarano,  1963)  although  recently  Holland  and  Giese  (1965) 
report  that  both  the  oogonia  and  the  pre-leptotene  primary  oocytes  of  the  sea  urchin 
synthesize  DNA  within  the  ovary.  These  results  imply  that  the  synthesis  of  DNA 
needed  for  subsequent  growth  and  maturation  of  the  oocytes  must  occur  after  the 
last  oogonial  division  and  no  later  than  the  early  premeiotic  changes  in  the  oocyte 
nucleus. 

Since  there  is  evidence  that  various  types  of  eggs  have  vast  reserves  of  cyto- 
plasmic  DNA  (Bieber  et  al,  1959)  or  deoxyribosides  (Hoff-J0rgensen  and 
Zeuthen,  1952)  and  some  indication  of  cytoplasmic  uptake  (Ficq,  1961a;  Gintsburg, 
1963)  of  H3-thymidine  into  the  oocytes,  the  question  arises,  is  the  entire  cyto- 
plasmic reserve  acquired  at  the  time  of  nuclear  DNA  synthesis  or  is  it  augmented 
throughout  development  of  the  oocyte?  It  is  pertinent  to  determine  whether  the 
developing  and  growing  oocyte  is  metabolically  stable  or  if  it  is  able  to  actively 
increase  its  DNA  reserve.  The  continuous  presentation  of  a  specifically  labeled 
nucleoside,  H3-thymidine,  in  vivo  is  one  approach  to  this  problem. 

In  contrast,  the  uptake  of  RNA  precursors  into  the  maturing  oocyte  appears  to 
be  widespread  during  oogenesis.  In  the  starfish,  H3-uridine  uptake  is  localized 
in  the  nucleus,  nucleolus  (Ficq,  1961b)  and  cytoplasm  (Geuskens,  1963),  in  the 
nucleus  and  nucleolus  of  the  sea  urchin  oocyte  (Ficq,  Aiello  and  Scarano,  1963) 
and  the  nuclear  sap,  chromosomes  and  cytoplasm  of  the  cricket  oocyte  (Favard- 
Sereno  and  Durand,  1963a).  The  use  of  H3-uridine  and  other  precursors  in 
conjunction  with  various  inhibitors  implicates  the  synthesis  of  at  least  three  types 
of  RNA  (m-RNA,  r-RNA  and  t-RNA)  during  maturation  of  the  starfish  and 
sea  urchin  oocyte  (Ficq,  1961a;  1962;  1964). 

Since  both  the  nucleolus  and  nucleus  in  Pectinaria  increase  in  size  and  activity 
as  the  oocyte  develops  (Tweedell,  1962),  a  study  of  the  incorporation  of  labeled 
uridine  into  the  RNA  of  the  nucleolus  and  nucleus  seemed  promising  (Tweedell. 
1964). 

516 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  517 

Fortunately,  the  growth  and  development  of  the  primary  oocyte  in  Pectinaria 
occurs  in  the  coelom  after  the  cells  leave  the  ovary.  The  utilization  of  these  labeled 
precursors  can  be  observed  in  vivo  within  the  ovary  and  during  individual  stages  of 
oocyte  formation  in  the  coelom.  These  results  are  compared  with  in  vitro  exposure 
of  the  oocytes  to  the  same  nucleosides. 

MATERIALS  AND  METHODS 

Specimens  of  the  marine  polychaete  annelid  Pectinaria  (=  Cistenidcs)  gouldii 
Verrill  (Hartman,  1941)  were  obtained  from  the  north  shore  of  Cape  Cod  during 
Tune,  July  and  August  of  1963  and  1964.  They  were  maintained  in  the  labora- 
tory as  described  earlier  (Tweedell,  1962). 

Incorporation  of  isotopes 

In  vivo.  Before  labeling,  the  animals  were  carefully  removed  from  their  sand 
test  and  the  sex  was  determined  microscopically  by  identification  of  the  gametes 
beneath  the  ventral  body  wall  or  the  parapodia.  The  animals  were  then  narcotized 
with  50%  ethyl  alcohol  added  dropwise  to  a  small  dish  of  sea  water  until  the  ani- 
mals were  flaccid.  Isotopes  were  injected  with  a  #27  hypodermic  needle  directly 
through  the  cephalic  plaque  (Fig.  1)  of  the  animals  into  the  coelomic  cavity. 
Narcotized  animals  were  injected  according  to  size  with  either  5  to  10  /xC.  of 
tritiated  thymidine  (1.9  C./millimole)  or  5  to  10  ,uC.  of  tritiated  uridine  (1.7 
C./millimole).  The  addition  of  a  small  amount  of  Nile  blue  sulphate  indicated 
that  most  of  the  injected  fluid  was  retained.  The  animals  were  then  returned  to 
their  tests  and  placed  in  normal  sea  water  where  they  recovered  quickly.  Animals 
were  sacrificed  or  the  eggs  were  harvested  and  fixed  at  intervals  of  ^-  1»  2,  4,  8, 
16,  24,  48  and  72  hours.  Additional  egg  harvests  were  made  at  2,  3,  6-7  and  19-21 
days  after  injection.  At  least  two  animals  were  examined  for  each  of  the  above 
intervals. 

In  vitro.  Gametes  were  also  shed  directly  into  sea  water  containing  10  ju,C.  of 
H3-thymidine  or  H3-uridine  per  ml.  of  sea  water.  Following  germinal  vesicle 
(G.V.)  breakdown  of  the  mature  oocytes,  they  were  removed,  washed  and  fixed 
at  intervals  of  2,  4  and  8  hours  after  exposure.  Thereafter  they  were  embedded 
in  paraffin  and  sectioned  at  4  p.. 

Recovery  of  free  oocytes.  Some  of  the  animals  injected  in  vivo  were  shed  and 
the  eggs  immediately  fixed  as  whole  mounts  on  coverslips  with  Kahle's  fixative 
using  a  modified  double  coverslip  sandwich  technique  (Tweedell,  1962).  Similar 
treatment  was  given  to  oocytes  which  were  labeled  by  shedding  directly  into  sea 
water  containing  the  isotopes.  At  the  moment  of  fixation,  the  cells  were  com- 
pressed by  placing  45  grams  pressure  on  the  coverslips  to  further  flatten  the  oocytes. 
After  fixation  the  whole  mounts  were  washed  and  dehydrated  to  100%  ethyl  alcohol 
when  they  were  air  dried.  The  coverslips  were  then  cemented  to  slides  with  the 
eggs  facing  upward.  Equivalent  animals  were  fixed  at  the  same  time  for  future 
exposure  of  the  oocytes  in  sections. 

Preparation  of  intact  animals.  The  majority  of  the  oocytes  labeled  in  vivo 
were  processed  for  sectioning  in  situ.  Prior  to  histological  preparation  of  adult 
animals  it  was  necessary  to  remove  the  sand  grains  from  the  digestive  tract.  Origi- 


518 


KENYON  S.  TWEEDELL 


A.M. 


FIGURE  1.  A  dorsal  dissection,  cut  in  the  midline  and  spread  laterally,  of  an  adult  Pecti- 
naria  ($),  showing  position  of  the  ovaries  and  attached  nephromixia.  CSP,  cephalic  spines; 
CP,  cephalic  plaque,  AM,  antennular  membrane;  G,  gill;  NM,  nephromixium ;  NP,  nephrodio- 
pore ;  MG,  mucus  gland ;  O V,  ovary ;  NO,  notopodium ;  S,  setae ;  MI,  middle  intestine ;  H,  heart. 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  519 

nally,  the  technique  of  Hanks  (1960)  was  repeated,  in  which  he  fed  living  animals 
ground  quahog  shells.  As  reported,  the  animals  readily  ingested  the  calcium  car- 
bonate. However,  the  required  decalcification  by  acetic  acid  in  the  intact  animal 
was  not  successful  and  was  further  complicated  by  the  collection  of  CO2  which  could 
not  be  removed  under  vacuum.  Alternate  materials,  dried  coffee  grounds,  corn 
meal,  powdered  charcoal,  ground  rice,  Dowex  resin  and  various  household  cereals 
were  tested.  In  all  cases,  the  materials  were  ground  and  screened  through  wire 
mesh  to  the  equivalent  size  of  large  sand  grains.  These  materials  were  placed  in 
5-inch  open  glass  tubes,  -J-inch  diameter,  that  were  covered  at  one  end  with  several 
layers  of  cheesecloth.  The  tubes  were  half-filled  with  the  test  material  and  placed 
cloth  end  down  in  a  test  tube  rack.  The  adult  animal  was  inserted  head  down  in 
the  tube  and  a  siphon  tube  of  slowly  running  sea  water  was  introduced  at  the  top 
to  insure  a  constant  sea  water  flow.  Animals  were  allowed  to  "feed"  in  this 
position  for  24  to  48  hours,  then  removed  and  left  to  "work"  in  running  sea  water 
for  24  hours  while  excess  ingested  products  were  eliminated.  Three  materials 
were  successful :  powdered  charcoal,  ground  white  rice  grains  and  a  protein  cereal 
(Kellogg's  Concentrate).  The  latter  two  were  readily  ingested  and  perfectly  com- 
patible with  histological  sectioning  while  the  charcoal  sections  tended  to  crumble. 
After  removal  from  their  tests  the  adult  animals  were  narcotized  and  then  fixed  in 
Kahle's  or  Kleinenberg's  fixative,  double  embedded  in  methyl  benzoate-parlodion 
paraffin  and  sectioned  at  5  /*,. 

Autoradiography.  Both  compressed  whole  oocytes  and  sections  were  prepared 
for  autoradiography  by  dipping  the  slides  in  Kodak  NTB2  or  Ilford  G-5  emulsions 
(diluted  50/50  with  distilled  water).  The  slides  were  incubated  in  light-tight  boxes 
with  "Drierite"  at  4°  C.  for  the  duration  of  exposure  from  two  to  three  weeks. 
Slides  were  developed  in  D-72  or  D-19  for  3  minutes  at  14°  C.,  placed  in  stop  bath 
for  15  seconds  and  fixed  for  3  minutes  at  14°  C.  The  slides  were  then  washed 
for  ^  hour  in  running  water. 

Coverslip  mounts  were  stained  in  gallocyanin  chrome-alum,  pH  1.7,  for  8-12 
hours  and  counterstained  with  eosin.  Sections  were  stained  in  Galligher's  haema- 
toxylin,  eosin-azure  II  (Gurr)  or  Jenner-Giemsa  solution. 

Serial  sections  of  oocytes  retained  within  the  whole  animal  or  sections  of  shed 
eggs  were  treated  with  RNase  or  DNase  prior  to  autoradiography.  DNase  was 
applied  to  sections  alternating  with  control  sections  in  a  concentration  of  0.1  mg./ml. 
prepared  in  0.05  M  Michaelis  veronal  acetate  buffer,  pH  6.8  with  0.0025  M  MgCl2 
added.  Incubation  was  at  37°  C.  for  2  hours. 

RNase  was  prepared  as  3  mg./ml.  in  0.05  M  Sorensen  phosphate  buffer  at  pH 
7.6.  The  sections  were  incubated  in  RNase  for  2  hours  at  48°  C.,  along  with  non- 
enzyme-treated  control  sections. 

OBSERVATIONS 

The  ovaries  are  minute,  well  vascularized  organs  located  along  the  anterior  sur- 
face of  two  pairs  of  prominent  yellow  brown  organs,  identified  as  nephromixia 
(Goodrich,  1945).  Externally,  the  paired  nephromixia  can  be  seen  through  the 
lateral  body  walls  of  the  2nd  and  3rd  setae-bearing  segments.  The  latter  glands, 
attached  to  the  ventral  body  wall  (Fig.  1)  first  loop  dorsally  at  the  midline,  then 


520 


KENYON  S.  TWEEDELL 


open  externally  through  dorso-lateral  nephridiopores  which  are  slightly  caudal  to 
the  abbreviated  setae  of  the  parapodia  on  the  same  segments. 

Viewed  from  a  dorsal  dissection,  the  ovaries  are  seen  anterior  and  slightly  dorsal 
to  the  nephromixia.  The  ciliated  ovaries  are  narrow  transparent  organs  enveloping 
lateral  ovarian  blood  vessels  which  flow  along  the  cephalic  edges  of  the  nephromixia. 
Lobe-shaped  fins  projecting  at  right  angles  to  the  main  axis  of  the  ovary  extend 
dorsad  and  somewhat  anteriorly  from  the  ovaries.  These  fins  are  sinuses  confluent 
with  the  ovarian  vessel  and  are  also  covered  with  cilia. 

The  germinal  portion  of  the  ovary  consists  of  budding  areas  located  at  the 
ventral  medial  end  of  each  of  four  ovaries  (Figs.  2,  3).  Sometimes  an  additional 
area  is  found  at  the  dorsal  lateral  end.  The  ovarian  cytology  seems  to  vary  accord- 
ing to  the  season.  Early  in  the  summer  the  ovaries  are  largely  composed  of  inter- 
phase  oogonial  cells  and  mitotic  figures  are  common,  whereas  in  August  the  ovaries 
have  a  large  proportion  of  oocytes  in  the  leptotene  stage  (Fig.  4). 


c.     N  H. 


FIGURE  2.  An  anterior  view  of  a  single  nephromixium  and  attached  ovary  (as  seen  in 
Figure  1).  D,  dorsal;  M,  medial;  C,  coelomostome ;  NM,  nephromixium;  OV,  ovary,  enclos- 
ing lateral  ovarian  vessel ;  GA,  germinal  area. 


The  generative  phase  (Raven,  1961)  of  oocyte  formation  begins  within  the 
germinal  area  of  the  ovary.  The  oocytes  are  budded  off  directly  into  the  coelomic 
cavity  from  the  germinal  area  at  the  cephalic  free  edge  of  the  ovaries.  Thus  a 
sample  of  the  coelomic  fluid  from  an  adult  female  yields  a  series  of  developmental 
phases  in  the  formation  of  the  primary  oocytes  after  they  leave  the  ovaries.  The 
developing  oocytes  are  moved  and  bathed  continuously  in  this  fluid  by  the  muscular 
pumping  of  the  adult.  A  diverse  selection  of  cell  types :  amoebocytes,  gregarines, 
histiocytes  and  others,  are  included  in  the  coelomic  fluid. 

Two  development  phases  of  oocyte  formation  within  the  coelom  can  be  recog- 
nized, a  packet  phase  that  is  followed  by  an  individual  oocyte  (solitary)  phase. 
The  packets  consist  of  small  cell  clumps,  about  18  to  20  microns  in  diameter, 
arranged  about  a  central  core  area.  They  appear  in  the  coelomic  fluid  after  being 
budded  from  the  surface  of  the  ovary  (see  Fig.  3). 

There  usually  are  16  to  32  oocytes  within  a  packet  and  the  individual  cell  is 
about  5.5  microns  in  diameter.  The  nucleus  fills  the  cell,  leaving  a  thin  rim  of 
cytoplasm,  and  the  chromosomes  are  generally  in  the  zygotene  stage.  The  single 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  521 

nucleolus  is  about  twice  the  size  of  those  within  the  ovary.  Thereafter  the  packets 
slowly  increase  in  size  from  growth  of  the  individual  cells  and  a  small  "core"  forms 
in  the  packet  center.  The  enlarged  nuclei,  entering  the  pachytene  stage,  now  have 
a  dominant  nucleolus  and  the  cytoplasm  shows  heavy  basophilia  (stainable  with 
alcian  blue,  gallocyanin  and  toluidine  blue)  at  the  basal  ends  of  the  cells  near  the 
center  of  the  packet.  The  largest  packets  consist  of  loosely  bound  cells  each 
measuring  10  to  20  microns  in  \vidth. 

During  the  second  growth  or  vegetative  phase,  the  packets  fragment  and  indi- 
vidual oocytes  appear  in  the  coelomic  fluid,  the  smallest  measuring  from  10  to  12.5 
microns.  These  cells  are  distinguished  by  a  large  germinal  vesicle  and  a  single 
amphinucleolus.  The  increase  in  size  of  these  solitary  oocytes  is  due  to  both 
nuclear  and  cytoplasmic  growth.  The  mature  oocyte  is  a  flattened  ellipse  which 
averages  48  by  56  microns  and,  since  it  is  flattened,  about  25  microns  in  thickness. 
At  this  time  the  germinal  vesicle  has  an  average  diameter  of  40  microns.  Thus, 
using  the  cell  diameter  as  a  crude  measurement  of  linear  growth,  the  cells  are  seen 
to  increase  about  ten  times  in  size  since  budding  from  the  ovary. 

As  the  oocyte  approaches  full  development,  the  diplotene  chromosomes  are  con- 
spicuously distributed  throughout  the  germinal  vesicle.  Increased  activity  of  the 
nucleolus  is  evidenced  by  the  formation  of  epinucleolar  and  intranucleolar  buds. 

Mechanical  stimulation  of  the  adult  usually  causes  artificial  shedding  when  part 
or  all  of  the  coelomic  fluid  with  its  cellular  components  is  ejected  from  posterior 
coelomoducts.  Approximately  12  to  18  minutes  after  shedding  into  sea  water 
(Austin,  1963;  Tweedell,  1962)  the  fully  developed  oocytes  undergo  germinal 
vesicle  breakdown,  become  spherical  and  develop  to  the  metaphase  of  the  first 
maturation  division  where  they  remain  unless  fertilized. 

There  are  indications,  however,  that  the  natural  spawning  process  follows  a 
different  sequence  of  events.  During  late  summer  induced  shedding  occasionally 
produces  oocytes  that  are  already  in  the  first  maturation  division.  However,  these 
cells  are  ejected  from  the  nephridiopores.  Ordinarily,  the  nephromixia  are  rela- 
tively empty,  but  during  late  summer  their  distal  ends  contain  heavy  concentrations 
of  oocytes.  Coelomic  oocytes  are  drawn  into  the  nephromixium  along  a  heavily 
ciliated  groove  that  lies  on  its  anterior  edge.  The  ciliary  action  transports  the 
oocytes  laterally  to  a  large  ciliated  funnel  (coelomostome)  through  which  they 
enter  the  nephromixium  (Fig.  2).  Upon  stimulation,  these  oocytes  are  readily 
observed  leaving  the  nephridiopores. 

In  addition,  sections  through  the  intact  animal  reveal  gravid  nephromixia  in 
which  the  enclosed  oocytes  are  almost  exclusively  in  a  post-G.V.  state  while  the 
germinal  vesicles  of  fully  developed  oocytes  within  the  coelom  are  still  intact  (Fig. 
5).  Presumably,  during  the  natural  spawning  process  G.V.  breakdown!  occurs 
within  the  nephromixia  and  the  oocytes  are  shed  through  the  nephridiopores. 

Some  of  the  cells  within  the  nephromixium  after  germinal  vesicle  breakdown 
are  often  still  in  late  diakinesis  (Fig.  6).  Chromosome  bivalents  are  widely  spread 
throughout  the  cytoplasm  and  the  nucleolus  is  absent.  Generally  it  is  possible  to 
distinguish  16  large  bivalents,  yet  as  many  as  9  additional  obscure,  wreakly  staining 
doublets  can  be  seen.  Austin  (1963)  reports  from  17  to  21  bivalents  from  his 
observations,  the  variation  being  attributed  to  faint,  minute  chromosomes.  Ob- 
viously a  detailed  karyotype  study  is  needed.  In  other  cells  within  the  nephro- 


522 


KENYON  S.  TWEEDELL 


PLATE  I 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  523 

mixium,  the  chromosomes  are  condensed  into  a  small  knot,  where  they  remain 
arrested  in  the  first  maturation  division. 

RESULTS 
H3-thymidine 

In  vivo  labeling.  Following  injection  of  tritiated  thymidine  into  the  living 
adult,  oocytes  were  harvested  by  shedding  and  prepared  as  whole  mounts  or 
embedded  and  sectioned.  Entire  animals  were  also  embedded  and  sectioned  intact. 

The  ovary.  Tritiated  thymidine  was  quickly  incorporated  into  the  ovary  after 
injection  into  the  coelom.  Localization  was  principally  over  the  nuclei  of  cells 
scattered  throughout  the  ovary.  The  first  uptake  was  detected  two  hours  after 
contact  with  H3-thymidine  (Fig.  8).  The  labeled  cells  were  generally  found  deep 
in  the  mid  region  of  the  ovary  rather  than  at  the  free  surface.  Since  the  label  in 
the  ovary  often  appeared  above  the  nuclei  of  cell  pairs,  these  cells  presumably  had 
just  completed  cell  division  and  the  resultant  oocytes  were  exposed  to  thymidine 
during  the  period  of  DNA  synthesis  (S  period).  Oocytes  associated  with  the 
budding  surface  of  the  ovary  did  not  show  appreciable  nuclear  label  at  this  time. 

After  12  hours  exposure  to  the  thymidine  some  of  the  deep-lying  cells  of  the 
ovary  were  intensely  labeled  while  those  along  the  surface  were  more  lightly 
marked  (Fig.  9).  Scattered  labeled  cells  appeared  throughout  the  ovary  and 
extended  to  the  free  border  where  they  budded  off.  Labeled  oocytes  were  also 
observed  in  ovaries  recovered  from  animals  24  and  48  hours  after  injection  of 
H3-thymidine  with  no  essential  difference. 

Heavy  nuclear  labeling  was  still  evident  in  ovarian  oocytes  taken  from  animals 
after  one-week  exposures.  The  major  change  from  earlier  ovarian  sections  was 
the  presence  of  more  labeled  cells  at  the  free  surface  of  the  ovary  (Fig.  10). 
Recently  released  labeled  oocytes  were  often  seen  adjacent  to  the  ovary.  In  gen- 
eral the  labeled  oocytes  were  more  widely  distributed  but  less  heavily  tagged  over 
the  nuclei. 

After  21  days  exposure  to  a  single  pulse,  grains  were  diffusely  scattered  over 
the  remaining  intact  oocytes.  This  distribution  suggested  that  the  incorporated 
thymidine  had  been  diluted  out  by  subsequent  mitoses  of  the  remaining  oogonia. 

The  actual  length  of  the  synthesis  period  prior  to  oocyte  growth  and  develop- 
ment was  undetermined  but  H3-thymidine  incorporation  in  the  ovary  did  indicate 
when  DNA  synthesis  took  place.  The  assumption  was  then  made  that  the  cells 
which  incorporate  thymidine  after  a  short  pulse  time  were  either  oogonia  or  pre- 

PLATE  I 

FIGURE  3.  Cross-section  of  a  normal  ovary  and  attached  ciliated  lobe.  Oocytes  are  budding 
from  free  edge  of  ovary  at  lower  left. 

FIGURE  4.     Developing  primary  oocytes  in  premeiotic  stages  within  the  ovary.     1920  X. 

FIGURE  5.  Post-G.V.  oocytes  within  lumen  of  nephromixium  on  the  right ;  pre-G.V.  coelo- 
mic  oocytes  to  the  left.  Arrow  indicates  wall  of  nephromixium  separating  pre-  and  post-G.V. 
oocytes.  H3-thymidine-injected,  2-hour  exposure.  No  uptake  in  either  pre-or  post-G.V.  oocytes. 
350  X. 

FIGURE  6.  Post-G.V.  oocytes  from  within  nephromixium  of  control  animal.  Optical  plane 
shows  portion  of  chromosomes  in  diakinesis.  1200  X. 

FIGURE  7.  Post-G.V.  oocytes  in  nephromixium  showing  absence  of  H3-thymidine  incor- 
poration after  a  two-hour  pulse.  580  X. 


524 


KENYON  S.  TWEEDELL 


PLATE  II 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  525 

meiotic  oocytes  in  the  "S"  period  preceding  growth  and  development  of  the  oocyte. 
Consequently,  the  developmental  age  of  subsequent  stages  found  with  nuclear  label 
was  extrapolated  at  successive  intervals  after  injection;  the  next  most  advanced 
stage  so  labeled  indicated  its  developmental  age. 

Coeloinic  oocytes.  After  a  short  exposure  period,  thymidine  was  also  readily 
incorporated  into  the  oocyte  packets  of  the  coelom.  The  especial  difference  between 
the  ovarian  oocytes  and  the  oocyte  packets  was  in  the  localization  of  the  radio- 
activity. The  packet  oocytes  were  mainly  labeled  above  the  cytoplasm  with  rela- 
tively little  nuclear  label. 

The  smallest  coelomic  oocyte  packets,  containing  cells  of  a  size  comparable  to 
ovarian  oocytes,  began  showing  a  label  primarily  over  the  cytoplasm  after  a  pulse 
of  30  minutes.  Thereafter,  recovery  of  shed  oocytes  from  animals  at  intervals  of 
2,  4,  8,  16  or  24  hours  after  injection  revealed  the  same  pattern  of  labeling.  An 
example  of  a  packet  recovered  after  24  hours  from  an  animal  injected  with  5  ju.C. 
is  seen  in  Figure  11.  The  nuclei  of  these  small  packet  cells  were  in  the  zygotene 
stage.  Larger  cell  packets  and  single  oocytes  were  noticeably  unlabeled.  Sections 
of  oocytes  in  situ  from  a  parallel  series  of  injected  animals  demonstrated  identical 
uptake  in  the  very  early  packets  of  oocytes  recovered  up  to  24  hours  after  injection. 

As  it  was  not  possible  to  effectively  chase  the  tritiated  thymidine  from  the  coelom 
of  the  living  animal,  it  was  not  certain  whether  these  early  cell  packets  incor- 
porated the  thymidine  before  or  after  leaving  the  ovary.  The  latter  possibility  was 
most  likely  since  ovarian  oocytes  at  the  budding  edge  of  the  ovary  did  not  become 
labeled  until  after  12  hours  injection.  Thus,  these  small  packets  were  distinctive 
since  they  were  the  only  coelomic  oocyte  stages  found  with  a  significant  grain  count 
after  2  to  24  hours  exposure  to  tritiated  thymidine. 

Examination  of  coelomic  oocytes  sectioned  in  situ  after  an  exposure  of  two 
days  indicated  a  significant  change  in  the  labeling  of  cell  packets.  A  few  larger 
oocyte  packets,  presumably  resulting  from  cellular  growth  of  younger  packets,  began 
exhibiting  a  heavy  cytoplasmic  label  with  very  few  grains  over  the  nucleus.  The 
additional  labeling  of  older  oocyte  packets  suggested  that  the  developing  oocytes 
had  acquired  the  H3-thymidine  during  the  early  packet  stage  and  that  cytoplasmic 
and  nuclear  growth  of  the  individual  oocytes  followed  subsequently. 

Both  small  and  medium-size  oocyte  packets  continued  to  show  a  predominately 
cytoplasmic  label  up  to  72  hours  after  exposure  to  labeled  thymidine  (Fig.  12)  ; 
others  had  a  light  nuclear  label. 

PLATE  II 

FIGURE  8.  Edge  of  ovary  after  a  two-hour  exposure  to  H3-thymidine.  Label  occurs  over 
isolated  cells  along  the  interior. 

FIGURE  9.  Ovary  from  H3-thymidine-injected  animal  sacrificed  after  12  hours.  Scattered 
oocytes  are  heavily  labeled  over  the  nuclei.  The  free  budding  edge  of  ovary  is  at  the  left.  A 
small  labeled  oocyte  packet  is  seen  in  insert.  234  X. 

FIGURE  10.  A  section  of  ovary  from  a  H3-thymidine-injected  animal  sacrificed  after  7  days, 
showing  scattered  distribution  of  labeled  oocytes.  585  X. 

FIGURE  11.  Single  coelomic  oocytes  which  are  unlabeled  after  a  two-hour  pulse  of  Hs- 
thymidine.  A  heavily  labeled  oocyte  packet  is  seen  at  the  lower  left.  375  X. 

FIGURE  12.  A  medium-size  packet  of  developing  oocytes  from  an  animal  sacrificed  72  hours 
after  H?'-thymidine  injection.  Incorporation  is  principally  over  the  cytoplasm. 

FIGURE  13.  Large  single  developing  oocytes  recovered  7  days  after  HMhymidine  injection. 
A  heavy  label  finally  appears  over  the  nucleus  and  cytoplasm.  The  edge  of  the  ovary  appears 
on  the  left.  Note  that  not  all  of  the  large  oocytes  are  labeled.  585  X. 


526  KENYON  S.  TWEEDELL 

After  a  prolonged  exposure  of  7  days,  many  small  and  medium  oocyte  packets 
were  labeled  as  they  had  been  earlier.  More  significantly,  new  oocyte  packets  with 
a  heavy  nuclear  label  appeared.  Since  cell  division  does  not  occur  until  germinal 
vesicle  breakdown,  these  packets  must  have  been  labeled  and  subsequently  released 
from  the  ovary. 

Animals  which  had  been  injected  with  H3-thymidine  were  also  sacrificed  after 
21  days.  Both  small  and  medium  cell  packets  and  a  few  late  oocyte  packets  were 
found  with  evidence  of  thymidine  uptake.  In  the  small  packets  the  grains  were 
scattered  over  the  cytoplasm  but  the  heaviest  concentration  occurred  over  the 
nucleus.  These  packets  possibly  represented  cells  that  had  taken  thymidine  into 
the  nucleus  at  an  earlier  time  and  the  labeled  material  had  moved  into  the  cyto- 
plasm secondarily.  Alternate  oocyte  sections  from  one  animal  exposed  in  situ  for 
21  days  were  pretreated  with  DNase  prior  to  emulsion  dipping.  The  DNase 
removed  both  the  nuclear  and  cytoplasmic  label  from  the  oocyte  packets. 

Single  oocytes.  Individual  coelomic  oocytes  in  the  final  growth  phase  failed 
to  indicate  any  incorporation  of  H3-thymidine  whether  they  were  harvested  early 
(£  to  16  hours),  as  seen  in  Figure  11,  or  after  prolonged  exposure  (1  to  2  days). 
The  same  results  were  obtained  from  compressed  egg  whole  mounts  and  from  a 
study  of  sections  of  8  entire  animals  sacrificed  at  2,  12  or  24  hours  after  injection 
with  either  5  or  10  //.C.  of  tritiated  thymidine.  The  solitary,  small  immature  co- 
elomic oocytes  were  completely  unlabeled  in  all  material  harvested  up  to  a  day 
after  injection.  The  larger  mature  cells  also  failed  to  take  in  the  precursor.  It 
was  also  found  that  mature  oocytes,  in  metaphase  I,  within  the  nephromixium 
showed  a  complete  absence  of  any  labeling  (Fig.  7). 

Occasional  single  oocytes,  in  their  early  growth  phase,  were  not  significantly 
tagged  until  48  to  72  hours  after  injection.  In  these  cases  the  grains  were  evenly 
distributed  over  the  entire  cell.  The  majority  of  these  single  oocytes,  however, 
remained  unlabeled.  A  sampling  of  50  fields  from  different  slides  showed  an 
average  of  9%  of  the  larger,  single  oocytes  were  tagged  at  72  hours.  The  average 
number  of  grains  per  labeled  cell  was  196  as  compared  to  a  background  count  of 
7  from  an  equivalent  area. 

Since  even  the  smallest  single  oocyte  failed  to  show  uptake  of  thymidine  after 
a  pulse  of  30  minutes,  it  was  assumed  that  the  small  number  of  older  oocytes  were 
labeled  at  the  packet  stage  or  earlier.  Therefore,  the  minimal  time  for  an  oocyte 
to  reach  full  size  appeared  to  be  from  48  to  72  hours. 

Isolated  single  oocytes,  recovered  at  still  longer  periods  after  injection  gave 
further  evidence  of  thymidine  redistribution.  A  study  of  three  different  H3- 
thymidine-injected  animals  sacrificed  and  sectioned  7  days  after  injection  showed 
again  that  individual  maturing  oocytes  were  labeled.  The  marked  cells  were  few 
and  scattered,  and  the  grains  appeared  with  equal  intensity  over  the  entire  cell. 
In  many  cases  a  heavily  labeled  oocyte  occurred  adjacent  to  a  group  of  entirely 
unlabeled  mature  oocytes  (Fig.  13).  Sections  through  the  center  of  the  oocyte 
indicated  little  if  any  of  the  label  was  concentrated  in  the  nucleus.  This  also  sug- 
gested that  the  radioactive  material  had  entered  the  cell  during  an  earlier  packet 
phase. 

In  animals  sacrificed  after  21  days  individual  oocytes  of  increasing  sizes  were 
also  tagged.  Many  of  the  larger  oocytes  were  still  unlabeled  but  a  survey  of  50 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  527 

fields  indicated  that  an  average  of  17%  of  the  largest  oocytes  had  the  labeled  pre- 
cursor. The  grain  count  averaged  228  per  cell  compared  to  9  for  an  equal  area 
of  background.  It  was  apparent  that  some  of  these  oocytes  approaching  maturity 
were  more  lightly  labeled  than  others.  The  grains  were  distributed  evenly  over 
the  nucleus  and  the  cytoplasm  in  the  more  heavily  labeled  cells.  Some  of  the 
lighter  marked  cells  showed  grains  only  over  the  cytoplasm. 

In  vitro  labeling.  The  previous  results  demonstrated  the  inability  of  solitary 
developing  coelomic  oocytes  or  mature  oocytes  to  incorporate  H3-thymidine  directly 
into  the  nucleus  but  indicated  a  possible  cytoplasmic  uptake  of  thymidine.  Con- 
ceivably, the  events  associated  with  the  breakdown  of  the  germinal  vesicle  and 
entrance  of  the  cell  into  metaphase  I  could  have  altered  the  pattern  of  thymidine 
incorporation;  this  seemed  to  justify  further  tests  on  the  oocytes  after  G.V. 
breakdown. 

Direct  shedding  of  the  oocyte  stages  into  H3-thymidine  provided  a  more  concise 
measure  of  the  time  of  uptake  into  the  early  packet  stages.  The  absence  of  the 
ovary  assured  that  the  earliest  stages  were  post-ovulatory  and  relatively  short 
pulse  times  could  be  tested  after  adequate  washing,  assuring  that  appearance  of 
a  label  over  a  particular  stage  would  represent  actual  uptake  at  that  developmental 
stage. 

Oocytes  were  shed  directly  into  sea  water  containing  the  precursors,  and  by 
regulating  the  time  of  initial  exposure  to  the  labeled  compounds,  it  was  possible 
to  compare  the  mature  primary  oocytes  before,  during  and  after  G.V.  breakdown. 
Initial  exposure  to  the  labeled  compounds  was  made  from  5  minutes  after  shedding 
(prior  to  G.V.  breakdown)  and  up  to  one  hour  post-shedding  (after  G.V.  break- 
down). The  cells  were  usually  pulsed  for  two  hours  but  in  some  cases  were  left 
in  the  isotopes  for  10  to  12  hours. 

Oocytes  placed  into  5  or  10  /^C.  of  thymidine/ml.  of  sea  water  between  5  and 
10  minutes  after  shedding  always  possessed  intact  germinal  vesicles  at  the  time 
they  were  introduced.  After  one  or  two  hours  they  were  washed,  fixed  and  pre- 
pared as  whole  mounts  or  embedded  and  sectioned. 

In  some  cases  G.V.  breakdown  did  not  occur  in  any  of  the  full  grown  oocytes 
but  this  was  attributed  to  a  lack  of  maturity.  In  other  experiments  up  to  80% 
of  the  cells  showed  G.V.  breakdown.  In  neither  instance  did  any  of  the  single 
oocytes  show  any  nucleoside  incorporation.  Prolonged  exposure  of  the  oocytes 
for  10  to  12  hours  had  no  effect. 

Oocytes  were  also  shed  into  plain  sea  water,  where  mature  oocytes  underwent 
G.V.  breakdown.  After  20  minutes  they  were  placed  into  sea  water  containing 
H3-thymidine.  The  results  were  the  same.  None  of  the  post-G.V.  oocytes  showed 
any  thymidine  incorporation. 

At  the  same  time  the  smaller  oocyte  packets  harvested  after  two  hours  exposure 
showed  moderate  to  heavy  labeling  over  the  cytoplasm,  confirming  what  had  been 
found  with  in  vivo  labeling.  None  of  the  packets  exhibited  any  significant  nuclear 
label,  which  verified  the  earlier  observations  that  nuclear  labeling  was  confined  to 
cells  within  the  ovary. 

In  order  to  substantiate  the  nature  of  the  thymidine  uptake  in  the  cytoplasm, 
sections  of  oocytes  which  had  been  pulsed  for  three  hours  were  exposed  to  DNase 


528 


KENYON  S.  TWEEDELL 


I  8 


PLATE  III 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  529 

prior  to  dipping  in  the  emulsion.     The  DNase  treatment  completely  eliminated  any 
labeling  in  the  cytoplasm  of  the  young  oocyte  packets. 

H3-uridine 

/;;  vivo  labeling.  Animals  were  pulsed  with  either  5  or  10  /xC.  of  H3-uridine, 
then  the  eggs  were  shed  and  sectioned  or  sectioned  in  situ. 

The  ovary.  Unlike  the  uptake  of  thymidine  in  the  ovary,  H3-uridine  incor- 
poration was  evenly  distributed  throughout  the  ovary.  As  soon  as  4  hours  after 
injection,  the  ovary  was  uniformly  labeled  over  the  entire  area.  Uridine  appeared 
to  be  taken  up  equally  in  the  nuclei  and  cytoplasm  of  the  ovarian  cells.  The  in- 
tensity of  the  label  increased  with  the  exposure  time  so  that  after  24  hours  the 
ovary  exhibited  a  dense,  diffuse  distribution  of  silver  grains.  Ovaries  from  animals 
sacrificed  after  48  to  72  hours  exposure  presented  a  heavy  nuclear  label  as  well  as 
a  moderate  concentration  of  grains  over  the  cytoplasm  of  ovarian  oocytes  (Fig.  14). 
After  prolonged  exposures  of  6  days  the  ovary  was  still  heavily  labeled,  particularly- 
over  the  cytoplasm  of  the  oocytes  nearest  the  periphery,  while  the  nuclear  concen- 
tration had  diminished. 

Coclomic  oocytes.  All  stages  of  coelomic  oocytes  exhibited  moderate  to  heavy 
nuclear  uptake  of  radioactive  uridine  after  short  pulses.  While  the  initial  labeling 
was  nuclear,  the  distribution  of  the  precursor  changed  with  increased  exposure. 

Oocyte  packets.  Oocytes  exposed  to  H3-uridine  in  the  early  phase  of  packet 
development  possessed  a  moderate  to  heavy  concentration  of  grains  over  their  nuclei 
after  two  hours  (Fig.  15).  Medium-sized  packets  were  also  labeled  primarily 
over  the  nucleus  although  a  light  scattering  of  grains  also  appeared  over  the  periph- 
eral or  outer  cytoplasm.  The  basal  ends  which  stain  heavily  for  basophilia  of  the 
oocytes  directed  toward  the  packet  center  demonstrated  little  uptake  of  uridine. 

After  a  4-hour  exposure,  the  nuclear  label  became  greatly  intensified  and  the 
cytoplasm  accrued  additional  grain  accumulation.  Small  compact  oocyte  packets 
with  prominent  nuclear  labeling  also  showed  diffuse  scattered  grains,  particularly 
around  the  peripheral  cytoplasmic  areas.  This  same  condition  was  found  in  oocyte 
packets  after  8  and  24  hours  exposure  to  the  uridine.  Significantly,  in  oocyte 
packets  of  larger  sizes,  the  granular  label  was  found  over  the  nuclei  with  scarcely 
a  trace  in  the  cytoplasm. 

It  was  very  apparent  that  the  cytoplasmic  label  first  detected  in  the  smaller 
oocyte  groups  was  intensely  distributed  over  all  cell  packets,  except  the  large  ter- 

PLATE  III 

FIGURE  14.  Portion  of  the  ovary  with  diffuse  labeling  of  oocytes  72  hours  after  H3-uridine 
injection.  An  oocyte  packet  is  superimposed.  750  X. 

FIGURE  15.  Oocyte  packet  with  strong  nuclear  and  light  cytoplasmic  label  adjacent  to  an 
individual  oocyte.  Shed  two  hours  after  H3-uridine  injection.  960  X. 

FIGURE  16.  Single  oocyte  with  labeled  nucleus  and  heavy  concentration  of  grains  over 
nucleolar  bud.  Two-hour  H3-uridine  pulse.  960  X. 

FIGURE  17.  A  comparison  of  the  heavy  cytoplasmic  label  in  oocyte  packets  with  the  pre- 
dominately nuclear  and  nucleolar  uptake  in  single  oocytes.  Sacrified  after  72  hours  H3-uridine 
injection.  936  X. 

FIGURE  18.  Coelomic  oocytes  after  two-hour  pulse  of  H3-uridine  and  subsequent  treat- 
ment with  RNase.  Most  cells  are  unlabeled  but  occasional  nuclei  resist  treatment  and  show 
retention  of  label.  936  X. 


530  KENYON  S.  TWEEDELL 

minal  oocyte  packets,  after  48  hours  exposure  to  H3-uridine.  Presumably,  the 
persistent  appearance  of  the  cytoplasmic  label  in  these  oocytes  either  represented 
utilization  of  H3-uridine  by  RNA  or  its  precursors  already  in  the  cytoplasm,  or 
resulted  from  the  movement  of  RNA  from  the  nucleus  into  the  cytoplasm. 

Oocytes  in  early  packet  stages  continued  to  display  heavy  cytoplasmic  and 
nuclear  label  when  the  animals  were  sacrificed  72  hours  after  injection.  Both 
small  and  intermediate  oocyte  packets  were  affected  alike,  as  seen  in  Figure  17. 
Again,  in  the  transition  to  larger  cell  packets  the  grains  were  still  limited  to  the 
nuclei  with  little  cytoplasmic  concentration. 

Single  oocytes.  After  a  short  pulse  (15  to  30  minutes)  single  cells  also  gave 
evidence  of  H3-uridine  uptake.  Two  hours  after  being  injected  and  shed,  the 
smaller  single  oocytes  appeared  with  a  heavy  nuclear  label  (Fig.  15)  while  larger 
oocytes  showed  a  less  dense  nuclear  labeling  as  seen  in  Figure  16.  In  some  cells 
sacrificed  after  4  hours  the  nuclear  and  nucleolar  label  was  so  intense  that  the  cells 
appeared  almost  black  in  these  areas.  There  was  often  a  concentration  of  grains 
around  the  nucleoli,  particularly  above  the  epinuclear  buds  (Fig.  16).  The  cyto- 
plasmic label  of  scattered  single  oocytes  varied  from  light  to  moderate. 

Single  oocytes  which  had  been  shed  and  recovered  8  hours  after  injection  of 
the  adult  animal  gave  the  same  pattern.  Medium  to  large  single  oocytes  were 
heavily  tagged  over  the  nucleus,  nucleolus  and  somewhat  over  the  cytoplasm.  In 
the  less  intensely  tagged  cells,  the  nuclei  were  principally  labeled. 

Following  24  hours  exposure  to  H3-uridine,  preparations  of  sectioned  oocytes 
in  situ  indicated  that  all  sizes  of  single  oocytes  had  a  strong  nuclear  incorporation 
with  scattered  grains  over  the  cytoplasm.  A  survey  of  50  fields  showed  that  81% 
of  these  cells  possessed  a  nuclear  label. 

Sections  from  H3-uridine  injected  animals  sacrificed  after  48  hours  exposure 
were  quite  similar  and  consistent  with  those  recovered  after  one  day. 

The  same  pattern  extended  to  single  growing  oocytes  after  72  hours  exposure 
to  uridine.  The  nuclear  labeling  of  the  smallest  single  oocytes  was  identical  to 
that  seen  in  the  larger  oocyte  packets.  The  concentration  appeared  more  intense, 
particularly  around  the  nucleolus,  as  the  oocytes  grew  larger  and  reached  a  peak 
in  the  mature  oocytes,  while  the  cytoplasmic  uptake  was  relatively  light.  This  dis- 
tribution is  just  opposite  to  that  seen  in  the  young  developing  oocyte  packets  which 
are  compared  in  Figure  17. 

Enzyme  treatment.  Pretreatment  of  the  egg  sections  with  RNase  eliminated 
both  the  cytoplasmic  and  nucleolar  label  after  a  two-hour  pulse  with  H3-uridine. 
While  the  nuclear  label  was  reduced,  scattered  single  oocytes  and  packets  still 
possessed  a  moderately  heavy  label  (Fig.  18).  Thus  it  appeared  that  a  large  part 
of  the  nuclear  label  was  not  susceptible  to  RNase  digestion. 

Essentially  similar  results  were  obtained  in  eggs  harvested  after  8  hours. 
Most  cells,  in  both  packets  and  single  oocyte  stages,  continued  to  show  a  moderate 
•concentration  of  nuclear  grains.  Scattered  single  oocytes  showed  a  heavier  label 
•over  the  nucleus  and  nucleolus  that  extended  into  the  cytoplasm  as  well.  Sections 
•of  the  same  eggs  were  pretreated  with  RNase  before  dipping  into  emulsion.  Again 
this  resulted  in  a  complete  loss  of  cytoplasmic  and  nucleolar  labeling  as  well  as 
the  nuclear  label  from  many  of  the  single  oocytes.  In  some  of  the  large  single 
•oocytes,  however,  a  heavy  nuclear  label  still  persisted  after  RNase  treatment. 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  531 

Consequently,  similar  sections  were  pretreated  with  DNase  but  this  action  did  not 
remove  any  of  the  nuclear,  nucleolar  or  cytoplasmic  labeling. 

Oocytes  were  harvested  and  animals  were  sacrificed  at  an  extended  interval 
of  6  days  after  injection  of  tritiated  uridine.  By  the  sixth  day  the  intensity  of 
the  label  had  increased  considerably.  As  indicated  previously  the  ovarian  oocytes 
were  evenly  and  densely  covered  by  fine  grains.  In  the  coelom,  heavy  labeling  was 
again  seen'over  the  cytoplasm  and  nuclei  of  the  smaller  single  oocytes.  In  larger 
oocytes  the  label  appeared  more  concentrated  in  the  karyoplasm  of  the  nucleus  and 
the  nuclear  membrane,  and  closely  adherent  to  the  nucleolar  perimeter.  This  con- 
centration became  more  accentuated  in  the  oocytes  approaching  maturation  size. 
In  addition,  the  cytoplasm  of  the  mature  oocytes  was  evenly  and  densely  covered 
by  fine  grains  as  shown  in  Figure  19.  The  outstanding  difference  noted  for  single 
oocytes  in  all  stages  of  growth  was  an  increase  in  grains  over  the  cytoplasm.  The 
amount  of  uptake  became  so  intense  that  it  equalled  the  original  nuclear  concen- 
tration. 

DISCUSSION  AND  CONCLUSIONS 

The  gametes  of  Pcctinaria  generally  were  retained  in  the  coelomic  cavity  over 
the  long  period  of  growth  and  maturation;  only  during  the  latter  part  of  August 
were  gametes  found  within  the  lumen  of  the  nephromixia.  This  suggested  that 
either  the  ciliated  funnels  of  the  nephromixia  rejected  the  gametes  until  the  spawn- 
ing period  or  that  some  mechanism  triggered  their  entrance  into  the  nephromixia. 
Howie  (1961a)  observed  that  changes  in  the  male  coelomic  gametes  of  Arenicola 
marina  allowed  them  to  be  taken  up  by  the  nephromixia  and  subsequently  shed. 
Further  experiments  with  females  (Howie,  1961b)  indicated  that  the  gonoducts 
would  accept  only  mature  oocytes  and  that  non-spawning  individuals  could  be 
induced  to  shed  with  injections  from  spawning  females. 

The  proposal  is  made,  based  upon  the  evidence  presented  here  and  the  observa- 
tions in  Arenicola  by  Howie,  that  the  mature  gametes  in  Pectinaria  are  selected  or 
allowed  to  enter  the  nephromixia  just  prior  to  spawning.  Full  maturation  of  these 
oocytes,  accomplished  while  the  oocytes  are  still  within  the  nephromixia,  is  accom- 
panied by  changes  in  the  nucleus  leading  to  G.V.  breakdown  and  arrestment  in 
metaphase  I  (Fig.  5).  It  is  important  to  note  that  upon  artificial  stimulation  or 
release  of  the  oocytes  from  the  coelomic  cavity,  the  same  stage  in  meiosis  is  reached. 
More  than  likely,  the  low  fertility  of  these  artificially  shed  eggs  (Austin,  1963 ; 
Tweedell,  1962)  is  due  to  premature  stimulation  {i.e.,  G.V.  breakdown)  of  these 
eggs  prior  to  their  complete  maturation. 

During  the  growth  or  vegetative  period  of  oocyte  development  in  Pcctinaria 
there  is  a  lack  of  thymidine  incorporation  into  the  germinal  vesicle,  a  condition 
that  extends  at  least  to  the  metaphase  of  the  first  maturation  division.  This  re- 
sponse is  the  same  as  that  in  oocytes  of  the  sea  urchin  (Ficq  et  al.,  1963b),  the 
starfish  (Geuskens,  1963),  the  sand  dollar  (Simmel  and  Karnovsky,  1961)  and 
the  cricket  (Favard-Sereno  and  Durand,  1963b). 

Nuclear  uptake  of  tritiated  thymidine  and  DNA  synthesis  is  apparently  re- 
stricted to  the  early  generative  stage  prior  to  the  growth  of  the  germinal  vesicle 
which  occurs  in  the  ovary  of  Pectinaria. 


532  KENYON  S.  TWEEDELL 

The  cytoplasm  of  the  early  oocyte  packets  of  Pectinaria  does  appear  to  utilize 
H3-thymidine,  presumably  in  the  synthesis  of  cytoplasmic  DNA.  Furthermore, 
the  labeled  material  is  probably  retained  in  the  cytoplasm  of  the  developing  oocyte 
to  maturity.  This  may  account  for  the  extra  DNA  content  that  has  been  reported 
for  many  eggs  (Grant,  1965).  In  Ilyanassa,  Collier  and  McCann-Collier  (1962) 
estimated  the  amount  of  DNA  of  the  ovarian  eggs  to  be  32  times  that  predicted  for 
chromosomal  DNA. 

Generally,  attempts  to  demonstrate  cytoplasmic  DNA  in  frog  oocytes  by  auto- 
radiography  have  been  unsuccessful  although  Ficq  (1961a)  did  find  that  the  cyto- 
plasm of  non-dividing  follicle  cells  became  labeled  but  not  the  oocyte  cytoplasm. 
In  the  cricket  oocyte,  however,  the  uptake  of  thymidine  by  follicular  cell  cytoplasm 
was  followed  by  the  transfer  of  radioactive  DNA  into  the  ooplasm  (Favard- 
Sereno  and  Durand,  1963b). 

The  heavy  concentration  of  radioactive  label  over  the  cytoplasm  of  mature 
oocytes  after  a  prolonged  exposure  to  H3-thymidine  might  be  attributed  to  the 
accumulation  of  breakdown  products  of  the  original  nucleoside.  However,  the 
differential  uptake  of  the  radioactive  thymidine  by  the  cells  renders  this  unlikely. 
Alternatively,  the  buildup  of  cytoplasmic  label  could  result  from  a  migration  of 
previously  labeled  nuclear  DNA  after  the  packets  had  reached  maturity.  Just  as 
possible,  the  collection  of  cytoplasmic  granules  could  result  from  cytoplasmic  uptake 
as  seen  in  the  early  oocyte  packet  phase  with  the  subsequent  fragmentation  and 
development  into  single  mature  coelomic  oocytes.  This  seems  likely  since  the  cyto- 
plasmic label  in  the  single  oocytes  appeared  only  after  progressively  longer  expo- 
sures to  the  H3-thymidine,  finally  occurring  over  17%  of  the  largest  oocytes  after 
21  days. 

The  rapid  uptake  of  H3-uridine  into  the  nuclear  sap  and  particularly  the 
nucleolus  of  the  growing  oocytes  of  Pectinaria  and  the  ultimate  transfer  of  the 
label  to  the  cytoplasm  of  older  oocytes  strongly  indicated  that  much  of  the  uridine 
was  used  in  the  production  of  nuclear  RNA.  It  also  supports  the  contention  that 
most  of  the  cellular  RNA  used  for  cytoplasmic  growth  originates  in  the  nucleus 
(Prescott,  1960). 

Zalokar  (1959,  1960)  found  that  after  one-  to  4-minute  exposures  of  Nenro- 
spora  hyphae  to  H3-uridine,  followed  by  centrifugation,  only  the  RNA  in  the 
stratified  nuclei  became  labeled.  He  indicated  that  at  least  99%  of  the  cellular 
RNA  originated  in  the  nucleus.  After  increased  exposures,  almost  all  of  the  label 
was  found  in  the  cytoplasm. 

Ficq  (1961a)  also  found,  in  autoradiographic  studies,  that  all  RNA  synthesis 
in  amphibian  oocytes  is  nuclear. 

During  the  diplotene  phase  of  the  amphibian  oocyte,  RNA  is  actively  synthesized 
along  the  chromosome.  In  an  autoradiographic  study  of  the  newt  oocyte,  Gall  and 
Callan  (1962)  found  a  progressive  incorporation  of  H3-uridine  which  moved  sequen- 
tially along  the  loops  of  the  lampbrush  chromosomes.  Microelectrophoretic  meas- 
urements of  the  base  composition  for  chromosomal  RNA  of  Tritirnts  oocytes  indi- 
cated low  guanine  and  cytosine,  similar  to  DNA,  and  suggested  m-RNA  (Edstrom 
and  Gall,  1963). 

Quite  similar  results  to  the  present  study  were  found  in  an  investigation  of 
iree  oocytes  from  the  sea  urchin  Lytechinus  pictus  by  Piatigorsky,  Ozaki  and  Tyler 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  533 

(1966).  There  was  an  appreciable  amount  of  C14  incorporation  in  RNA  of  the 
oocytes  after  15-  to  60-niinute  exposures,  measured  both  quantitatively  and  by 
autoradiography.  Labeling  was  found  in  the  germinal  vesicle  and  quite  heavily 
in  the  nucleolus  where  uptake  was  almost  entirely  blocked  by  actinomycin  D.  In 
mature  eggs  very  little,  if  any,  incorporation  of  C14-uridine  could  be  measured  and 
none  \vas  detected  by  labeling. 

It  is  likely  that  much  of  the  labeled  nuclear  RNA  in  Pcctinaria  oocytes  is  m- 
RNA.  Edstrom  et  al.  (1961)  have  shown  that  the  base  composition  of  nuclear 
RNA  in  the  starfish  oocyte  resembles  DNA  and  is  probably  m-RNA.  Likewise, 
Ficq  (1961a,  1961b,  1964;  Ficq  et  al.,  1963)  has  long  proposed  that  the  uptake 
of  H3-uridine  and  H3-cytidine  in  the  nucleoplasm  of  various  oocytes  is  due  to  a 
high  molecular  weight,  metabolically  active  RNA,  in  close  contact  with  DNA, 
that  could  be  messenger  RNA. 

More  direct  evidence  of  messenger,  i.e.,  chromosomal,  RNA  was  obtained  by 
Sirlin,  Jacob  and  Kato  (1962).  When  a  specific  block,  such  as  thioacetamide,  is 
applied  to  the  nuclei  of  chironomid  larvae,  the  nucleolar  RNA  is  inhibited  from 
uptake  of  H3-uridine  and  free  m-RNA  appears  instead.  This  labeled  m-RNA  was 
observed  directly  in  the  nucleus  and  followed  to  the  cytoplasm. 

Further  indication  that  H3-uridine  was  incorporated  into  nuclear  and  nucleolar 
RNA  in  the  present  study  resulted  when  the  nucleolar  label  in  all  oocytes  and  part 
of  the  nuclear  label  in  most  oocytes  was  removed  when  they  were  submitted  to 
RNase  prior  to  autoradiography.  Similar  autoradiographs  of  H3-uridine  uptake 
into  the  nuclear  sap  and  nucleoli  of  oocytes  have  been  shown  in  the  cricket  ( Favard- 
Sereno  and  Durand,  1963a),  the  sea  urchin  (Ficq  et  al.,  1963),  the  starfish 
Geuskens,  1963)  and  the  toad  (Ficq,  1964). 

The  failure  of  RNase  to  remove  the  entire  nuclear  label  from  some  of  the 
larger,  single  oocytes  of  Pectinaria  may  be  significant  since  H3-uridine  may  act  as 
a  precursor  for  DNA.  During  vitellogenesis  in  the  cricket  oocyte,  uridine  is 
incorporated  into  DNA  in  the  same  manner  as  the  uptake  of  thymidine  (Favard- 
Sereno  and  Durand,  1963b).  Bieliavsky  and  Tencer  (1960)  noted  that  uridine 
is  selectively  taken  into  DNA  of  amphibian  embryos  at  gastrulation.  Collier 
(1963a.  1965)  also  found  Cli-uridine  acts  as  a  major  precursor  in  the  synthesis 
of  DNA  in  the  Ilyanassa  embryo. 

The  same  retention  of  nuclear  label  was  found  in  H3-uridine-labeled  oocytes  of 
Pectinaria  after  pre-treatment  with  DNase.  If  some  of  the  H3-uridine  is  con- 
verted to  DNA,  the  marked  compound  must  be  tied  up,  possibly  as  a  RNA-DNA 
hybrid.  This  awaits  further  investigation. 

The  tremendous  increase  in  size,  and  particularly  the  budding  activity  of  the 
nucleoli,  during  oocyte  development  and  growth  in  Pectinaria  also  suggest  an 
active  production  of  RNA.  Previous  results  show  that  both  the  epinucleolar  and 
intranucleolar  buds  fluoresce  bright  red  with  acridine  orange  (Tweedell,  1962), 
another  indication  of  RNA.  Nucleolar  extrusion  and  emission  of  nucleolar  prod- 
ucts into  the  cytoplasm  is  well  documented  for  other  oocytes  (Raven,  1961). 
Consequently,  the  heavy  labeling  of  the  nucleolar  buds  in  the  primary  oocyte  of 
Pectinaria  supported  this  viewpoint. 

The  evidence  for  nucleolar  incorporation  of  RNA  precursors  into  developing 
oocytes  has  been  well  documented  (Ficq,  1961,  1962,  1964;  Ficq  et  al.,  1963; 


534  KENYON  S.  TWEEDELL 

Favard-Sereno  and  Durand,  1963a;  Ozban  et  al.,  1964).  Furthermore,  Brown 
and  Gurdon  (1964)  found  that  there  is  a  total  absence  of  ribosomal  RNA  synthesis 
in  embryos  of  an  anucleolate  mutant  of  Xenopiis  lacvis,  even  though  other  RNA  was 
being  synthesized. 

Yet  the  relationship  between  the  exact  site  of  nucleolar  RNA  synthesis,  the 
chromosome  and  the  organizer  DNA  has  not  been  too  clear. 

Zalokar  (1962)  believes  the  nucleoli  are  only  temporary  storage  places  for 
newly  formed  RNA.  Following  incorporation  of  H3-uridine  into  RNA  of  oocyte 
nuclei  of  Blatella  germanica,  he  found  that  the  RNA  was  detected  in  the  chromo- 
somal region  peripheral  to  the  nucleolus.  The  nucleoli  remained  unlabeled  but 
chromosomes  were  labeled  whtn  moderate  amounts  of  actinomycin  were  applied. 
In  experiments  on  the  salivary  gland  nuclei  of  chironomid  larvae  (Sinittia  sp.) 
Sirlin  et  al.  (1962)  pulse-labeled  the  cells  with  H3-uridine  and  pretreated  with 
TRB,  a  general  RNA  inhibitor.  The  nucleoli  were  heavily  labeled  while  the 
nucleolar  organizer  remained  unlabeled.  They  presented  evidence  for  an  extrinsic 
nucleolar  RNA  (chromosomal  RNA)  and  an  intrinsic  nucleolar  RNA,  and  pro- 
posed that  the  chromosomal  RNA,  not  organizer  DNA,  primed  the  intrinsic 
nucleolar  RNA. 

On  the  other  hand,  when  isolated  amphibian  oocytes  were  exposed  in  vitro  to 
H3-uridine,  H3-cytidine,  incorporation  into  the  lampbrush  chromosomes  took  place 
(Izawa  et  al.,  1963).  However,  prior  application  of  actinomycin  D  blocked  uptake 
in  both  the  chromosomal  loops  and  the  nucleolus.  From  this  they  concluded  that 
RNA  synthesis  in  both  areas  is  DNA-dependent. 

The  type  of  RNA  synthesized  and  the  sequential  movement  of  RNA  within 
the  nucleus  are  still  very  controversial. 

One  model  for  the  origin  of  RNA  synthesis  (Perry,  1965)  proposes  that  the 
nucleolus  accounts  for  roughly  two-thirds  of  the  cytoplasmic  RNA  (r-RNA  species) 
while  extranucleolar  synthesis  accounts  for  m-RNA  and  t-RNA. 

It  is  germane  to  examine  which  kinds  of  RNA  are  involved  in  the  labeling  of 
the  oocyte  nucleus  and  particularly  the  nucleolus.  All  three  types  of  RNA  (t-RNA, 
r-RNA  and  m-RNA)  have  been  recognized  in  the  nucleoli  of  oocytes  and  other 
cells  (Sirlin  et  al.,  1963).  Direct  evidence  for  two  RNA  types  in  the  nucleolus  was 
obtained  from  fractionation  of  the  nucleoli  of  pea  seedlings  (Birnstiel  and  Chip- 
chase,  1963),  where  it  was  found  that  56%  of  the  extractable  RNA  is  t-RNA  (4s), 
the  remainder  consisting  of  ribosomal  species. 

At  least  two  types  of  nuclear  RNA,  which  varied  in  their  solubility  and  specific 
activity,  have  been  found  in  the  starfish  oocyte  nucleus  and  nucleolus  (Vincent, 
1954,  1957).  Vincent  and  Baltus  (1960)  later  found  that  the  oocyte  nucleolus 
of  the  starfish  binds  C14-leucine  to  an  "activation"  RNA  that  is  attached  to  a  4.5s 
protein,  presumably  used  in  the  synthesis  of  cellular  proteins. 

Possible  supporting  evidence  of  nucleolar  production  of  t-RNA  was  presented 
by  Fleissner  and  Borek  (1962)  in  the  transmethylation  of  (C14  methyl)  methionine 
to  ribonucleic  acid.  Sirlin,  Jacob  and  Tandler  (1963)  were  able  to  show  by 
autoradiography  preferential  uptake  of  (methyl  C1*)  methionine  into  the  nucleolus 
of  the  salivary  glands  of  a  chironomid.  When  puromycin  was  applied  to  block 
the  incorporation  of  methionine  into  the  protein  of  the  chromosomes  and  cytoplasm, 
the  latter  showed  little  or  no  uptake.  The  remaining  nucleolar  label  was  entirely 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION  535 

removed  by  RNase.  This  was  interpreted  as  the  transfer  of  the  methyl  group  to 
t-RNA  of  the  nucleolus. 

The  same  formation  of  nucleolar  RNA  from  (methyl  C14)  methionine  has  been 
obtained  in  developing  oocytes  of  the  toad  (Ozban,  Tandler  and  Sirlin,  1964). 
The  increase  of  incorporation  into  RNase-sensitive  nucleolar  material  was  propor- 
tional to  oocyte  growth,  e.i/.,  cytoplasmic  growth.  In  older  larger  oocytes,  almost 
all  nucleolar  label  derived  in  the  presence  of  puromycin  was  RNase-sensitive. 

In  contrast,  Ficq  (1961)  found  t-RNA  was  formed  in  the  cytoplasm  of  am- 
phibian oocytes  from  labeled  cytidine.  More  recently,  Ficq  (1966)  combined 
methyl-C14-methionine  with  enzymatic  digestion  by  ribonuclease  on  the  oocytes 
of  newts  and  salamanders.  The  methylating  activity  was  reported  in  the  cyto- 
plasm as  well  as  the  nucleolus ;  she  did  not  find  the  localization  of  t-RNA  to  be 
preferentially  nucleolar. 

Birnstiel,  Fleissner  and  Borek  (1963)  have  reported  that  RNA  methylases 
are  concentrated  in  the  nucleoli  of  pea  nuclei  where  the  enzymes  are  believed  to 
alter  t-RNA  by  the  incorporation  of  methyl  groups  into  the  component  bases. 

Davidson,  Allfrey  and  Mirsky  (1964)  concluded  that  during  the  lampbrush 
stage  of  oogenesis  the  oocytes  of  Xenopiis  laevis  produce  huge  quantities  of  ribo- 
somal  RNA.  After  injection  of  H3-uridine,  they  found  that  over  90%  of  the  RNA 
recovered  was  of  the  ribosomal  type.  In  the  amphibian  most  of  this  RNA  is  pro- 
duced in  early  oocytes  rather  than  in  ovulated  eggs.  After  isolation  of  RNA  from 
immature  (ovarian)  oocytes  of  Xenopus  with  P32  the  immature  oocytes  produced 
an  abundance  of  two  ribosomal  species  whereas  no  labeled  ribosomal  RNA  was 
found  in  ovulated  eggs  (Brown  and  Littna,  1964). 

The  nucleolar  and  cytoplasmic  RNA  in  the  starfish  oocyte  have  very  similar 
base  ratios  (Edstrom  ct  al.,  1961),  suggesting  that  ribosomal  RNA  is  a  result  of 
this  nucleolar  activity.  In  oocytes  of  Triturus,  Edstrom  and  Gall  (1963)  similarly 
reported  there  is  an  overall  resemblance  between  nucleolar  and  cytoplasmic  RNA. 
Both  are  rich  in  guanine-cytosine  compared  to  relatively  low  G-C  content  of  chro- 
mosomal RNA.  Furthermore,  Ficq  (1964)  finds  that  H3-cytidine,  H3-5-methyl- 
cytocine  and  FP-uridine  are  preferentially  incorporated  into  RNA  of  the  oocyte 
nucleolus  of  the  sea  urchin  and  toad ;  this  uptake  is  completely  blocked  by  actino- 
mycin  D. 

Radiography  and  extraction  of  RNA  from  the  sea  urchin  oocytes  (Gross  ct  al., 
1965)  confirm  the  contention  that  H3-uridine  is  incorporated  into  several  types  of 
RNA  during  oocyte  development.  One  week  after  injection  of  H3-uridine  into 
Arbacia  punctulata  the  whole  ovarian  region  became  radioactive.  The  smaller 
ovarian  oocytes  were  heavily  labeled  in  both  nuclei  and  cytoplasm  while  the  larger 
oocytes  had  radioactivity  primarily  in  the  cytoplasm.  The  number  of  ootids  with 
a  cytoplasmic  label  varied  from  f  of  the  cells  intermediate  in  the  acinus  of  the 
ovary  to  only  a  few  mature  ootids  from  the  central  lumen.  A  few  were  heavily 
labeled  in  the  cytoplasm  and  nucleus.  Some  of  the  central  ootids  contained  labeled 
nuclei  although  "rarely  with  significant  radioactivity."  Apparently  these  were 
labeled  during  late  maturation  stages. 

Subsequent  extraction  of  RNA  from  these  central  ootids  yielded  three  major 
RNA  components.  Two  were  28S  and  18S  ribosomal  species ;  the  third  was  4S 


536  KENYON  S.  TWEEDELL 

material,  some  of  which  was  t-RNA.  Another  fraction  was  presumed  to  be  m- 
RNA. 

The  relatively  light  cytoplasmic  uptake  of  H3-uridine  into  oocytes  of  Pectinaria 
after  short  pulses  or  moderate  exposures  indicated  that  little  uridine  was  taken 
directly  into  cytoplasmic  RNA.  There  was,  however,  a  slow  increase  in  cyto- 
plasmic lahel  after  two  days  that  increased  dramatically  after  a  six-day  exposure, 
which  might  represent  a  latent  uptake  of  uridine  into  ribosomal  RNA.  However, 
if  nucleolar  RNA  and  ribosomal  RNA  are  related,  the  cytoplasmic  buildup  could 
be  due  to  a  migration  of  nuclear  RNA  into  the  cytoplasm.  Indeed,  the  nuclear 
and  nucleolar  label  does  diminish  as  the  cytoplasmic  activity  increases,  a  situation 
similar  to  that  in  the  cricket  occytes  (Favard-Sereno  and  Durancl,  1963a)  and  in 
the  starfish  oocytes  (Edstrom  et  al.,  1961). 

We  can  conclude,  then,  that  H3-uridine  is  incorporated  into  several  types  of 
RNA,  both  nuclear  and  nucleolar  in  origin,  during  oocyte  development.  Presum- 
ably the  nuclear  label  is  due  to  m-RNA  and  ribosomal  RNA  does  appear  to  be 
derived  directly  from  nucleolar  RNA. 

SUMMARY 

1.  The  development  and  growth  of  the  primary  oocytes  in  the  coelomic  fluid 
are  described.     Oocytes  progress  from  a  packet   stage   to   a   single  oocyte  stage 
accompanied  by  vegetative  growth  of  the  germinal  vesicle.     Evidence  presented 
indicates  that  the  germinal  vesicle  breaks  down  and  the  oocyte  reaches  the  first 
maturation  division  prior  to  shedding  under  natural  conditions. 

2.  Nuclear  uptake  of  H3-thymidine  is  confined  to  the  ovarian  cells  following 
the  last  oogonial  division  in  the  premeiotic  phase.     There   is  evidence  that  free 
coelomic  oocyte  packets  incorporate  H3-thymidine  directly  into  the  cytoplasm.     The 
cytoplasmic  label  is  removed  by  treatment  with  DNase. 

3.  Short  pulses  of  H3-uridine  are  taken  up  diffusely  by  the  ovarian  oocytes, 
while  small  packets  and   single  oocytes  incorporate   H3-uridine  primarily   in   the 
nucleus  and  in  the  nucleolus.     Extended  exposure  from  4  to  48  hours  indicates 
that  some  of  the  nuclear  uridine  moves  into  the  cytoplasm  in  the  packet  oocytes. 
Individual  oocytes  show  strong  nuclear  labeling  up  to  72  hours  after  injection. 
Particularly  long  exposures  of  6  days  indicate  that  the  uridine  accumulates  in  the 
cytoplasm  in  the  largest  oocytes. 

4.  Treatment  with  RNase  removes  most  of  the  nuclear  and  cytoplasmic  H3- 
uridine  label.     Some  of  the  nuclear  label  is  resistant  and  is  not  removed  by  RNase 
or  DNase. 

LITERATURE  CITED 

AUSTIN,    C.    R.,    1963.     Fertilization   in   Pectinaria    (=  Cistcnides')    gouldii.     Biol.    Bull.,    124: 

115-124. 
BIEBER,  S.,  J.  A.  SPENCE  AND  G.  H.  HITCHINGS,  1959.     Nucleic  acids  and  their  derivatives  and 

the  development  of  Rana  pipicns.     I.  Oogenesis.     Exp.  Cell  Res.,  16:  202-214. 
BIELIAVSKY,   N.,  AND  R.  TENCER,   1960.     fitude  de  1'incorporation  de  1'uridine  tritiee  dans   les 

oeufs  d'amphibiens.     Exp.  Cell  Res.,  21:  279-285. 
BIRNSTIEL,  M.  L.,  AND  M.  I.  H.   CniPCHASE,   1963.     The  chemical  and  physical   fractionation 

of  nucleoli.     Fed.  Proc.,  22 :  473. 
BIRNSTIEL,  M.  L.,  E.  FLEISSNER  AND  E.  BOREK,  1963.     Nucleolus :  A  center  of  RNA  methyla- 

tion.     Science,  142:  1577-1580. 


OOCYTE  AND  NUCLEOSIDE  INCORPORATION 

BROWN,  D.  D.,  AND  J.  B.  GURDON,  1964.     Absence  of  ribosomal  RNA  synthesis  in  the  anucleo- 

late  mutant  of  Xcnopus  laci'is.     Proc.  Nat.  Acad.  Sci.,  51:   139-146. 
BROWN,  D.   D.,   AND  E.   LITTNA,   1964.     Variation   in   the   synthesis   of   stable   RNA's   during 

oogenesis  and  development  of  Xciwpus  lacris.     J.  Mol.  Biol.,  8:  688-695. 
COLLIER,  J.  R.,  1963.     The  incorporation  of  uridine  into  the  deoxyribonucleic  acid  of  the  Ilya- 

nassa  embryo.     E.vp.  Cell  Res.,  32 :  442-447. 
COLLIER,  J.  R.,  1965.     Morphogenetic  significance  of  biochemical  patterns  in  mosaic  embryos. 

In:  The  Biochemistry  of  Animal  Development,  Vol.  1,  R.  Weber,  Ed.,  Academic  Press, 

New  York,  pp.  203-244. 
COLLIER,  J.  R.,  AND  M.  MCCANN-COLLIER,  1962.     The  deoxyribonucleic  acid  content  of  the  egg 

and  sperm  of  Ilyanassa  obsoleta.     E.vp.  Cell  Res.,  27:  553-559. 
DAVIDSON,  E.  H.,  V.  G.  ALLFREY  AND  A.  E.  MIRSKY,  1964.     On  the  RNA  synthesized  during 

the  lampbrush  phase  of  amphibian  oogenesis.     Proc.  Nat.  Acad.  Sci.,  52:   501-508. 
EDSTROM,  J.  E.,  AND  J.  G.  GALL,  1963.     The  base  composition  of  ribonucleic  acid  in  lampbrush 

chromosomes,  nucleoli,  nuclear  sap  and  cytoplasm  of   Tritunis  oocytes.     /.  Cell  Biol., 

19:  279-284. 

EDSTROM,  J.  E.,  W.  GRAMPP  AND  N.  SCHOR,  1961.     The  intracellular  distribution  and  hetero- 
geneity of  ribonucleic  acid  in  starfish  oocytes.     /.  Biophy.  Biochcm.  Cytol.,  11:  549-557. 
FAVARD-SERENO,   C.,   AND  M.   DURAND,    1963a.     L'utilization   de   nucleosides    dans   1'ovaire   du 

grillon  et  ses  variations  au  cours  de  1'ovogenese.     I.  Incorporation  dans  1'ARN.     Dev. 

Biol.,  6:  184-205. 
FAVARD-SERENO,   C.,   AND   M.   DURAND,    1963b.     L'utilization   de   nucleosides   dans    1'ovaire   du 

grillon   et   ses    variations   au   cours    de   1'ovogenese.      II.    Incorporation    dans    1'ADN. 

Dev.  Biol.,  6:206-218. 
FICQ,  A.,   1961a.     Metabolisme   de  1'oogenese  chez   les   amphibiens.     In:    Symposium   on   germ 

cells  and  earliest  stages  of  development.     S.  Ranzi,  Ed.,  Fondazione  A.   Baselli,  Lst. 

Lombardo,  Milano. 
FICQ,  A.,   1961b.     Localization  of  different  types  of  ribonucleic  acids    (RNA'S)    in  amphibian 

oocytes.     E.rp.  Cell  Res..  23:  427-429. 
FICQ,   A.,   1962.     Localization   d'un  acide   ribonucleique    (RNA)    de   transfer   dans   les   oocytes 

d'asteries.     E.rp.  Cell  Res.,  28  :  543-548. 
FICQ,  A.,  1964.     Effets  de  1'actinomycine  D  et  de  la  puromycine  sur  le  metabolisme  de  1'oocyte 

en  croissance.     E.vp.  Cell  Res.,  34:  581-594. 
FICQ,   A.,   1966.     Sites   de  methylation  des  acides   ribonucleiques   dans   les   oocytes   d'Urodeles. 

Arch.  Biol.,  77 :47-58. 
FICQ,  A.,   F.  AIELLO  AND  E.   SCARANO,   1963.     Metabolism  des  acides   nucleiques   dans   1'oeuf 

d'oursin  en  developpement.     E.vp.  Cell  Res.,  29:  128-136. 
FLEISSNER,  E.,  AND  E.   BOREK,   1962.     A  new  enzyme   of  RNA   synthesis :    RNA   methylase. 

Proc.  Nat.  Acad.  Sci.,  48:  1199-1203. 
GALL,  J.  G.,  AND  H.  G.  CALLAN,  1962.     H3-uridine  incorporation  in  lampbrush  chromosomes. 

Proc.  Nat.  Acad.  Sci.,  48:  562-570. 
GEUSKENS,  M.,   1963.     Accumulation  nucleolaire   d'acide  ribonucleique    (RNA)    dans   1'oocyte 

d'asterie.    E.vp.  Cell  Res.,  30:  322-330. 
GINTSBERG,  G.  I.,  1963.     Autoradiographic  study  of  the  uptake  of  H3  labeled  thymidine  in  the 

process  of  oogenesis   (Russian).     Zhnrnal  Obschchei  Biol..  24:  71-73. 
GOODRICH,  E.  S.,  1945.     The  study  of  nephridia  and  genital  ducts  since  1895.     Quart.  J.  Micr. 

Sci.  London,  86  (2-4)  :  113-392. 
GRANT,  P.,  1965.     Informational  molecules  and  embryonic  development.     In:  The  Biochemistry 

of  Animal  Development,  Vol.  1,  R.  Weber,  Ed.,  Academic  Press,  New  York,  pp.  483- 

593. 
GROSS,  P.  R.,  L.  I.  MALKIN  AND  M.  HUBBARD,  1965.     Synthesis  of  RNA  during  oogenesis  in 

the  sea  urchin.     /.  Mol.  Biol.,  13:  463-481. 
HANKS,  J.  E.,  1960.     A  method  for  preparing  the  marine  polychaete  Pectinaria  gouldii  (Verrill) 

for  histological  study.     Trans.  Amer.  Micr.  Soc.,  79/4:  470-471. 

HARTMAN,  O.,  1941.     Polychaetous  annelids.     Part  4.     Pectinariidac  with  a  review  of  all  spe- 
cies from  the  western  hemisphere.     Allan  Hancock  Pacific  Expedition,  7:  325-345. 
HOFF-J0RGENSEN,  E.,  AND  E.  ZEUTHEN,  1952.     Evidence  of  cytoplasmic  deoxyribosides  in  the 

frog's  egg.     Nature,  169:  245-246. 


538  KENYON  S.  TWEEDELL 

HOLLAND,  N.  D.,  AND  A.  C.  GIESE,  1965.  An  autoradiographic  investigation  of  the  gonads  of 
the  purple  sea  urchin  (Strongylocentrotus  purpnratus).  Biol.  Bull.,  128:  241-258. 

HOWIE,  D.  I.  D.,  1961a.  The  spawning  of  Arenicola  marina  (L.).  II.  Spawning  under  experi- 
mental conditions.  /.  Mar.  Biol.  Assoc.,  41 :  127-144. 

HOWIE,  D.  I.  D.,  1961b.  The  spawning  of  Arenicola  marina  (L.).  III.  Maturation  and  shed- 
ding of  the  ova.  /.  Mar.  Biol.  Assoc.,  41 :  771-783. 

IZAWA,  M.,  V.  G.  ALLFREY  AND  A.  E.  MIRSKY,  1963.  The  relationship  between  RNA  synthesis 
and  loop  structure  in  lampbrush  chromosomes.  Proc.  Nat.  Acad.  Sci.,  49:  544-551. 

OZBAN,  N.,  C.  J.  TANDLER  ANoJ.  L.  SIRLIN,  1964.  Methylation  of  nucleolar  RNA  during  develop- 
ment of  the  amphibian  oocyte.  /.  Embryol.  E.vp.  Morph.,  12:  373-380. 

PERRY,  R.  P.,  1965.  The  nucleolus  and  the  synthesis  of  ribosomes.  Nat.  Cancer  Instit.  Mono- 
graph, 18:  325-340. 

PIATIGORSKY,  J.,  H.  OzAKi  AND  A.  TYLER,  1966.  RNA-  and  protein-synthesizing  capacity  of 
isolated  oocytes  of  the  sea  urchin  Lytechinns  pictiis.  Dcv.  Biol.  (in  press). 

PRESCOTT,  D.  M.,  1960.  Nuclear  function  and  nuclear-cytoplasmic  interactions.  Ann.  Rev. 
Physiol.,22:  17-44. 

RAVEN,  C.  P.,  1961.  Oogenesis :  The  Storage  of  Developmental  Information.  Pergamon  Press, 
N.  Y. 

SIMMEL,  E.  B.,  AND  D.  A.  IvARNovsKY,  1961.  Observations  on  the  uptake  of  tritiated  thymidine 
in  the  pronuclei  of  fertilized  sand  dollar  embryos.  /.  Biophy.  Biochcm.  Cytol.,  10: 
59-65. 

SIRLIN,  J.  L.,  J.  JACOB  AND  K.  I.  KATO,  1962.  The  relation  of  messenger  to  nucleolar  RNA. 
Exp.  Cell  Res.,  27:  355-359. 

SIRLIN,  J.  L.,  J.  JACOB  AND  C.  J.  TANDLER,  1963.  Transfer  of  the  methyl  group  of  methionine 
to  nucleolar  ribonucleic  acid.  Biochem.  J.,  89 :  447-452. 

TWEEDELL,  KENYON  S.,  1962.  Cytological  studies  during  germinal  vesicle  breakdown  of  Pec- 
tinaria  gouldii  with  vital  dyes,  centrifugation  and  fluorescence  microscopy.  Biol.  Bull., 
123:  424-449. 

TWEEDELL,  KENYON  S.,  1964.  Incorporation  of  H3-thymidine  and  H3-uridine  by  oocytes  of 
Pectinaria  gouldii.  Biol.  Bull,  127:  394. 

VINCENT,  W.  S.,  1954.  P32  incorporation  into  starfish  oocyte  nucleoli.  Biol.  Bull.,  107:  326- 
327. 

VINCENT,  W.  S.,  1957.     Heterogeneity  of  nuclear  ribonucleic  acid.     Science,  126:  306-307. 

VINCENT,  W.  S.,  AND  E.  BALTUS,  1960.     A  function  for  the  nucleolus.     Biol.  Bull,  119:  299-300. 

WILLIAMS,  J.,  1965.  Chemical  constitution  and  metabolic  activities  of  animal  eggs.  In:  The 
Biochemistry  of  Animal  Development,  Vol.  1,  Academic  Press,  New  York,  pp.  13-71. 

ZALOKAR,  M.,  1959.     Nuclear  origin  of  ribonucleic  acid.     Nature,  183:  1330. 

ZALOKAR,  M.,  1960.  Sites  of  protein  and  ribonucleic  acid  synthesis  in  the  cell.  Exp.  Cell  Res., 
19:  559-576. 

ZALOKAR,  M.,  1962.  The  role  of  the  nucleolus  in  the  production  of  ribonucleic  acid.  Genetics, 
47 :  996. 


INDEX 


Abstracts  of  papers  presented  at  the  Marine 
Biological  Laboratory,  378. 

Acartia,   predicting  development  rate  of  eggs 
of,  457. 

Acidity  tolerance  of  oyster  and  clam  embryos 
and  larvae,  427. 

Actinomycin  D,  role  of  in  development  of  sea 
urchin  embryos,  388   (abstract). 

Activity,  lysosomal,  in  clam  mantle  epithelium, 
76. 

Adaptations  to  temperature  by  Euglena,  83. 

Adipose  tissue  thermogenesis  during  arousal  of 
bats  from  hibernation,  94. 

Aggregation   size   in    Dictyostelium,    effect   of 
light  on,  446. 

Aging  and  motility  of  Arbacia  sperm,  251. 
Musca,  wing  beat  frequency  and  duration  in, 
479. 

Alanine,  uptake  of  by  Ophiactis,  172. 

Alcohol,    effects    of    on    sperm    motility    and 
respiration,  166. 

Alkalinity,  effect  of  on  development  of  oyster 
and  clam  embryos  and  larvae,  427. 

Alga,  development  of  in  closed  aquarium  sys- 
tem, 487. 

Amino  acid  incorporation  into  embryonic  cell- 
free  Arbacia  preparations,  391   (abstract). 

Amino  acids,   influence   of  on  uptake  and  in- 
corporation of  valine,  glutamic  acid,  and 
arginine  in  sea  urchin  eggs,  204. 
uptake  of  by  Ophiactis,  172. 

Annual  Report  of  the  Marine  Biological  Lab- 
oratory, 1. 

Anomalies  in  x-irradiated  mouse  embryos,  145. 

Amphibian  gastrulae,  role  of  NaCl  in  sequen- 
tial induction  in,  415. 

Annelid,    oocyte    development   and    nucleoside 
incorporation  in,  516. 

APLEY,  M.  L.     See  W.  R.  HUNTER,  392  (ab- 
stract). 

Aquarium  system,  closed,  use  of  in  vitro  cul- 
ture of  sea  weeds,  487. 

Aquatic  invertebrates,  uptake  of  organic  ma- 
terial by,  172. 

Arbacia  sperm,  aging  and  motility  of,  251. 
effects  of  glycerol  on  motility  and  respira- 
tion of,  166. 


Arenicola,  mechanism  of  burrowing  in,  369. 

Arginine,   influence  of  amino  acids  on  uptake 
and  incorporation  of,  in  sea  urchin  eggs, 
204. 
uptake  of  by  Ophiactis,  172. 

Artificial  culture  of  sea  weeds,  487. 

Argyrome  of  new  species  of  Euplotes,  437. 

ARNOLD,  J.  M.  Squid  lens  development  in 
compounds  that  affect  microtubules,  383 
(abstract). 

Arousal  of  bats  from  hibernation,  brown  adi- 
pose tissue  thermogenesis  during,  94. 

Artemia,  genetics  of,  230. 

reproductive    capacity    of    after     successive 
P-32  doses,  261. 

Assay  of  starfish  shedding  substance,  104. 

Asterias,  mechanism  of  spawning  reaction  in, 
104. 

Asterina,  mechanism  of  spawning  reaction  in, 
104. 

AUCLAIR,  W.,  and  B.  W.  SIEGEL.  Cilia  re- 
generation in  the  sea  urchin  embryo,  379 
(abstract). 

AUSTIN,  C.  R.  See  D.  G.  WHITTINGHAM,  412 
(abstract). 

Autoradiography  of  Pectinaria  oocytes,  516. 

Axial  polarity  in  regenerating  planarians,  de- 
termination of,  323. 

Axon  resting  potential  of  lobster,  effects  of 
some  inhibitors  on,  382  (abstract). 

Axons  of  squid,  effects  of  divalent  cations  on, 
411  (abstract). 

B 

BAL,  A.,  P.  L.  KRUPA  AND  G.  H.  COUSINEAU. 
Ribonucleic  acid  in  membranes  of  devel- 
oping cells,  384  (abstract). 

BAL,  A.  K.     See  G.  H.  COUSINEAU,  388  (ab- 
stract). 
P.  L.  KRUPA,  395  (abstract). 

BALL,  E.  G.     See  J.  S.  HAYWARD,  94. 

BANCROFT,  F.  C.,  R.  C.  TERWILLIGER  AND 
K.  E.  VAN  HOLDE.  Investigations  of  the 
subunit  structure  of  Limulus  hemocyanin, 
384  (abstract). 

BARCLAY,  N.  E.    See  H.  SATO,  405  (abstract) . 

BARNETT,  G.  R.  See  A.  J.  D.  DE  LORENZO, 
380  (abstract). 


539 


540 


INDEX 


EARTH,  L.   G.     The  role  of  sodium  chloride 
in  sequential  induction  of  the  presumptive 
epidermis  of  Rana  gastrulae,  415. 
Bats,     brown     tissue     thermogenesis     during 

arousal  of,  from  hibernation,  94. 
BEERS,  C.  D.     Distribution  of  Urceolaria  on 
the   spines   of  the   sea   urchin    Strongylo- 
centrotus,  219. 

Behavior  during  feeding  of  Pisaster,  127. 
and  settling  mechanism  of  Hydractinia  pla- 

nulae,  410  (abstract). 

Behavioral   inhibition  in  Tubular ia,   394    (ab- 
stract), 
sequences  in  feeding  response  of  Hydra,  4 

(abstract). 
BELL,  A.  L.     The  fine  structure  of  the  eye  of 

the  scallop,  Pecten,  385   (abstract). 
BENNETT,  M.  V.  L.     See  G.  D.  PAPPAS,  381 

(abstract). 

BEVELANDER,  G.,  AND  H.  NAKAHARA.     Corre- 
lation of  lysosomal  activity  and  ingestion 
by  the  mantle  epithelium,  76. 
BHATNAGAR,  P.  L.     See  M.  ROCKSTEIN,  479, 
Bioluminescence,    endogenous    diurnal    rhythm 

of,  in  dinoflagellates,  115. 
Bipolar  head   formation   in   regenerating  pla- 

narians,  323. 
Birefringence  of  squid  giant  axon,  effects  of 

temperature  on.  390   (abstract). 
Bladder,  urinary,  of  crab,  functions  of,  272. 
Blood  cells,  mechanical  forces  as  cause  of  cel- 
lular damage  in  freezing  and  thawing  of, 
197. 

changes  in  x-irradiated  mouse  embryos,  145. 
ionic  concentrations  of  crab,  272. 
Botryllus,    inland    culture    of,    and    use    of    in 

genetic  experiments,  398   (abstract). 
Bovine  prolactin,  effects  of  on  salt  fluxes  in 

fresh-water-adapted    Fundulus,    362. 
BOWEN,  S.  T.,  J.  HANSON,  P.  DOWLING  AND 
M.-C.    POON.     The   genetics   of  Artemia. 
VI,  230. 
BRANDT,    P.   W.     See   P.    B.   DUNHAM,   389 

(abstract) . 

A.  SELVERSTON,  407    (abstract). 
BRANHAM,  J.  M.     Motility  and  aging  of  Ar- 

bacia  sperm,  251. 

Breeding  experiments  with  Artemia,  230. 
Brine  shrimp,  effect  of  P-32  on  reproduction 

of,  261. 

mutations  in,  230. 

Brittle  star,  uptake  of  amino  acids  by,  172. 
Bromide  fluxes  in  hypophysectomized  Fundu- 
lus, 362. 

Brown  shrimp,  effect  of  temperature  on  post- 
larval  growth  of,  186. 

BRUNER-LORAND,    J.      See    L.    LORAND,    397 
(abstract). 


BRYAN,  J.     See  H.  SATO,  405   (abstract). 
Burrowing  of  Arenicola,  mechanism  of,  369. 


Caffeine,    effects    of    on    response    of    dogfish 

melanophores  to  MSH,  470. 
CALABRESE,  A.,  AND  H.  C.  DAVIS.     The  pH 
tolerance  of  embryos  and  larvae  of  Mer- 
cenaria  and  Crassostrea,  427. 

Calanus  eggs,  predicting  rate  of  development 
of,  457. 

CAPEN,  R.  L.     See  W.  J.  GROSS,  272. 

Capture-recapture  study  of  Polinices  popula- 
tion, 292. 

Carbon  dioxide,  effect  of  on  motility  of  Ar- 
bacia  sperm,  251. 

Cardioactive  compounds  from  Mercenaria 
heart,  393  (abstract). 

CAROI.A.X,  M.  H.,  H.  SATO  AND  S.  INOUE. 
Further  observations  on  the  thermody- 
namics of  the  living  mitotic  spindle,  385 
(abstract). 

CASSIDY,  J.   D.     Ultrastructural  relationships 
lu'Uveen   the   developing   oocyte   and   aux- 
iliary  cells    in   adult    Artemia,    385    (ab- 
stract). 
See  SR.  M.  R.  SCHMEER,  405   (abstract). 

Cataract  formation  in  x-irradiated  mouse  em- 
bryos, 145. 

GATHER,  J.  N.  Induction  of  the  shell  gland 
by  transplanted  polar  lobes  in  Ilyanassa, 
386  (abstract). 

Cell  mass  of  Euglena  grown  at  different  tem- 
peratures, 83. 

Cellular  damage  in  freezing  and  thawing,  197. 

Ceratium,  endogenous  diurnal  rhythm  of  bio- 
luminescence  in,  115. 

CHANDLER,  A.     See  R.  RUGH,  145. 

Chemotaxis  in  slime  molds,  effects  of  light  on, 
446. 

Chick  embryos,  diurnal  patterns  of  metabolic 
activity  in,  308. 

Chloramphenicol-treatment  of  regenerating 
planarians,  323. 

Chlorophyll-bodies  in  new  species  of  Euplotes, 
437. 

Chromatophore  response  in  Loligo,  410  (ab- 
stract). 

Cilia,  regeneration  of  in  sea  urchin  embryos, 
379  (abstract). 

Ciliate,   distribution   of   on   sea   urchin   spines, 

219. 
hymenostome,  destruction  of  Tubularia  gono- 

phores  by,  391    (abstract), 
new  species  of,  437. 

Cistenides  (Pectinaria)  oocytes,  development 
and  nucleoside  incorporation  of,  516. 


INDEX 


541 


CLAFF,  C.  L.,  A.  A.  CRESCENZI  AND  A.  P. 
RICHMOND.  Respiration  studies  with  the 
shark,  386  (abstract). 

Clam  embryos  and  larvae,  pH  tolerance  of, 
427. 

CLARK,  J.  I.  See  R.  E.  STEPHENS,  409  (ab- 
stract). 

Cleavage  inhibition  in  Arbacia  caused  by  estra- 
diol,  cytological  study  of,  412  (abstract). 

CLEMENT,  A.  C.  Cleavage  and  differentiation 
of  the  vegetal  half  of  the  Ilyanassa  egg 
after  removal  of  most  of  the  yolk  by  cen- 
trifugal force,  387  (abstract). 

Clotting  of  blood,  role  of  transglutaminase  in, 
397  (abstract). 

Coelomic  fluid,  occurrence  of  starfish  spawn- 
ing substance  in,  104. 

pressure,  role  of  in  burrowing  of  Arenicola, 
369. 

Collagen,    development    of    in    Fundulus,    399 

(abstract). 

from    cuticle    of    marine    worms,    390    (ab- 
stract). 

Collagens  of  echinoderms,  396    (abstract). 

COLLINS,  S.  E.     See  T.  M.  THABES,  411   (ab- 
stract). 
C.  R.  WYTTENBACH,  412   (abstract). 

Color  changes  in  Palaemonetes,  contribution 
of  abdominal  nerve  cord  to,  388  (ab- 
stract). 

COLTON,  J.  B.,  JR.  See  T.  J.  M.  SCHOPF,  406 
(abstract). 

Congo  eel  blood  cells,  mechanical  forces  as 
cause  of  cellular  damage  in,  after  freezing 
and  thawing,  197. 

Contamination  with  P-32,  effects  of  on  repro- 
duction of  Artemia,  261. 

Control  of  pigment  in  dogfish,  470. 

COOK,  J.  R.  Adaptations  to  temperature  in 
two  closely  related  strains  of  Euglena,  83. 

COOPER,  W.  R.    See  R.  RUGH,  145. 

COPELAND,  D.  E.  Electron  microscopy  of  the 
gas-secreting  gland  of  Physalia,  387  (ab- 
stract). 

Copepod  eggs,  predicting  development  rate  of, 
457. 

Corneal  changes  in  x-irradiated  mouse  em- 
bryos, 145. 

epithelial  cells  of  dogfish,  protein  synthesis 
in,  413   (abstract). 

Correlation  of  lysosomal  activity  and  ingestion 
by  clam  mantle  epithelium,  76. 

Cortical  protein  from  sea  urchin  eggs,  382 
(abstract). 

COUCH,  E.  F.,  M.  FINGERMAN  AND  E.  W. 
STOOL.  The  contribution  of  the  abdomi- 
nal nerve  cord  to  the  chromatic  physiol- 


ogy   of    the    prawn,     Palaemonetes,    388 
(abstract). 

COUCH,  E.  F.  See  M.  FINGERMAN,  390  (ab- 
stract). 

COUSINEAU,  G.  H.,  P.  L.  KRUPA  AND  A.  K. 
BAL.  The  challenge  of  Actinomycin  D  on 
early  development  in  sea  urchin  embryos, 
388  (abstract). 

COUSINEAU,  G.  H.     See  A.  K.  BAL,  384  (ab- 
stract). 
P.  L.  KRUPA,  395  (abstract). 

Crab  urinary  bladder,  functions  of,  272. 

Crassostrea  embryos  and  larvae,  pH  tolerance 
of,  427. 

CRESCENZI,  A.  A.  See  C.  L.  CLAFF,  386 
(abstract). 

Crossvein  of  Drosophila  wing,  effects  of  tem- 
perature on,  331,  346. 

Crustacean,   effect  of  temperature   on   growth 

of,   186. 

genetics  of,  230. 

reproduction,  effects  of  P-32  on,  261. 
urinary  bladder,  functions  of,  272. 

Culture  of  sea  weeds  in  recirculating  aquarium 
system,  487. 

CUMMINS,  J.  T.     See  J.  A.  STRAND,  487. 

Cycles  of  reproduction  in   Pisaster,   127. 

Cyclic  metabolic  variations  in  chick  embryos, 
308. 

Cytology  of  new  species  of  Euplotes,  437. 

Cytoplasmic  accumulation  of  uridine  in  Pec- 
tinaria  oocytes,  516. 

D 

DMAC,  DMF  and  DMSO,  effects  of  on  sperm 

motility  and  respiration,  166. 
DNA  synthesis,  role  of  in  determining  polar- 
ity in  regenerating  planarians,   323. 
in    dogfish    peripheral    blood,    in    vitro,    401 

(abstract). 

in  Pectinaria  oocytes,  516. 
Damage  in  freezing  and  thawing,  mechanical 

forces  as  factor  in,  197. 
Darkening  of  dogfish  skin  in  vitro,  in  response 

to  MSH,  470. 
Darkness,    effect    of    on    aggregation    size    in 

slime  mold,  446. 
role   of  in   development   of  Ulva   in  closed 

aquarium  system,  487. 
in    endogenous    diurnal    rhythm    of    bio- 
luminescence  in  dinoflagellates,    115. 
DAVIS,  H.  C.     See  A.  CALABRESE,  427. 
Density  of  Polinices  population,  292. 

of   population   as    factor    in   aggregation    of 

slime  mold,  446. 

Description  of  new  species  of  Euplotes,  437. 
of  Notocotylus,  501. 


542 


INDEX 


DESHA,  D.  L.     See  J.  A.   MILLER,  JR.,  398 

(abstract). 
Determination  of  axial  polarity  in  regenerating 

planarians,  323. 

Developing  chick  embryos,  patterns  of  metabo- 
lism in,  308. 

oysters  and  clams,  pH  tolerance  of,  427. 
Rana,  role  of  NaCl  in  sequential  induction 

in,  415. 

Development  of  Fundulus,  effect  of  low  tem- 
perature on,  379  (abstract), 
of  Ilyanassa  shell   gland  after  transplanta- 
tion of  polar  lobes,  386  (abstract), 
vegetal  halves  after  removal  of  yolk,  387 

(abstract). 

of  oocytes  in  Pectinaria,  516. 
rate  of  copepod  eggs,  prediction  of,  457. 
of  Ulva  in  vitro,  487. 
Dictyostelium,  effect  of  light  on  aggregation 

size  in,  446. 
Differences  in  pH  tolerance  of  oyster  and  clam 

larvae,  427. 
Differentiation    in    explanted    Rana    gastrulae 

tissues  treated  with  NaCl,  415. 
of  Ulva  in  closed  aquarium  system,  487. 
Digenetic  trematode  parasite   of  eider   ducks, 

morphology  of,  501. 

DILLER,  W.  F.,  AND  D.  KOUNARIS.     Descrip- 
tion   of    a    zoochlorella-bearing    form    of 
Euplotes,    437. 
Dilution,    role    of    in    motility    and    aging    of 

Arbacia  sperm,  251. 
Dinoflagellates,  endogenous  diurnal  rhythm  of 

bioluminescence  in,  115. 
Directin,   chemical   and  biological   studies   on, 

381   (abstracts). 

Dispersal  in  population  of  Polinices,  292. 
Distribution   of  oyster  and  clam  larvae,   role 

of  pH  in,  427. 

of  spawning  substance  in  starfish,  104. 
of  Urceolaria  on  sea  urchin  spines,  219. 
Diurnal    patterns    of    metabolic    variations    in 

chick  embryos,  308. 
rhythm  of  bioluminescence  in  dinoflagellates, 

115. 

tidal  migration  rhythm  of  diatom,  400   (ab- 
stract). 

Division    rate    of    Euglena    at    different    tem- 
peratures, 83. 
Dogfish    skin    melanophores,    response    of    to 

MSH,  470. 
Dopamine,  effects  of  on  Mercenaria  heart,  408 

(abstract). 

DOWLING,  P.     See  S.  T.  BOWEN,  230. 
Drosophila,  effects  of  temperature  on,  331,  346. 
Ducks,  trematode  parasite  of,  501. 
Dugesia,  regenerating,  role  of  DNA  in  deter- 
mination of  axial  polarity  in,  323. 


DUHAMEL,  I.     See  R.  RUGH,  145. 

DUNHAM,  J.  E.,  G.  W.  HARRINGTON  AND  G. 
G.  HOLZ,  JR.  Phytoplankton  sources  of 
the  eicosapentaenoic  and  docosahexaenoic 
fatty  acids  characteristic  of  marine  Meta- 
zoa,  389  (abstract). 

DUNHAM,  P.  B.,  J.  P.  REUBEN  AND  P.  W. 
BRANDT.  Ionic  fluxes  in  crayfish  muscle 
fibers  before  and  after  swelling  of  the 
TTS,  389  (abstract). 

Duration  of  wing  beat  in  aging  Musca,  479. 

E 

Echinoderm,     distribution     of    Urceolaria    on 

spines  of,  219. 
feeding  behavior  and  reproductive  cycles  in, 

127. 

mechanism  of  spawning  in,  104. 
uptake  of  amino  acids  by,  177. 
eggs,  uptake  and  incorporation  of  amino 

acids  by,  204. 
sperm,   effects   of   glycerol    on   motility   and 

respiration  of,   166. 
motility  and  aging  of,  251. 
Ecology  of  Pisaster,  127. 

of  Polinices,  292. 

Eel  blood  cells,  mechanical  forces  as  cause  of 
cellular  damage  in  freezing  and  thawing 
of,  197. 

Effect  of  hypophysectomy  on  sodium  metabo- 
lism of  Fundulus  gill  and  kidney,  155. 
of    temperature    on    growth    of    postlarval 

Penaeus,  186. 

Effects  of  glycerol  on  sperm  motility,  166. 
of  hypophysectomy  and  bovine  prolactin  on 

salt  fluxes  in  Fundulus,  362. 
of  temperature  on  Drosophila,  331,  346. 
Eggs,  copepod,  predicting  development  of,  457. 
sea    urchin,    uptake    and    incorporation    of 

amino  acids  by,  204. 
Eider  ducks,  trematode  parasite  of,  501. 
Elasmobranch,  response  of  melanophores  of  to 

MSH  in  vitro,  470. 

Electron  microscopy  of  clam  mantle  epithe- 
lium, 76. 

Embryos,  chick,   diurnal   patterns   of  metabo- 
lism in,  308. 
Crassostrea  and   Mercenaria,   pH  tolerance 

of,  427. 

mouse,  x-irradiation  of,  in  utero,   145. 
Rana,  role  of  NaCl  in  sequential  induction 

in,  415. 
Endocrine  responses  of  dogfish  skin  in  vitro, 

470. 
Endogenous  diurnal  rhythm  of  bioluminescence 

in  dinoflagellates,  115. 

Epidermis  of  Rana  gastrulae,  role  of  NaCl  in 
induction  of,  415. 


INDEX 


543 


Epifaunation  of  sea  urchin  spines,  219. 
Epithelium  of  clam  mantle,  correlation  of  lyso- 

somal  activity  and  ingestion  in,  76. 
Eptesicus,  brown  tissue  thermogenesis  during 

arousal  of  from  hibernation,  94. 
Estimates  of  population  density  for  Polinices, 

292. 

Ethylene  glycol,  effects  of  on  sperm  respira- 
tion and  motility,   166. 
Euglena,     strain     differences     in     temperature 

adaptation  in,  83. 

Euplotes,  description  of  new  species  of,  437. 
Euryhaline  fish,  salt  fluxes  in,  362. 
EVANS,  D.  H.     See  W.  T.  W.  POTTS,  362. 
Extraction   of  organisms   from   sand   samples, 

new  method  for,  413   (abstract). 
Eye,  human,  new  method  for  determination  of 

quantum  efficiency  of,  402  (abstract), 
anomalies    in    x-ir radiated    mouse    embryos, 

145. 
color  mutations  in  Artemia,  230. 

F 

Factors  affecting  response  of  dogfish  melano- 
phores  to  MSH,  470. 

Fatty  acids  in  phytoplankton,  389   ("abstract). 

Fecundity  of  Artemia  treated  with  successive 
doses  of  P-32,  261. 

Feeding  behavior  in  Pisaster,  127. 

Fertilized  sea  urchin  eggs,  uptake  and  incor- 
poration of  amino  acids  by,  204. 

Fertilizing  capacity  of  aging  Arbacia  sperm, 
251. 

Fine  structure  of  living  squid  sperm  head,  404 
(abstract). 

FlNGERMAN,     M.,     E.     F.     COUCH     AND    E.     W. 

STOOL.  Analysis  of  the  melanin-dispers- 
ing and  red  pigment-dispersing  hormones 
of  the  prawn  and  the  fiddler  crab,  390 
(abstract). 

FINGERMAN,  M.  See  E.  F.  COUCH,  388  (ab- 
stract) . 

FISH,    effect    of    hypophysectomy    on    sodium 

metabolism  of  gill  and  kidney  of,   155. 
fresh-water-adapted,    salt   fluxes   in,    362. 

FISHMAN,  L.,  AND  M.  LEVY.  Collagen  from 
the  cuticles  of  marine  worms,  390  (ab- 
stract). 

FISHMAN,  L.     See  M.  LEVY,  396   (abstract). 

Flagellate,  temperature  adaptation  in,  83. 

FLEMING,  W.  R.     See  J.  G.  STANLEY,  155. 

FLICKINGER,  R.  A.     See  D.  M.  KOHL,  323. 

Flight  ability  in  aging  Musca,  479. 

Fly,  aging,  duration  and  frequency  of  wing 
beat  in,  479. 

Food  conversion  of  Penaeus  at  different  tem- 
peratures, 186. 


FORBES,    W.    F.      See    S.    LERMAN,    396    (ab- 
stract). 
FORMAN,    D.    S.      Reversible   changes    in    the 

birefringence  of  the  squid  giant  axon  with 

temperature,  390    (abstract). 
Freezing  and   thawing,   mechanical    forces   as 

cause  of  cellular  damage  in,  197. 
Frequency  of  wing  beat  in  aging  Musca,  479. 
Fresh-water-adapted  Fundulus,  salt  fluxes  in, 

362. 
Frog    gastrulae,    role    of    NaCl    in    sequential 

induction  in,  415. 

Fruitfly,   effects  of  temperature  on,   331,   346. 
Functions  of  crab  urinary  bladder,   272. 
Fundulus,    effect    of   hypophysectomy    on    salt 

metabolism  of  gill  and  kidney  of,   155. 
fresh-water-adapted,   salt  fluxes   in,   362. 


Gametes  of  starfish,  mechanism  of  spawning 
of,  104. 

Gastropod,  density  and  dispersal  in,  292. 

Gastrulae  of  Rana,  role  of  NaCl  in  sequential 
induction  in,  415. 

GEENS,  M.,  M.  JAMES  AND  G.  G.  HOLZ,  JR. 
Destruction  of  the  male  gonophores  of 
Tubularia  by  a  hymenostome  ciliate  of  the 
genus  Paranophrys,  391  (abstract). 

GELFANT,  S.  See  T.  PEDERSON,  401  (ab- 
stract). 

Genetics  of  Artemia,  230. 

GIBBINS,  J.  R.  See  L.  G.  TILNEY,  378  (ab- 
stract). 

Gill  metabolism  of  sodium  in  Fundulus,  effect 
of  hypophysectomy  on,  155. 

Glenodinium,  endogenous  diurnal  rhythm  of 
bioluminescence  in,  115. 

Glutamic  acid,  influence  of  amino  acids  on 
uptake  and  incorporation  of  in  sea  urchin 
eggs,  204. 

Glycerol,  effects  of  on  sperm  motility,   166. 

Glycine,   uptake   of  by   Ophiactis,    172. 

Gonad  indices  of  Pisaster,  127. 

Gonads  of  starfish,  sensitivity  of  to  nerve 
extract,  104. 

Gonyaulax,  endogenous  diurnal  rhythm  of  bio- 
luminescence  in,  115. 

GRANT,  D.  C.     See  W.  R.  HUNTER,  292. 

GRIFFITH,  G.  W.     See  Z.  P.  ZEIN-£LDIN,  186. 

GROSCH,  D.  S.  The  reproductive  capacity  of 
Artemia  subjected  to  successive  contamina- 
tions with  radiophosphorus,  261. 

GROSS,  W.  J.,  AND  R.  L.  CAPEN.  Some  func- 
tions of  the  urinary  bladder  in  crabs,  272. 

GROSSMAN,  A.,  AND  W.  TROLL.  The  incorpora- 
tion of  C-14  lysine  and  C-14  phenylalanine 
into  embryonic  cell-free  Arbacia  prepara- 
tions, 391  (abstract). 


544 


INDEX 


Growth  of  clam  and  oyster  embryos  and  larvae, 

effect  of  pH  on,  427. 

and  development  of  Pectinaria  oocytes,  516. 
of  Euglena  at  different  temperatures,  83. 
of  sea  weeds  in  vitro,  487. 

H 

HANSON,  J.     See  S.  T.  BOWEN,  230. 

HARRINGTON,  G.  W.  See  J.  E.  DUNHAM,  389 
(abstract). 

HASTINGS,  J.  W.  See  G.  T.  REYNOLDS,  403 
(abstract). 

Hatching  rate  in  Fundulus,  as  affected  by  tem- 
perature, 380  (abstract). 

HAYS,  R.  L.,  AND  A.  I.  LANSING.  Isolation 
of  surface  membranes  of  Strongylocen- 
trotus  eggs,  392  (abstract). 

HAYWARD,  J.  S.,  AND  E.  G.  BALL.  Quantita- 
tive aspects  of  brown  adipose  tissue  ther- 
mogenesis  during  arousal  from  hiberna- 
tion, 94. 

Heart  tissue  of  bat,  metabolism  of  during 
arousal  from  hibernation,  94. 

Heat,  effect  of  on  growth  of  postlarval   Pe- 

naeus,  186. 

genesis  of  brown  adipose  tissue  of  bats  dur- 
ing arousal  from  hibernation,  94. 
sensitivity  of  Drosophila  pupae,  331,  346. 

HEGYELI,  A.    Chemical  studies  of  directin,  381 

(abstract). 
See  R.  JOHNSSON-HEGYELI,  382   (abstract). 

HEIDGER,  P.  M.  See  J.  A.  MILLER,  JR.,  398 
(abstract). 

Hemocyanin,  Limulus,  subunit  structure  of, 
384  (abstract). 

Hepatic  tissue  of  Pisaster,  cyclic  changes  in, 
127. 

Hibernation  of  bats,  brown  adipose  tissue 
thermogenesis  during  arousal  from,  94. 

HILLE,  B.,  AND  R.  MILKMAN.  A  quantitative 
description  of  some  temperature  effects  on 
Drosophila,  346. 

HILLE,  B.     See  R.  MILKMAN,  331. 

Histochemistry  of  clam  mantle  epithelium,  76. 

Histo-incompatibility  between  strains  of  Hy- 
dractinia,  393  (abstract). 

Histology  of  clam  mantle  epithelium,  76. 
of  Pectinaria  oocyte  development,  516. 
of  Pisaster  tissues,  127. 

HOFMAN,  F.  See  N.  B.  RUSHFORTH,  403 
(abstract). 

VAN  HOLDE,  K.  E.  See  F.  C.  BANCROFT,  384 
(abstract). 

HOLZ,   G.   G.,  JR.     See  J.  E.   DUNHAM,  389 

(abstract). 
M.  GEENS,  391   (abstract). 

Housefly,  duration  and  frequency  of  wing  beat 
in,  479. 


HUNTER,  W.  R.,  AND  M.  L.  APLEY.  Quanti- 
tative aspects  of  early  life-history  in  the 
salt-marsh  pulmonate  snail  Melampus  and 
their  evolutionary  significance,  392  (ab- 
stract). 

HUNTER,  W.  R.,  AND  D.  C.  GRANT.  Esti- 
mates of  population  density  and  dispersal 
in  the  naticid  gastropod  Polinices,  with  a 
discussion  of  computational  methods,  292. 

Hydrostatic  pressure,  role  of  in  burrowing  of 
Arenicola,  369. 

Hyperbaric  oxygen,  role  of  in  development  of 
Tubularia,  398  (abstract). 

Hypophysectomy,  effect  of  on  sodium  metabo- 
lism of  Fundulus  gill  and  kidney,  155. 
on   salt  fluxes   in  fresh-water-adapted   Fun- 
dulus, 362. 


Ice  formation  in  eel  red  blood  cells,   197. 

In   vitro  dogfish   skin,   response   of   to   MSH, 
470. 

Incorporation     of    nucleosides     in     Pectinaria 

oocytes,  516. 

of  valine,  glutamic  acid  and  arginine  in  sea 
urchin  eggs,  influence  of  amino  acids  on, 
204. 

Induction    in    Rana    gastrulae,    role    of    NaCl 
in,   415. 

Influence  of  individual  amino  acids  on  uptake 
and  incorporation  of  valine,  glutamic  acid 
and  arginine  by  sea  urchin  eggs,  204. 
of    light    on    aggregation    size    in    Dictyo- 

stelium,  446. 

of  salinity  on  uptake  of  amino  acids  by 
Ophiactis,  172. 

Ingestion  by  clam  mantle  epithelium,  76. 

Inheritance  of  mutations  in  Artemia,  230. 

INDUE,    S.      See    R.    M.    CAROLAN,    385    (ab- 
stract). 

H.  SATO,  405    (abstract). 
R.  E.  STEPHENS,  409  (abstract). 

Integration  of  Drosophila  temperature  effects, 
346. 

Invertebrate    sperm,    effects    of    glycerol    on 
motility   and   respiration   of,    166. 

Invertebrates,  lysine  uptake  by,  172. 

Ionic  concentrations,  intracellular,  determina- 
tion by  ultramicro  flame  photometry,  394 
(abstract), 
effects  in  response  of  dogfish  melanophores 

to  MSH  in  vitro,  470. 

fluxes  in  crayfish  muscle  fibers,  389  (ab- 
stract). 

Ions,  role  of  in  sequential  induction  in  Rana, 
415. 

Irradiation  of  mice  in  utero,  145. 


INDEX 


545 


IVKER,  F.  S.  Histo-incompatibility  and  stolon 
overgrowth  between  interbreeding  strains 
of  Hydractinia,  393  (abstract). 


JACOBOWITZ,  D.,  AND  M.  A.  SPIRTES.  Chro- 
matographic  studies  on  cardioactive  com- 
pounds extracted  from  Mercenaria  hearts, 
393  (abstracts). 

JACOBOWITZ,  D.  See  M.  A.  SPIRTES,  408 
(abstract). 

JAMES,  M.     See  M.  GEENS,  391   (abstract). 

JOHNSON,  L.  G.  Diurnal  patterns  of  meta- 
bolic variations  in  chick  embryos,  308. 

JOHNSSON-HEGYELI,  R.,  AND  A.  HEGYELI.  In 
vitro  and  in  vivo  studies  of  directin,  382 
(abstract). 

JOSEPHSON,    R.    K.,    AND   J.    F.    UHRICH.      The 

stalk  conducting  system  mediating  be- 
havioral inhibition  in  the  hydroid  Tubu- 
laria,  394  (abstract). 

K 

KANATANI,  H.,  AND  M.  OHGURI.  Mechanism 
of  starfish  spawning,  104. 

KANE,  R.  E.  See  R.  E.  STEPHENS,  382  (ab- 
stract). 

KARNOVSKY,  M.  J.  See  J.-P.  REVEL,  380 
(abstract). 

KATONA,  S.     See  M.  G.  KELLY,  115. 

KATZ,  G.  M.  Intracellular  ionic  concentra- 
tions determined  by  ultramicro  flame  pho- 
tometry, 394  (abstract). 

KELLY,  M.  G.,  AND  S.  KATONA.  An  endoge- 
nous diurnal  rhythm  of  bioluminescence 
in  a  natural  population  of  dinoflagellates, 
115. 

KEMPTON,  R.  T.  Morphological  comments  on 
blood  pressure  relationships  in  Squalus 
branchial  arteries,  395  (abstract). 

Kidney  metabolism  of  sodium  in  Fundulus, 
effect  of  hypophysectomy  on,  155. 

Killifish,  fresh-water-adapted,  salt  fluxes  in, 
362. 

Kinetic  analysis  of  Drosophila  temperature 
effects,  331,  346. 

Kinetics  of  new  species  of  Euplotes,  437. 

KOHL,  D.  M.,  AND  R.  A.  FLICKINGER.  The 
role  of  DNA  synthesis  in  the  determina- 
tion of  axial  polarity  of  regenerating  pla- 
narians,  323. 

KONIJN,  T.  M.,  AND  K.  B.  RAPER.  The  in- 
fluence of  light  on  the  size  of  aggregations 
in  Dictyostelium,  446. 

KOUNARIS,  D.     See  W.  F.  DILLER,  437. 

KRUPA,  P.  L.,  A.  K.  BAL  AND  G.  H.  Cousi- 
NEAU.  The  fine  structure  of  the  redia  of 


the     trematode     Cryptocotyle,     395     (ab- 
stract). 

KRUPA,  P.  L.    See  A.  K.  BAL,  384  (abstract). 
G.  H.  COUSINEAU,  388   (abstract). 


LD/50  x-ray  exposure  of  mice  in  utero,   145. 

Laboratory-held  shrimp,  effect  of  temperature 
on  postlarval  growth  of,  186. 

LANSING,  A.  I.  See  R.  L.  HAYS,  392  (ab- 
stract). 

Larvae  of  Mercenaria  and  Crassostrea,  pH 
tolerance  of,  427. 

Length  of  sea  urchin  spine,  relation  of  to  dis- 
tribution of  Urceolaria  thereon,  219. 

Lenticular  gamma  crystallin,  characteristics 
of,  396  (abstract). 

LERMAN,  L.  See  A.  WATANABE,  411  (ab- 
stract). 

LERMAN,  S.,  W.  F.  FORBES  AND  S.  ZIGMAN. 
Further  characteristics  of  lenticular 
gamma  crystallin,  396  (abstract). 

LERMAN,  S.     See  S.  ZIGMAN,  413   (abstract). 

LERNER,  A.  B.     See  G.  SZABO,  410  (abstract). 

Lethality  of  x-rays  to  mouse  embryos,  145. 

LEVY,  M.,  AND  L.  FISHMAN.  Collagens  of 
echinoderms,  396  (abstract). 

LEVY,  M.     See  L.  FISHMAN,  390    (abstract). 

Life-history  of  Notocotylus,  501. 

Light,    influence    of    on    aggregation    size    in 

Dictyostelium,  446. 
role   of  in  development   of  Ulva   in   closed 

aquarium  system,  487. 
in    endogenous    diurnal    rhythm    of    bio- 
luminescence  in  dinoflagellates,  115. 
-emitting    particles    of    Gonyaulax,    identity 
and  photon  yield  of,  403  (abstract). 

Liver  of  bat,  metabolism  of  during  arousal 
from  hibernation,  94. 

LORAND,  L.,  et  al.  Transglutaminase  and 
blood  clotting,  397  (abstract). 

DE  LORENZO,  A.  J.  D.,  AND  G.  R.  BARNETT. 
Fine-structural  basis  for  chemical  and 
electrotonic  transmissions  in  a  parasympa- 
thetic  ganglion,  380  (abstract). 

Lugworm,  mechanism  of  burrowing  in,  369. 

Lysosomal  activity  in  clam  mantle  epithelium, 
76. 

Lytechinus  eggs,  uptake  and  incorporation  of 
amino  acids  by,  204. 

M 

MSH,  response  of  dogfish  skin  melanophores 
to,  in  vitro,  470. 

Macrocallista  mantle  epithelium,  correlation  of 

lysosomal  activity  and  ingestion  in,  76. 
Alagnesium  fluxes  in  crab,  272. 


546 


INDEX 


MALAWISTA,  S.,  AND  H.  SATO.  Vinblastine 
and  griseofulvin  reversibly  disrupt  the 
living  mitotic  spindle,  397  (abstract). 

Malic  dehydrogenase  isozymes,  intracellular 
distribution  of  in  red  and  white  halves  of 
sea  urchin  eggs,  400  (abstract). 

Malignant  cells,  chemistry  of  agent  directing 
growth  of  in  vitro,  381  (abstract). 

Mantle  epithelium,  correlation  of  lysosomal 
activity  and  ingestion  by,  76. 

Marine  sea  weeds,  in  vitro  culture  of,  487. 

Maturation  of  Pectinaria  oocytes,   516. 

of  starfish  oocytes  under  influence  of  spawn- 
ing substance,  104. 

MAUZEY,  K.  P.  Feeding  behavior  and  repro- 
ductive cycles  in  Pisaster,  127. 

MCLAREN,  I.  A.  Predicting  development  rate 
of  copepod  eggs,  457. 

Mechanical  forces  as  cause  of  cellular  damage 
by  freezing  and  thawing,  197. 

Mechanism  of  burrowing  in  Arenicola,  369. 
of  starfish  spawning,  104. 

Medium  for  culture  of  sea  weeds  in  recircu- 
lating  aquarium  system,  487. 

Melanophores  of  dogfish  skin,  response  of  to 
MSH,  470. 

Membranes  of  sea  urchin  eggs,  isolation  of, 
392  (abstract). 

Mercenaria  embryos  and  larvae,  pH  tolerance 
of,  427. 

Metabolic  variations  in  chick  embryos,  308. 

Metabolism  of  bats  during  arousal  from  hiber- 
nation, 94. 

of  Euglena  at  different  temperatures,  83. 
of  sodium  by  Fundulus  gill  and  kidney,  effect 
of  hypophysectomy  on,   155. 

METS,  L.     See  G.  PATTON,  400   (abstract). 

Microtubules,  possible  role  of  in  development 

of  squid  lens,  383    (abstract). 
role  of  in  development  of  sea  urchin  mesen- 
chyme,  378   (abstract). 

MILKMAN,  R.,  AND  B.  HILLE.  Analysis  of 
some  temperature  effects  on  Drosophila 
pupae,  331. 

MILKMAN,  R.,  AND  J.  PEDERSON.  Inland  cul- 
ture of  Botryllus :  Genetic  crosses,  398 
(abstract). 

MILKMAN,  R.     See  B.  HILLL,  346. 

MILLER,  F.  S.  See  J.  A.  MILLER,  JR.,  398 
(abstract). 

MILLER,  J.  A.,  JR.,  et  al.  Hyperbaric  oxygen 
and  succinic  dehydrogenase  in  the  em- 
bryology of  Tubularia,  398  (abstract). 

MITCHELL,  B.  S.,  AND  G.  SZABO.  The  effect 
of  phenylthiourea  on  the  embryology  of 
Fundulus,  398  (abstract). 

Mitosis,  sulfhydryl  balance  in,  409   (abstract). 


Mitotic  delay  in  sea  urchin  eggs,  radiation-in- 
duced, recovery  from,  404   (abstract), 
spindle,  effects  of  D2O  on,  405    (abstract), 
reversible  disruption  of  by  vinblastine  and 

griseofulvin,  397    (abstract), 
thermodynamics   of,   384    (abstract). 
Molecular  mechanisms  of  Drosophila  temper- 
ature effects,  331. 

Mollusc,  density  and  dispersal  in,  292. 
embryos  and  larvae,  pH  tolerance  of,  427. 
mantle  epithelium,   correlation  of  lysosomal 

activity  and  ingestion  in,  76. 
sperm,   effects   of   glycerol    on   motility   and 

respiration   of,    166. 
Morphology  of  Artemia  mutants,  230. 
of  Notocotylus,  501. 
of  Ulva  in  vitro,  487. 
Mosaics  in  Artemia,  230. 
Motility  and  aging  of  Arbacia  sperm,  251. 
of  sperm,  effects  of  glycerol  and  other  or- 
ganic solutes  on,  166. 
Mouse  embryos,  x-irradiation  of,   145. 
MOZLEY,  S.  C.     Distribution  and  responses  to 
salinity    of   larval    chironomids    from    the 
upper  Pocasset  River,  399  (abstract). 
MULLER,  K.  J.     See  H.  SATO,  404  (abstract). 
Musca,    aging,    wing   beat    duration    and    fre- 
quency in,  479. 
Mustelus,    response    of    melanophores    of    to 

MSH  in  vitro,  470. 
Mutations  in  Artemia,  230. 
Mytilus  sperm,  effects  of  glycerol  on  motility 

and  respiration  of,  166. 

Myxamoebae,  effect  of  light  on  aggregation 
size  in,  446. 

N 

NADOL,  J.  B.,  JR.  The  development  of  an 
ordered  array  of  collagen  in  Fundulus, 
399  (abstract). 

NAKAHARA,  H.     See  G.  BEVELANDER,  76. 

Natural  population  of  dinoflagellates,  diurnal 
rhythm  of  bioluminescence  in,  115. 

Nerve  extract,  mechanism  of  action  of,  in  star- 
fish spawning,  104. 

New  species  of  Euplotes,  description  of,  437. 
of  Notocotylus,  501. 

Notocotylidae,  further  studies  on,  409  (ab- 
stract). 

Notocotylus,  life-history  and  morphology  of, 
501." 

NOVALES,  B.  J.     See  R.  R.  NOVALES,  470. 

NOVALES,  R.  R.,  AND  B.  J.  NOVALES.  Factors 
influencing  the  response  of  isolated  dog- 
fish skin  melanophores  to  melanocyte- 
stimulating  hormone,  470. 

Nuclear  and  nucleolar  uptake  of  thymidine  in 
Pectinaria  oocytes,  516. 


INDEX 


547 


Nucleoside  incorporation  in  Pectinaria  oocytes, 

516. 
Nutrition  of  Pisaster,   127. 


OHGURI,  M.     See  H.  KANATANI,  104. 
ONG,  H.  H.     See  L.  LORAND,  397  (abstract). 
Oocyte  development  and  nucleoside  incorpor- 
ation in  Pectinaria,  516. 

Oocytes,   starfish,  maturation  of,   under   influ- 
ence of  spawning  substance,   104. 
Ophiactis,   uptake  of  amino  acids  by,   172. 
Organic  material,  uptake  of  by  marine  inver- 
tebrates, 172. 
solutes,    effects    of    on    sperm    motility    and 

respiration,  166. 
Osmotic  effects  in  response  of  dogfish  melano- 

phores  to  MSH  in  vitro,  470. 
relations  in  Fundulus,  effect  of  hypophysec- 

tomy  on,  155,  362. 
in  Pachygrapsus,  272. 
Ova,  sea  urchin,  uptake  and  incorporation  of 

amino  acids  by,  204. 
Ovarian  cytology  of  Pectinaria,   516. 
Ovaries  of  Pisaster,  seasonal  changes  in,  127. 
Oxygen   consumption   of   bats   during   arousal 

from  hibernation,  94. 
of  chick  embryos,  patterns  of,  308. 
of  Euglena  at  different  temperatures,  83. 
of  invertebrate  sperm  treated  with  glycerol, 

166. 
Oyster  embryos  and  larvae,  pH  tolerance  of, 

427. 
OZAKI,  H.     See  A.  TYLER,  204. 


pH,  role  of  in  development  of  Ulva  in  closed 

aquarium  system,  487. 
change,  effect  of  on  motility  and  aging  of 

Arbacia  sperm,  251. 

tolerance  of  oyster  and  clam  embryos  and 
larvae,  427. 

Pachygrapsus,  functions  of  urinary  bladder  of, 
272. 

Pallial  fluid  of  clam,  role  of  in  shell  formation, 
76. 

PALMER,  J.  D.,  AND  F.  E.  ROUND.  The  di- 
urnal nature  of  the  tidal  migration  rhythm 
of  the  diatom  Hantzschia,  400  (abstract). 

PAPPAS,  G.  D.,  AND  M.  V.  L.  BENNETT.  The 
fine  structure  of  vesicles  associated  with 
excitatory  and  inhibitory  junctions,  381 
(abstract). 

Parasite  of  eider  ducks,  morphology  and  life- 
history  of,  501. 

Particulate  uptake  by  clam  mantle  epithelium, 
76. 


Patterns  of  metabolic  variations  in  chick  em- 
bryos, 308. 

PATTON,  G.,  L.  METS  AND  C.  VILLEE.  Intra- 
cellular  distribution  of  malic  dehydro- 
genase  isozymes  in  developing  red  and 
white  halves  of  sea  urchin  eggs,  400  (ab- 
stract) . 

Pectinaria  oocytes,  development  and  nucleoside 
incorporation  in,  516. 

PEDERSON,  J.  See  R.  MILKMAN,  398  (ab- 
stract). 

PEDERSON,  T.,  AND  S.  GELFANT.  Autoradio- 
graphic  studies  of  DNA  synthesis  in  cul- 
tures of  peripheral  blood  from  the  smooth 
dogfish,  Mustelus,  401  (abstract). 

Penaeus,  effect  of  temperature  on  growth  of, 
186. 

Peridinium,  endogenous  rhythm  of  biolumines- 
cence  in,  115. 

Periodicity  of  feeding  in  Pisaster,   127. 

in    oxygen   consumption    of   chick   embryos, 
308. 

Permeability   relations  of   Pachygrapsus,   272. 

Phenylthiourea,  effect  of  on  development  of 
Fundulus,  398  (abstract). 

Photo-inhibition  of  rhythm  of  luminescence  in 
dinoflagellates,  115. 

Photoperiod,  role  of  in  development  of  Ulva 
in  recirculating  aquarium  system,  487. 

PIATIGORSKY,  J.     See  A.  TYLER,  204. 

Pigment  cells  of  dogfish,  response  of  to  MSH 

in  vitro,  470. 
-dispersing    hormones,    analysis    of    by    gel 

filtration,  390  (abstract), 
nerve    fibers    in    sand    flounder,    evidence 
against,  406    (abstract). 

Pigmentation  differences  in  various  races, 
after  ultraviolet  radiation,  378  (abstract). 

Pinocytosis  in  clam  mantle  epithelium,  76. 

Pisaster,  feeding  behavior  and  reproductive 
cycles  in,  127. 

Pituitary    extract,    response    of    dogfish    skin 

melanophores  to,  in  vitro,  470. 
removal,  effect  of  on  salt  fluxes  in  Fundulus, 

362. 

on  salt  metabolism  of  Fundulus  gill  and 
kidney,  155. 

Planarians,  regenerating,  determination  of 
axial  polarity  in,  323. 

PLATT,  C.     See  H.  SATO,  405  (abstract). 

Polarity  in  regenerating  planarians,  role  of 
DNA  synthesis  in  determination  of,  323. 

Polinices,  density  and  dispersal  in,  292. 

Pollution,  possible  effect  of  on  development  of 
clam  and  oyster  larvae,  427. 

Polychaete,  mechanism  of  burrowing  in,  369. 
oocytes,  development  of,  516. 

POON,  M.-C.    See  S.  T.  BOWEN,  230. 


548 


INDEX 


Population  density  of  Polinices,  292. 

of  Urceolaria  on  sea  urchin  spines,  219. 
differences  in  genetics   of  Artemia,   230. 
of    dinoflagellates,    diurnal    rhythm    of    bio- 
luminescence  in,   115. 

expansion   in   Euglena   at   different   temper- 
atures, 83. 

size  as  factor  in  aggregation  of  slime  mold, 
446. 

Populations  of  Artemia,  effects  of  succesive 
doses  of  P-32  on,  261. 

Postlarval  growth  of  Penaeus,  effect  of  tem- 
perature on,  186. 

POTTS,  W.  T.  W.,  AND  D.  H.  EVANS.  The  ef- 
fects of  hypophysectomy  and  bovine  pro- 
lactin  on  salt  fluxes  in  fresh-water- 
adapted  Fundulus,  362. 

POTTS,  W.  T.  W.  See  J.  T.  STANGEL,  408 
(abstract). 

Predation  relations  of  Pisaster,  127. 

Predicting  development  rate  of  copepod  eggs, 
457. 

Pre-implantation  mouse  embryos,  x-irradiation 
of,  145. 

Pressure,  role  of  in  burrowing  of  Arenicola, 
369. 

Presumptive  epidermis  of  Rana  gastrulae,  role 
of  NaCl  in  sequential  induction  of,  415. 

Proboscis,  role  of  in  burrowing  of  Arenicola, 
369. 

Prolactin,  bovine,  effects  of  on  salt  fluxes  in 
fresh-water-adapted  Fundulus,  362. 

Protein  content  of  Euglena  at  different  tem- 
peratures, 83. 
synthesis  in  regenerating  planarians,  323. 

Protozoan,  description  of  new  species  of,  437. 
distribution  of  on  sea  urchin  spines,  219. 
temperature  adaptation  in,  83. 

Pseudocalanus  eggs,  predicting  development 
rate  of,  457. 

Pyloric  caeca  of  Pisaster,  seasonal  changes  in, 
127. 


Quantitative    aspects    of    Drosophila    temper- 
ature effects,  346. 
of  life-history  of  Melampus,  392  (abstract). 

R 

RNA  content  of  Euglena  grown  at  different 

temperatures,  83. 

in  membranes  of  developing  cells,  384   (ab- 
stract), 
synthesis  in  Pectinaria  oocytes,  516. 

in   regenerating   planarians,   323. 
RAAB,  J.     The  effects  of  NaCl  on  respiration 
of  Squalus  rectal  gland  in  vitro,  401   (ab- 
stract). 


Radial  nerve  extract  of  starfish,  mechanism 
of  action  of,  in  starfish  spawning,  104. 

Radiocarbon,  uptake  of  by  regenerating  plan- 
arians, 323. 

Radiophosphorus,  effects  of  on  reproduction 
of  Artemia,  261. 

Rana  gastrulae,  role  of  NaCl  in  sequential 
induction  in,  415. 

RAPER,  K.  B.    See  T.  M.  KONIJN,  446. 

Rate  of  development  of  copepod  eggs,  predic- 
tion of,  457. 

Recirculation  aquarium  system,  use  of  for 
culture  of  sea  weeds,  487. 

Red  blood  cells  of  eels,  mechanical  damage  to 
after  freezing  and  thawing,  197. 

Regenerating  planarians,  determination  of 
axial  polarity  in,  323. 

REITE,  O.  B.  Mechanical  forces  as  a  cause  of 
cellular  damage  by  freezing  and  thawing, 
197. 

Renal  metabolism  of  sodium  in  Fundulus,  ef- 
fect of  hypophysectomy  on,  155. 

Reproduction  of  Ulva  in  closed  aquarium  sys- 
tem, 487. 

Reproductive   capacity   of   irradiated   Artemia, 

261. 
cycles  in  Pisaster,  127. 

Reserpine  and  guanethidine,  effects  of  on  Cam- 
panularia  hydranths,  411,  412  (abstracts). 

Respiration  of  bats  during  arousal  from  hiber- 
nation, 94. 

of   chick   embryo,    diurnal    pattern    of   vari- 
ations in,  308. 

of  Euglena  at  different  temperatures,  83. 
of  shark,  386  (abstract), 
of  sperm,  effects  of  glycerol  on,   166. 

Response  of  isolated  dogfish  melanocytes  to 
MSH,  470. 

REUBEN,  J.  P.     See  P.  B.  DUNHAM,  389  (ab- 
stract). 
A.  SELVERSTON,  407   (abstract). 

REVEL,  J.-P.  Fine  structure  of  intercellular 
contacts  in  the  sponge,  Microciona,  402 
(abstract). 

REVEL,  J.-P.,  AND  M.  J.  KARNOVSKY.  Fine 
structure  of  tight  junctions,  380  (ab- 
stract). 

REYNOLDS,  G.  T.  Determination  of  the  quan- 
tum efficiency  of  the  human  eye  by  a  new 
method,  402  (abstract). 

REYNOLDS,  G.  T.,  et  al.  The  identity  and 
photon  yield  of  scintillons  of  Gonyaulax, 
403  (abstract). 

REYNOLDS,  G.  T.  See  A.  W.  SENFT,  407  (ab- 
stract). 

Rhythm  of  bioluminescence  in  dinoflagellates, 

115. 
of  metabolic  activity  in  chick  embryos,  308. 


IX  HEX 


549 


RICHMOND,  A.  P.  See  C.  L.  CLAFF,  386  (ab- 
stract). 

RocKFORi),  S.    See  S.  ZIGMAN,  413  (abstract). 

ROCKSTEIN,  M.,  AND  P.  L.  BHATNAGAR.  Dura- 
tion and  frequency  of  wing  beat  in  the 
aging  house  fly  Musca,  479. 

Role  of  NaCl  in  sequential  induction  of  Rana 
gastrulae,  415. 

ROUND,  F.  E.  See  J.  D.  PALMER,  400  (ab- 
stract). 

RUGH,  R.,  et  al.  Sequelae  of  the  LD/50  ex- 
posure of  the  pre-implantation  mouse  em- 
bryo, 145. 

RULE,  N.  G.     See  L.  LORAND,  397  (abstract). 

RUSHFORTH,     N.     B.,    AND    F.     HoFMAN.       Be- 

havioral  sequences  in  the  feeding  response 
of  Hydra,  403   (abstract). 
RUSTAD,  R.   C.     Recovery  from  radiation-in- 
duced  mitotic   delay   in   sea   urchin   eggs, 
404  (abstract). 


Salinity,  influence  of  on  uptake  of  amino  acids 

by  Ophiactis,  172. 
role  of  in  distribution  of  larval  chironomids, 

399  (abstract), 
relations    in    fresh-water-adapted    Fundulus, 

362. 

in  Pachygrapsus,  272. 
Salt   fluxes   in   fresh-water-adapted   Fundulus, 

362. 
metabolism    of    Fundulus    gill    and    kidney, 

effect  of  hypophysectomy  on,  155. 
SATO,  H.,  AND  K.  J.  MULLER.    An  analysis  of 
living  sperm  head  fine  structure  through 
polarized  UV  microbeam  irradiation,  404 
(abstract). 
SATO,   H.,   et  al.     The   effect  of   DoO   on   the 

mitotic   spindle,  405    (abstract). 
SATO,  H.   See  R.  M.  CAROLAN,  385  (abstract). 
S.  MALAWISTA,  397  (abstract). 
G.  T.  REYNOLDS,  403   (abstract). 
Schistosoma,  visualization  of  radioactivity  in, 
407  (abstract). 

SCHMEER,     SR.     M.     R.,     AND     J.      D.      CASSIDY. 

Mercenene :  preliminary  analysis  of  in- 
duced focal  changes  in  the  Krebs-2  car- 
cinoma fine  structure,  405  (abstract). 

SCHOPF,  T.  J.  M.,  AND  J.  B.  COLTON,  JR. 
Bottom  temperatures  and  faunal  provinces  : 
continental  shelf  from  Hudson  Canyon  to 
Nova  Scotia,  406  (abstract). 

SCOTT,  G.  T.,  AND  K.  K.  WONG.  Evidence 
against  the  presence  of  functional  pigment- 
dispersing  nerve  fibers  in  the  sand  flounder 
Scophthalmus,  406  (abstract). 


Sea  urchin  eggs,  uptake  and  incorporation  of 

amino  acids  by,  204. 
sperm,   effects   of  glycerol   on  motility  and 

respiration  of,  166. 
motility  and  aging  of,  251. 
spines,  distribution  of  Urceolaria  on,  219. 

Sea  weeds,  in  vitro  culture  of,  487. 

Seasonal   changes  in   Pisaster  tissues,   127. 
in  respiration  of  chick  embryos,  308. 

SELVERSTON,  A.,  P.  W.  BRANDT  AND  J.  P. 
REUBEN.  Swelling  of  the  tubular  system 
in  twitch  fibers  of  Carcinus,  407  (ab- 
stract). 

Semen,  Arbacia,  effects  of  glycerol  on,  166. 
motility  and  aging  of,  251. 

Senescence  and  motility  of  Arbacia  sperm,  251. 

Senescent  housefly,  duration  and  frequency  of 
wing  beat  in,  479. 

SENFT,  A.  W.,  AND  G.  T.  REYNOLDS.  Vis- 
ualization of  radioactivity  in  Schistosoma 
by  means  of  an  image  intensifier,  407 
(abstract). 

SENFT,  J.  P.  The  effects  of  some  inhibitors 
on  the  temperature-dependent  component 
of  the  lobster  axon  resting  potential,  382 
(abstract). 

Sequelae  of  x-irradiation  of  mice  in  utero,  145. 

Sequential  induction  in  Rana  gastrulae,  role 
of  NaCl  in,  415. 

Serotonin  levels  in  Campanularia  colonies,  ef- 
fects of  reserpine  and  guanethidine  sulfate 
on,  411  (abstract). 

Sex  differences  in  duration  and  frequency  of 

wing  beat  in  aging  Musca,  479. 
in    potency   of   starfish    shedding    substance, 
104. 

Sexual  mosaics  in  Artemia,  230. 

SHANKLIN,  D.  R.  The  effect  of  15°  C.  on  the 
stages  of  normal  development  of  Fundu- 
lus, 379  (abstract). 

Rate  of  hatching  of  Fundulus  at  20°,  and 
the  effect  of  prior  exposure  at  15°,  380 
(abstract). 

Shedding  of  starfish  gametes,  mechanism  of, 
104. 

Shell-forming  epithelium  of  clam,  histochemis- 
try  and  electron  microscopy  of,  76. 

Shrimp,    effect   of   temperature    on   postlarval 

growth  of,  186. 
brine,  mutations  in,  230. 
P-32  treatment  of,  261. 

SIEGEL,  B.  W.  See  W.  AUCLAIR,  379  (ab- 
stract). 

Size  of  aggregations  in  Dictyostelium,  effect 

of  light  on,  446. 

of  egg,  role  of  in  developmental  rate,  in 
copepods,  457. 


550 


INDEX 


of  sea  urchin,  relation  of  to  distribution  of 
Urceolaria  on  its  spines,  219. 

Skin  of  dogfish,  response  of  to  MSH  in  vitro, 
470. 

Slime  mold,  effect  of  light  on  aggregration 
size  of,  446. 

SMITH,  R.     See  R.  RUGH,  145. 

Snail  population,  density  and  dispersal  in,  292. 

Sodium,  role  of  in  response  of  dogfish  melano- 

phores  to  MSH  in  vitro,  470. 
chloride,   role   of  in   sequential   induction   in 

Rana  gastrulae,  415. 
in  in  vitro  respiration  of   Squalus   rectal 

gland,  401    (abstract), 
fluxes  in  crab,  272. 

in  hypophysectomized  Fundulus,  362. 
metabolism  of  Fundulus  gill  and  kidney,  ef- 
fect of  hypophysectomy  on,  155. 
turnover   in   dogfish  tissues,   in  vivo  deter- 
mination of,  408   (abstract). 

Solutes,  organic,  effects  of  on  sperm  motility 
and  respiration,  166. 

Somateria,  trematode  parasite  of,  501. 

SOMOGYI,  C.     See  R.  RUGH,  145. 

Sorocarp  formation  in  slime  mold,  effect  of 
light  on,  446. 

Spawning  of  starfish,  mechanism  of,  104. 

Sperm,   Arbacia,   motility   and   aging   of,   251. 
motility  and  respiration,  effects  of  glycerol 
on,  166. 

SPIRTES,  M.  A.,  AND  D.  JACOBOWITZ.  Effects 
of  dopamine  on  Mercenaria  heart,  408 
(abstract). 

SPIRTES,  M.  A.  See  D.  JACOBOWITZ,  393  (ab- 
stract). 

Squalus,  blood  pressure  relationships  in  bran- 
chial arteries  of,  395    (abstract), 
response   of   melanophores   of   to    MSH,    in 
vitro,  470. 

STANFORD,  G.    See  R.  RUGH,  145. 

STANGEL,  J.  T.,  AND  W.  T.  W.  POTTS.  In 
vivo  determination  of  sodium  turnover  in 
tissues  in  the  smooth  dogfish,  Mustelus, 
408  (abstract). 

STANLEY,  J.  G.,  AND  W.  R.  FLEMING.  The 
effect  of  hypophysectomy  on  sodium 
metabolism  of  the  gill  and  kidney  of 
Fundulus,  155. 

Starfish,    feeding    behavior    and    reproductive 

cycles  in,  127. 

uptake  of  amino  acids  by,  177. 
spawning,  mechanism  of,   104. 

Statistical   analysis   of   developmental    rate   of 

copepod  eggs,  457. 
study  of  Polinices  population,  292. 

STEINBACH,  H.  B.  The  effects  of  glycerol  and 
other  organic  solutes  on  motility  and 


respiration  of  some  invertebrate  spermat- 
ozoa, 166. 

STEPHENS,  G.  C.,  AND  R.  A.  VIRKAR.  Uptake 
of  organic  material  by  aquatic  inverte- 
brates, 172. 

STEPHENS,  R.  E.,  AND  R.  E.  KANE.  Studies 
on  a  major  protein  from  isolated  sea 
urchin  egg  cortex,  382  (abstract). 

STEPHENS,  R.  E.,  S.  INDUE  AND  J.  I.  CLARK. 
Sulfhydryl  balance  in  mitosis :  The  effect 
of  mercaptoethanol  on  spindle  birefring- 
ence, 409  (abstract). 

STOOL,  E.   W.     See  E.   F.   COUCH,  388    (ab- 
stract). 
M.  FINGERMAN,  390  (abstract). 

Strain  differences  in  temperature  adaptation 
of  Euglena,  83. 

STRAND,  J.  A.,  J.  T.  CUMMINS  AND  B.  E. 
VAUGHAN.  Artificial  culture  of  marine 
sea  weeds  in  recirculation  aquarium  sys- 
tems, 487. 

Strongylocentrotus,  distribution  of  Urceolaria 
on  spines  of,  219. 

STUNKARD,  H.  W.  Further  studies  on  di- 
genetic  trematodes  of  the  family  Noto- 
cotylidae,  409  (abstract). 
The  morphology  and  life -history  of  Noto- 
cotylus,  a  digenetic  trematode  of  eider 
ducks,  501. 

Successive  P-32  contaminations,  effects  of  on 
Artemia  reproduction,  261. 

Sucrose  in  sequential  induction  in  Rana  gas- 
trulae, 415. 

Survival  of  Artemia  after  successive  doses  of 

P-32,  261. 

of  mouse  embryos   after   x-irradiation,    145. 
of  oyster  and  clam  larvae,  effect  of  pH  on, 

427. 

of   postlarval    Penaeus,    in   relation   to   tem- 
perature, 186. 

SWEENEY,  A.  R.  See  G.  T.  REYNOLDS,  403 
(abstract). 

Synthesis  of  DNA,  role  of  in  determination  of 
polarity  in  regenerating  planarians,  323. 

Systematics  of  Notocotylus,  501. 

SZABO,  G.  Effects  of  ultraviolet  irradiation 
with  special  reference  to  racial  differences 
in  coloration,  378  (abstract). 

SZABO,  G.,  AND  A.  B.  LERNER.  Chromato- 
phore  response  in  Loligo,  410  (abstract). 

SZABO,  G.  See  B.  S.  MITCHELL,  398  (ab- 
stract). 


TASAKI,    I.      See    A.    WATANABE,    411     (ab- 
stract). 


INDEX 


551 


Taxonomy    of    bioluminescent    dinoflagellates, 

115.  " 

of  Notocotylus,  501. 
TEITELBAUM,     M.       Behavior     and     settling 

mechanism    of    planulae    of    Hydractinia, 

410  (abstract). 
Teleost,  effect  of  hypophysectomy  on  gill  and 

kidney  sodium  metabolism  of,   155. 
fresh-water-adapted,   salt   fluxes   in,   362. 
Temperature,  effect  of  on  development  rate  of 

copepod  eggs,  457. 

on  growth  of  postlarval  Penaeus,  186. 

on  metabolism  of  chick  embryos,  308. 
ocean,   in   relation   to  faunal   provinces,   406 

(abstract), 
role   of  in   development   of  Ulva   in   closed 

aquarium  system,  487. 
adaptation  in  Euglena,  83. 
effects  on  Drosophila,  331,  346. 
TERWILLIGER,    R.    C.      See    F.    C.    BANCROFT, 

384  (abstract). 

Testes   of   Pisaster,   seasonal   changes   in,   127. 
THABES,  T.  M.,  C.  R.  WYTTENBACH  AND  S. 

E.  COLLINS.    The  effects  of  reserpine  and 

guanethidine    sulfate    on    serotonin    levels 

in  Campanularia  colonies,  411    (abstract). 
THABES,  T.  M.     See  C.  R.  WYTTENBACH,  412 

(abstract). 
Thawing   and   freezing,   mechanical   forces   as 

cause  of  cellular  damage  in,  197. 
Thermogenesis    of   bat   brown    adipose    tissue 

during  arousal  from  hibernation,  94. 
Thymidine  incorporation  in  Pectinaria  oocytes, 

516. 
TILNEY,  L.  G.,  AND  J.   R.  GIBBONS.     Micro- 

tubules  and  morphogenesis,  378  (abstract). 
Tissues  of  bat,  metabolism  of  during  arousal 

from  hibernation,  94. 
Tolerance  to  pH  change  of  oyster  and  clam 

embryos  and  larvae,  427. 
Tortanus,  predicting  development  rate  of  eggs 

of,  457. 

Trematode  parasite  of  eider  ducks,  morphol- 
ogy and  life-history  of,  501. 
TROLL,  W.    See  A.  GROSSMAN,  391  (abstract). 
TRUEMAN,  E.  R.    The  mechanism  of  burrow- 
ing   in    the    polychaete    worm,    Arenicola, 

369. 
Tube    feet,    occurrence    of    starfish    spawning 

substance  in,  104. 

TUTTLE,  J.     See  S.  ZIGMAN,  413    (abstract). 
TWEEDELL,    K.    S.      Oocyte    development    and 

incorporation    of    H3-thymidine    and    H3- 

uridine  in  Pectinaria,  516. 
Twitch  fibers  of  Carcinus,  swelling  of  tubular 

system  in,  407   (abstract). 
TYLER,    A.,   J.    PIATIGORSKY   AND   H.    OZAKI. 

Influence  of  individual  amino  acids  on  up- 


take and  incorporation  of  valine,  glutamic 
acid  and  arginine  by  unfertilized  and 
fertilized  sea  urchin  eggs,  204. 

U 

UHRICH,   J.   F.     See   R.    K.   JOSEPHSON,   394 

(abstract). 

Ultrastructural  basis  for  transmission  in  para- 
sympathetic  ganglion,  380    (abstract). 
Ultrastructure  of  Artemia  oocytes  and  auxili- 
ary cells,  385    (abstract), 
of  changes  induced  by  mercenene  in  carci- 
noma, 405   (abstract), 
of  clam  mantle  epithelium,  76. 
of  Cryptocotyle  redia,  395    (abstract), 
of  intercellular  contacts  in  Microciona,  402 

(abstract). 

of  Physalia  gas-secreting  gland,  387  (ab- 
stract). 

of  scallop  eye,  385    (abstract), 
of  tight  junctions,   380    (abstract), 
of   vesicles    associated   with   excitatory   and 

inhibitory  junctions,  381    (abstract). 
Ulva,  in  vitro  culture  of,  487. 
Unfertilized   sea   urchin  eggs,   uptake  and  in- 
corporation of  amino  acids  by,  204. 
Uptake    of    organic    material    by    aquatic    in- 
vertebrates,  172. 

of  valine,  glutamic  acid  and  arginine  by  sea 
urchin  eggs,  influence  of  amino  acids  on, 
204. 

URAYAMA,  T.    See  L.  LORAND,  397  (abstract). 
Urceolaria,     distribution     of     on     sea     urchin 

spines,  219. 

Urea,  effects  of  on  sperm  motility  and  respira- 
tion, 166. 
Uridine    incorporation    in    Pectinaria    oocytes, 

516. 

Urinary  bladder  of  crabs,  function  of,  272. 
Urine   excretion   of   sodium   in   hypophysecto- 
mized  Fundulus,  155. 


Valine,  influence  of  amino  acids  on  incorpor- 
ation of,  in  sea  urchin  eggs,  204. 
uptake  of  by  Ophiactis,   172. 

Variations  of  metabolism  in  chick  embryos, 
308. 

VAUGHAN,  B.  E.     See  J.  S.  STRAND,  487. 

Viability  of  Artemia  after  successive  doses  of 
P-32,  261. 

VILLEE,  C.     See  G.   PATTON,  400    (abstract). 

VIRKAR,  R.  A.     See  G.  C.  STEPHENS,  172. 

W 

WATANABE,  A.,  L  TASAKI  AND  L.  LERMAN. 
A  study  of  the  effects  of  divalent  cations 
on  squid  giant  axoms,  411  (abstract). 


552 


INDEX 


Water   entry,   role  of  in  response   of  dogfish 

melanophores  to  MSH,  470. 
relations    in   fresh-water-adapted    Fundulus, 

362. 
in  Pachygrapsus,  272. 

WHITTINGHAM,  D.  C,  AND  C.  R.  AUSTIN. 
Cytological  studies  on  the  inhibition  of 
early  cleavage  by  estradiol  in  Arbacia, 
412  (abstract). 

Wild  populations  of  Artemia,  mutations  in, 
230. 

Wing  beat  frequency  and  duration   in   aging 

Musca,  479. 

veins  of  Drosophila,  effects  of  temperature 
on  development  of,  331,  346. 

WONG,  K.  K.  See  G.  T.  SCOTT,  406  (ab- 
stract). 

WYTTENBACH,  C.  R.,  T.  M.  THABES  AND  S.  E. 
COLLINS.  The  physiological  effects  of 
reserpine  and  guanethidine  sulfate  on 
Campanularia  hydranths,  412  (abstract). 


WYTTENBACH,  C.  R.    See  T.  M.  THABES,  411 
(abstract). 

X 

X-irradiation   of  mice   in   utero,   145. 


Yolk,     role    of    in    rate    of    development    of 
copepod  eggs,  457. 


ZEIN-ELDIN,  Z.  P.,  AND  G.  W.  GRIFFITH.  The 
effect  of  temperature  upon  the  growth  of 
laboratory-held  postlarval  Penaeus,  186. 

ZIGMAN,  S.,  et  al.  Protein  synthesis  in  dog- 
fish cornea  epithelial  cells,  413  (abstract). 

ZIGMAN,  S.     See  S.  LERMAN,  396  (abstract). 

ZINN,  D.  J.  A  new  method  for  the  extraction 
of  living  Thalassopsammon  from  inter- 
tidal  and  subtidal  marine  sands,  413  (ab- 
stract). 

Zoochlorella-bearing  form  of  Euplotes,  437. 


Volume  131  Number  1 


THE 

BIOLOGICAL  BULLETIN 


PUBLISHED  BY 


THE  MARINE  BIOLOGICAL  LABORATOR          VV 


Editorial  Board 

JOHN  B.  BUCK,  National  Institutes  of  Health          JOHN  H.  LOCHHEAD,  University  of  Vermont 
PHILIP  B.  DUNHAM,  Syracuse  University  ROBERTS  RUGH,  Columbia  University 

SALLY  HUGHES-SCHRADER,  Duke  University          MELVIN  SPIEGEL'  Dartm0^  College 

_  WM.  RANDOLPH  TAYLOR,  University  of 

LEBBIE  H.  HYMAN,  Amencan  Museum  of 

Natural  History 

ANNA  R.  WHITING,  Oak  Ridge  National 
SHINYA  INOUE,  Dartmouth  College  Laboratory 

J.  LOGAN  IRVIN,  University  of  North  Carolina         CARROLL  M.  WILLIAMS,  Harvard  University 

DONALD  P.  COSTELLO,  University  of  North  Carolina 
Managing  Editor 


AUGUST,  1966 


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