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THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE  BIOLOGICAL  LABORATORY 

Editorial  Board 


E.  G.  CONKLIN,  Princeton  University 

E.  N.  HARVEY,  Princeton  University 

SELIG  HECHT,  Columbia  University 

LEIGH  HOADLEY,  Harvard  University 

L.  IRVING,  Swarthmore  College 

M.  H.   JACOBS,  University  of  Pennsylvania 

H.   S.  JENNINGS,   Johns  Hopkins  University 


FRANK  R.  LILLIE,  University  of  Chicago 
CARL  R.  MOORE,  University  of  Chicago 
GEORGE  T.  MOORE,  Missouri  Botanical  Garden 
G.  H.  PARKER,  Harvard  University 
A.  C.  REDFIELD,  Harvard  University 
F.  SCHRADER,  Columbia  University 
DOUGLAS  WHITAKER,  Stanford  University 


H.  B.  STEINBACH,  Washington  University 
Managing  Editor 


VOLUME  91 

AUGUST  TO  DECEMBER,  1946 


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,  $1.75.  Subscription  per  volume  (three 
issues),  $4.50. 

Communications  relative  to  manuscripts  should  be  sent  to  the 
Managing  Editor,  Marine  Biological  Laboratory,  Woods  Hole, 
Massachusetts,  between  July  1  and  September  1,  and  to  the  De- 
partment of  Zoology,  Washington  University,  St.  Louis,  Missouri, 
during  the  remainder  of  the  year. 


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


1ANCASTFR  PRESS.  INC.,  LANCASTER,  PA 


CONTENTS 


No.   1.     AUGUST,  1946 

PAGE 

ANNUAL  REPORT  OF  THE  MARINE  BIOLOGICAL  LABORATORY  ...  1 

POMERAT,  C.  M.,  AND  C.  M.  WEISS 

The  influence  of  texture  and  composition  of  surface  on  the  attachment 

of  sedentary  marine  organisms 57 

SCOTT,  SISTER  FLORENCE  MARIE 

The  developmental  history  of  Amaroecium  constellatum.     II.  Organo- 

genesis  of  the  larval  action  system 66 

GIESE,  ARTHUR  C. 

Comparative  sensitivity  of  sperm  and  eggs  to  ultraviolet  radiations    .        81 
CARRIKER,  MELBOURNE  ROMAINE 

'  Observations  on  the  functioning  of  the  alimentary  system  of  the  snail 

Lymnaea  stagnalis  appressa  Say .  .      88 

CHEN,  TZE-TUAN 

Temporary  pair  formation  in  Paramecium  bursaria .  112 

No.  2.     OCTOBER,  1946 

WEISZ,  PAUL  B. 

The  space-time  pattern  of  segment  formation  in  Artemia  salina.  .  119 

JAKUS,  M.  A.,  AND  C.  E.  HALL 

Electron  microscope  observations  of  the  trichocysts  and  cilia  in  Para- 
mecium    141 

PEASE,  DANIEL  C. 

Hydrostatic  pressure  effects  upon  the  spindle  figure  and  chromosome 
movement.  II.  Experiments  on  the  meiotic  divisions  of  Tradescantia 
pollen  mother  cells 145 

BROWN,  FRANK  A.  JR.,  AND  LORRAINE  M.  SAIGH 

The  comparative  distribution  of  two  chromatophorotropic  hormones 
(CDH  and  CBLH)  in  Crustacean  nervous  systems 170 

MORRISON,  PETER  R. 

Physiological  observations  on  water  loss  and  oxygen  consumption  in 
Peripatus 181 

KOZLOFF,  EUGENE  N. 

Studies  on  ciliates  of  the  family  Ancistrocomidae  Chatton  and  Lowff 
(order  Holotricha,  suborder  Thigmotricha).  III.  Ancistrocoma  pelse- 
neeri  Chatton  and  Lwoff,  Ancistrocoma  dissimilis  sp.  nov.,  and  Hypo- 
comagalma  pholadidis  sp.  nov ....  189 

KOZLOFF,  EUGENE  N. 

Studies  on  ciliates  of  the  family  Ancistrocomidae  Chatton  and  Lwoff 
(order  Holotricha,  suborder  Thigmotricha).  IV.  Heterocineta  janickii 
Jarocki,  Heterocineta  goniobasidis  sp.  nov.,  Heterocineta  fluminicolae 

sp.  nov.,  and  Enerthecoma  properans  Jarocki 200 

iii 


60544 


iv  CONTENTS 

ABSTRACTS  OF  SCIENTIFIC  PAPERS  PRESENTED  AT  THE  MARINE  BIOLOGICAL 
LABORATORY,  SUMMER  OF  1946 210 

PAPERS  PRESENTED  AT  THE  MEETING  OF  THE  SOCIETY  OF  GENERAL  PHYSI- 
OLOGISTS    .  .  236 

No.  3.     DECEMBER,  1946 

WHITING,  P.  W. 

A  strongly  intersexual  female  in  Habrobracon  .  .  243 

TOBIAS,  J.  M.,  AND  J.  J.  KOLLROS 

Loci  of  action  of  DDT  in  the  cockroach  (Periplaneta  americana) .  247 

BEERS,  C.  D. 

Tillina  magna:  Micronuclear  number,  encystment  and  vitality  in  diverse 
clones;  capabilities  of  amicronucleate  races.  .  .  .  256 

SCOTT,  ALLAN 

The  effect  of  low  temperature  and  of  hypotonicity  on  the  morphology 

of  the  cleavage  furrow  in  Arbacia  eggs.  .  .  272 

BODENSTEIN,  DIETRICH 

Developmental  relations  between  genital  ducts  and  gonads  in  Droso- 
phila 288 

LEHMAN,  H.  E. 

A  histological  study  of  Syndisyrinx  franciscanus,  gen.  et  sp.  nov.,  an 
endoparasitic  rhabclocoel  of  the  sea  urchin,  Strongylocentrotus  francis- 
canus   295 

SPOOR,  W.  A. 

A  quantitative  study  of  the  relationship  between  the  activity  and  oxygen 
consumption  of  the  goldfish,  and  its  application  to  the  measurement  of 
respiratory  metabolism  in  fishes . 


Vol.  91,  No.   1  August,  1946 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE  MARINE  BIOLOGICAL  LABORATORY 


THE  MARINE  BIOLOGICAL  LABORATORY 
FORTY-EIGHTH   REPORT,   FOR  THE  YEAR   1945 — FIFTY-EIGHTH   YEAR 

I.     TRUSTEES  AND  EXECUTIVE  COMMITTEE  (AS  OF  AUGUST  14,  1945)   ....     .1 

STANDING  COMMITTEES 
II.     ACT  OF  INCORPORATION  

III.  BY-LAWS  OF  THE  CORPORATION  

IV.  REPORT  OF  THE  TREASURER 

V.     REPORT  OF  THE  LIBRARIAN  

VI.     REPORT  OF  THE  DIRECTOR  11 

Statement    11 

Addenda : 

1 .  Publications  from  this  Laboratory  during  the  years  1941-1945.  .  13 

2.  The  Staff 33 

3.  Investigators  and  Students 35 

4.  Tabular  View  of  Attendance,  1941-1945  41 

5.  Subscribing  and  Co-operating  Institutions   42 

6.  Evening  Lectures  42 

7.  Shorter  Scientific  Papers   43 

8.  Members  of  the  Corporation   44 


I.     TRUSTEES 

EX    OFFICIO 

FRANK  R.  LILLIE,  President  Emeritus  of  the  Corporation,  The  University  of  Chicago 
LAWRASON  RIGGS,    President  of  the  Corporation,  120  Broadway,  New  York  City 
E.  NEWTON  HARVEY,  Vice  President  of  the  Corporation,  Princeton  University 
CHARLES  PACKARD,  Director,  Marine  Biological  Laboratory 
OTTO  C.  GLASER,  Clerk  of  the  Corporation,  Amherst  College 
DONALD  M.  BRODIE,  Treasurer,  522  Fifth  Avenue,  New  York  City 


EMERITUS 


E.  G.  CONKLIN,  Princeton  University 
B.  M.  DUGGAR,  University  of  Wisconsin 
W.  E.  GARREY,  Vanderbilt  University 
R.  A.  HARPER,  Columbia  University 
Ross  G.  HARRISON,  Yale  University 

H.  S.  JENNINGS,  University  of  California 

F.  P.  KNOWLTON,  Syracuse  University 


2  MARINE  BIOLOGICAL  LABORATORY 

R.  S.  LILLIE,  The  University  of  Chicago 

*C.  E.  McCLUNG,  University  of  Pennsylvania 

S.  O.  MAST,  Johns  Hopkins  University 

A.  P.  MATHEWS,  University  of  Cincinnati 

*T.  H.  MORGAN,  California  Institute  of  Technology 

W.  J.  V.  OSTERHOUT,  Rockefeller  Institute 

G.  H.  PARKER,  Harvard  University 

W.  B.  SCOTT,  Princeton  University 

TO  SERVE   UNTIL   1949 

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

P.  B.  ARMSTRONG,  Syracuse  University 

L.  G.  BARTH,  Columbia  University 

S.  C.  BROOKS,  University  of  California 

W.  C.  CURTIS,  University  of  Missouri 

H.  B.  GOODRICH,  Wesleyan  University 

A.  C.  REDFIELD,  Harvard  University 

C.  C.  SPEIDEL,  University  of  Virginia 

TO   SERVE   UNTIL    1948 

ERIC  G.  BALL,  Harvard  University  Medical  School 

R.  CHAMBERS,  Washington  Square  College,  New  York  University 

EUGENE  F.  DuBois,  Cornell  University  Medical  College 

COLUMBUS  ISELIN,  Woods  Hole  Oceanographic  Institution 

C.  W.  METZ,  University  of  Pennsylvania 

H.  H.  PLOUGH,  Amherst  College 

E.  W.  SINNOTT,  Yale  University 

W.  R.  TAYLOR,  University  of  Michigan 

TO    SERVE    UNTIL    1947 

W.  C.  ALLEE,  The  University  of  Chicago 

G.  H.  A.  CLOWES,  Lilly  Research  Laboratory 

P.  S.  GALTSOFF,  U.  S.  Fish  and  Wild  Life  Service 

L.  V.  HEILBRUNN,  University  of  Pennsylvania 

LAURENCE  IRVING,  Swarthmore  College 

J.  H.  NORTHROP,  Rockefeller  Institute 

A.  H.  STURTEVANT,  California  Institute  of  Technology 

LORANDE  L.  WOODRUFF,  Yale  University 

TO    SERVE    UNTIL    1946 

DUGALD  E.  S.  BROWN,  New  York  University 

E.  R.  CLARK,  University  of  Pennsylvania 

OTTO  C.  GLASER,  Amherst  College 

E.  N.  HARVEY,  Princeton  University 

M.  H.  JACOBS,  University  of  Pennsylvania 

A.  K.  PARPART,  Princeton  University 
FRANZ  SCHRADER,  Columbia  University 

B.  H.  WILLIER,  Johns  Hopkins  University 

EXECUTIVE  COMMITTEE  OF  THE  BOARD  OF  TRUSTEES 

LAWRASON  RIGGS,  Ex  officio.  Chairman 
E.  N.  HARVEY,  Ex  officio 

*  Deceased. 


ACT  OF  INCORPORATION 

D.  M.  BRODIE,  Ex  officio 
CHARLES  PACKARD,  Ex  officio 

P.  B.  ARMSTRONG,  to  serve  until  1947 

L.  G.  EARTH,  to  serve  until  1946 

P.  S.  GALTSOFF,  to  serve  until  1947 

WM.  RANDOLPH  TAYLOR,  to  serve  until  1946 

THE  LIBRARY  COMMITTEE 
A.  C.  REDFIELD,  Chairman 

E.  G.  BALL 
S.  C.  BROOKS 
M.  E.  KRAHL 
J.  W.  MAYOR 

THE  APPARATUS  COMMITTEE 

E.  P.  LITTLE,  Chairman 

C.  L.  CLAFF 
G.  FAILLA 
S.  E.  HILL 

A.  K.  PARPART 

THE  SUPPLY  DEPARTMENT  COMMITTEE 

D.  A.  MARSLAND,  Chairman 
P.  B.  ARMSTRONG 

P.  S.  GALTSOFF 
R.  T.  KEMPTON 
CHARLES  PACKARD 

THE  EVENING  LECTURE  COMMITTEE 

F.  M.  LANDIS,  Chairman 
CHARLES  PACKARD 

THE  INSTRUCTION   COMMITTEE 

H.  B.  GOODRICH,  Chairman 
W.  C.  ALLEE 
S.  C.  BROOKS 
VIKTOR  HAMBURGER 
CHARLES  PACKARD,  Ex  officio 

THE  BUILDINGS  AND  GROUNDS  COMMITTEE 

E.  G.  BALL,  Chairman 
D.  P.  COSTELLO 
MRS.  E.  N.  HARVEY 
ROBERTS  RUGH 

MRS.  C.  C.  SPEIDEL 


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 


4  MARINE  BIOLOGICAL  LABORATORY 

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

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. 


III.     BY-LAWS   OF  THE  CORPORATION   OF   THE   MARINE 

BIOLOGICAL  LABORATORY 

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

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

III.  The  Annual  Meeting  of  the  members  shall  be  held  on  the  second  Tuesday  in 
August  in  each  year,  at  the  Laboratory  in  Woods  Hole,  Massachusetts,  at  11:30  A.M., 
and  at  such  meeting  the  members  shall  choose  by  ballot  a  Treasurer  arid  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  By-laws,  no  notice  of  the  Annual  Meeting  need  be  given.     Notice  of  any  special 
meeting  of  members,  however,  shall  be  given  by  the  Clerk  by  mailing  notice  of  the  time 
and  place  and  purpose  of  such  meeting,  at  least  fifteen   (15)  days  before  such  meeting, 
to  each  member  at  his  or  her  address  as  shown  on  the  records  of  the  Corporation. 

VII.  The  Annual  Meeting  of  the  Trustees  shall  be  held  on  the  second  Tuesday  in 
August  in  each  year,  at  the  Laboratory  in  Woods   Hole,   Mass.,  at   10  A.M.     Special 
meetings  of  the  Trustees  shall  be  called  by  the  President,  or  by  any  seven  Trustees,  to  be 
held  at  such  time  and  place  as  may  be  designated,  and  the  Secretary  shall  give  notice 
thereof  by  written  or  printed  notice,  mailed  to  each  Trustee  at  his  address  as  shown  on 
the  records  of  the  Corporation,  at  least  one    (1)    week  before  the  meeting.     At   such 
special  meeting  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 ;  and  in  addition  there  shall  be  two  groups  of  Trustees  as  follows : 


REPORT  OF  THE  TREASURER  5 

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

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

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

IX.  The  Trustees  shall  have  the  control  and  management  of  the  affairs  of  the  Cor- 
poration; they  shall  elect  a  President  of  the  Corporation  who  shall  also  be  Chairman  of 
the  Board  of  Trustees;  and  shall  also  elect  a  Vice  President  of  the  Corporation  who  shall 
also  be  the  Vice  Chairman  of  the  Board  of  Trustees ;  they  shall  appoint  a  Director  of 
the  Laboratory;  and  they  may  choose  such  other  officers  and  agents  as  they  may  think 
best ;  they  may  fix  the  compensation  and  define  the  duties  of  all  the  officers  and  agents ; 
and  may  remove  them,  or  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  Corpora- 
tion upon  such  terms  and  conditions  as  they  may  think  best. 

X.  Any  person  interested  in  the  Laboratory  may  be  elected  by  the  Trustees  to  a  group 
to  be  known  as  Associates  of  the  Marine  Biological  Laboratory. 

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


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

Gentlemen: 

Herewith  is  my  report  as  Treasurer  of  the  Marine  Biological  Laboratory  for 
the  year  1945. 

The  accounts  have  been  audited  by  Messrs.  Seamans,  Stetson,  and  Tuttle,  certi- 
fied public  accountants.  A  copy  of  their  report  is  on  file  at  the  Laboratory  and 
inspection  of  it  by  members  of  the  Corporation  will  be  welcomed. 

The  principal  summaries  of  their  report — The  Balance  Sheet,  Statement  of 
Income  and  Expense,  and  Current  Surplus  Account — are  appended  hereto  as 
Exhibits  A,  B,  and  C. 

The  following  are  some  general  statements  and  observations  based  on  the  de- 
tailed reports : 


6  MARINE  BIOLOGICAL  LABORATORY 

I.  Assets 

1.  Endowment  Assets 

As  of  December  31,  1945,  the  total  book  value  of  all  the  Endowment  Assets, 
including  the  Scholarship  Funds,  was  $966,772.16,  a  loss  for  the  year  of  $17,128.41. 
The  decline  was  due,  as  in  the  last  two  years,  to  losses  on  the  mortgage  participa- 
tions on  New  York  City  realty  held  in  the  Trust  Funds. 

At  the  end  of  the  year  $831,993.01  was  invested  in  marketable  securities  (bonds, 
preferred  stocks  and  common  stocks)  with  a  market  value  of  $910,162.31.  $125,- 
753.85  was  invested  in  mortgage  participations  on  New  York  City  real  estate. 
$9,025.30  was  in  uninvested  principal  cash. 

The  Treasurer's  estimate  of  the  actual  value  of  the  $125,753.85  in  mortgage 
notes  and  participations  held  on  December  31  is  $85,750.00.  With  the  market 
value  of  $910,162.31  on  marketable  securities  and  the  $9,025.30  in  cash  this  makes 
a  total  current  valuation  of  $1,004,937.61  compared  with  total  book  value  of 
$966,772.16. 

The  increase  for  the  year  in  market  values,  $75,454.79,  is  largely  due  to  the 
rise  in  common  stock  prices. 

t 

2.  Plant  Assets 

There  were  no  changes  of  any  consequence  in  Plant  Assets  during  the  year. 
The  Reserve  Fund  was  increased  nearly  $10,000.00  to  a  total  of  $26,830.71  by  the 
transfer  of  the  Crane  Co.  dividends,  part  of  the  General  Biological  Supply  House 
dividends  and  other  items  of  current  income. 

3.  Current  Assets 

The  total  of  current  assets  increased  $10,730.68  during  1945  to  a  total  of 
$212,970.35.  Current  Liabilities  at  the  end  of  the  year  were  $2,754.70.  Current 
Surplus  increased  $12,277.94  to  a  total  of  $196,337.90. 

II.  Income  and  Expenditures 

The  return  to  more  normal  operations  for  the  Laboratory  last  summer  resulted 
in  larger  totals  for  both  income  and  expense  items.  Total  income  was  $182,818.23, 
total  expense  including  depreciation  reserves  of  $26,968.12  was  $173,044.95,  giving 
a  net  surplus  for  the  year  of  $9,773.28. 

EXHIBIT  A 

MARINE  BIOLOGICAL  LABORATORY  BALACE  SHEET,  DECEMBER  31,   1945 

Assets 
Endowment  Assets  and  Equities : 

Securities  and  Cash  in  Hands  of  Central  Hanover  Bank  and 

Trust  Company,  New  York,  Trustee   $    950,130.04 

Securities  and  Cash  in  Minor  Funds  16,642.12 

$   966,772.16 


REPORT  OF  THE  TREASURER 

Plant  Assets : 

Land     $    111,425.38 

Buildings    1,326,345.54 

Equipment     187,837.87 

Library     337,266.01 


Less  Reserve  for  Depreciation 

Reserve  Fund,  Securities  and  Cash 
Book  Fund,  Securities  and  Cash    . 


$1,962,874.80 
677,140.22 


Current  Assets : 

Cash     

Accounts  Receivable    

Inventories : 

Supply   Department    $      44,441.66 

Biological    Bulletin     20,1 17.40 


$1,285,734.58 

26,830.71 
18,282.46 


$     30,467.02 
20,396.05 


64,559.06 


$1,330,847.75 


Investments  : 

Devil's   Lane   Property    $     46,556.99 

Gansett   Property    1,749.92 

Stock  in  General  Biological  Supply  House, 

Inc 12,700.00 

Other  Investment  Stocks    20,095.00 

Retirement    Fund    11,517.82 


Prepaid    Insurance 
Items  in  Suspense 


92,619.73 

4,033.08 
895.41 


$   212,970.35 

Total    Assets    $2,510,590.26 

Liabilities 

Endowment  Funds : 

Endowment    Funds     $    948.646.S2 

Reserve  for  Amortization  of  Bond  Premiums..  1,483.22 


Minor    Funds 


Plant  Funds : 

Mortgage  Notes   Payable    

Donations  and  Gifts   $1,172,564.04 

Other    Investments    in    Plant    from    Gifts    and 

Current    Funds  153,283.71 


Current  Liabilities  and  Surplus : 

Accounts    Payable    

Items  in  Suspense    

Reserve  for  Repairs  and  Replacements 
Current  Surplus   


$    950,130.04 
16,642.12 


$       5,000.00 


$1,325,847.75 


$       2,754.70 

1,799.63 

12,078.12 

196,337.90 


$   966,772.16 


$1,330,847.75 


$    212,970.35 
Total    Liabilities    $2,510,590.26 


MARINE  BIOLOGICAL  LABORATORY 
EXHIBIT  B 

MARINE  BIOLOGICAL  LABORATORY  INCOME  AND  EXPENSE, 
YEAR  ENDED  DECEMBER  31,   1945 

Total  Net 

Expense  Income          Expense  Income 

Income : 


General   Endowment  Fund    

$  32,214.07 

$  32,214.07 

Library   Fund    

9,479.18 

9,479.18 

Donations   

755.00 

755.00 

Instruction    -.  

$    9,554.39 

7,220.00 

$    2,334.39 

Research    

4,550.59 

17,434.24 

12,883.65 

Evening  Lectures   

86.35 

86.35 

Biological   Bulletin   and   Membership   Dues. 

6,393.65 

8.775.63 

2,381.98 

Supply    Department     

39,255.03 

47,812.56 

8,557.53 

Mess     

24,146.52 

20,750.36 

3,396.16 

Dormitories    

27,443.23 

14,547.91 

12,895.32 

(Interest  and  Depreciation  charged  to  above 

3  Departments)    

(25,574.03) 

25,574.03 

Dividends,  General  Biological  Supply  House, 

Inc  

14.732.00 

14.732.0C 

Dividends,  Other  Investment   Stocks    

725.00 

725.00 

Rents  : 

Bar   Neck   Property    

767.65 

6,000.00 

5,232.35 

Janitor    House    

30.89 

360.00 

329.11 

Danchakoff    Cottages    

240.86 

275.00 

34.14 

Sale  of  Library  Duplicates,  Micro  Film,  etc. 

344.74 

344.74 

Microscope  and  Apparatus  Rental   

1,372.54 

1,372.54 

Sundry    Income    

20.00 

20.00 

Maintenance  of  Plant  : 

Buildings   and   Grounds    

23,642.27 

23,642.27 

Apparatus  Department    

4,911.52 

4,911.52 

Chemical    Department    

2,265.30 

. 

2,265.30 

Library   Expense    

6,487.95 

/ 

6,487.95 

Workmen's  Compensation  Insurance    .... 

526.63 

526.63 

Truck    Expense    

238.60 

238.60 

Bay  Shore  Property   

92.78 

92.78 

Great  Cedar  Swamp   

21.00 

21.00 

General  Expenses : 

Administration    Expense    15,168.99  15,168.99 

Endowment  Fund  Trustee  and  Safe-Keep- 
ing      1,028.45  1,028.45 

Bad  Debts    375.97  375.97 

Special  Repairs  on  account  of   1944   Hurri- 
cane  Damage    4,297.24  4,297.24 

Interest    125.00  125.00 

Reserve  for  Depreciation   26,968.12  26,968.12 


$173,044.95    $182,818.23    $104,862.04    $114,635.32 
Excess  of  Income  over   Expense  carried   to 

Current  Surplus   9,773.28  9,773.28 


$182,818.23  $114,635.32 


REPORT  OF  THE  LIBRARIAN 
EXHIBIT  C 

MARINE   BIOLOGICAL   LABORATORY.    CURRENT    SURPLUS    ACCOUNT, 
YEAR  ENDED  DECEMBER  31,  1945 

Balance  January  1,  1945  $184,059.96 

Add: 

Excess  of  Income  over  Expense  for  Year  as  shown,  in  Exhibit  B . .  $  9,773.28 

Gain  on  Gansett  Lots  Sold  464.18 

Bad   Debts   Recovered    82.23 

Mortgage  Payable,  Transferred  to  Plant  Funds   5,000.00 

Reserve  for  Depreciation  Charged  to  Plant  Funds   26.968.J2        42,287.81 


$226,347.77 
Deduct : 

Payments  from  Current  Funds  during  Year  for  Plant  Assets  as 
shown  in  Schedule  IV: 

Buildings     $  7,402.65 

Equipment    4,462.50 

Library    7,500.43 


$19,365.58 
Less  Received  for  Plant  Assets  Sold   5,600.00 


$13,765.58 

Pensions    Paid    $  3,460.00 

Loss  on  Retirement  Fund  Securities  847.32 


$  4,307.32 
Less  Retirement  Fund  Income  311.79 


$  3,995.53 
Transfers  to  Reserve  Fund  : 

Portion  of  Dividends  from  General  Biological  Sup- 
ply House,   Inc $  2,500.00 

Dividends  from  Crane  Co 625.00 

Income  from  Operation  and  Sale  of  Property  445- 

51  W.  23rd  and  450-2  W.  24th  Sts.,  N.  Y.  C.        8,947.72 
Gansett  Property  Profits,  1944   176.04      12,248.76        30,009.87 


Balance,  December  31,  1945    $196,337.90 

Respectfully  submitted, 

DONALD  M.  BRODIE, 

Treasurer 


V.     REPORT  OF  THE  LIBRARIAN 

The  sum  $12,262.54  appropriated  to  the  library  in  1945  was  expended  as 
follows:  books,  $469.99;  serials,  $2,625.79;  binding,  $577.60;  express,  $43.22; 
supplies,  $147.84;  salaries,  $7,262.54  ($1,150  of  this  sum  was  contributed  by  the 
Woods  Hole  Oceanographic  Institution)  ;  back  sets,  $1,104.98;  insurance,  $45.00; 


10  MARINE  BIOLOGICAL  LABORATORY 

sundries,  $5.00 ;  total,  $12,281 .96.  The  cash  receipts  of  the  library  totalled  $344.74 : 
for  microfilms,  $220.34  ($62.17  expenses  paid  by  the  library  and  accounted  above 
under  "supplies")  ;  sale  of  duplicates,  $122.74;  sale  of  the  "Serial  List,"  Biological 
Bulletin  supplement  number,  $1.66.  This  sum,  $344.74,  reverts  to  the  laboratory 
and  does  not  include  rent  payments  for  library  readers  which  are  collected  by  the 
main  office.  There  were  49  library  readers  accommodated  in  the  library  during 
the  summer  of  1945. 

Of  the  Carnegie  of  New  York  Fund,  $126.35  was  expended  for  the  completion 
of  one  journal  and  the  partial  completion  of  another. 

The  sum  appropriated  by  the  Woods  Hole  Oceanographic  Institution  in  1945 
for  purchases  was  $800.  A  balance  of  $949.39  remaining  from  1944  made  an 
available  total  of  $1,749.39.  Of  this  sum  $947.12  was  expended,  leaving  a  balance 
of  $802.27  towards  future  purchases.  In  addition  to  the  above,  the  Woods  Hole 
Oceanographic  Institution  contributed  $1,150  (see  above  under  salaries). 

During  1945  the  library  received  902  current  journals:  279  (8  new)  by  sub- 
scription to  the  Marine  Biological  Laboratory;  30  (7  new)  to  the  Woods  Hole 
Oceanographic  Institution;  exchanges  352  (6  new;  145  reinstated  foreign)  and  58 
(35  foreign  reinstated)  with  the  Woods  Hole  Oceanographic  Institution  publica- 
tions; 177  as  gifts  to  the  former  and  6  to  the  latter.  The  library  acquired  206 
books :  77  by  purchase  of  the  Marine  Biological  Laboratory ;  44  by  purchase  of 
the  Woods  Hole  Oceanographic  Institution;  10  gifts  by  the  authors;  46  gifts  by 
the  publishers ;  20  by  miscellaneous  donors  and  9  from  Miss  Jane  Strong.  There 
were  22  back  sets  of  serial  publications  completed:  14  purchased  by  the  Marine 
Biological  Laboratory  (one  with  the  Carnegie  Fund)  ;  2  by  the  Woods  Hole 
Oceanographic  Institution ;  5  by  exchange  of  the  "Biological  Bulletin"  and  one  by 
exchange  of  duplicate  material.  Partially  completed  sets  were  54:  purchased  by 
the  Marine  Biology  Laboratory,  27  (1  by  the  Carnegie  Fund)  ;  purchased  by  the 
Woods  Hole  Oceanographic  Institution,  2 ;  by  exchange  with  the  "Biological 
Bulletin,"  2;  by  gift  and  exchange  of  duplicate  material,  23. 

The  reprint  additions  to  the  library  were  4,620;  current  of  1944,  604;  current 
of  1945,  64;  and  of  previous  dates,  3,952.  A  total  of  6,390,  2,130  not  duplicates 
of  our  holdings,  were  presented  to  the  library ;  4,295  by  Mrs.  Meigs ;  67  by  Dr. 
H.  G.  Cassidy;  627  by  the  University  of  Utah;  26  by  Dr.  B.  M.  Davis;  and  1,375 
by  Dr.  L.  C.  Wyman.  The  large  collection  of  Dr.  Carrey's  reprints  presented  last 
year  have  not  as  yet  been  counted  nor  started  on  the  way  toward  cataloguing. 

At  the  end  of  the  year  1945  the  library  contained  53,990  bound  volumes  and 
137,674  reprints. 

Readers  of  the  library  report  will  be  glad  to  note  the  number  of  foreign  ex- 
changes that  have  already  been  reinstated  during  1945,  both  for  the  "Biological 
Bulletin"  and  for  the  Woods  Hole  Oceanographic  Institution  publications :  145  for 
the  former  and  35  for  the  latter.  Next  year's  report  will  probably  show  the  pre-war 
number  reinstated  save  only  for  Germany  and  perhaps  for  Russia  since  we  get  very 
poor  response  from  that  country.  Nor  have  we  heard  anything  in  regard  to  the 
German  journals  on  order  with  Otto  Harrassowitz  which  are  apparently  stalled 
if  not  destroyed  in  Leipzig.  The  library  committee  that  is  working  with  the  State 
Department  to  get  these  released  has  nothing  so  far  to  report  to  this  library. 


REPORT  OF  THE  DIRECTOR 

VI.     REPORT  OF  THE  DIRECTOR 

To  THE  TRUSTEES  OF  THE  MARINE  BIOLOGICAL  LABORATORY: 
Gentlemen: 

I  herewith  submit  a  report  on  the  fifty-eighth  session  of  the  Marine  Biological 
Laboratory. 

1.  Attendance 

Since  1943  our  total'  attendance  has  increased  from  the  low  point  reached  that 
year.  In  1944  it  was  53  per  cent  of  the  pre-war  average  of  490;  in  1945  it  was 
63  per  cent.  This  increase  is  found  among  the  independent  investigators  and  the 
students;  the  beginning  investigators  and  research  assistants,  who,  as  I  explained 
in  the  last  report,  belong  in  one  group,  are  still  sparsely  represented.  In  1945 
there  were  only  36,  whereas  the  pre-war  average  was  130.  The  advance  registra- 
tion for  1946  shows  an  encouraging  increase  in  this  group.  Many  of  the  appli- 
cants are  veterans  who  are  taking  advantage  of  Government  funds  provided  under 
the  G.  I.  Bill  of  Rights. 

2.  Building  Repairs 

One  of  the  inevitable  effects  of  the  war  has  been  the  deterioration  of  our  build- 
ings. Lack  of  materials  and  labor  has  up  until  now  prevented  all  but  the  most 
essential  repairs  from  being  made.  Fortunately,  we  are  now  able  to  begin  to  put 
our  house  in  order,  in  spite  of  the  shortage  of  some  critical  materials.  To  deter- 
mine what  work  should  be  done,  a  Committee  on  Special  Repairs,  under  the  able 
leadership  of  Mr.  C.  L.  Claff,  conducted  a  thorough  survey  of  all  of  the  buildings 
and  made  detailed  recommendations.  The  report,  a  model  of  completeness  as 
drawn  up  by  Mr.  Claff,  calls  for  the  ultimate  expenditure  of  approximately  $145,000 
for  present  repairs  and  future  desirable  improvements  not  only  in  the  buildings  but 
also  in  equipment  for  the  Apparatus  Department  and  the  Supply  Department. 

The  Executive  Committee  voted  to  expend  the  entire  Reserve  Fund,  amounting 
to  $25,000,  and  all  but  a  minimum  of  the  current  cash  on  hand  for  making  the  most 
urgently  needed  repairs  at  once.  It  also  laid  plans  for  securing  outside  funds  with 
which  to  complete  the  changes  called  for  in  the  report,  and  to  purchase  apparatus. 
In  addition,  funds  for  a  new  building  and  for  additional  endowment  are  to  be 
sought. 

Many  of  the  essential  repairs  have  already  been  made.  The  Mess  kitchen, 
never  properly  restored  after  the  Navy  occupation,  is  now  in  good  condition,  and 
improvements  in  the  dining  room  have  been  made.  The  Botany  Building,  unused 
for  several  seasons,  has  been  put  to  rights  with  new  plumbing,  wall  tables,  shelves, 
and  other  fixtures.  Replacements  have  been  made  in  the  Supply  Department  and 
Rockefeller  Building;  hot  water  systems  are  installed  in  the  residences  heretofore 
not  so  provided ;  and  much  painting  has  been  done.  This  work  was  accomplished 
in  large  measure  by  our  permanent  staff,  under  the  direction  of  Mr.  MacNaught. 
All  the  men  worked  faithfully  and  energetically,  and  have  completed  the  assigned 
tasks  in  a  most  satisfactory  manner.  It  is  hoped  that  waterproofing  of  the  Crane 
and  Brick  Buildings  may  be  completed  before  the  1946  season  begins.  As  soon  as 
this  has  been  satisfactorily  finished,  those  Laboratory  rooms  which  have  been  dam- 
aged by  water  can  be  made  presentable. 


12  MARINE  BIOLOGICAL  LABORATORY 

3.  The  Housing  Problem 

An  unexpected  outcome  of  war-time  activities  is  the  housing  shortage.  Before 
the  war  our  residences  and  the  houses  in  the  village  could  accommodate  450  to  500 
persons  during  the  course  of  a  summer.  But  when  the  Oceanographic  Institution 
emharked  on  extensive  defense  projects,  the  number  of  its  workers  increased  from 
comparatively  few  to  upwards  of  250,  most  of  whom  are  year  round  residents. 
They  now  occupy  most  of  the  available  houses  in  the  village ;  some  are  forced  to 
live  as  far  away  as  North  Falmouth  and  Hyannis.  As  a  result  of  this  crowding 
we  shall  be  unable,  in  the  summer  of  1946,  to  take  care  of  more  than  375  investi- 
gators and  students — that  is,  about  100  less  than  our  pre-war  average.  Indeed, 
we  can  accommodate  this  number  only  because  the  authorities  of  the  U.  S.  Fish 
and  Wild  Life  Commission  have  granted  us  the  use  of  a  part  of  the  Fisheries 
residence.  For  their  cooperation  in  this,  and  in  many  other  ways,  the  Laboratory 
is  grateful. 

When  it  is  possible  once  more  to  build  houses,  some  of  this  pressure  for  living 
space  will  be  relieved.  To  encourage  investigators  to  have  homes  here  in  Woods 
Hole,  the  Laboratory  has  opened  up  the  Devil's  Lane  tract,  situated  a  mile  and 
more  from  the  center  of  the  village,  between  the  State  Road  to  Falmouth  and  the 
railroad.  About  100  lots  will  presently  be  available. 

In  the  meantime,  the  number  of  applicants  for  research  space  will  undoubtedly 
increase,  and  we  shall  be  unable  to  find  places  for  all  cmalified  investigators  who 
wish  to  come.  The  Administration  thus  faces  the  unwelcome  prospect  of  having 
to  choose  between  applicants.  The  Executive  Committee  has  ruled  that  investi- 
gators, instructors,  and  students  should  have  preference  over  Library  readers  in 
the  residences  and  at  the  Mess.  But  some  further  method  of  selection  must  be 
followed  until  the  housing  shortage  is  relieved. 

4.  Financial  Problems 

The  report  of  the  Treasurer  shows  that  our  financial  condition  is  sound;  that 
is,  we  are  free  from  debt,  and  have  about  $57,000  in  the  Reserve  and  Current  Cash 
accounts.  But  most  of  this  has  already  been  ear-marked  to  pay  for  the  most 
necessary  repairs,  and  for  foreign  journals  not  yet  delivered.  We  shall  still  need 
a  larger  amount  for  other  needed  repairs  and  replacements.  When  these  have 
been  made  we  can  say  that  our  regular  income  from  all  sources  is  sufficient  to 
maintain  the  Laboratory  on  its  present  basis.  But  in  order  to  expand  our  research 
facilities  we  must  have  additional  funds.  It  is  estimated  that  $30,000  each  year 
should  be  spent  for  this  purpose. 

5.  Gifts 

Mr.  Allen  R.  Memhard  has  provided  a  fund  of  $1,000,  the  income  of  which 
may  be  awarded  to  a  qualified  student  who  has  completed  the  Embryology  course. 

Mrs.  Adele  K.  Strieker  has  presented  to  the  Laboratory  the  sum  of  $50  in 
memory  of  her  son,  Capt.  George  J.  Strieker,  who  worked  here  during  the  sum- 
mers of  1933  and  1934. 

Dr.  A.  C.  Redfield  contributed  $100  for  a  hedge  and  trees  to  be  planted  to  the 
east  of  the  Stone  Building. 

Donations  for  current  purposes  received  during  the  year  were  as  follows : 
Mrs.  E.  B.  Meigs,  $25.00;  Dr.  William  D.  Curtis,  $100.00;  M.  B.  L.  Associates, 
$630.00. 


REPORT  OF  THE  DIRECTOR  13 

6.  Deaths 

This  year  \ve  have  sustained  irreparable  losses  by  death ;  Dr.  T.  H.  Morgan, 
Trustee  since  1897,  whose  scientific  achievements  and  devotion  to  this  Laboratory 
from  its  earliest  days  contributed  greatly  to  its  growth  in  usefulness  and  influence, 
and  Dr.  C.  E.  McClung,  elected  Trustee  in  1913,  active  in  all  Laboratory  affairs, 
especially  in  the  building  up  of  our  great  Library. 

7.  Election  'of  Trustees 

At  the  meeting  of  the  Corporation,  held  August  14,  1945.  the  following  were 
elected  Trustees  Emeriti:  Dr.  F.  P.  Knowlton,  elected  Trustee  in  1922;  Dr.  R.  S. 
Lillie,  elected  Trustee  in  1921. 

The  following  were  elected  Trustees:  Dr.  P.  B.  Armstrong,  Professor  of 
Anatomy,  College  of  Medicine,  Syracuse  University ;  Dr.  A.  K.  Parpart,  Associate 
Professor  of  Biology,  Princeton  University. 

8.  Publications 

The  Executive  Committee  voted  to  print  in  this  Report  a  list  of  papers,  based 
wholly  or  in  part  on  work  done  at  this  Laboratory,  and  published  during  the  years 
1941-1945.  A  similar  list,  which  appeared  in  the  Annual  Report  of  1908,  covered 
the  years  from  the  beginning  of  the  Laboratory  in  1888  to  1907.  It  is  hoped  that 
eventually  a  complete  compilation  of  titles  to  include  the  intervening  years  may 
be  made. 

Appended  as  parts  of  this  Report  are : 

1.  Publications  from  this  Laboratory  during  the  years   1941-1945. 

2.  The  Staff. 

3.  Investigators  and  Students. 

4.  Tabular  View  of  Attendance,  1941-1945. 

5.  Subscribing  and  Cooperating  Institutions. 

6.  Evening  Lectures. 

7.  Shorter  Scientific  Papers. 

8.  Members  of  the  Corporation. 

Respectfully  submitted, 

CHARLES  PACKARD, 

Director 

( 

1.  PUBLICATIONS  FROM   THE  MARINE  BIOLOGICAL  LABORATORY,    1941-1945 

Note:  An  asterisk  before  a  title  indicates  that  the  work  was  done  only  in  part  at  this  Laboratory. 

ABELL,  R.  G.  On  the  comparative  permeability  of  blood  capillary  sprouts,  newly  formed  capil- 
laries, and  older  capillaries.  Anat.  Rcc.,  82:  1942. 

ABELL,  R.  G.     See  also  Zweifach,  Abell,  Chambers,  and  Clowes,  1945. 

ABELL,  R.  G.  AND  I.  H.  PAGE.  *Behavior  of  the  arterioles  in  hypertensive  rabbits  and  in  normal 
rabbits  following  injections  of  angiotonin.  Biol.  Bull.,  81:  1941  (abs.). 

ABELL,  R.  G.  AND  I.  H.  PAGE.  *The  reaction  of  peripheral  blood  vessels  to  angiotonin,  renin, 
and  other  pressor  agents.  Jour.  E.rp.  Med.,  75 :  1942. 

ADDISON,  W.  H.  F.  *The  distribution  of  elastic  tissue  in  the  arterial  pathway  to  the  carotid 
bodies  in  the  adult  dog.  Bio!.  Bull.,  81:  1941  (abs.). 

ADDISON,  W.  H.  F.  *Histologic  methods  adapted  for  rat  tissues.  A  Chapter  in  "The  Rat  in 
Laboratory  Investigation,"  Lippincott,  1942. 

ADDISON,  W.  H.  F.     The  hypophysis  of  the  goose-fish.     Anat.  Rcc..  85:  1943  (abs.). 


14  MARINE  BIOLOGICAL  LABORATORY 

ADDISON,  W.  H  F.     *The  extent  of  the  carotid  pressoreceptor  area  in  the  cat  as  indicated  by 

its  special  elastic-tissue  wall.     Anat.  Rec.,  91 :  1945. 
ADDISON,  W.  H.  F.     *The  arterial  relations  of  the  glomus  caroticum  in  the  rabbit.     Anat.  Rec., 

88:  1944  (abs.). 
ALBAUM,  H.  G.  AND  BARRY  COMMONER.     The  relation  between  the  four-carbon  acids  and  the 

growth  of  bat  seedlings.     Blol.  Bull,  80:  1941. 

ALLEE,  W.  C.     *Integration  of  problems  concerning  protozoan  populations  with  those  of  gen- 
eral biology.    Amer.  Nat.,  75:  1941. 
ALLEE,  W.  C.  AND  RUTH  M.  MERWIN.     The  effect  of  carbon  dioxide  on  the  rate  of  cleavage 

in  frog's  eggs.    Anat.  Rec.  Suppl,  81:  1941   (abs.). 
ALLEE,  W.  C.  AND  RUTH  M.  MERWIN.     The  effect  of  low  concentration  of  carbon  dioxide  on 

the  cleavage  rate  in  frog's  eggs.    Ecology,  24:  1943. 
ALLEE,  W.  C,  A.  J.  FINKEL  AND  H.  R.  GARNER.     Factors  affecting  rate  of  cleavage  in  Arbacia; 

the  accelerating  action  of  copper.     Anat.  Rec.  SuppL,  81:  1941   (abs.). 
ALLEE,  W.  C.,  A.  J.  FINKEL  AND  H.  R.  GARNER.     Copper  and  the  acceleration  of  cleavage. 

Jour.  Cell.  Comp.  Physiol,  20:  1942. 
ALLEE,  W.  C.,  A.  J.  FINKEL,  H.  R.  GARNER,  R.  M.  MERWIN  AND  G.  E.  EVANS.     Some  effects 

of  homotypic  extracts  on  the  rate  of  cleavage  of  Arbacia  eggs.     Biol.  Bull.,  83 :  1942. 
ALLEE,  W.  C.  AND  MARJORIE  B.  DOUGLIS.     A  dominance  order  in  the  hermit  crab,  Pagurus 

longicarpus  Say.    Ecology,  26 :  1945. 
ALSUP,  F.  W.     Photodynamic  action  in  the  eggs  of  Nereis  limbata.     Jour.  Cell.  Comp.  Physiol., 

17:  1941. 

ALSUP,  F.  W.     Photodynamic  studies  on  Arbacia  eggs.     Biol.  Bull.,  81  :   1941. 
ALSUP,  F.  W.     The  effects  of  light  alone  and  photodynamic  action  on   relative  viscosity  of 

Amoeba  protoplasm.    Physiol.  Zool.,  15:  1942. 
ANDERSON,  T.  F.     *The  study  of  colloids  with  the  electron  microscope.    Advances  in   Colloid 

Science,  1 :  1942. 
ANDERSON,  T.  F.     *The  application  of  the  electron  microscope  to  biology.     Collecting  Net,  17 : 

1942. 
ANDERSON,  T.  F.     See  also  Harvey,  E.  B.  and  Anderson,  1943;  Luria,  Delbruck  and  Anderson, 

1943 ;  Richards,  Steinbach  and  Anderson,  1943. 
ANDERSON,  T.  F.  AND  A.  G.  RICHARDS.     Nature  through  the  electron  microscope.    Scientific 

Month.,  55 :  1942. 
ANDREW,  WARREN.     The  reticular  nature  of  glia  fibers  in  the  cerebrum  of  the  frog  and  in  the 

higher  vertebrates.    Jour.  Comp.  Neural.,  79 :  1943. 
ANGERER,  C.  A.    Sec  also  Hartman,  Lewis,  Brownell,  Sheldon  and  Angerer.     Physiol.  Zool., 

17:  1944. 
ANGERER,  C.  A.  AND  H.  ANGERER.     *Weight  variations  of  muscles  of  adrenalectomized  frogs  in 

normal  and  hypotonic  Ringer  solutions.     Amer.  Jour.  Physiol.,  133:   1941. 
ANGERER,  C.  A.  AND  K.  M.  WILBUR.     *The  action  of  various  types  of  electric  fields  on  the 

relative  viscosity  of  plasmagel  of  Amoeba  proteus.     Physiol.  Zool.,  16 :   1943. 
BAILEY,  BASIL,  P.  KORAN  AND  H.  C.  BRADLEY.     The  autolysis  of  muscle  of  highly  active  and 

less  active  fish.     Biol.  Bull,  83 :  1942. 

BAKER,  GLADYS  E.     *Studies  in  the  genus  Physalacria.     Bull.  Torrey  Bot.  Club,  68 :  1941. 
BAKER,  H.  D.     *Notes  on  Salasiella  from  Mexico.    Nautilus,  54:   1941. 
BAKER,  H.  D.     *Zonitid  snails  from  Pacific  Islands.     Bull.  Bishop  Museum,  Honolulu,   166: 

1941. 

BAKER,  H.  D.     *Some  Haplotrematidae.    Nautilus,  54:  1941. 
BAKER,  H.  D.     *Puerto  Rican  Oleacinidae.      Nautilus,  55  :  1941. 
BAKER,  H.  D.     ^Outline  of  American  Oleacininae  and  new  species  from   Mexico.    Nautilus, 

55:  1941. 

BAKER,  H.  D.     *A  new  genus  of  Mexican  helicids.    Nautilus,  56:   1942. 
BAKER,  H.  D.     *A  new  genus  of  Chinese  Microcystinae.    Nautilus,  56 :  1942. 
BAKER,  H.  D.     *Some  Antillean  helicids.    Nautilus,  56:  1943. 

BAKER,  H.  D.     *The  mainland  genera  of  American  Oleacininae.    Proc.  Acad.  Nat.  Sci.,  Phila- 
delphia, 95 :  1943. 

BAKER,  R.  F.    See  Cole  and  Baker,  1941. 
BALL,  E.  G.     A  blue  chromoprotein  found  in  the  eggs  of  the  goose-barnacle.     Jour.  Biol.  Chcm., 

152:  1944. 


REPORT  OF  THE  DIRECTOR  15 

BALL,  ERNEST.     The  effects  of  synthetic  growth  substances  on  the  shoot  apex  of  Tropaeolum 

majus.     Amer.  Jour.  Bot.,  31  :  1944. 
BALLARD,  W.  W.     The  mechanism   for  synchronous   spawning   in   Hydractinia   and   Pennaria 

B iol  Bull,  82  :  1942. 

BALLENTINE,  ROBERT.     See  Parpart  and  Ballentine,  1943. 
BARNES,  W.  A.  AND  O.  B.  FURTH.     Roentgen  rays  in  single  and  parabiotic  mice.    Amer.  Jour. 

Roentgenol.  and  Radium  Therap.,  69:   1943. 
BARRON,  E.  S.  G.  AND  J.  M.  GOLDINGER.     Pyruvate  metabolism  in  fertilized  and  non-fertilized 

sea  urchin  eggs.     Biol.  Bull.,  81  :  1941. 

EARTH,  L.  G.     *Neural  differentiation  without  organizer.    Jour.  Exp.  Zool.,  87:  1941. 
BARTH,  L.  G.     *Oxygen  consumption  of  the  amphibian  gastrula.     Physiol.  Zool.,  15 :  1942. 
BARTH,  L.  G.     *Colloid  chemistry  of  development.     A  chapter  in  Alexander's  Colloid  Chem- 
istry.    New  York,  1944. 
BARTH,  L.  G.     The  determination  of  the  regenerating  hydranth  in  Tubularia.    Physiol.  Zool., 

17:  1944. 

BARTH,  L.  G.     Sec  also  Goldin  and  Earth,  1941. 

BARTLETT,  J.  H.     *Transient  Anode  Phenomena.     Trans.  Electrochem.  Soc.,  87  :   1945. 
BARTLETT,  J.  H.     *Periodic  phenomena  at  anodes.     Phys.  Rev.,  67 :  1945. 
BEAMS,  H.  W.     See  Evans,  Beams  and  Smith,  1941. 
BEERS,  C.  D.     The  role  of  bacteria  in  the  excystment  of  the  ciliate  Didinium  nasutum.     Biol. 

Bull.,  89:  1945  (abs.). 
BELDA,  W.  H.     *Permeability  to  water  in  Pelomyxa  carolinensis.     1.   Changes   in  volume  of 

P.  carolinensis  in  solutions  of  different  osmotic  concentration.     The  Salesianum,  37:   1942. 
BELDA,  W.  H.    2.  The  contractile  vacuoles  of  P.  carolinensis.     The  Salesianum,  37  :  1942. 
BELDA,   W.   H.     3.  The  permeability  constant  for   water   in   P.   caroliensis.     The  Salesianum, 

38:  1943. 
BERGER,  C.  A.     *Some  criteria  for  judging  the  degree  of  polyploidy  of  cells  in  the  resting  stage. 

Amer.  Nat.,  75:  1941. 

BERGER,  C.  A.     *Reinvestigation  of  polysomaty  in  Spinacia.     Bot.  Gaz.,  102:   1941. 
BERGER,  C.  A.     ^Multiple  chromosome  complexes  in  animals  and  polysomaty  in  plants.     Cold 

Spring  Harbor  Symposia,  9:  1941. 

BERGER,  C.  A.     *Experimental  studies  on  the  cytology  of  Allium.     Torreya  (abs.)  44 :  1944. 
BERGER,  C.  A.  AND  E.  R.  WITKUS.     *A  cytological  study  of  c-mitosis  in  the  polysomatic  plant 

Spinacia  oleracea,  with  comparative  observations  on  Allium  cepa.     Bull.  Torrey.  Bot.  Club, 

70f:  1943. 
BERGER,  C.  A.  AND  E.  R.  WITKUS.     *Veratrine,  a  new  polyploidy  inducing  agent.    Jour.  Hered., 

35:  1944. 
BERGER,  C.  A.,  E.  R.  WITKUS  AND  B.  J.  SULLIVAN.     *The  cytological  effects  of  benzene  vapor. 

Bull.  Torrey  Bot.  Club,  71  :  1944. 
BERTHOLF,  L.  M.     Accelerating  metamorphosis  in  the  tunicate,  Styela  partita.     Biol.  Bull.,  89 : 

1945  (abs.). 
BERTHOLF,  L.  M.  AND  S.  O.  MAST.     Metamorphosis  in  the  larva  of  the  tunicate,  Styela  partita. 

Biol.  Bull.,  87:  1944  (abs.). 
BEVELANDER,  GERRIT.     ^Radioactive  phosphate  absorption  by  dentin  and  enamel.    Jour.  Dent. 

Res.,  24:  1945. 
BEVELANDER,  GERRIT.     *The  histochemical  localization  of  alkaline  phosphatase  in  the  developing 

tooth.     Jour.  Cell.  Comp.  Physiol.,  26 :  1945. 
BEVELANDER,  GERRIT.     *The  localization  of  phosphatase  in  the  cyclic  growth  of  the  hair,.    Anat. 

Rcc.,  91 :  1945. 
BIRMINGHAM,   LLOYD.     Regeneration   in  the   early   zooid   of   Amaroucium   constellatum.     Biol. 

Bull,  81:  1941   (abs.). 
BLISS,  A.  F.     Derived  photosensitive  pigments   from   invertebrate   eyes.     Jour.   Gen.   Physiol, 

26:  1943. 
BOCHE,  R.  D.  AND  J.  B.  BUCK.     Studies  on  the  hydrogen  ion  concentration  of  insect  blood  and 

their  bearing  on  in  vitro  cytological  technique.     Physiol.  Zool,  15 :  1942. 
BODIAN,  DAVID.     *Cytological  aspects  of  synaptic  function.     Physiol.  Rev.,  22 :   1942. 
BODIAN,   DAVID.     *Poliomyelitic  changes  in  tnultinucleated  neurons,  with  special   reference  to 

the  site  of  action  of  virus  in  the  cell.     Bull.  Johns  Hopkins  Hosp.,  77  :  1945. 


16  MARINE  BIOLOGICAL  LABORATORY 

BODIAN,  DAVID  AND  R.  C.  MELLORS.     *The  regenerative  cycle  of  motor  neurons,  with  special 

reference  to  phosphatase  activity.    Jour.  E.\-p.  Mcd.,  81 :  1945. 
BOELL,  E.  J.  AND  L.  L.  WOODRUFF.     Respiratory  metabolism  and  mating  types  in  Paramoecium 

calkinsi.    Jour.  E.vpcr.  Zoo!.,  87:  1941. 
BOTSFORD,  E.  FRANCES.     The  effect  of  physostigmine  on  the  responses  of  earthworm  body  wall 

preparations  to  successive  stimuli.     Biol.  Bull.,  80:  1941. 
BRADLEY,  H.  C.    Sec  Bailey,  Koran  and  Bradley,  1942. 
BROOKS,  M.  M.     *Infrared  spectrophotometric   studies   on  hemoglobin   as  affected  by   cyanide, 

methylene  blue,  and  carbon  monoxide.     Amcr.  Jour.  PhysioL,  132:   1941. 
BROOKS,  M.  M.     Effects  of  CO  and  methylene  blue  upon  O~  consumption  of  shark  blood.     Proc. 

Soc.  Exp.  Biol.  Mcd.,  46:  1941. 

BROOKS,  M.  M.     Interpretations  of  effects  of  CO  and  CN  on  oxidations  in  living  cells.     Col- 
lecting Net,  16:'  1941. 
BROOKS,  M.  M.     Further  interpretations  of  the  effects  of  CO  and  CN  on  oxidations  in  living 

cells.     Biol.  Bull.,  81:  1941. 

BROOKS,  M.  M.     The  effect  of  thiamine  chloride  on  the  oxygen  consumption  and  the  develop- 
ment of  Arbacia  punctulata  at  different  stages.     Biol.  Bull.,  83 :  1942. 
BROOKS,  M.  M.     Mechanism  of  fertilization  of  eggs.     Federation  Proc.,  2 :  1943. 
BROOKS,   M.   M.     Methylene  blue,  potassium  cyanide,   and  carbon   monoxide  as   indicators   for 
studying   the   oxidation-reduction   potentials   of   developing   marine   eggs.     Biol.    Bull.,   84 : 
1943. 
BROOKS,  M.  M.     *E1  mecanismo  de  accion  del  azul  de  metileno  en  las  celulas  vivas.     Adas 

Acad.  Nacional  de  Ciensa  c.ractas,  fisicas  y  naliialcs  de  Lima,  Peru,  7  :  1944. 
BROOKS,   M.   M.     *The  effect   of  methylene  blue   on   performance   efficiency   at   high   altitudes. 

Jour.  Aviation  Mcd.,  16:  1945. 
BROOKS,   M.   M.     *Oxidation-reduction   studies   on   Penicillium   notatum   and   other   organisms. 

Biol.  Bull.,  89 :  1945. 
BROOKS,  M.  M.     Electrode  potential  measurements  of  Penicillium  notatum.     Federation  Proc., 

4:  1945. 

BROOKS,  M.  M.     Mechanism  of  fertilization  of  eggs.    Federation  Proc.,  5:  1945. 
BROOKS,  M.  M.  AND  S.   C.  BROOKS.     ^Permeability  of  Living   Cells.     Gebriider   Borntraeger, 

Berlin.     Preprinted  by  Edwards  Bros.,  1945. 
BROOKS,  S.  C.     Intake  and  loss  of  phosphate  ions  by  eggs  and  larvae  of  Arbacia  and  Asterias. 

Biol.  Bull.,  83  :  1942. 

BROOKS,  S.  C.     Intake  and  loss  of  ions  by  living  cells.     1.  Eggs  and  larvae  of  Arbacia  punctu- 
lata and  Asterias  forbesi  exposed  to  phosphate  and  sodium  ions.     Biol.  Bull.,  84:  1943. 
BROOKS,  S.  C.     2.  Early  changes  of  phosphate  content  of  Fundulus  eggs.     Biol.  Bull.,  84 :  1943. 
BROOKS,  S.  C.     *The  permeability  of  cells.     Science,  100:  1944. 
BROOKS,  S.  C.     Sec  also  Brooks  and  Brooks,  1945. 
BROWN,  D.  E.  S.     Sec  Marsland  and  Brown,  1941,  1942;  Marsland,  Johnson  and  Brown,  1941, 

1942;  Hiatt.  Brown,  Quinn  and  MacDuffie,  1945. 
BROWN,  F.  A.  AND  O.  CUNNINGHAM.     Upon  the  presence  and  distribution  of  a  chromatophoro- 

tropic  principle  in  the  central  nervous  system  of  Limulus.     Biol.  Bull.,  81 :  1941. 
BROWN,  F.  A.  AND  V.  J.  WULFF.     Chromatophore  types  in  Crago  and  their  endocrine  control. 

Jour.  Cell.  Comp.  PhysioL,  18:  1941. 
BROWNELL,   K.   A.     Sec   Hartman,   Lewis,   Brownell,    Shelden   and   Walther,    1941 ;    Hartman, 

Lewis,  Brownell,  Angerer  and  Shelden,  1944. 

BUCHSBAUM,  RALPH  AND  R.  WILLIAMSON.     The  rate  of  elongation  and  constriction  of  dividing 
sea  urchin  eggs  as  a  test  of  a  mathematical  theory  of  cell  division.     PhysioL  ZooL,  16 :  1943. 
BUCK,  J.  B.     *Micromanipulation  of  salivary  gland  chromosomes.    Jour.  Hered.,  33 :  1942. 
BUCK,  J.  B.    Sec  also  Boche  and  Buck,  1942. 
BUCK,  J.  B.  AND  A.  M.  MELLAND.     ^Methods  for  isolating,  collecting,  and  orienting  salivary 

gland  chromosomes  for  diffraction  analysis.     Jour.  Hercd.,  33:   1942. 

BUDINGTON,  R.  A.     The  ciliary  transport-system  of  Asterias  forbesii.     Biol.  Bull.,  83 :  1942. 
BULLOCK,  T.  H.     Neuromuscular  facilitation  in  scyphomedusae.    Jour.   Cell.   Comp.  PhysioL, 

22:  1943. 

BULLOCK,  T.  H.     The  giant  nerve  fiber  system  in  balanoglossids.     Jour.   Comp.  Neural. ,  80: 
1944. 


REPORT  OF  THE  DIRECTOR  17 

BULLOCK,  T.  H.     Problems  in  the  comparative  study  of  brain  waves.     Yale  Jour.  Biol.  Mcd., 

17:  1945. 
BULLOCK,  T.  H.     Organization  of  the  giant  nerve  fiber  system  in  Neanthes  virens.    Biol.  Bull., 

89 :  1945. 
'BULLOCK,  T.  H.     Anatomical  organization  of  the  nervous  system  of  Enteropneusta.     Quart.  Jour. 

Micr.  Set.,  86 :  1945. 
BULLOCK,    T.    H.   AND    D.    NACHMANSOHN.     Choline   esterase    in    primitive    nervous    systems. 

Jour.  Cell.  Comp.  Physiol,  20:  1942. 
CABLE,  R.  M.    Sec  also  Hunninen  and  Cable,  1941,  1943. 

CABLE,  R.  M.  AND  A.  V.  HUNNINEN.     Studies  on  Deropristis  inflata,  its  life  history  and  affi- 
nities to  trematodes  of  the  family  Acanthocolpidae.     Biol.  Bull..  82 :   1942. 
CABLE,  R.  M.  AND  A.  V.  HUNNINEN.     Studies  on  the  life  history  of  Siphodera  vinaledwardsii. 

Jour.  Parasitol.,  28 :  1942. 
CAHEN,  R.  L.     *The  effects  of  morphine  on  the  cortical  activity  of  the  rat.     Yale  Jour.  Biol. 

Mcd.,  16:  1944. 
CAHEN,  R.  L.     *The  influence  of  morphine  on  tissue  permeability  and  the  spreading  effect  of 

hyaluronidase.     Yale  Jour.  Biol.  Mcd.,  16 :  1944. 
CAHEN,  R.  L.     *Urinary  17  ketosteroids  in  metabolism.     1.  Standardized  chemical  estimation. 

Jour.  Biol.  Chem.,  152:  1944. 
CANNAN,  R.  K.     *The  hydrogen  ion  dissociation  curve  of  beta-lactoglobulin.     Jour.  Biol.  Chem., 

142:  1942. 
CANNAN,  R.  K.     *The  dicarboxylic  amino  acids  in  protein  hydrolysates.    Jour.  Biol.  Chem., 

152:  1944. 
CARSON,  H.  L.     A  comparative  study  of  the  apical  cell  of  the  insect  testis.     Jour.  Morph.,  77 : 

1945. 
CHAMBERS,  ROBERT.     The  intrinsic  expansibility  of  the  fertilization  membrane  of  echinoderm 

ova.    Jour.  Cell.  Comp.  Physiol.,  19:  1942. 
CHAMBERS,   ROBERT.     Electrolytic   solutions  compatible   with   the   maintenance   of  protoplasmic 

structures.     Biol.  Symposia,  10:  1943. 

CHAMBERS,  ROBERT.     Post  war  Biology  rehabilitation.     Science.  100:   1944. 
CHAMBERS,  ROBERT.     Some  physical  properties  of  protoplasm.     Chapter  in  Alexander's  Colloid 

Chemistry,  1944. 
CHAMBERS,   ROBERT.     ^Rehabilitation  of  the  biological   sciences   in  the   post-war  period.     Am. 

Nat.,  79:  1945. 
CHAMBERS,  ROBERT,  B.  W.  ZWEIFACH,  B.  E.  LOWENSTEIN  AND  R.  E.  LEE.     Vaso-excitor  and 

vaso-depressor  substances  as  "toxic"  factors  in  experimentally  induced  shock.     Proc.  Soc. 

Exp.  Biol.  Med.,  56:  1944. 
CHAMBERS,  ROBERT,  B.  W.  ZWEIFACH  AND  B.  E.  LOWENSTEIN.     ^Circulatory  reactions  of  rats, 

traumatized  in  the  Noble-Collip  drum.     Am.  J.  Physiol.,  139:  1943. 
CHAMBERS,  ROBERT,  B.  W.  ZWEIFACH   AND  B.  E.  LOWENSTEIN.     *The  peripheral  circulation 

during  the  tourniquet  syndrome  in  the  rat.     Ann.  Surg.,  120:  1944. 
CHASE,  A.  M.     *Effect  of  azide  on  Cypridina  luciferin.     Collecting  Net,  16 :  1941. 
CHASE,  A.  M.     Observations  on  luminescence  in  Mnemiopsis.     Biol.  Bull.  81:  1941   (abs.). 
CHASE,  A.  M.     *The  reaction  of  Cypridina  luciferin  with  azide.     Jour.  Cell  Comp.  Physiol.,  19 : 

1942. 
CHASE,  A.  M.     *The  absorption  spectrum  of  luciferin  and  oxidized  luciferin.     Jour.  Biol.  Chem., 

150:  1943. 

CHASE,  A.  M.     *The  visible  absorption  band  of  reduced  luciferin.     Jour.  Biol.  Chem.,  159:  1945. 
CHENEY,  R.  H.     Myofibrillar  modifications  induced  by  caffeine  in  cardiac  muscle  of  the  frog. 

Jour.  Comp.  Physiol.,  18:  1941. 
CHENEY,  R.  H.     Caffeine  effect  in  fertilization  and  development  of  Arbacia  eggs.     Biol.  Bull., 

83:  1942  (abs.). 

CHENEY,  R.  H.     Oxygen  consumption  of  caffeinized  Arbacia  eggs.     Biol.  Bull.,  83:  1942.  (abs.). 
CHENEY,  R.  H.     Development  of  Arbacia  eggs  in  caffeinized  sea  water.    Anat.  Rec.,  84:  1942 

(abs.). 
CHENEY,  R.  H.     Inhibitory  effect  of  caffeine  on  oxygen  consumption  in  Arbacia  eggs.     Anat. 

Rec.,  84:  1942  (abs.). 
CHENEY,  R.  H.     *Variation  in  reproductive  phenomena  by  caffeine.    Federation  Proc.,  3 :  1944 

(abs.). 


18  MARINE  BIOLOGICAL  LABORATORY 

CHENEY,  R.  H.  The  effects  of  caffeine  on  oxygen  consumption  and  cell  division  in  the  fertilized 
egg  of  the  sea  urchin.  Jour.  Gen.  Physiol.,  29:  1945. 

CHURNEY,  LEON.     The  osmotic  properties  of  the  nucleus.     Biol.  Bull.,  82 :  1942. 

CLAFF,  C.  L.  *Glass  electrode  for  determination  of  hydrogen  ion  activity  of  small  quantities 
of  culture  media.  Science,  94:  1941. 

CLAFF,  C.  L.,  VIRGINA  C.  DEWEY  AND  G.  W.  KIDDER.  *Feeding  mechanisms  in  three  species 
of  Bresslaua.  Biol.  Bull.,  81  :  1941. 

CLAFF,  C.  L.  AND  O.  SWENSON.  *Micro  glass  electrode  technique  for  determination  of  hydrogen- 
ion  activity  of  blood  and  other  biological  fluids.  Jour.  Biol.  Chcin.,  152:  1944. 

CLARK,  E.  R.  AND  ELEANOR  L.  CLARK.  ^Microscopic  observations  on  the  formation  of  cartilage 
and  bone  in  the  living  mammal.  Amcr.  Jour.  Anat.,  70:  1942. 

CLARK,  E.  R.  AND  ELEANOR  L.  CLARK.  *Caliber  changes  in  minute  blood  vessels  observed  in 
living  mammal.  Amer.  Jour.  Anat.,  73:  1943. 

CLARK,  E.  R.  AND  ELEANOR  L.  CLARK     *The  formation  of  venae  comites.    Anat.  Rec.,  85:  1943. 

CLARK,  E.  R.  AND  ELEANOR  L.  CLARK.  *Growth  and  behavior  of  epidermis  as  observed  mi- 
croscopically in  the  living,  in  chambers  introduced  in  the  rabbit's  ear.  Anat.  Rcc.,  88 :  1944. 

CLOWES,  G.  H.  A.  *Interactions  of  biologically  significant  substances  in  surface  films,  with 
especial  reference  to  two-dimensional  solutions  and  association  complexes  formed  by  car- 
cinogenic hydrocarbons  and  sterols.  Publication  21.  Amer.  Assoc.  Advance.  Sci.,  1942. 

CLOWES,  G.  H.  A.  See  also  Krahl,  Keltch,  Neubeck  and  Clowes,  1941 ;  Keltch,  Baker,  Krahl 
and  Clowes,  1941;  Davis,  Baker  and  Clowes,  1941,  1942;  Krahl,  Jandorff  and  Clowes,  1942; 
Powell,  Krahl  and  Clowes,  1942;  Hutchens,  Keltch,  Krahl  and  Clowes,  1942;  Zweifach, 
Abell,  Chambers  and  Clowes,  1945. 

COLE,  K.  S.     Rectification  and  inductance  in  the  squid  giant  axon.     Jour.  Gen.  Ph\siol.,  25:  1941. 

COLE,  K.  S.     See  also  Guttman  and  Cole,  1941 ;  Curtis  and  Cole,  1942,  1944. 

COLE,  K.  S.  AND  R.  F.  BAKER.  Transverse  impedance  of  the  squid  giant  axon  during  current 
flow.  Jour.  Gen.  Physiol.,  24:  1941. 

COLE,  K.  S.  AND  R.  F.  BAKER.  Longitudinal  impedance  of  the  squid  giant  axon.  Jour.  Gen. 
Physiol.,  24 :  1941. 

COLE,  K.  S.  AND  H.  J.  CURTIS.  Membrane  potential  of  the  squid  giant  axon  during  current  flow. 
Jour.  Gen.  Physiol.,  24:  1941. 

COLE,  K.  S.  AND  H.  J.  CURTIS.  *Electrical  Physiology :  Electrical  resistance  and  impedance  of 
cells  and  tissues.  Chapter  in  Medical  Physics.  Chicago,  1944. 

COLE,  K.  S.  AND  R.  H.  COLE.  *Dispersion  and  absorption  in  dielectrics.  1.  Alternating  current 
characteristics.  Jour.  Cheni.  Phys.,2:  1941. 

COLE,  K.  S.  AND  R.  H.  COLE.     *2.  Direct  current  characteristics.     Jour.  Chcm.  Phys.  10 :  1942. 

COLWIN,  LAURA  H.  Binary  fission  and  conjugation  in  Urceolaria  synapta,  with  special  refer- 
ence to  the  nuclear  phenomena.  Jour.  Morph.,  75:  1944. 

COMMONER,  BARRY.     Sec  Albaum  and  Commoner,  1941. 

CORNMAN,  IVOR.  Characteristics  of  the  acceleration  of  Arbacia  cleavage  in  hypotonic  sea- 
water.  Biol.  Bull.  81:  1941  (abs.). 

CORNMAN,  IVOR.  Sperm  activation  by  Arbacia  egg  extractives,  with  special  reference  to 
echinoehrome.  Biol.  Bull.  80:  1941. 

CORNMAN,  IVOR.  Acceleration  of  cleavage  of  Arbacia  eggs  by  hypotonic  sea  water.  Biol.  Bull., 
84:  1943. 

COSTELLO,  D.  P.     ^Advances  in  Zoology  during  1940.    hid.  Engineer.  Chcm.,  19:  1941. 

COSTELLO,  D.  P.  Segregation  of  ooplasmic  constituents.  Jour.  Elisha  Mitchell  Sci.  Soc.,  61  : 
1945. 

COSTELLO,  D.  P.  Experimental  studies  in  germinal  localization  in  Nereis.  1.  The  development 
of  isolated  blastomeres.  Jour.  E.rp.  Zool.,  100:  1945. 

COSTELLO,  D.  P.  WITH  G.  I.  LAVIN.  Ultra  violet  photomicroscopy  of  the  Nereis  and  Asterias 
egg.  Anat.  Rcc.  Suppl.,  87:  1943.  (abs.). 

CROUSE,  HELEN  V.  *Translocations  in  Sciara ;  their  bearing  on  chromosome  behavior  and  sex 
determination.  Univ.  Missouri  Res.  Bull.,  379:  1943. 

CROUSE,  HELEN  V.     See  also  Ris  and  Crouse,  1945. 

CROWELL,  SEARS.  A  comparison  of  shells  utilized  by  Hydractinia  and  Podocoryne.  Ecology, 
26 :  1945. 

CUNNINGHAM,  O.     Sec  Brown  and  Cunningham,  1941. 


REPORT  OF  THE  DIRECTOR  19 

CURTIS,  H.  J.     See  also  Cole  and  Curtis,  1941. 

CURTIS,  H.  J.  AND  K.  S.  COLE.     Membrane  resting  and  action  potentials'  from  the  squid  giant 

axon.     Jour.  Cell.  Comfy.  Physiol.,  19:  1942. 
DAVSON-,  HUGH.     See  also  Shapiro  and  Davson,  1941. 
DAVSON,  HUGH   AND  J.   M.   REINER.     Ionic  permeability;   an  enzyme-like  factor  concerned  in 

the  migration  of  sodium  through  the  cat  erythrocyte  membrane.    Jour.  Cell.  Comp.  Physiol 

20:  1942. 

DELBRUCK,  M.  See  Luria,  Delbruck  and  Anderson,  1943. 

DENT,  J.  N.     *The  embryonic  development  of  Plethodon  cinereus  as  correlated  with  the  dif- 
ferentiation and  functioning  of  the  thyroid  gland.     Jour.  Morph.  71 :  1942. 
DENT,  J.  N.     See  also  Lynn  and  Dent,  1941. 
DEWEY,  VIRGINIA  C.      See  also  Claff,  Dewey  and  Kidder,  1941. 
DEWEY,  VIRGINIA  C.  AND  G.  W.  KIDDER.     The  possibility  of  thiamin  synthesis  by  ciliates.    Biol. 

Bull.,  81:  1941.  (abs.). 

DOUGLIS,  MARJORIE  B.     See  Alice  and  Douglis,  1945. 
DZIEMIAN,  A.  J.     *The  permeability  and  the  lipid  content  of  immature  red  cells.     Jour.  Cell. 

Comp.  Physiol.,  20 :  1942. 
EVANS,  T.  C.,  H.  W.  BEAMS,  AND  M.  E.  SMITH.     Effects  of  roentgen  radiation  on  the  jelly  of 

the  Arbacia  egg.     Biol.  Bull.  80:  1941. 
EVANS,  T.  C.  AND  J.  C.  SLAUGHTER.     Effect  of  sea  water  on  the  radiosensitivity  of  Arbacia 

sperm.     Biol.  Bull.,  81 :  1941   (abs.). 
EVANS,  T.  C.  AND  J.  C.  SLAUGHTER,  E.  P.  LITTLE  AND  G.  FAILLA.     Influence  of  the  medium  on 

radiation  injury  of  sperm.     Radiology,  39:  1942. 
FAILLA,  G.     See  Evans,  Slaughter,  Little  and  Failla,   1942. 

FAUST,  E.  C.     *Clinical  Parasitology.     Co-author  with  C.  F.  Craig,  Lea  and  Febiger,  1945. 
FINKEL,  A.  J.  See  Alice,  Finkel  and  Garner,  1941,   1942;  Alice,  Finkel,  Garner,  Merwin  and 

Evans,  1942 
FISHER,  K.  C.     The  fractionation  of  cellular  respiration  by  the  use  of  narcotics.     Biol.  Bull.,  81 : 

1941  (abs.). 

FREEDMAN,  W.  B.  AND  R.  WALKER.  *Size,  development,  and  innervation  of  labyrinth  sensory 
areas  in  Squalus.  Jour.  Comp.  Neur.,  77  :  1942. 

FREIS,  E.  F.  B.  Some  neurohumoral  evidence  for  double  innervation  of  xanthophores  in  kill- 
fish.  Biol.  Bull.,  82  :  1942. 

FRISCH,  J.  A.  *The  rate  of  pulsation  of  the  posterior  contractile  vacuole  in  Paramoecium 
woodruffi  and  P.  calkinsi.  Anat.  Rec.  89:  1944  (abs.). 

FRISCH,  J.  A.     *The  rate  of  adaptation  of  P.  caudatum  to  sea  water.     Anat.  Rec.  89:  1944  (abs.). 

FROEHLICH,  ALFRED.     The  influence  of  drugs  on  heat  narcosis.     Biol.  Bull.  89:  1945   (abs.). 

FURTH,  JACOB.     *The  teaching  of  experimental  pathology.      Arch.  Path.,  34 :  1942. 

FURTH,  JACOB.     Neoplastic  growth.     Ann.  Rev.  Physiol.,  6:  1944. 

FURTH,  JACOB,  R.  K.  COLE  AND  M.  C.  BOON.  *The  effect  of  maternal  influence  upon  spontane- 
ous leukemia  of  mice.  Cancer  Research,  2 :  1942. 

FURTH,  JACOB  AND  M.  C.  BOON.  ^Enhancement  of  leukemogenic  action  of  methylcholanthrene 
by  pre-irradiation  with  X-rays.  Science,  98  :  1943. 

FURTH,  JACOB,  M.  C.  BOON  AND  N.  KALISS.  *On  the  genetic  character  of  neoplastic  cells  as 
determined  in  transplantation  experiments :  with  notes  on  the  somatic  mutation  theory. 
Cancer  Research,  4  :  1944. 

GABRIEL,  M.  L.     The  effect  of  temperature  on  vertebral  variations  is  Fundulus.     Biol.  Bull.,  83 : 

1942  (abs.). 

GABRIEL,  M.  L.     Factors  affecting  the  number  and  form  of  vertebrae  in  Fundulus.    Jour.  Exp. 

Zoo!.,  95 :  1944. 
GALTSOFF,  P.  S.     Storage  and  distribution  of  manganese  in  Ostrea  virginica.     Collecting  Net,  16  : 

1941. 
GALTSOFF,  P.  S.     Accumulation  of  manganese  and  the  sexual  cycle  in  Ostrea  virginica.     Physiol. 

Zool.,  15 :  1942. 

GALTSOFF,  P.  S.     Reaction  of  the  oyster  to  free  chlorine.     Biol.  Bull.,  89:  1945   (abs.). 
GARNER,  H.  R.    See  Alice,  Finkel  and  Garner,  1941,  1942. 
CARREY,  W.  E.     Action  of  acetylcholine  on  the  heart  of  Limulus.     Amer.  Jour.  Physiol.,  133 : 

1941. 


20  MARINE  BIOLOGICAL  LABORATORY 

CARREY,  W.  E.     An  analysis  of  the  action  of  acetylcholine  on  the  cardiac  ganglion  of  Limulus. 

Amer.  Jour.  Physiol.  136:  1942. 

GATES,  R.  R.     Tests  of  nuceoli  and  cytoplasmic  granules  in  marine  eggs.     Biol.  Bull.,  81 :  1941. 
GATES,  R.  R.     *Nucleoli  and  phylogeny.     Collecting  Net,  17 :  1942. 
GATES,  R.  R.     *Chromosome  numbers  in  mammals  and  man.     Science,  96 :  1942. 
GATES,  R.  R.     *Symbols  for  human  genes.     Science,  95 :  1942.t 
GATES,  R.  R.     *Nucleoli  and  related  nuclear  structures.     Bot.  Rev.,  8 :  1942. 
GATES,  R.  R.     *Our  ancestors,  ourselves,  our  descendants.     Medical  Genetics  and  Eugenics,  2 : 

1943. 
GATES,  R.  R.     (with  G.  N.  PATHAK).     *Variations  in  the  offspring. of  tetraploid  Oenotheras. 

Amer.  Naturalist,  78  :  1944. 
GIDGE,  NATALIE  M.  -AND  S.  M.  ROSE.     The  role  of  larval  skin  in  promoting  limb  regeneration 

in  adult  Anura.    Jour.  Exp.  Zool.,  97 :  1944. 
GIESE,  A.  C.     *Studies  on  nutrition  of  dim  and  bright  variants  of  a  species  of  luminous  bacteria. 

Jour.  Bact.,  46:  1943. 
GIESE,  A.  C.     *The  action  of  azide  on  luminescence,  respiration,  and  growth  of  the  luminous 

bacteria.     Jour.  Cell.  Comp.  Physiol.,  26  :  1945. 

GIESE,  A.  C.     ""Ultraviolet  radiations  and  life.     Physiol.  Zool.,  18 :  1945. 
GIESE,  A.  C.     *Effects  of  ultraviolet  radiations  on  luminescence  and  respiration  of  Achromobac- 

ter  fischeri.     Jour.  Cell.  Comp.  Physiol.,  17:  1941. 
GIESE,  A.  C.  AND  E.  L.  TATUM.     *The  effect  of  some  vitamins  of  the  B-complex  on  respiration 

of  mutants  of  Neurospora.     Biol.  Bull.,  83:  1942  (abs.). 
GILBERT,  P.  W.    *The  urogenital  system  of  the  male  frilled  shark,  Chlamydoselachus  anguineus. 

Anat.  Rec.,  84:  1942  (abs.). 

GILBERT,  P.  W.     *The  morphology  of  the  male  urogenital  system  of  the  frilled  shark  Chlamy- 
doselachus anguineus.     Jour.  Morph.,  3:  1943. 
GILMAN,  L.  C.       *Mating  types  in  diverse  races  of  Paramoecium  caudatum.     Biol.  Bull.,  80: 

1941. 

GLASER,  O.  C.     Protein  metabolism  and  embryonic  growth  rate.     Biol.  Bull.,  83:  1942  (abs.). 
GOLDIN,  A.     Factors  influencing  regeneration  and  polarity  determination  in  Tubularia  crocea. 

Biol.  Bull,  82  :  1942. 

GOLDIN,  A.  A  quantitative  study  of  the  interrelationships  of  oxygen  and  hydrogen  ion  concen- 
tration in  influencing  Tubularia  regeneration.     Biol.  Bull.,  82 :  1942. 
GOLDIN,  A.     See  also  Spiegelman  and  Goldin,  1944. 
GOLDIN,  A.  AND  L.  G.  BARTH.     Regeneration  of  coenosarc  fragments  removed  from  the  stem 

of  Tubularia  crocea.     Biol.  Bull,  81 :  1941. 
GOLDINGER,  J.  M.     Sec  Barren  and  Goldinger,  1941. 
GOODCHILD,  CHAUNCEY.     *Additional  observations  on  the  life  history  of    Gorgodera  amplicava. 

Jour.  Parasitol,  31  :  1945. 
GOODRICH,  H.  B.,  N.  D.  JOSEPHSON,  J.  P.  TRINKHAUS  AND  JEANNE  M.  SLATE.     *The  cellular 

expression  and  genetics  of  two  new  genes  in  Lebistes  reticulatus.     Genetics,  29 :  1944. 
GRANICK,  SAM.    See  Michaelis  and  Granick,  1945. 

GRAVE,  B.  H.     The  sexual  cycle  of  the  shipworm,  Teredo  navalis.     Biol.  Bull,  82:  1942  (abs.). 
GRAVE,  CASWELL.     Further  studies  of  metamorphosis  of  ascidian  larvae.     Biol.  Bull,  81 :   1941 

(abs:). 
GRAVE,  CASWELL.     The  "eye  spot"  and  light  response  of  the  larva  of  Cynthia  partita.     Biol  Bull, 

81:  1941  (abs.). 

GRAVE,   CASWELL,  AND  S.   O.   MAST.     The  larva  of   Styela    (Cynthia)    partita;    structure,   ac- 
tivities, and  duration  of  life.    Jour.  Morph.,  75 :  1944. 
GRAY,  I.  E.     *Changes  in  weight  and  water  content  during  the  life  cycle  of  the  wood-eating 

beetle,  Passalus  cornutus.     Biol  Bull,  86:  1944. 
GROSCH,  D.  S.     *The  relation  of  cell  size  and  organ  size  to  mortality  in  Habrobracon.     Growth, 

9:  1945. 
GUTTMAN,  RITA.     *Action  of  potassium  and  narcotics  on  rectification  in  nerve  and  muscle. 

Jour.  Gen.  Physiol,  28  :  1944. 
GUTTMAN,  RITA  AND  K.   S.  COLE.     The  rectifying  property  of  the  giant  axon  of  the  squid. 

Collecting  Net,  16:  1941. 
GUTTMAN,  RITA  AND  K.   S.  COLE.     Electrical   rectification   in   single  nerve   fibers.     Proc.  Soc. 

Exp.  Biol.Afed.,48:  1941. 


REPORT  OF  THE  DIRECTOR  21 

HAGEK,  R.  P.     *Sex  linkage  of  stubby  in  Habrobracon.     Biol.  Bull.,  81:   1941   (abs.). 
HAMILTON,  H.  L.     *The  influence  of  hormones  on  the  differentiation  of  melanophores  in  birds. 

Biol.  Bull,  81:  1941   (abs.). 
HAMILTON,  H.  L.  AND  B.  H.  WILLIER.     ^Developmental  Physiology.     Ann.  Rev.  Physiol     4- 

1942. 
HARNLY,  M.  H.     *Wing  form  and  gene  function  in  nine  genotypes  of  Drosophila  melanogaster. 

Biol.  Bull.,  82 :  1942. 
HARRIS,  D.  L.     The  osmotic  properties  of  cytoplasmic  granules  of  the  sea  urchin  egg.     Biol. 

Bull.,  85 :  1943. 
HARTMAN,  F.  A.,  L.  A.  LEWIS,  K.  A.  BROWNELL,  F.  F.  SHELDEN  AND  R.  F.  WALTHER.     Some 

blood  constituents  of  the  normal  skate.     Physiol.  Zool.  14:  1941. 
HARTMAN,  F.  A.,  F.  F.  SHELDEN,  AND  E.  L.  GREEN.     Weights  of  interrenal  glands  of  elasmo- 

branchs.     Anat.  Rcc.,  87  :  1943. 
HARTMAN,  F.  A.,  L.  A.  LEWIS,  K.  A.  BROWNELL,  C.  A.  ANGERER  AND  F.  F.  SHELDEN.     Effect  of 

interrenalectomy  on  some  blood  constituents  in  the  skate.     Physiol.  Zool.,  17  :   1944. 
HARVEY,  ETHEL  BROWNE.     Relation  of  the  size  of  "halves"  of  the  Arbacia  punctulata  egg  to 

centrifugal  force.     Biol.  Bull.,  80:  1941. 
HARVEY  ETHEL  BROWNE.     The  cytology  of  fertilization  and  cleavage  of  Arbacia  punctulata. 

Turtox  Neivs,  19:  1941. 

HARVEY  ETHEL  BROWNE.     Cross  fertilization  of  echinoderms.     Science,  94 :   1941. 
HARVEY  ETHEL  BROWNE.     Vital  staining  of  the  centrifuged  Arbacia  egg.     Biol.  Bull.,  81 :  1941. 
HARVEY  ETHEL  BROWNE.     Maternal  inheritance  in  echinoderm  hybrids.     Biol.  Bull.,  81 :   1941. 

(abs.). 
HARVEY  ETHEL  BROWNE.     Rate  of  breaking  and  size  of  the  "halves"  of  the  Arbacia  egg  when 

centrigued  in  hypo-  and  hypertonic  sea  water.     Biol.  Bull.,  85  :  1943. 
HARVEY,  ETHEL  BROWNE.     *Early  biological  photomicrographs.     Jour.  Biol.  Photograph.  Assoc., 

13:  1945. 
HARVEY  ETHEL  BROWNE.     Stratification  and  breaking  of  the  Arbacia  egg  when  centrifuged  in 

single  salt  solutions.     Biol.  Bull.,  89 :  1945. 
HARVEY  ETHEL  BROWNE.     Development  of  granule-free  fractions  of  Arbacia  eggs.     Biol.  Bull., 

89:  1945  (abs.). 
HARVEY  ETHEL  BROWNE  AND  T.  F.  ANDERSON.     The  spermatozoan  and  fertilization  membrane 

of  Arbacia,  as  shown  by  the  electron  microscope.     Biol.  Bull.,  85:   1943. 
HARVEY  ETHEL  BROWNE  AND  G.  I.  LAVIN.     The  chromatin  in  the  living  Arbacia  egg;  and  the 

cytoplasm  of  the  centrifuged  egg  as  photographed  by  ultraviolet  light.     Biol.   Bull.,  86 : 

1944. 
HARVEY,  E.  N.     Stimulation  of  cells  by  intense  flashes  of  ultraviolet  light.     Jour.  Gen.  Physiol., 

25 :  1942. 
HARVEY,  E.  N.     Note  on  the  red  luminescence  and  the  red  pigment  of  the  "railroad  worm." 

Jour.  Cell.  Comp.  Physiol.,  25 :  1945. 

HARVEY,  E.  N.  AND  H.  SHAPIRO.     The  recovery  period   (relaxation)   of  marine  eggs  after  de- 
formation.    Jour.  Cell.  Comp.  Physiol.,  17  :  1941. 
HARVEY,  E.  N.  AND    F.  J.  M.  SICHEL.     The  response  of  single  striated  muscle  fibers  to  intense 

flashes  of  ultraviolet  light.     Jour.  Cell.  Comp.  Physiol.,  19:  1942. 
HARVEY,  E.  N.  AND  F.  J.  M.  SICHEL.     A  method  of  recording  the  dimensions  of  muscle  fiber 

striations  during  contraction.     Federation  Proc.,  1  :  1942. 
HARVEY,   E.   N.  AND  F.  J.   M.   SICHEL.     High   speed  linear   photography.     Jour.   Cell.   Comp. 

Physiol.,  25 :  1945. 
HARVEY,  E.  N.,  D.  K.  BARNES,  W.  D.  MCELROY,  A.  H.  WHLTELY,  D.  C.  PEASE  AND  K.  W. 

COOPER.     *Bubble  formation  in  animals.     I.  Physical  factors.     Jour.  Cell.  Comp.  Physiol., 

24:  1944. 
HASSETT,  C.  C.     *Photodynamic  action  in  the  flagellate   Peranema  trichophorum  with   special 

reference  to  motor  response  to  light.     Physiol.  Zool.,  17 :  1944. 
HAYASHI,  TERU.     Dilution  medium  and  survival   of  the  spermatozoa  of  Arbacia  punctulata. 

I.  Effect  of  the  medium  on  fertilizing  power.     Biol.  Bull.,  89:  1945. 
HAYWOOD,   CHARLOTTE.     The   permeability   of   the   toadfish   liver   to    inulin,    with    and   without 

choleretics.     Federation  Proc.,  2:  1943  (abs.). 
HAYWOOD,  CHARLOTTE,  VIRGINIA  C.  DICKERSON,  AND  MARGARET  C.  COLLINS.     *The  secretion 

of  dye  by  the  fish  liver.    Jour.  Cell.  Comp.  Physiol.,  25 :  1945. 


MARINE  BIOLOGICAL  LABORATORY 

HEILBRUNN,  L.  V.     *An  Outline  of  General  Physiology.     2nd  Ed.  Saunders.     1943. 

HEWATT,  W.  G.     A  method  of  narcotizing  Holothurians.     Science,  97  :   1943. 

HIATT,   E.    P.   AND   G.    P.    QUINN.     *The   distribution   of   quinine,    quinidine,   cinchonine,    and 

cinchonidine  in  fluids  and  tissues  of  dogs.     Jour.  Pharmacol.  Exp.  Thcrap.,  83  :  1945. 
HIATT,  E.  P.,  D.  E.  S.  BROWN,  G.  P.  QUINN  AND  K.  MACDUFFIE.     *The  blocking  action  of 

the  cinchona  alkaloids  and  certain  related  compounds  on  the  cardioinhibitory  vagus  endings 

of  the  dog.    Jour.  Pharmacol.  Exp.  Therap.,  85 :  1945. 
HIBBARD,  HOPE  AND  G.  I.  LAVIN.     *A  study  of  the  Golgi  apparatus  in  chicken  gizzard  epithelium 

by  means  of  the  quartz  microscope.    Biol.  Bull.,  89:  1945. 
HIESTAND,  W.  A.     Oxygen  consumption  of  the  sea  cucumber  as  a  function  of  oxygen  tension 

and  hydrogen  ion  concentration  of  the  surrounding  medium.     Trans.  Wisconsin  A  cad.  Sci- 
ences, Arts  and  Letters,  32  :  1941. 
HIESTAND,  W.  A.     Action  of  certain  drugs  on  the  sea  star,  Asterias  forbesii.     Proc.  Soc.  Exp. 

Biol.  Med.,  52  :  1943. 
HILL,  S.  E.     The  relation  between  protoplasmic  streaming  and  the  action  potential   in  Nitella 

and  Chara.    Biol.  Bull.,  81:  1941   (abs.). 
HOLLINGSWORTH,  JOSEPHINE.     Activation  of  Cumingia  and  Arbacia  eggs  by  bivalent  cations. 

Biol.  Bull,  81:  1941. 

HOPKINS,  D.  L.    See  Mast  and  Hopkins,  1941. 
HORN,  ANNABELLE.     *Proof  for  multiple  allelism  of  sex  differentiating  factors  in  Habrobracon. 

Amer.  Nat.,  77:  1943. 
HOUCK,  C.  R.     The  effects  of  bichloride  of  mercury  upon  the  luminescence  and  respiration  of 

the  luminous  bacterium,  Achromobacter  fischeri.     Jour.  Cell.  Comp.  Physiol.,  20 :  1942. 
HUNNINEN,  A.  V.    See  also  Cable  and  Hunninen,  1941,  1942. 
HUNNINEN,  A.  V.  AND  R.  M.  CABLE.     Life  history  of  Lecithaster  confusus.     Jour.  ParasitoL, 

29:  1943. 
HUNNINEN,-  A.  V.  AND  R.  M.  CABLE.     The  life  history  of  Podocotyle  atomon.     Trans.  Amcr. 

Microscop.  Soc.,  62:  1943. 
HUTCHENS,  J.  O.     The  utilization  of  ammonia  by  Chilomonas  paramoecium.     Biol.   Bull.,  81  : 

1941  (abs.). 

HUTCHENS,  J.  O.,  A.  K.  KELTCH,  M.  E.  KRAHL  AND  G.  H.  A.  CLOWES.  Studies  on  cell  metab- 
olism and  cell  division.  VI.  Observations  on  the  glycogen  content,  carbohydrate  consump- 
tion, lactic  acid  production,  and  ammonia  production  of  eggs  of  Arbacia  punctulata.  Jour. 

Gen.  Physiol.,  25  :  1942. 
HUTCHENS,  J.  O.,  M.  J.  KOPAC  AND  M.  E.  KRAHL.     The  cytochrome  content  of  centrifugally 

separated  fractions  of  unfertilized  Arbacia  eggs.    Jour.  Cell.  Comp.  Physiol.,  20 :  1942. 
IRVING,  LAURENCE.     See  Root  and  Irving,  1941. 

JACOBS,  M.  H.    Sec  also  Netzky  and  Jacobs,  1941  ;  Stewart  and  Jacobs,  1941. 
JACOBS,  M.  H.  AND  DOROTHY  R.  STEWART.     Catalysis  of  ionic  exchange  by  bicarbonates.     Biol. 

Bull.,  81  :  1941. 
JACOBS,  M.  H.  AND  DOROTHY  R.  STEWART.     The  role  of  carbonic  anhydrase  in  certain  exchange 

involving  the  erythrocyte.    Jour.  Gen.  Physiol.,  25 :  1942. 

JACOBS,  M.  H.  AND  DOROTHY  R.  STEWART.     *A  biological  method  for  the  quantitative  estima- 
tion of  certain  organic  bases.     Amer.  Jour.  Med.  Sci.,  206 :  1943. 
JACOBS,  M.  H.  AND  DOROTHY  R.  STEWART.     *Osmotic  equilibria  between  the  erythrocyte  and 

a  complex  external  solution.     Amer.  Jour.  Med.  Sci.,  209:   1945. 
JACOBS,  M.  H.  AND  J.  D.  HELM.     *Some  apparent  differences  between  the  erythrocytes  of  white 

and  negro  subjects.    Jour.  Cell.  Comp.  Physiol.,  22:  1943. 
JACOBS,  M.  H.,  DOROTHY  R.  STEWART  AND  MARY  K.  BUTLER.     *Some  effects  of  tannic  acid  on 

the  cell  surface.    Amer.  Jour.  Med.  Sci.,  205 :  1943. 
JAEGER,  LUCENA.     Glycogen  utilization  by  the  amphibian  gastrula   in   relation   to   invagination 

and  induction.    Jour.  Cell.  Comp.  Physiol.,  25 :  1945. 
JANDORF,  B.  J.  AND  M.  E.  KRAHL.     Studies  on  cell  metabolism  and  division.     VIII.  The  diphos- 

phopyridine  nucleotide  content  of  eggs  of  Arbacia  punctulata.     Jour.   Gen.  Physiol.,  25 : 

1942. 

JONES,  E.  R.     *The  morphology  of  Enterostomula  gram.    Jour.  Morph.,  68:  1941. 
JONES,  E.  R.  AND  W.  J.  HAYES,  JR.     *Microdalyellia  gilesi,  a  new  Turbellarian  worm  from 

Mountain  Lake,  Va.     Amer.  Midland  Naturalist,  26:  1941. 


REPORT  OF  THE  DIRECTOR  23 

KALISS,  NATHAN.     Sec  Furth,  Boon  and  Kaliss,  1944. 

KAWATA,  N.     See  Steinbach,  Spiegelman  and  Kawata,  1944. 

KAYLOR,  C.  T.     *Studies  on  experimental  haploidy  in  salamander  larvae.     Blol.  Bull.,  81 :  1941. 

KAYLOR,  C.  T.     *Sex  differentiation  in  two  androgenetic  salamander  larvae.    Anat    Rec     87  • 

1943. 
KELTCH,  ANNA  K.    See  Krahl,  Keltch,  Neubeck  and  Clowes,  1941 ;   Hutchens,  Keltch,  Krahl 

and  Clowes,  1942. 
KIDDER,  G.  W.     *Growth  studies  on  ciliates.     VII.  Comparative  growth  characteristics  of  four 

species  of  sterile  ciliates.     Biol.  Bull.,  80 :  1941. 

KIDDER,  G.  W.     See  also  Claff,  Dewey  and  Kidder,  1941  ;  Burt,  Kidder  and  Gaff,  1941. 
KNOWLTON,  F.  P.     Observations  on  the  dual  contraction  of  crustacean  muscle.     Biol.  Bull.,  82  : 

1942. 
KNOWLTON,  F.  P.     An  investigation  of  inhibition  by  direct  stimulation  of  the  turtle's  heart. 

Amer.  Jour.  Physiol.,  135:  1942. 
KNOWLTON,  F.  P.     The  action  of  certain  drugs  on  crustacean  muscle.    Jour.  Exp.  Pharmacol. 

Exp.  Therap.,  75:  1942. 
KNOWLTON,  F.  P.     """Inhibition  of  the  turtle's  atria  by  single  induction  shocks  applied  directly. 

Amer.  Jour.  Physiol.,  140:  1943. 
KOPAC,  M.  J.     Disintegration  of  the  fertilization  membrane  of  Arbacia  by  the  action  of  an 

"enzyme."     Jour.  Cell.  Comp.  Physiol.,  18:  1941. 
KOPAC,  M.  J.      See  also  Hutchens,  Kopac  and  Krahl,  1942. 
KRAHL,  M.  E.     See  also  Hutchens,  Keltch,  Krahl  and  Clowes,  1942;  Jandorf  and  Krahl,  1942; 

Hutchens,  Kopac  and  Krahl,  1942. 

KRAHL,  M.  E.,  A.  K.  KELTCH,  C.  E.  NEUBECK  AND  G.  H.  A.  CLOWES.     Studies  on  cell  metab- 
olism and  cell  division.     V.  Cytochrome  oxidase  activity  in  eggs  of  Arbacia  punctulata. 

Jour.  Gen.  Physiol.,  24:  1941. 
KRAHL,  M.  E.,  B.  J.  JANDORF  AND  G.  H.  A.  CLOWES.     Studies  on  cell  metabolism  and  cell 

division.     VII.  Observations  on  the  amount  and  possible  function  of  diphosphothiamine  in 

eggs  of  Arbacia  punctulata.     Jour.  Gen.  Physiol.,  25  :   1942. 
KRASNOW,  FRANCES.     *The  physiological   significance  of  phospholipid  in  human   saliva.     Jour. 

Dental  Res.,  24:  1945. 

LANCEFIELD,  REBECCA  C.     *Studies  on  the  antigenic  composition  of  Group  A  hemolytic  strepto- 
cocci.    I.  Effects  of  proteolytic  enzymes  on  streptococcal  cells.     Jour.  Exp.  Med.,  78 :  1943. 
LANCEFIELD,  REBECCA  C.  AND  W.  A.  STEWART.     *II.  The  occurrence  of  strains  in  a  given  type 

containing  M  but  no  T  antigen.     Jour.  Exp.  Med.,  79:  1944. 
LAVIN,   G.  J.     Some  observations  with  a  simplified  quartz  microscope.     Biol.  Bull.,  83:    1942 

(abs.). 

LAVIN,  G.  J.     *Simplified  ultraviolet  microscopy.     Rec.  Scientific  Instruments,  14:   1943. 
LAVIN,  G.  J.     See  also  Costello  and  Lavin,  1943;  Harvey  and  Lavin,  1944;  Hibbard  and  Lavin, 

1945. 

LAZAROW,  ARNOLD.     *The  chemical  organisation  of  the  cytoplasm.     Biol.  Bull.,  87:  1944. 
LEE,  R.  E.     The  occurrence  of  female  sword-fish  in  southern  New  England  waters,  with  a 

description  of  their  reproductive  condition.     Copeia,  1942. 
LEE,  R.  E.     The  hypophysis  of  the  broad-billed  sword-fish,  Xiphias  gladius.     Biol.  Bull     82 : 

1942. 
LEE,  R.  E.     Notes  on  the  color  changes  of  the  sea  robin,  with  special  reference  to  the  erythro- 

phores.     Jour.  Exp.  Zool,  91 :  1942. 
LEE,  R.  E.     Pituitary  function  in  the  chromatic  physiology  of  Opsanus  tau.     Biol.  Bull     83  • 

1942  (abs.). 
LEE,  R.  E.     *A  quantitative  survey  of  the  invertebrate  bottom  fauna  in  Menemsha  Bight.     Biol 

Bull.  86:  1944. 
•LEE,  R.  E.    See  also  Chambers,  Zweifach,  Lowenstein  and  Lee,  1944;  Zweifach,  Lee,  Hyman 

and  Chambers,  1944. 
LEFEVRE,  P.  F.     Certain  chemical  factors  influencing  artificial  activation  of  Nereis  eggs     Biol 

Bull,  89 :  1945. 

LEVY,  MILTON  AND  A.  H.  PALMER.     *Amino  peptidase.     Jour.  Biol.  Chein.,  150 :  1943. 
LEWIS,  L.  A.     See  Hartman,  Lewis,  Brownell,  Shelden  and  Walther,  1941 ;   Hartman,  Lewis, 

Brownell,  Angerer  and  Shelden,  1944. 


24  MARINE  BIOLOGICAL  LABORATORY 

LEWIS,  MARGARET  R.     *The  failure  of  purified  penicillin  to  retard  the  growth  of  sarcoma  in 

mice.    Science,  100:  1944. 
LEWIS,    MARGARET    R.     *A    study    of    inducement    and    transplantability    of    sarcoma    in    rats. 

Growth,  9 :  1945. 

LEWIS,  W.  H.     The  superficial  gel  layer  and  its  role  in  development.     Blol.  Bull.,  87 :  1944. 
LIEBMAN,  EMIL.     *The  coelomocytes  of  Lumbricidae.    Jour.  Morph.,  71 :   1942. 
LILLIE,  F.  R.     Further  experiments  on  artificial  parthenogenesis  in  starfish  eggs,  with  a  review. 

Physiol.  Zool,  14:  1941. 

LILLIE,  F.  R.     The  Woods  Hole  Marine  Biological  Laboratory.     Univ.  Chicago  Press,  1944. 
LILLIE,  R.  S.     *The  problem  of  synthesis  in  Biology.     Philosoph.  Science,  9:  1942. 
LILLIE,  R.  S.     *Living  systems  and  non-living  systems.     Philosoph.  Science,  9 :  1942. 
LILLIE,  R.  S.     *The.  psychic  factor  in  living  organisms.     Philosoph.  Science,  10:  1943. 
LILLIE,  R.  S.     *Vital  organization  and  the  psychic  factor.     Philosoph.  Science,  11 :  1944. 
LILLIE,  R.  S.     *General  Biology  and  Philosophy  of  the  Organism.     Univ.  Chicago  Press,  1945. 
LITTLE,  E.  P.    See  Evans,  Slaughter,  Little  and  Failla,  1942. 
LLOYD,  D.  P.  C.     *Activity  in  neurons  of  the  bulbospinal  correlation   system.    Jour.  Neuro- 

physiol.,  4:  1941. 

LOEWI,  OTTO.     *Chemical  transmission  of  nerve  impulses.     Science  Progress,  4 :  1945. 
LOEWI,  OTTO.     ^Aspects  of  the  transmission  of  the  nervous  impulse.     Jour.  Mt.  Sinai  Hasp., 

12:  1945. 
LUCAS,  A.  M.  AND  J.  SNEDECOR.     Coordination  of  ciliary  movement  in  the  Modiolus  gill.     Biol. 

Bull.,  81:  1941   (abs.). 
LUCRE,  BALDUIN,  A.  K.  PARPART  AND  R.  A.  RICCA.     Failure  of  choleic  acids  in  carcinogenic 

hydrocarbons  to  alter  permeability  of  marine  eggs  and  of  mammalian  erythrocytes.     Cancer 

Research,  1:  1941. 
LURIA,  S.  E.,  M.  DELBRUCK  AND  T.  F.  ANDERSON.     Electron  microscope  studies  of  bacterial 

viruses.    Jour.  Bad.,  46 :  1943. 
LYNN,  W.  G.     *The  embryology  of  Eleutherodactylus  nubicola,  an  anuran  which  has  no  tadpole 

stage.     Carnegie  Contrib.  to  Embryol.,  30:  1942. 
LYNN,  W.  G.  AND  J.  M.  DENT.     Notes  on  Plethodon  cinereus  and  Hemidactylium  scutatum  on 

Cape  Cod.     Copeia,  1941. 
MACLEAN,  BERNICE.     Sec  Shapiro,  1945. 

MARSLAND,  D.  A.     *Protoplasmic  streaming.     Chronica  Botan.,  6:   1941. 
MARSLAND,  D.  A.     ^Protoplasmic  streaming.     Collecting  Net,  16:   1941. 
MARSLAND,   D.   A.     *Protoplasmic   streaming   in   relation   to   gel    structure   in   the   cytoplasm. 

Chapter  in  The  Structure  of  Protoplasm,  Iowa  State  College  Press,  1942. 
MARSLAND,  D.  A.     The  contractile  mechanism  in  unicellular  chromatophores.     Biol.  Bull.,  83 : 

1942  (abs.). 

MARSLAND,  D.  A.     ^Quieting  Paramoecium  for  the  elementary  student.     Science,  98 :   1943. 
MARSLAND,  D.  A.     Mechanism  of  pigment  displacement  in  unicellular  chromatophores.    Biol. 

Bull.,  87 :  1944. 

MARSLAND,  D.  A.     *Principles  of  Modern  Biology.     Holt  and  Co.,  New  York,  1945. 
MARSLAND,  D.  A.  AND  D.  E.  S.  BROWN.     The  action  of  pressure  on  sol-gel  equilibria.    Anat. 

Rec.,  81:  Suppl.,  1941. 
MARSLAND,  D.  A.  AND  D.  E.  S.  BROWN.     The  effects  of  pressure  on  sol-gel  equilibria,  with 

special  reference  to  myosin  and  other  protoplasmic  gels.    Jour.  Cell.  Comp.  Physiol.,  20 : 

1942. 

MARSLAND,  D.  A.  AND  R.  RUGH.     *Effects  of  pressure  on  maturation,  cleavage,  and  early  de- 
velopment of  the  frog's  egg.     Anat.  Rec.,  82 :  1942. 

MARSLAND,  D.  A.  AND  R.  RUGH.     *The  effect  of  hydrostatic  pressure  upon  the  early  develop- 
ment of  the  frog's  egg.     Proc.  Amer.  Philosoph.  Soc.,  86:  1943. 
MARTIN,  W.  E.     Cerama  solemyae,  probably  a  blood  fluke  from  the  marine  pelecypod,  Solemya 

velum.     Jour.  Parasitol,  30 :  1944. 
MARTIN,  W.   E.     Studies  on  trematodes  of  Woods   Hole.     IV.  Additional   observations  upon 

Cercaria  loossi  developing  in  an  Annelid.     Trans.  Amcr.  Micro.  Soc.,  63:  1944. 
MARTIN,  W.  E.    Two  new  species  of  marine  cercariae.     Trans.  Amcr.  Micro.  Soc.,  64:   1945. 
MAST,  S.  O.     *The  hydrogen  ion  concentration  of  the  content  of  the  food  vacuoles  and  the 

cytoplasm  in  Amoeba  and  other  phenomena  concerning  the  food  vacuoles.    Biol.  Bull..  83 : 

1942. 


REPORT  OF  THE  DIRECTOR  25 

MAST,  S.  O.     A  new  peritrich  belonging  to  the  genus  Ophridium.     Trans.  Amer.  Mic.  Soc.,  63  : 

1944. 

MAST,  S.  O.      Sec  also  Bertholf  and  Mast,  1944;  Grave  and  Mast,  1944. 
MAST,  S.  O.  AND  D.  L.  HOPKINS.     *Regulation  of  the  water  content  of  Amoeba  mira  and 

adaptation  to  changes  in  the  osmotic  concentration  of  the  surrounding  medium.    Jour.  Cell. 

Comp.  Physiol,  17:  1941. 
MAST,  S.  O.  AND  D.  M.  PACE.     *The  effect  of  phosphorus  on  metabolism  in  Chilomonas  para- 

moecium.     Jour.  Cell.  Comp.  Physiol.,  20 :  1942. 
MAST,  S.  O.  AND  W.  G.  BOWEN.     *The  food  vacuole  in  the  Peritricha,  with  special  reference 

to  the  hydrogen  ion  concentration  of  its  content  and  of  the  cytoplasm.     Biol.  Bull.,  87 :  1944. 
MCELROY,  W.  D.     See  Harvey,  Barnes,  McElroy,  Whitely,  Pease  and  Cooper,  1944. 
MELLAND,  A.  M.     See  Buck  and  Melland,  1942. 

MENKIN,  VALY.     *Studies  on  the  chemical  basis  of  fever.     Biol.  Bull..  87  :  1944. 
MERWIN,  RUTH  M.     See  Alice  and  Merwin,  1941 ;  Alice,  Finkel,  Garner,  Merwin  and  Evans, 

1942;  Alice  and  Merwin,  1943. 
METZ,   C.   B.     *The  inactivation  of  fertilizin  and  its  conversion  to   the   "univalent"   form  by 

X-rays  and  ultraviolet  light.     Biol.  Bull.,  82 :  1942. 

METZ,  C.  B.     *The  agglutination  of  starfish  sperm  by  fertilizin.     Biol.  Bull.,  89 :  1945. 
MEYERHOF,  BETTINA.     See  Nachmansohn  and  Meyerhof,  1941. 

MEYERHOF,  OTTO.     *Nature,  function,  and  distribution  of  the  phosphagens  in  the  animal  king- 
dom.    Collecting  Net,  16:  1941. 
MICHAELIS,  L.  AND  SAM  GRANiCK.     *Metachromasy  of  basic  dye  stuffs.     Jour.  Amer.  Chem. 

Soc.,  67 :  1945. 
MILLER,  J.  A.     Some  effects  of  covering  the  perisarc  upon  tubularian  regeneration.    Biol.  Bull., 

83:   1942. 

MIRSKY,  A.  E.     See  Pollister  and  Mirsky,  1942,  1943. 
MOOG,  FLORENCE.     The  influence  of  temperature  on  reconstitution   in  Tubularia.     Biol.   Bull., 

81:  1941  (abs.). 
MOOG,  FLORENCE.     Some  effects  of  temperature  in  the  regeneration  of  Tubularia.    Biol.  Bull., 

83:  1942  (abs.). 

MOOG,  FLORENCE.     See  also  Spiegelman  and  Moog,  1944. 
MOOG,  FLORENCE  AND  S.  SPIEGELMAN.     Effects  of  some  respiratory   inhibitors  on  respiration 

and  reconstitution  in  Tubularia.     Proc.  Soc.  Exp.  Biol.  Med.,  49:  1942. 
MORGAN,  T.  H.     *Further  experiments  in  cross-  and  self-fertilization  of  Ciona  at  Woods  Hole 

and  Corona  del  Mar.     Biol.  Bull.,  80:  1941. 

MORGAN,  T.  H.     Cross-  and  self-fertilization  in  the  Ascidian,  Styela.     Biol.  Bull.,  82 :   1942. 
MORGAN,   T.   H.     Cross-   and   self-fertilization   in   the   Ascidian,    Molgula   manhattensis.     Biol. 

Bull.,  82 :  1942. 
MUIR,  R.  M.     Effect  of  radiation  from  radioactive  isotopes  on  the  protoplasm  of  Spirogyra. 

Jour.  Cell.  Comp.  Physiol.,  19:  1942. 
NACHMANSOHN,    DAVID.     On   the   mechanism   of   transmission   of   nerve   impulses.     Collecting 

Net,  17 :  1942. 
NACHMANSOHN,  DAVID.     On  the  energy  source  of  the  nerve  action  potential.     Biol.  Bull.,  87: 

1944. 
NACHMANSOHN,  DAVID  AND  B.  MEYERHOF.     Relation  between  electrical  changes  during  nerve 

activity  and  concentration  of  choline  esterase.     Jour.  Neurophysiol.,  4:  1941. 
NACHMANSOHN,  DAVID  AND  H.  B.  STEINBACH.     On  the  localization  of  enzymes  in  nerve  fibers. 

Science,  95:   1942. 
NACHMANSOHN,  DAVID  AND  H.  B.  STEINBACH.     Localization  of  enzymes  in  nerves.     1.  Suc- 

cinic  dehydrogenase  and  vitamin  B!.     Jour.  Neurophysiol.,  5 :  1942. 
NAVEZ,  A.  E.,  J.  D.  CRAWFORD,  D.  BENEDICT  AND  A.  B.  DuBois.     On  the  metabolism  of  the 

heart  of  Venus  mercenaria.     Biol.  Bull.,  81 :  1941. 

NETSKY,  M.  G.  AND  M.  H.  JACOBS.     Some  effects  of  desoxycorticosterone  and  related  com- 
pounds on  the  mammalian  red  cell.     Biol.  Bull.,  81 :  1941. 
NEUBECK,  C.  E.      See  Krahl,  Keltch,  Neubeck  and  Clowes,  1941. 
O'BRIEN,  J.  P.     ^Studies  on  the  effects  of  X-rays  on  regeneration  in  the  fragmenting  oligo- 

chaete,  Nais  paraguayensis.     Growth,  6 :  1942. 
OLSON,  MAGNUS.     Histology  of  the  radula  protractor  muscles  of  Busycon  canaliculatum.     Biol. 

Bull,  82 :  1942. 


26  MARINE  BIOLOGICAL  LABORATORY 

OPPENHEIMER,  JANE  M.     The  anatomical  relationships  of  abnormally  located  Mauther's  cells 

in  Fundulus  embryos,     four.  Com  p.  Neural.,  74:  1941. 

ORMSBEE,  R.  A.     *The  normal  growth  of  Tetrahymena  geleii.     Biol.  Bull.,  82  :  1942. 
OSBORN,   C.   M.     Studies  on  the  growth   of  integumentary   pigment   in   the   lower   vertebrates. 

*I.  The  origin  of  artificially  developed  melanophores  on  the  normally  unpigmented  ventral 

surface  of  the  summer  flounder.     Biol.  Bull.,  81:  1941. 
OSBORN,  C.  M.     II.  The  role  of  the  hypophysis  in  melanogenesis  in  the  common  catfish.     Biol. 

Bull.,  81:  1941. 
OSTERHOUT,  W.  J.  V.     Some  properties  of  protoplasmic  gels.     I.  Tension  in  the  chloroplast  of 

Spirogyra.     Jour.  Gen.  Physiol.,  29 :  1945. 
PACE,  D.  M.    See  Mast  and  Pace,  1942. 

PACKARD,  CHARLES.     *Roentgen  radiation  in  biological  research.     Radiology,  45 :  1945. 
PACKARD,  CHARLES  AND  F.  M.  EXNER.     *Comparison  of  physical  and  biological  methods  of 

depth  dose  measurement.     Radiology,  44 :   1945. 
PARK,  THOMAS.     The  laboratory  population  as  a  test  of  a  comprehensive  ecological  system. 

Quart.  Rev.  Biol.,  16:  1941. 
PARK,   THOMAS,   ELLA   V.   GREGG   AND   CATHERINE   Z.    LUTHERMAN.     *Studies    in   population 

physiology.     X.  Interspecific  competition  in  populations  of  granary  beetles.     Physiol.  Zool., 

14:  1941. 
PARKER,  G.  H.     Melanophore  bands  and  areas  due  to  nerve  cutting,  in  relation  to  the  protracted 

activity  of  nerves.     Jour.  Gen.  Physiol.,  24:  1941. 
PARKER,  G.  H.     The  methods  of  excitation  of  melanophores  in  the  skin  of  the  catfish  Ameiurus. 

Science,  93:  1941. 

PARKER,  G.  H.     Limited  responses  of  melanophores  as  determined  by  activating  agents.     Sci- 
ence, 93:  1941. 

PARKER,  G.  H.     The  responses  of  catfish  melanophores  to  ergotamine.     Biol.  Bull.,  81 :  1941. 
PARKER,  G.  H.     The  organization  of  the  melanophore  system  in  bony  fishes.     Biol.  Bull.,  81  : 

1941. 
PARKER,  G.  H.     Hypersensitization  of  catfish  melanophores  to  adrenalin  by  denervation.    Biol. 

Bull.,  81:  1941. 
PARKER,  G.  H.     The  method  of  activation  of  melanophores  and  the  limitations  of  melanophore 

responses  in  the  catfish  Ameiurus.     Proc.  Amcr.  Philosoph.  Soc.,  85:  1941. 
PARKER,   G.    H.     The   activity   of   peripherally    stored   neurohumors    in   catfishes.    Jour.    Gen. 

Physiol.,  25:  1941. 
PARKER,  G.  H.     *Color  changes  in  Mustelus  and  other  elasmobranch  fishes.    Jour.  Exp.  Zool., 

89:  1942. 
PARKER,  G.  H.     *Sensitization  of  melanophores  by  nerve  cutting.     Proc.  Nat.  Acad.  Sci.,  28: 

1942. 
PARKER,  G.  H.     Methods  of  estimating  the  effects  of  melanophore  changes  in  animal  coloration. 

Biol.  Bull.,  84:  1943. 

PARKER,  G.  H.     Color  changes  in  the  American  eel,  Anguilla  rostrata.    Anat.  Rcc.,  87:   1943. 
PARKER,  G.  H.     *The  time  factor  in  chromatophore  responses.    Proc.  Amer.  Philosoph.  Soc., 

87 :  1944. 
PARKER,  G.  H.     Melanophore  activators  in  the  common  American  eel  Anguilla  rostrata.    Jour. 

Exp.  Zool.,  98:  1945. 

PARPART,  A.  K.     Lipoprotein  complexes  in  the  egg  of  Arbacia.     Biol.  Bull.,  81 :  1941. 
PARPART,  A.  K.     The  preparation  of  red  cell  membranes.    Jour.  Cell.  Comp.  Physiol.,  19:  1942. 
PARPART,  A.  K.     See  also  Lucke,  Parpart  and  Ricca,  1941 ;  Chase,  Lorenz,  Parpart  and  Gregg, 

1944. 
PARPART,  A.  K.  AND  R.  BALLENTINE.     *Hematocrit  determination  of  red  cell  volume.    Science, 

98:  1943. 

PEASE,  D.  C.    Sec  Harvey,  Barnes,  McElroy,  Whitely,  Pease  and  Cooper,  1944. 
PIERCE,  MADELENE  E.     Response  of  melanophores  of  the  skin  to  injections  of  adrenalin,  with 

special  reference  to  body  weight  of  the  animal.     Jour.  Exp.  Zool.,  86:  1941. 
PLOUGH,  H.  H.     *Spontaneous  mutability  in  Drosophila.     Cold  Spring  Harbor  Svmposia,  9 : 

1941. 

PLOUGH,  H.  H.     ^Temperature  and  evolution.     Biol.  Symposia,  6 :  1942. 
POLLISTER,  A.  W.     *Mitochondrial   orientations  and  molecular  patterns.     Physiol.   Zool.,   14 : 

1941. 


REPORT  OF  THE  DIRECTOR  27 

POLLISTER,  A.  W.  AND  A.  E.  MiRSKY.     *Nucleoproteins  of  cell  nuclei.     Proc.  Nat.  Acad.  Sci., 

28:  1942. 
POLLISTER,  A.  W.  AND  A.  E.  MIRSKY.     *Studies  on  the  chemistry  of  chromatin.     Trans.  N.  Y. 

Acad.  Sci.,  5  :  1943. 
POLLISTER,  A.  W.  AND  A.  E.  MIRSKY.     *Fibrous  nucleoproteins  of  chromatin.     Biol.  Symposia, 

10:  1943. 
POLLISTER,  A.  W.  AND  P.   F.   POLLISTER.     *Relation  between   centriole  and   centromere   in  a 

typical  spermatogenesis  of  viviparid  snails.     Ann.  N.  Y.  Acad.  Sci.,  45 :  1943. 
PRICE,  DOROTHY.     *A  comparison  of  the  reactions  of  male  and  female  rat  prostate  transplants. 

Anat.  Rec.,  82  :  1942. 
PROSSER,  C.  L.     An  analysis  of  the  action  of  acetylcholine  on  hearts,  particularly  in  Arthopods. 

Biol.  Bull.,  83 :  1942. 
PROSSER,   C.  L.     Single  unit  analysis  of  the  heart  ganglion  discharge   in   Limulus   polyphemus. 

Jour.  Cell.  Comp.  Physiol.,  21 :  1943. 
PROSSER,  C.  L.  AND  G.  L.  ZIMMERMAN.     Comparative  pharmacology  of  myogenic  and  neuro- 

genic  hearts.     Biol.  Bull.,  81:  1941   (abs.). 
QUINN,  GERTRUDE  P.     Sec  Hiatt  and  Quinn,  1945. 
RECKNAGEL,  RICHARD.     See  Wilbur  and  Recknagel,  1943. 
REID,   W.   M.     *The  relationship  between  glycogen  depletion   in  the   nematode   Ascaridia  galli 

and  the  elimination  of  the  parasite  by  the  host.     Amcr.  Jour.  Hyg.,  41  :  1945. 
REID,  W.  M.     *In  vivo  and  in  vitro  glycogen  utilization  in  the  fowl  nematode  Ascaridia  galli. 

Biol.  Bull.,  89:  1945  (abs.). 
REINHARD,  E.  G.     A  hermit  crab  as  intermediate  host  of  Polymorphus.     Jour.  Parasitol.,  30 : 

1944. 
REINHARD,  E.  G.     Paguritherium  alatum,  an  entoniscian  parasite  of  Pagurus  longicarpus.    Jour. 

Parasitol.,  31 :  1945. 

RICCA,  R.  A.     See  Lucke,  Parpart  and  Ricca,  1941. 
RICHARDS,  A.  G.     The  interfibrillar  material  in  the  central  nervous  system  of  mosquito  larvae. 

Biol.  Bull,,  83:  1942  (abs.). 
RICHARDS,  A.  G.     *Lipid  nerve  sheaths  in  insects  and  their   probable   relation  to   insecticide 

action.     Jour.  N.  Y.  Entomol.  Soc.,  51 :  1943. 
RICHARDS,  A.  G.     *The  structure  of  living  insect  nerves  and  nerve  sheaths  as  deduced  from  the 

optical  properties.     Jour.  N.  Y.  Entomol.  Soc.,  52 :  1944. 
RICHARDS,  A.  G.  AND  JANE  L.  WEYGANT.     *The  selective  penetration  of  fat  solvents  into  the 

nervous  system  of  mosquito  larvae.    Jour.  N.  Y.  Entomol.  Soc.,  53 :  1945. 
RICHARDS,  A.  G.,   H.  B.   STEINBACH   AND  T.  F.   ANDERSON.     Electron  microscope   studies  of 

squid  giant  nerve  axoplasm.     Jour.  Cell.  Comp.  Physiol.,  21  :  1943. 
Ris,  HANS.     *A  cytological  and  experimental  analysis  of  the  meiotic  behavior  of  the  univalent 

X-chromosome  in  the  bearberry  aphid  Tamalia.     Jour.  Exp.  Zool.,  90 :  1942. 
Ris,  HANS.     *A  quantitative  study  of  anaphase  movement  in  the  aphid  Tamalia.     Biol,  Bull., 

85:  1943. 
Ris,  HANS  AND  HELEN  GROUSE.     *Structure  of  the  salivary  gland  chromosomes  of  Diptera. 

Proc.  Nat.  Acad.  Sci,,  31 :  1945. 
ROBBIE,  W.  A.     Balanced  centerwell  solutions  for  manometric  experimentation.     Biol.  Bull.,  89 : 

1945  (abs.). 

ROGICK,  MARY  D.     Resistance  of  fresh  water  Bryozoa  to  dessication.     Biodynamica,  3:   1941. 
ROGICK,  MARY  D.     Supplementary  note  on  the  effect  of  the  1938  hurricane.     Ohio  J.  Sci,,  41  : 

1941. 
ROGICK,  MARY  D.     ^Studies  on  fresh-water  Bryozoa  XV.     Hyalinella  punctata  growth  data. 

Ohio  Jour.  Sci.,  45 :  1945. 

ROGICK,  MARY  D.     Field  trips  with  a  long-range  purpose.     Amcr.  Biol.  Teacher,  7 :  1945. 
ROGICK,  MARY  D.     Studies  on  marine  Bryozoa.     I.  Aeverrillia  setigera.     Biol.  Bull.,  89 :  1945. 
ROGICK,  MARY  D.     *Studies  on  fresh-water  Bryozoa.     XVI.  Fredericella  australiensis.     Biol. 

Bull.,  89 :  1945. 

ROGICK,  MARY  D.     "Calcining"  specimens.     Amcr.  Biol.  Teacher,  8 :  1945. 
ROOT,   R.   W.  AND   LAURENCE   IRVING.     The   equilibrium  between   hemoglobin   and   oxygen   in 

whole  and  hemolyzed  blood  of  the  tautog,  with  a  theory  of  the  Haldane  effect.     Biol.  Bull., 

81 :  1941. 


MARINE  BIOLOGICAL  LABORATORY 

ROSE,  S.  M.     *A  method  for  inducing  limb  regeneration  in  adult  Anura.     Proc.  Soc.  Exp.  Biol. 

Med.,  49 :  1942. 

ROSE,  S.  M.     *Causes  for  loss  of  regenerative  power  in  adult  Anura.    Anat.  Rec.,  89:  1944. 
ROSE,  S.  M.     *Methods  for  initiating  limb  regeneration  in  adult  Anura.     Jour.  Exp.  Zool.,  95  : 

1944. 
ROSE,  S.  M.     *The  effect  of  NaCl  in  simulating  regeneration  of  limbs  of  frogs.    Jour.  Morph., 

77:  1945. 
ROSE,  S.  M.  AND  FLORENCE  C.  ROSE.     The  role  of  a  cut  surface  in  Tubularia  regeneration. 

Physiol.  Zool.,  14:  1941. 
ROSE,  S.  M.     See  also  Gidge  and  Rose,  1944. 
RUGH,  ROBERT.     See  Marsland  and  Rugh,  1942. 
RUNYON,  E.  H.     Aggregation  of  separate  cells  of  Dictyostelium  to  form  a  multicellular  body. 

Biol.  Bull,  83:  1943  (abs.). 
SANDOW,   ALEXANDER.     Studies  of  the  muscle   twitch   recorded  by   electronic   methods.     Biol. 

Bull,  89:  1945  (abs.). 
SAYLES,  L.  P.     Regeneration  in  the  polychaete,  Clymenella  torquata.     Biol  Rev.  of  the   City 

College,  N.  Y .,  3 :  1941. 
SAYLES,  L.  P.     Buds  induced  in  Clymenella  torquata  by  implants  of  nerve  cord  and  neighboring 

tissues  derived  from  the  mid-body  region  of  worms  of  the  same  species.     Biol.  Bull,  82  : 

1942. 

SAYLES,  L.  P.     Implants  consisting  of  young  buds,  formed  in  anterior   regeneration   in   Cly- 
menella, plus  the  nerve  cord  of  the  adjacent  old  part.    Jour.  Exp.  Zool,  94:  1943. 
SCHAEFFER,  MORRIS.     *Preparation  of  influenza  virus.     Proc.  Soc.  Exp.  Biol  Med.,  51 :   1942. 
SCHALLEK,  WILLIAM.     The  reaction  of  certain  Crustacea  to  direct  and  to  diffuse  light.     Biol 

Bull,  84:  1943. 
SCHALLEK,    WILLIAM.     Action    of   potassium    on   bound    acetylcholine    in    lobster    nerve    cord. 

Jour.  Cell  Comp.  Physiol,  26 :  1945. 
SCHARRER,  BERTA.     *Neurosecretion.     II.  Neurosecretory  cells  in  the  central  nervous  system  of 

cockroaches.     Jour.  Comp.  Neural,  74:   1941. 

SCHARRER,  BERTA.     III.  The  cerebral  organ  of  the  nemerteans.    Jour.  Comp.  Neurol,  74:  1941. 
SCHARRER,  BERTA.     IV.  Localization  of  neurosecretory  cells  in  the  central  nervous  system  of 

Limulus.     Biol  Bull,  81:  1941. 

SCHARRER,  BERTA.     *Endocrines  in  Invertebrates.    Physiol.  Re^'.,  21 :  1941. 
SCHARRER,  BERTA.     *Experimental  tumors  after  nerve  section  in  an  insect.    Proc.  Soc.  Biol. 

Med.,  60:  1945. 

SCHARRER,  BERTA.    See  also  Scharrer  and  Scharrer,  1945. 
SCHARRER,  BERTA  AND  E.  SCHARRER.     Neurosecretion  VI.     A  comparison  between  the  inter- 

cerebralis-cardiacumallatum  system  of  the  insects  and  the  hypothalamo-hypophyseal  system 

of  the  vertebrates.     Biol  Bull,  87:  1944. 
SCHARRER,    ERNST.     Neurosecretion    I.     The    nucleus    preopticus    of    Fundulus.     Jour.    Comp. 

Neurol,  74:  1941. 
SCHARRER,  ERNST.     The  capillary  bed  of  the  central  nervous  system  of  certain  invertebrates. 

Biol  Bull,  87  :  1944. 

SCHARRER,  ERNST.     *The  blood  vessels  of  the  nervous  tissue.     Quart.  Rev.  Biol,  19 :  1944. 
SCHARRER,  ERNST.     Capillaries  and  mitochrondria  in  neurophil.     Jour.  Comp.  Neurol,  1945. 
SCHARRER,  ERNST.     See  also  Scharrer  and  Scharrer,  1944. 

SCHARRER,  ERNST,  S.  L.  PALAY  AND  R.  G.   NILGES.     Neurosecretion  VIII.     The  Nissl   sub- 
stance in  secreting  nerve  cells.     Anat.  Rec.,  92 :  1945. 

SCHARRER,  ERNST  AND  BERTA  SCHARRER.     Neurosecretion.     Physiol  Rev.,  25:   1945. 
SCHECHTER,  VICTOR.     Experimental  studies  upon  the  egg  cells  of  the  clam,  Mactra  solidissima, 

with  special  reference  to  longevity.    Jour.  Exp.  Zool,  86:  1941. 
SCHECHTER,  VICTOR.     *Oxygen  as  a  factor  in  the  polarity  of   Corymorpha   palma.     Physiol. 

Zool,  14:  1941. 
SCHECHTER,  VICTOR.     *Tolerance  of  the  snail,  Thais  floridana  to  waters  of  low  salinity  and 

the  effect  of  size.    Ecology,  24  :  1943. 
SCHECHTER,   VICTOR.     *Two   flatworms   from   the   oyster-drilling   snail,   Thais   floridana.     Jour. 

Parasitol,  29 :  1943. 
SCHMITT,  F.  O.     *Structural  proteins  of  cells  and  tissues.     In  Advances  in  Protein  Chemistry, 

1:  1945. 


REPORT  OF  THE  DIRECTOR  29 

SCOTT,  ALLAN.     Reversal  of  sex  production  in  Micromalthus.     Biol.  Bull.,  83:   1942   (abs.). 
SCOTT,  SISTER  FLORENCE  MARIE.     The  early  embryonic  development  of  Amaroecium  constel- 

latum.     Biol.  Bull,  83:  1942  (abs.). 
SCOTT,    SISTER    FLORENCE    MARIE.     The    developmental    history    of    Amaroecium    constellatum. 

1.  Early  embryonic  development.     Biol.  Bull.,  88:  1945. 
SEVAG,  M.  G.     *Immuno-catalysis.    C.  C.  Thomas,  Springfield,  111.,  1945. 
SHAEFFER,  A.  A.     A  fourteen  day  rhythm  in  the  left-right  spiralling  ratio  of  Flabellula  citata. 

Biol.  Bull. ,83:  1942  (abs.). 
SHANES,  A.  M.     Current,  voltage,  and  resistance  characteristics  of  injured  nerves.     Biol.  Bull 

87:  1944  (abs.). 
SHANES,  A.  M.     Frog  nerve  as  a  generator  of  current  and  voltage.     Jour.  Cell.  Comp.  Ph\sioL, 

24:  1944. 
SHANES,  A.  M.     Evidence  of  a  metabolic  effect  by  potassium  in  lowering  the  injury  potential 

of  invertebrate  nerve.     Biol.  Bull,  89:  1945   (abs.). 
SHAPIRO,  H.  H.  AND  BERNICE  L.  MACLEAN.     *Transplantation  of  developing  tooth  germs  in 

the  mandible  of  the  cat.    Jour.  Dental  Res.,  24:  1945. 

SHAPIRO,  HERBERT.     Oxygen  utilization  by  starfish  eggs.     Amcr.  Jour.  Physiol,  133:   1941. 
SHAPIRO,  HERBERT.     Metabolism  and  fertilization  in  the  starfish  egg.     Collecting  Net,  16:  1941. 
SHAPIRO,  HERBERT.     Centrifugal  elongation  of  cells,  and  some  conditions  governing  the  return 

to  sphericity,  and  cleavage  time.     Jour.  Cell  Comp.  Physiol,  18 :  1941 
SHAPIRO,  HER^ERT.     Water  permeability  of  the  Chaetopterus  egg  before  and  after  fertilization. 

Jour.  Cell  Comp.  Physiol,  18  :  1941. 
SHAPIRO,  HERBERT  AND  HUGH  DAVSON.     Permeability  of  the  Arbacia  egg  to  potassium.     Biol 

Bull,  81:  1941. 

SHAPIRO,  HERBERT.     Metabolism  and  fertilization  in  the  starfish  egg.     Biol  Bull,  81 :  1941. 
SHAPIRO,   HERBERT.     The  speed  of  membrane  formation.     Anat.  Rcc.,  81  :    1941. 
SHAPIRO,  HERBERT.     See  also  Harvey  and  Shapiro,  1941. 
SHAW,  MYRTLE.     Sec  Sickles  and  Shaw,  1941. 
SHELDEN,  F.  F.     See  Hartman,  Lewis,  Brownell  and   Shelden,    1941 ;    Hartman,   Shelden  and 

Green,  1943;  Hartman,  Lewis,  Brownell,  Angerer,  and  Shelden,  1944. 
SICHEL,  F.  J.  M.     *The  relative  elasticity  of  the  sarcolemma  and  of  the  entire  skeletal  muscle 

fiber.    Amer.  Jour.  Physiol,  133 :  1941. 
SICHEL,  F.  J.  M.     Sec  also  Harvey  and  Sichel,  1942,  1945. 
SICKLES,  GRACE  M.  AND  MYRTLE  SHAW.     The  production  of  specific  antisera  for  enzymes  that 

decompose  pneumococcus  carbohydrates  types  3  and  8.     Jour.  Bacterial,  42:  1941. 
SLAUGHTER,  J.  C.     See  Evans,  Slaughter,  Little,  and  Failla,  1942. 
SLIFER,  ELEANOR  H.     *A  mutant  stock  of  Drosophila  with  extra  sex-combs.    Jour.  Exp.  Zool 

90:  1942. 
SLIFER,  ELEANOR  H.     *The  internal  genitalia  of  some  previously  unstudied  species  of  female 

Acrididae.    Jour.  Morph.  72:  1943. 
SLIFER,  ELEANOR  H.     *The  internal  genitalia  of  female  Tetrigidae,  Eumastacidae,  and  Proscopi- 

idae.    Jour.  Morph.,  73  :  1943. 

SMELSER,  G.  K.     The  oxygen  consumption  of  eye  muscles  of  thyroid-ectomized  and  thyroxin  in- 
jected guinea  pigs.     Amer.  Jour.  Physiol,  142:  1944. 
SMITH,  M.  E.     See  Evans,  Beams,  and  Smith,  1941. 
SOSA,  J.  M.     *Woods  Hole,  Acropolis  de  los  biologos.     El.  Dia.,  1943. 
SOSA,  J.  M.     *Quince  meses  en  los  Estados  Unidos  de  Norte  America.     Anales  Facultad  de 

Med.  Montevideo,  30:  1945. 
SPIEGELMAN,  S.     Mass  and  time  relationships  in  the  regeneration  of  Tubularia.     Biol  Bull,  83 : 

1942  (abs.). 

SPIEGELMAN,  S.     See  also  Moog  and  Spiegelman,  1942 ;  Steinbach  and  Spiegelman,  1943,  1944. 
SPIEGELMAN,    S.    AND   A.    GOLDIN.     A   comparison    of    regeneration    and    respiration    rates    of 

Tubularia.     Proc.  Soc.  Exp.  Biol  Med.  55 :  1944. 
STEINBACH,  H.  B.      Chloride  in  the  giant  axons  of  the  squid.     Jour.  Cell  Comp.  Physiol,  17 : 

1941. 
STEINBACH,    H.    B.     See   also    Nachmansohn   and    Steinbach,    1942 ;    Richards,    Steinbach    and 

Anderson,  1943. 


30  MARINE  BIOLOGICAL  LABORATORY 

STEIXBACH,  H.  B.  AND  S.  SPIEGELMAN.     The  sodium  and  potassium  balance  in  squid  nerve  axo- 

plasm.    Jour.  Cell.  Comp.  Physiol,  22 :  1943. 
STEINBACH,  H.  B.  AND  N.  KAWATA.     The  recovery  of  the  cut  surface  of  the  scallop  muscle. 

Fed.  Proc.,  3  :  1944. 
STEINBACH,   H.   B.,   S.   SPIEGELMAN  AND  N.  KAWATA.     Rectification  and  injury   potential   in 

squid  axons.     Fed.  Proc.,  3 :  1944. 
STEINBACH,  H.  B.,  S.  SPIEGELMAN  AND  N.  KAWATA.     The  effects  of  potassium  and  calcium  on 

the  electrical  properties  of  squid  axons.    Jour.  Cell.  Comp.  Physiol.  24:  1944. 
STERN,   K.   G.     *Oxidases,    Peroxidases,   and   Catalase.     Symposium   on   respiratory    enzymes. 

Madison,  Wis.,  1942. 
STERN,  K.  G.     Physical-chemical  studies  on  chromosomal  nuceloproteins.    Biol.  Bull.,  89 :  1945 

(abs.). 
STERN,  K.  G.  AND  S.  F.  VELICK.     The  effect  of  centrifugation  upon  the  oxygen  consumption  of 

Arbacia  eggs.    Biol.  Bull,  81 :  1941. 
STEWART,  DOROTHY  R.    See  Jacobs  and  Stewart,  1941,  1942,  1945 ;  Jacobs,  Stewart  and  Butler, 

1943. 
STEWART,  DOROTHY  R.  AND  M.  H.  JACOBS.     The  role  of  carbonic  anhydrase  in  the  catalysis  of 

ionic  exchanges  by  bicarbonates.     Biol.  Bull.,  81 :  1941  (abs.). 
STEWART,  W.  A.    See  Lancefield  and  Stewart,  1944. 
STILES,   K.  A.     *Handbook   of  microscopic   characteristics  of   tissues  and   organs.     Blakiston, 

1943. 

STILES,  K.  A.     ^Laboratory  explorations  in  general  zoology.     Macmillan,  1943. 
STOREY,  ALMA  G.     *Gametophytes  of  Marattia  sambucina  and  Macroglossum   Smithii.    Bot. 

Gas.,  103 :  1942. 

STOKEY,  ALMA  G.     *The  gametophyte  of  Dipteria  conjugata.     Bot.  Gas.,  106:   1945. 
STUNKARD,  H.  W.     Specificity  and  host-relations  in  the  trematode  genus  Zoogonus.     Biol.  Bull., 

81 :  1941. 
STUNKARD,  H.  W.     Pathology  and  immunity  to  infection  with  heterophyid  trematodes.    Biol. 

Bull.,  81 :  1941. 
STUNKARD,  H.  W.     Studies  on  pathology  and  resistance  in  terms  and  dogs  infected  with  the 

heterophyid  trematode,  Cryptocotyle  lingua.     Trans.  Amer.  Microscop.  Soc.,  61 :   1942. 
STUNKARD,  H.  W.    The  morphology  and  life  history  of  the  digenetic  trematode,  Zoogonoides 

laevis.    Biol.  Bull.,  85 :  1943. 
TAFT,   C.   H.     The  effects  of  a  mixture   of  high  molecular   alkyl-dimethyl-benzyl-ammonium 

chlorides  on  the  isolated  heart  of  Limulus  polyphemus.     Proc.  and   Trans.   Texas  A  cad. 

Set.,  28 :  1945. 

TAFT,  C.  H.     The  action  of  amino  acids  on  color  changes  in  Fundulus.     Science,  101 :  1945. 
TAFT,  C.  H.     *Action  of  quitenine  on  the  living  Tautog  and  Toadfish.     Biol.  Bull.,  89:   1945 

(abs.). 
TAFT,  C.  H.  AND  J.  A.   PLACE.     *The  comparative  effects  of  the   subcutaneous   injection  of 

quitenine  on  the  kidneys  of  glomerular  and  aglomerular  fish.     Texas  Rep.  on  Biol.  Mcd.  2 : 

1944. 

TAYLOR,  W.  R.     Reappearance  of  rare  New  England  marine  algae.     Rhodora,  43:  1941. 
TAYLOR,  W.  R.     *Notes  on  the  marine  algae  of  Texas.     Mich.  Acad.  Sci.  Artes.  and  Letter,  26 : 

1941. 
TAYLOR,  W.  R.     Tropical  marine  algae  of  the  Arthur  Schott  Herbarium.     Field  Mus.  Nat. 

Hist.  Bot.,  230  :  1942. 
TAYLOR,  W.  R.     *Marine  algae  of  the  Allan  Hancock  Expedition  to  the  Caribbean,  1937.    Allan 

Hancock  Atlantic  Exped.  2:  1942. 

TAYLOR,  W.  R.     *Marine  algae  from  Haiti.     Mich.  Acad.,  28 :  1943. 
TAYLOR,  W.  R.     *The  collecting  of  seaweeds  and  fresh  water  algae.     Instructions  to  naturalists 

in  the  armed  forces  for  botanical  work.     1944  2nd  ed.  1945. 
TAYLOR,  W.  R.     *William  Gilson  Farlow,  promotor  of  phycological  research  in  America.    Far- 

lowia,  2 :  1945. 
TAYLOR,  W.  R.     *Pacific  marine  algae  of  the  Allan  Hancock  Expeditions  to  the  Galapagos 

Islands.    Allan  Hancock  Pacific  Exp.  12 :  1945. 
TEWINKEL,  Lois  E.     Structures  concerned  with  yolk  absorption  in  Squalus  acanthias.     Biol. 

Bull,  81:  1941  (abs.). 


REPORT  OF  THE  DIRECTOR  31 

TE\VINKEL,  Lois  E.     Observations  on  later  phases  of  embryonic  nutrition  in  Squalus  acanthias. 

Jour.  Morph.,  73:  1943. 
TE\VINKEL,  Lois  E.     Embryonic  nourishment  in  the  spiny  dogfish.     Wards  Nat.  Hist.  Bull., 

19:  1945. 

THIVY,  FRANCESA.     A  new  species  of  Ectochaete  from  Woods  Hole.     Biol.  Bull.,  83 :  1942. 
THIVY,  FRANCESA.     New  records  of  some  marine  Chaetophoraceae  and  Chaetosphaeridiaceae 

for  North  America.     Biol.  Bull.,  85 :  1943. 

TRACER,  WILLIAM.     *The  nutrition  of  invertebrates.     Physiol.  Rev.,  21 :   1941. 
TRACER,  WILLIAM.     *Studies  on  conditions  affecting  the  survival  in  vitro  of  a  malarial  para- 
site.   Biol.  Bull.,  81:  1941  (abs.). 

TRINKHAUS,  J.  P.     See  Goodrich,  Josephson,  Trinkhaus  and  Slate,   1944. 
TROMBETTA,  VIVIAN  (Mrs.  Roland  Walker).     The  cytonuclear  ratio.     Bot.  Rev.,  8:  1942. 
VON  DACH,  HERMAN.     Respiration  of  a  colorless  flagellate,  Astasia  klebsii.     Biol.   Bull.,  82 : 

1942. 
VON  SALLMAN,  L.  J.  K.     *Hydrogen  ion  concentration  of  the  vitreous  in  the  living  eye.     Arch. 

Ophthalmol.,  33 :  1945. 
WALKER,  ROLAND  AND  G.  C.  BENNET.     *Size  relations  in  the  optic  system  of  telescope-eyed 

goldfish.     Trans.  Connecticut  Acad.  Art  and  Sci.,  36:  1945. 
WALKER,  ROLAND.     See  also  Freedman  and  Walker,  1942. 

WARREN,  C.  O.     *The  role  of  bicarbonate  in  the  action  of  serum  in  supporting  tissue  respira- 
tion.   Jour.  Biol.  Chem.,  156  :  1944. 
WARREN,  C.  O.     *The  effect  of  thiouracil  on  the  respiration  of  bone  marrow  and  leucocytes 

in  vitro.    Amcr.  Jour.  Physiol.,  71  :  1944. 

WATERMAN,  A.  J.     The  action  of  drugs  on  the  compound  ascidian,  Perophora  viridis,  as  in- 
dicated by  the  activity  of  the  intact  heart.     Physiol.  Zool.,  15:  1942. 
WATERMAN,  A.  J.     Further  study  of  the  action  of  drugs  on  the  heart  of  the  compound  ascidian. 

Physiol.  Zool.,  16 :  1943. 
WATTERSON,  R.  L.     *Some  aspects  of  pigment  deposition  in  feather  germs  of  chick  embryos. 

Biol.  Bull.,  81:  1941  (abs.). 
WATTERSON,   R.   L.     Asexual   reproduction   in   the   colonial   tunicate    Botryllus   schlosseri,   with 

special  reference  to  the  developmental  history  of  intersiphonal  band  of  pigment  cells.     Biol. 

Bull.  88 :  1945. 
WEIDENREICH,    FRANZ.     *The    brachycephalization    of    recent    mankind.     Southwestern.    Jour. 

Anthropol,  1 :  1945. 
WENRICH,  D.  H.     *The  morphology  of  some  protozoan  parasites  in  relation  to  microtechnique. 

Jour.  Parasitol.,  27:  1941. 
WENRICH,  D.  H.     *Observations  on  the  food  habits  of  Entamoeba  muris  and  Entamoeba  ra- 

narum.     Biol.  Bull.,  81:  1941. 
WENRICH,  D.  H.     Morphology  of  the  intestinal  trichomonad  flagellates  in  man  and  of  similar 

forms  in  monkeys,  cats,  dogs,  and  rats.     Jour.  Morph.,  74 :  1944. 
WENRICH,  D.  H.     Comparative  morphology  of  the  trichomonad  flagellates  of  man.     Amcr.  Jour. 

Trap.  Med.,  24 :  1944. 
WENRICH,    D.    H.     Nuclear    structure    and    nuclear    division    in    Dientamoeba    fragilis.    Jour. 

Morph.,  74:  1944. 
WENRICH,  D.  H.     Studies  on  Dientamoeba  fragilis.     IV.  Further  observations  with  an  outline 

of  present  day  knowledge  of  this  species.     Jour.  Parasitol.,  30 :  1944. 
WHALEY,  W.  G.  AND  C.  Y.  WHALEY.     A  developmental  analysis  of  inherited  leaf  patterns  in 

Tropaeolum.     Amcr.  Jour.  Botany,  29 :  1942. 
WHITING,  ANNA  R.     *X-ray  sensitivity  of  first  meiotic  prophase  and  metaphase  in  Habrobracon 

eggs.     Genetics,  27  :  1942. 
WHITING,  ANNA  R.     *Effects  of  X-rays  on  hatchability  and  on  chromosomes  of  Habrobracon 

eggs  treated  in  first  meiotic  prophase  and  metaphase.    Amcr.  Nat.,  79 :  1945. 
WHITING,  P.  W.     *The  cytogenetics  of  sex  determination.     Proc.  7th  Internat.  Genctical  Cong., 

1941. 
WHITING,  P.  W.     *Sex  determination  in  Habrobracon.     Proc.  7th  Internat.  Genctical   Cong., 

1941. 
WHITING,    P.    W.     *Multiple    alleles    in    complementary    sex    determination    of    Habrobracon. 

Genetics,  28:  1943. 


32  MARINE  BIOLOGICAL  LABORATORY 

WHITING,   P.  W.     *Intersexual   females   and  intersexuality   in   Habrobracon.     Biol.   Bull.,  85 : 

1943. 

WHITING,  P.  W.     *Androgenesis  in  the  parasitic  wasp  Habrobracon.    Jour.  Hered.,  34:  1944. 
WHITING,  P.  W.     *The  problem  ®f  reversal  of  male  haploidy  by  selection.     Biol.  Bull.,  89: 

1945  (abs.). 

WHITING,  P.  W.     *The  evolution  of  male  haploidy.     Quart.  Rev.  Biol.,  20 :  1945. 
WICHTERMAN,  RALPH.     *Pure  line  mass  cultures  for  demonstrating   the  mating   reaction  and 

conjugation  in  Paramoecium.     Turtox  News,  22 :   1944. 
WICHTERMAN,    RALPH.     *Recent    discoveries    of   nuclear   processes    and    sexual    phenomena    in 

Paramoecium.     Turtox  News,  22  :  1944. 
WICHTERMAN,  RALPH.     A  modified  petri  dish  for  continuous  temperature  observation.     Science, 

101:  1945. 
WTIERCINSKI,  F.  J.     An  experimental  study  of  intracellular  pH  in  the  Arbacia  egg.     Biol.  Bull., 

81:  1941. 
WIERCINSKI,  F.  J.     An  experimental  study  of  protoplasmic  pH  determination.     I.  Amoebae  and 

Arbacia  punctulata.     Biol.  Bull.,  86  :  1944. 
WILBUR,  K.  M.     The  stimulating  action  of  citrates  and  oxalates  on  the  Nereis  egg.     Physiol. 

Zoo/.,  14:  1941. 

WILBUR,  K.  M.    See  also  Angerer  and  Wilbur,  1943. 
WILBUR,  K.  M.  AND  R.  O.  RECKNAGEL.     The  radiosensitivity  of  eggs  of  Arbacia  punctulata  in 

various  salt  solutions.     Biol.  Bull.,  85 :  1943. 
WILHELMI,  R.  W.     *The  application  of  the  precipitin  technique  to  theories  concerning  the  origin 

of  vertebrates.     Biol.  Bull.,  82 :  1942. 

WILLIAMSON,  R.  R.     See  Buchsbaum  and  Williamson,  1943. 
WILLIER,   B.   H.     *Melanophore  control   of  the   sexual  dimorphism  of  feather  pigmentation  in 

the  Barred  Plymouth  Rock.     Biol.  Bull.,  87 :  1944. 

WILLIER,  B.  H.  AND  MARY  E.  RAWLES.     *Genotypic  control  of  feather  color  pattern  as  demon- 
strated by  the  effects  of  a  sex-linked  gene  upon  the  melanophores.     Genetics,  29 :  1944. 
WILLIER,  B.  H.  AND  MARY  E.  RAWLES.     Melanophore  control  of  the   sexual  dimorphism  of 

feather  pigmentation  pattern  in  the  Barred  Plymouth  Rock  fowl.     Yale  Jour.  Biol.  Med., 

17:  1944. 

WITKUS,  ELEANOR  R.     *Some  hints  on  smear  technique.     Turtox  News,  23 :  1945. 
WITKUS,  ELEANOR  R.     *Endomitotic  tapetal  cell  divisions  in  Spinacia.    Amer.  Jour.  Botany, 

32:  1945. 

WITKUS,  ELEANOR  R.     *Endomitosis  in  plants.     Biol.  Bull.,  89:  1945   (abs.). 
WITKUS,  ELEANOR  R.     Sec  also  Berger  and  Witkus,  1943 ;  Berger,  Sullivan  and  Witkus,  1944. 
WOODRUFF,  L.  L.     Sec  Boell  and  Woodruff,  1941. 
WOODWARD,  ALVALYN  E.  AND  J.  M.  CONDRIN.     *Physiological  studies  on  hibernation   in  the 

chipmunk.    Physiol.  Zool,  18:  1945. 
WRINCH,  DOROTHY.     *The  native  protein  theory  of  the  structure  of  protoplasm.     Cold  Spring 

Harbor  Symposia,  9:  1941. 

WRINCH,  DOROTHY.     Proteins  in  action.     Collecting  Net,  16:  1941. 
WRINCH,  DOROTHY.     Further  implication  of  flexible  protein  frameworks.     Collecting  Net,  16 : 

1941. 
WRINCH,  DOROTHY.     The  structure  of  biologically   active  membranes.     Biol.   Bull.,   83:    1942 

(abs.). 
WRINCH,    DOROTHY.     *Native    proteins,    flexible    frameworks    and    cytoplasmic    organization. 

Nature,  150:  1942. 

WRINCH,  DOROTHY.     *Growth  and  form.     Isis,  34:  1943. 
WRINCH,  DOROTHY.     Native  protein  crystallography  and  diffraction  patterns.     Biol.  Bull.,  87  : 

1944. 

WRINCH,  DOROTHY.     Fourier  transforms  and  structure  factors.    Phys.  Rev.,  67 :  1945. 
WRINCH,  DOROTHY.     A  tetrahedral  framework  for  native  proteins?     Biol.  Bull.,  89:  1945. 
WULFF,  V.  J.      Sec  Brown  and  Wulff,  1941. 
YNTEMA,  C.  L.     *An  experimental  study  on  the  origin  of  the  sensory  neurones  and  sheath  cells 

of  the  IXth  and  Xth  cranial  nerves  in  Amblystoma  punctatum.     Jour.  Exp.  Zool.,  92:  1943. 
YNTEMA,  C.  L.     ^Experiments  on  the  origin  of  the  sensory  ganglia  of  the  facial  nerv»e  in  the 

chick.     Jour.  Comp.  Ncur.,  81  :  1944. 


REPORT  OF  THE  DIRECTOR  33 

YNTEMA,  C.  L.  AND  W.  S.  HAMMOND.     ""Depletions  and  abnormalities  in  the  cervical   sympa- 
thetic system  of  the  chick  following  extirpation  of  neural  crest.     Jour.   E.r/>.  Zoo/.,   100 : 

1945. 
ZINN,  D.  J.  AND  R.  W.  PERNAK.     Mystacocarida,  a  new  order  of  Crustacea  from  intcrtidal 

beaches  in   Massachusetts  and   Connecticut.     Smithsonian  Miscellaneous   Collections,    103: 

1943. 
ZWEIFACH,  B.  J.,  R.  E.  LEE,  C.  HYMAN  AND  R.  CHAMBERS.     *Omental  circulation  in  morph- 

inized  dogs  subjected  to  graded  hemorrhage.     Annals  Surg.,  120:   1944. 
ZWEIFACH,  B.  J.     See  also  Chambers  and  Zweifach,  1944. 
ZWEIFACH,  B.  J.,  R.  G.  ABELL,  R.  CHAMBERS  AND  G.  H.  A.  CLOWES.     Role  of  the  decompensa- 

tory  reactions  of  peripheral  blood  vessels  in  tourniquet  shock.     Surg.  Gyn.  Obstct.,  80 :  1945. 

2.     THE  STAFF,  1945 

CHARLES  PACKARD,  Director,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts. 

SENIOR  STAFF  OF  INVESTIGATION 

E.  G.  CONKLIN,  Professor  of  Zoology,  Emeritus,  Princeton  University. 

FRANK  R.  LILLIE,  Professor  of  Embryology,  Emeritus,  The  University  of  Chicago. 

RALPH  S.  LILLIE,  Professor  of  General  Physiology.  Emeritus.  The  University  of  Chicago. 

C.  E.  McCLUNG,  Professor  of  Zoology,  Emeritus,  University  of  Pennsylvania. 

S.  O.  MAST,  Professor  of  Zoology,  Emeritus,  Johns  Hopkins  University. 

A.  P.  MATHEWS,  Professor  of  Biochemistry,  Emeritus.  University  of  Cincinnati. 

T.  H.  MORGAN,  Director  of  the  Biological  Laboratory,  California  Institute  of  Technology. 

G.  H.  PARKER,  Professor  of  Zoology,  Emeritus,  Harvard  University. 

ZOOLOGY 

I.  CONSULTANTS 

T.  H.  BISSONNETTE,  Professor  of  Biology,  Trinity  College. 
L.  L.  WOODRUFF,  Professor  of  Protozoology,  Yale  University. 

II.  INSTRUCTORS 

F.  A.  BROWN,  Associate  Professor  of  Zoology,  Northwestern    University,  in  charge  of 

course. 

T.  H.  BULLOCK,  Assistant  Professor  of  Neurology  University  of  Missouri  Medical  School. 
W.  D.  BURBANCK,  Associate  Professor  of  Biology,  Drury  College. 
C.  G.  GOODCHILD,  Associate  Professor  of  Biology,  Southwest  Missouri  State  Teachers 

College. 

JOHN  H.  LOCH  HE  AD,  Instructor  in  Zoology,  University  of  Vermont. 
MADELENE  E.  PIERCE,  Assistant  Professor  of  Zoology,  Vassar  College. 
W.  M.  REID,  Assistant  Professor  of  Biology,  Monmouth  College. 
MARY  D.  ROGICK,  Professor  of  Biology,  College  of  New  Rochelle. 

III.     LABORATORY  ASSISTANT 
ANTOIN  BACA,  Duke  University  Medical  School. 

EMBRYOLOGY 

I.     CONSULTANTS 
H.  B.  GOODRICH,  Professor  of  Biology,  Wesleyan  University. 


34  MARINE  BIOLOGICAL  LABORATORY 

II.     INSTRUCTORS 

W.  W.  BALLARD,  Professor  of  Zoology,  Dartmouth  College. 

DONALD  P.  COSTELLO,  Professor  of  Zoology,  University  of  North  Carolina. 

VIKTOR  HAMBURGER,  Professor  of  Zoology,  Washington  University,  in  charge  of  course. 

JANE  M.  OPPENHEIMER,  Assistant  Professor  in  Biology,  Bryn  Mawr  College. 

III.     RESEARCH  ASSISTANT 
MARJORIE  HOPKINS,  University  of  California. 

IV.     LABORATORY  ASSISTANTS 

CATHERINE  HENLEY,  The  Johns  Hopkins  University. 
ELEANOR  LERNER,  Washington  University. 

PHYSIOLOGY 

I.  CONSULTANTS 

WILLIAM  R.  AMBERSON,  Professor  of  Physiology,  University  of  Maryland,  School  of 

Medicine. 

HAROLD  C.  BRADLEY,  Professor  of  Physiological  Chemistry,  University  of  Wisconsin. 
WALTER  E.  CARREY,  Professor  of  Physiology,  Vanderbilt  University  Medical  School. 
MERKEL  H.  JACOBS,  Professor  of  Physiology,  University  of  Pennsylvania. 

II.  INSTRUCTORS 

ROBERT  BALLENTINE,  Lecturer  in  Zoology,  Columbia  University  (absent  in  1945). 

AURIN  CHASE,  Assistant  Professor  of  Biology,  Princeton  University. 

ARTHUR  C.  GIESE,  Associate  Professor  of  Biology,  Stanford  University  (absent  in  1945  I. 

E.  S.  GUZMAN  BARROX,  Associate  Professor  of  Biochemistry,  The  University  of  Chicago. 

RUDOLF  T.  KEMPTOX,  Professor  of  Zoology,  Vassar  College  (absent  in  1945). 

ARTHUR  K.  PARPART,  Associate  Professor  of  Biology,  Princeton  University,  in  charge 

of  course. 
ROBERT  RAMSEY,  Associate  Professor  of  Physiology,  Medical  College  of  Virginia. 

BOTANY 

I.  CONSULTANTS 

S.  C.  BROOKS,  Professor  of  Zoology,  University  of  California. 

B.  M.  DUGGAR,  Professor  of  Plant  Physiology.  University  of  Wisconsin. 

II.  INSTRUCTORS 

WM.  RANDOLPH  TAYLOR,  Professor  of  Botany,  University  of  Michigan,  in  charge  of 

course. 
HANNAH  CROASDALE,  Technical  Assistant,  Dartmouth  College. 

EXPERIMENTAL  RADIOLOGY 

G.  FAILLA,  Memorial  Hospital,  New  York  City. 

L.  ROBINSON  HYDE,  Phillips  Exeter  Academy,  Exeter,  N.  H. 

LIBRARY 

PRISCILLA  B.  MONTGOMERY  (MRS.  THOMAS  H.  MONTGOMERY,  JR.),  Librarian 
DEBORAH  LAWRENCE  MRS.  ELON  H.  JESSUP  MARY  A.  ROHAN 


REPORT  OF  THE  DIRECTOR 

APPARATUS  DEPARTMENT 

E.  P.  LITTLE,  Phillips  Exeter  Academy,  Exeter,  N.  H.,  Manager 
J.  D.  GRAHAM  DOROTHY  LEFEVRE 

CHEMICAL  DEPARTMENT 
E.  P.  LITTLE,  Phillips  Exeter  Academy,  Exeter,  N.  H.,  Manager 

SUPPLY  DEPARTMENT 

JAMES  Mclxxis,   Manager 

D.  J.  Zixx.  Naturalist 

RUTH  CROWELL  GRACE  M.  KENNERSON 

M.  B.  GRAY  W.  E.  KAHLER  F.  N.  WHITMAN 

A.  M.  HILTON  G.  LEHY 

GENERAL  OFFICE 

F.  M.  MACNAUGHT,  Business  Manager 
POLLY  L.  CROWELL  MRS.  LILA  S.  MYERS 

* 

GENERAL  MAINTENANCE 

T.  E.  LARKIN,  Superintendent 

W.  C.  HEMENWAY  G.  T.  NICKELSON,  JR. 

R.  W.  KAHLER  T.  E.  TAWELL 

A.  J.  PIERCE 

THE  GEORGE  M.  GRAY  MUSEUM 
GEORGE  M.  GRAY,  Curator  Emeritus 


3.     INVESTIGATORS  AND  STUDENTS 
•  Independent  Investigators,  1945 

ABELL,  RICHARD  G.,  Assistant  Professor  of  Anatomy,  University  of  Pennsylvania. 

ADDISON,  WILLIAM   H.   F.,   Professor  of   Normal   Histology   and    Embryology,   University   of 

Pennsylvania. 

ANDERSON,  RUBERT  S.,  Assistant  Professor  of  Physiology,  University  of  Maryland. 
ANFINSON,  CHRISTIAN  B.,  JR.,  Instructor  in  Biological  Chemistry,  Harvard  Medical  School. 
ARMSTRONG,  PHILIP  B.,  Professor  of  Anatomy,  Syracuse  University. 
ARONOFF,  SAMUEL,  Instructor,  University  of  Chicago. 
AXELRAD,  ARTHUR  A.,  Investigator,  McGill  University. 

BALL,  ERIC  G.,  Associate  Professor  of  Biological  Chemistry,  Harvard  Medical  School. 
BALLARD,  W.  W.,  Professor  of  Zoology,  Dartmouth  College. 
BALLENTINE,  ROBERT,  Instructor,  Columbia  University. 

BARRON,  E.  S.  GUZMAN,  Associate  Professor  of  Biochemistry,  The  University  of  Chicago. 
EARTH,  L.  G.,  Associate  Professor  of  Zoology,  Columbia  University. 
BEERS,  CHARLES  DALE,  Professor  of  Zoology,  University  of  North  Carolina. 
BERGER,  CHARLES  A.,  Professor  of  Cytology,  Fordham  University. 
BERTHOLF,  LLOYD  M.,  Professor  of  Biology,  Western  Maryland  College. 
BEVELANDER,  GERRIT,  Associate  Professor  of  Anatomy,  New  York  University. 
BLISS,  ALFRED  F.,  Instructor  in  Physiology  and  Pharmacology,  Union  University. 
BODIAN,  DAVID,  Associate  in  Epidemiology,  Johns  Hopkins  University. 


36  MARINE  BIOLOGICAL  LABORATORY 

BRONK,  DETLEV  W.,  Professor  of  Biophysics,  Johnson  Foundation. 

BROOKS,  MATILDA  M.,  Research  Associate  in  Biology,  University  of  California. 

BROOKS,  SUMNER  C.,  Professor  of  Zoology,  University  of  California. 

BROWN,  DUGALD  E.  S.,  Professor  of  Physiology,  New  York  University. 

BROWN,  FRANK  A.,  JR.,  Associate  Professor  of  Zoology,  Northwestern  University. 

BROWNELL,  KATHARINE  A.,  Research  Associate,  Ohio  State  University. 

BUDINGTON,  ROBERT  A.,  Professor  of  Zoology,  Emeritus,  Oberlin  College. 

BULLOCK,  THEODORE  H.,  Assistant  Professor  of  Anatomy,  University  of  Missouri. 

BURBANCK,  WILLIAM  D.,  Associate  Professor  of  Biology,  Drury  College. 

BURKHOLDER,  PAUL  R.,  Professor  of  Botany.  Yale  University. 

CHAMBERS,  ROBERT,  Research  Professor  of  Biology,  New  York  University. 

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

CHENEY,  RALPH  H.,  Chairman  Biology  Department,  Long  Island  University. 

CHIDESTER,  F.  E.,  Research  Worker,  Lee  Foundation. 

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

CLARK,  ELEANOR  L.,  Voluntary  Research  Worker,  University  of  Pennsylvania. 

CLARK,  ELIOT  R.,  Professor  of  Anatomy,  University  of  Pennsylvania. 

CLAUDE,  ALBERT,  The  Rockefeller  Institute  for  Medical  Research. 

CLEMENT,  A.  C.,  Associate  Professor  in  Biology.  College  of  Charleston. 

CLOWES,  G.  H.  A.,  Director  of  Research,  Lilly  Research  Laboratories. 

CONKLIN,  EDWIN  G.,  Professor  of  Zoology,  Emeritus,  Princeton  University. 

COPELAND,  MANTON,  Professor  of  Biology,  Bowdoin  College. 

COSTELLO,  DONALD  P.,  Professor  of  Zoology,  University  of  North  Carolina. 

CRAMPTOX,  HENRY  E.,  Professor  Emeritus.  Columbia  L'niversity. 

CROASDALE,  HANNAH  T.,  Technical  Assistant,  Dartmouth  College. 

CROUSE,  HELEN  V.,  Research  Associate,  University  of  Pennsylvania. 

CROWELL,  SEARS,  Assistant  Professor  of  Zoology,  Miami  University. 

FROEHLICH,  ALFRED,  Associate,  May  Institute  for  Medical  Research. 

FURCHGOTT,  ROBERT  F.,  Research  Associate,  Cornell  L^niversity. 

FURTH,  JACOB,  Professor  of  Pathology,  Cornell  University. 

GAFFRON,  HANS,  Assistant  Professor  of  Biochemistry,  Research  Associate,  University  of  Chicago. 

GALTSOFF,  PAUL  S.,  Senior  Biologist,  U.  S.  Fish  and  Wildlife  Service. 

CARREY,  W.  E.,  Professor  of  Physiology,  Emeritus,  Vanderbilt  University,  School  of  Medicine. 

GLASER,  OTTO  C.,  Professor  of  Biology,  Amherst  College. 

GOODCHILD,  DR.  C.  G.,  Associate  Professor  of  Biology,  State  Teachers  College. 

GORBMAN,  AUBREY,  Instructor  in  Biology,  Wayne  University. 

GOULD,  HARLEY  N.,  Professor  of  Biology,  H.  Sophie  Newcomb  College. 

GRAND,  C.  G.,  Research  Associate,  New  York  University. 

HAMBURGER,  VIKTOR,  Professor  of  Zoology,  Washington  University. 

HARTMAN,   FRANK   A.,   Professor   and   Chairman   of   Department   of    Physiology,    Ohio    State 

University. 

HARVEY,  ETHEL  BROWNE,  Independent  Investigator  Biology  Department,  Princeton  University. 
HARVEY,  E.  NEWTON,  Professor  of  Physiology,  Princeton  University. 
HAYASHI,  TERU,  Instructor  in  Zoology,  University  of  Missouri. 
HAYWOOD,  CHARLOTTE,  Professor  of  Physiology,  Mount  Holyoke  College. 
HEILBRUNN,  L.  V.,  Professor  of  Zoology,  University  of  Pennsylvania. 
HIBBARD,  HOPE,  Professor  of  Zoology,  Oberlin  College. 
HICKSON,  ANNA  KELTCH,  Research  Chemist,  Eli  Lilly  &  Company. 
HOPKINS,   HOYT   S.,   Associate   Professor   of   Physiology,    New    York    University,    College    of 

Dentistry. 

HUBER,  WOLFGANG,  Senior  Research  Chemist,  Winthrop  Chemical  Company. 
JACOBS,  M.  H.,  Professor  of  General  Physiology,  University  of  Pennsylvania. 
JAEGER,  LUCENA,  Research  Associate,  Columbia  University. 

JENKINS,  GEORGE  B.,  Professor  of  Anatomy,  Emeritus,  George  Washington  University. 
JOHLIN,  J.  M.,  Associate  Professor  of  Biochemistry,  Vanderbilt  University. 
JOHNSON,  FRANK  H.,  Assistant  Professor  of  Biology,  Princeton  University. 
KRAHL,  M.  E.,  Instructor  in  Pharmacology,  Columbia  University. 
LANDIS,   EUGENE   M.,    Professor   of    Physiology   and    Head    of    Department,    Harvard    Medical 

School. 


REPORT  OF  THE  DIRECTOR  37 

LAVIN,  GEORGE  I.,  In  charge  of  Spectroscopic  Laboratory,   Rockefeller   Institute  for   Medical 

Research. 

LEE,  RICHARD  E.,  Student,  Columbia  University. 
LIEBEN,  FRITZ,  Research  Fellow,  Johns  Hopkins  University. 
LILLIE,  RALPH  S.,  Professor  of  Physiology,  Emeritus,  University  of  Chicago. 
LOCH  HEAD,  JOHN  H.,  Assistant  Professor  of  Zoology,  University  of  Vermont. 
McCLUNG,  C.  E.,  Professor  of  Zoology,  Emeritus,  University  of  Pennsylvania. 
MACLEAN,  BERNICE  L.,  Assistant  Professor,  Department  Biological  Sciences,  Hunter  College. 
MAC.ALHAES,  HULDA,  Instructor  in  Zoology,  Duke  University. 
MARKS,  MILDRED  H.,  Student,  Massachusetts  Institute  of  Technology. 
MARSLAND,  DOUGLAS  A.,  Associate  Professor  of  Biology,  New  York  University. 
MAST,  S.  O.,  Professor  of  Zoology,  Emeritus,  Johns  Hopkins  University. 
MATHEWS,  ALBERT  P.,  Professor  of  Biochemistry,  Emeritus,  University  of  Cincinnati. 
MATTHEWS,  SAMUEL  A.,  Professor  of  Biology,  Williams  College. 
MEMHARD,  ALLEN  R.,  Crescent  Road,  Riverside,  Connecticut. 
MENKIN,  VALY,  Assistant  Professor  of  Pathology,  Duke  University. 
METZ,  CHARLES  W.,  Director  Zoological  Laboratory,  University  of  Pennsylvania. 
MICHAELIS,  LEONOR,  Member  Emeritus,  Rockefeller  Institute  for  Medical  Research. 
NACHMANSOHN,  DAVID,  Research  Associate  in  Neurology,  Columbia  University. 
NORTHROP,  JOHN  H.,  Member  of  the  Institute,  Rockefeller  Institute  for  Medical  Research. 
OPPENHEIMER,  JANE  M.,  Assistant  Professor  of  Biology,  Bryn  Mawr  College. 
OSTERHOUT,  W.  J.  V.,  Member  Emeritus,  Rockefeller  Institute  for  Medical  Research. 
PARPART,  ARTHUR  K.,  Associate  Professor  of  Biology,  Princeton  University. 
PIERCE,  MADELEXE  E.,  Associate  Professor  of  Zoology,  Vassar  College. 
RAMSEY,  ROBERT  W.,  Associate  Professor  of  Physiology,  Medical  College  of  Virginia. 
RANKIN,  JOHN  S.,  JR.,  Assistant  Professor  of  Zoology,  University  of  Connecticut. 
REID,  W.  MALCOLM,  Assistant  Professor  of  Biology,  Monmouth  College. 
RIKER,  WALTER  F.,  JR.,  Instructor  in  Medicine  and  Pharmacology,  Cornell  University  Medical 

School. 

Ris,  HANS,  Assistant  in  Physiology,  Rockefeller  Institute  for  Medical  Research. 
ROBBIE,  WILBUR  A.,  Research  Associate,  State  University  of  Iowa. 
ROGICK,  MARY  DORA,  Professor  of  Biology,  College  of  New  Rochelle. 
SAMPSON,  MYRA  M.,  Professor  of  Zoology,  Smith  College. 
SANDOW,  ALEXANDER,  Assistant  Professor  of  Biology,  New  York  University. 

SCHAEFFER,  A.  A.,  Professor  of  Biology,  Temple  University. 

SCHARRER,  ERNST  A.,  Assistant  Professor  of  Anatomy,  Western  Reserve  University  School  of 
Medicine. 

SCOTT,  SISTER  FLORENCE  MARIE,  Professor  of  Zoology,  Seton  Hill  College. 

SCOTT,  GEORGE  T.,  Instructor,  Oberlin  College. 

SHANES,  ABRAHAM  M.,  Assistant  Professor  of  Physiology.  New  York  University  College  of 
Dentistry. 

SHAPIRO,  HARRY  H.,  Assistant  Professor  of  Anatomy,  Columbia  University. 

SLIFER,  ELEANOR  H.,  Assistant  Professor  of  Zoology,  State  University  of  Iowa. 

SMITH,  DIETRICH  CONRAD,  Associate  Professor  of  Physiology,  University  of  Maryland,  School 
of  Medicine. 

STERN,  KURT  G.,  Lecturer  in  Department  of  Chemistry,  Polytechnic  Institute  of  Brooklyn. 

STEWART,  DOROTHY  R.,  Fellow  in  Physiology,  University  of  Pennsylvania. 

STOKEY,  ALMA  G.,  Professor  of  Plant  Science,  Emeritus,  Mount  Holyoke  College. 

STUNKARD,  H.  W.,  Professor  of  Biology,  New  York  University. 

TAFT,  CHARLES  H.,  Associate  Professor  of  Pharmacology,  Medical  Branch,  University  of  Texas. 

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

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

THIVY,  FRANCESCA,  Professor  of  Biology,  Women's  Christian  College. 

VILLEE,  CLAUDE  A.,  Assistant  Professor  of  Zoology,  University  of  North  Carolina. 

WAINIO,  WALTER  W.,  Assistant  Professor  of  Physiology,   New   York  University,   College  of 
Dentistry. 

WARREN,  CHARLES  O.,  Assistant  Professor  of  Physiology,  Cornell  University  Medical  College. 

WENRICH,  D.  H.,  Professor  of  Zoology,  University  of  Pennsylvania. 

WHITING,  ANNA  R.,  Visiting  Investigator,  University  of  Pennsylvania. 


MARINE  BIOLOGICAL  LABORATORY 

WHITING,  P.  W.,  Associate  Professor  of  Zoology.  University  of  Pennsylvania. 

WICHTERMAN,  RALPH,  Assistant  Professor  of  Biology,  Temple  University. 

WILLIER,    B.    H.,    Professor   of   Zoology   and   Director   of   the    Biological    Laboratories,    Johns 

Hopkins  University. 

WINSOR,  CHARLES  P.,  Research  Associate,  Princeton  University. 
WITKUS,  ELEANOR  R.,  Instructor  in  Botany  and  Bacteriology,  Fordham  University. 
WOODWARD,  ALVALYN  E.,  Assistant  Professor,  University  of  Michigan. 
WOODWARD,  ARTHUR  A.,  JR.,  Research  Assistant,  University  of  Pennsylvania. 
WOOLEY,  D.  W.,  Associate.  Rockefeller  Institute  for  Medical  Research. 
WRINCH,  DOROTHY,  Lecturer  in  Physics,  Smith  College. 

YNTEMA,  CHESTER  L.,  Assistant  Professor  of  Anatomy,  Cornell  University  Medical  College. 
ZWEIFACH,  BENJAMIN  W.,  Research  Associate  in  Biology,  New  York  University. 

Beginning  Investigators,  1945 

BROWN,  ELLEN,  Commonwealth  Fellow,  University  of  California  Medical  School. 

BROWN,  VIRGINIA  H.,  Graduate  Student,  Ohio  State  University. 

COYNE,  CHRISTOPHER  J.,  Student,  University  of  Pennsylvania. 

DAVIDSON,  MARGARET  E.,  Demonstrator  and  Assistant  to  Dr.  Berrill,  McGill  University. 

KRUGELIS,  EDITH  J.,  Graduate  Student,  Columbia  University. 

LERNER,  ELEANOR,  Fellow  in  Zoology,  Washington  University. 

LOVELACE,  ROBERTA,  Teaching  Fellow,  University  of  North  Carolina. 

MILLER,  HELMA  C.,  Assistant,  Johns  Hopkins  University. 

SCHNEYER,  LEON  H.,  Instructor,  New  York  University,  College  of  Dentistry. 

WILSON,  WALTER,  Graduate  Student,  University  of  Pennsylvania. 

Research  Assistants,  1945 

ABRAMSKY,  TESS,  Research  Assistant,  Rockefeller  Institute  for  Medical  Research. 

BRUNELLI,  ELEANOR  L.,  Research  Assistant,  New  York  University,  College  of  Dentistry. 

DEFALCO,  ROSE  H.,  Research  Assistant,  University  of  Pennsylvania. 

FISCHL,  MATHILDA,  Research  Assistant  in  Medicine,  Cornell  University. 

FRANZ,  RUTH  ESTELLE,  Research  Assistant,  Yale  University. 

GARZOLI,  RAY  F.,  Graduate  Student,  University  of  California. 

GOULD,  DAVID,  Research  Technician,  New  York  University. 

HARLOW,  JANET,  Technician,  Syracuse  University. 

HELFMAN,  MYRNA,  Technician,  New  York  University. 

HENLEY,  CATHERINE,  Graduate  Teaching  Assistant,  Johns  Hopkins  University. 

HONEGGER,  CAROL,  Student,  Temple  University. 

LAWLER,  H.  CLAIRE,  Research  Assistant,  New  York  University. 

LEVIN,  ISAAC,  Research  Assistant,  Princeton  University. 

LEVY,  BETTY,  Research  Assistant,  Rockefeller  Institute  for  Medical  Research. 

LOOFBOURROW,  G.  N.,  Instructor,  Rhode  Island  State  College. 

MCVEIGH,  IDA,  Research  Assistant  in  Botany,  Yale  University. 

METZ,  DELILAH  B.,  Research  Assistant,  Eli  Lilly  &  Co. 

MINER,  KARYL,  Research  Assistant,  New  York  University. 

MITCHELL,  CONSTANCE,  Research  Assistant,  University  of  Pennsylvania. 

PETTENGILL,  OLIVE  S.,  Student,  Temple  University. 

QUINN,  GERTRUDE  P.,  Research  Assistant,  New  York  University. 

ROTHENBERG,  M.  A.,  Research  Assistant  in  Biochemistry,  College  of  Physicians  and  Surgeons. 

UHLMAN,  GLORIA  E.,  Research  Assistant,  Yale  University. 

WALTERS,  C.  PATRICIA,  Research  Assistant,  Eli  Lilly  &  Co. 

WARNER,  CHARLOTTE,  Medical  Student,  University  of  Pennsylvania. 

ZACKS,  SUMNER  L,  Student,  Brookline  High  School. 

Library  Readers,  1945 

AMBERSON,  WILLIAM  R.,  Professor  of  Physiology,  University  of  Maryland. 
BECK,  LYLE  V.,  Associate  Professor  of  Physiology,  Hahnemann  Medical  College. 


REPORT  OF  THE  DIRECTOR  39 

BENDICH,  AARON,  Member  War  Research  Division,  Neurological  Institute,  New  York. 

BLOCK,  RICHARD  J.,  Associate,  New  York  Medical  College. 

BREHME,  KATHERINE  S.,  Lecturer,  Cornell  University  Medical  College. 

CAHEN,  RAYMOND  L.,  Research  Assistant,  Yale  University,  Medical  School. 

CARSON,  HAMPTON  L.,  Instructor  in  Zoology,  Washington  University. 

CASSIDY,  HAROLD  G.,  Assistant  Professor  of  Chemistry,  Yale  University. 

COLWIN,  LAURA  HUNTER,  Instructor,  Pennsylvania  College  for  Women. 

FRIEDEMANN,  ULRICH  H.,  Head  of  Department  of  Bacteriology,  Brooklyn  Jewish  Hospital. 

FRISCH,  JOHN  A.,  Professor  of  Biology,  Canisius  College. 

GATES,  R.  RUGGLES,  Professor  Emeritus,  University  of  London. 

GUREWICH,  VLADIMIR,  Assistant  Visiting  Physician,  Bellevue  Hospital. 

KABAT,  ELVIN  A.,  Research  Associate  in  Biochemistry,  College  of  Physicians  and  Surgeons. 

KAYLOR,  CORNELIUS  T.,  Assistant  Professor  of  Anatomy,  Syracuse  University. 

KELLER,  RUDOLPH,  Researcher,  Robinson  Foundation,  New  York. 

KRASNOW,  FRANCES,  Head  of  Department  of  Research,  Guggenheim  Dental  Foundation. 

LANGE,  MATHILDE  M.,  Professor  of  Zoology,  Head  of  Department  of  Biology,  W'heaton  College. 

LOEWI,  OTTO,  Research  Professor  of  Pharmacology,  New  York  University,  College  of  Medicine. 

MARINELLI,  LEONIDAS,  Physicist,  Memorial  Hospital. 

MAVOR,  JAMES  W.,  Professor  of  Biology,  Union  College. 

MAYER,  MANFRED  M.,  Scientific  Staff,  War  Research  Division,  Columbia  University. 

METZ,  CHARLES  B.,  Instructor  in  Biology,  Wesleyan  University. 

MEYERHOF,  DR.  OTTO,  Research  Professor  of  Biochemistry,  University  of  Pennsylvania. 

MOLDAVER,  JOSEPH.  Research  Associate  in  Neurology,  Columbia  University. 

MOORE,  JOHN  A.,  Assistant  Professor  of  Zoology,  Barnard  College. 

MOSCHCOWITZ,  ELI,  Assistant  Professor  of  Chemical  Medicine,  Columbia  University. 

OSEASOHN,  ROBERT  O.,  Long  Island  College  of  Medicine. 

PERRY,  BARBARA  H.,  Graduate  Student  and  Teaching  Fellow  in  Zoology,  Smith  College. 

PONDER,  ERIC,  Research,  Nassau  Hospital. 

RAMSDEN,  ETHEL  J.,  Instructor  in  Biology,  Montclair  Teachers  Colle;-ir. 

ROBINSON,  MILES  H.,  Instructor  in  Pharmacology,  University  of  Pennsylvania. 

RYAN,  FRANCIS  J.,  Assistant  Professor,  Columbia  University. 

SCOTT,  ALLAN,  Assistant  Professor  of  Biology,  Union  College. 

STRAUSS,  WILLIAM  L.,  JR.,  Associate  Professor  of  Anatomy,  Johns  Hopkins  University. 

VoxDACH,  HERMAN,  Assistant  Professor  of  Physiology,  Georgetown  Medical  School. 

WALLACH,  JACQUES  B.,  Long  Island  College  of  Medicine. 

ZORZOLI,  ANITA,  Assistant  Instructor,  New  York  University. 

Students,  1945 
BOTANY 

BARRACLOUGH,  MARY  EDITH,  Student,  Smith  College. 

DIETZ,  ALMA,  Assistant  in  Biology,  American  International  College. 

GARDNER,  ELIZABETH  B.,  Radcliffe  College. 

MOUL,  EDWIN  THEODORE,  Botany  Assistant,  University  of  Pennsylvania. 

SMITH,  MATTIE  Lot',  Student,  Radcliffe  College. 

EMBRYOLOGY 

BEACH,  JANET,  Student,  University  of  Connecticut. 

BERNIER,  GERMAINE,  University  of  Montreal,  Quebec,  Canada. 

BERRY,  BETH  SINCLAIR,  Student,  Rockford  College,  Illinois. 

CARTER,  MARJORIE  ESTELLE,  Teacher,  Georgia  State  Women's  College. 

CHIRICO,  ANNA  MARIE,  Student.  Seton  Hill  College. 

CLARK,  CARL  CYRUS,  Student,  Amherst  College.  • 

COPINGER,  ANNE  STEVENS,  Goucher  College. 

EHRLICH,  MIRIAM,  Knox  College. 

Izzo,  MARY  JANE,  University  of  Rochester. 

KELL,  AMY,  University  of  Illinois. 

LEVIN,  ILANE  B.,  Goucher  College. 


40  MARINE  BIOLOGICAL  LABORATORY 

LODICO,  DOROTHY  GERALDINE,  University  of  Rochester. 

LOVELACE,  LOLLIE  ROBERTA,  Teaching  Fellow,  University  of  North  Carolina. 

MARKER,  MURIEL  JOSEPHINE,  Student,  Colby  College. 

MEZGER,  LISELOTTE,  Student,  Bryn  Mawr  College. 

MILLER,  HELMA  C.,  Graduate  Assistant,  Johns  Hopkins  University. 

PERKINS,  BARBARA  BURNHAM,  University  of  Connecticut. 

RAYMOND,  BARBARA,  Student,  Swarthmore  College. 

RICE,  MARY  ESTHER,  Assistant  in  Biology  Laboratory,  Drew  University. 

ROBERTS,  ELIZABETH  S.,  Assistant  in  Biology,  Wilson  College. 

RUDERMAN,  CLAIRE,  Teaching  Assistant,  University  of  Rochester. 

THORBY,  JEAN  ADELAIDE,  Student,  Rockford  College. 

UPHOFF,  DELTA  Ev  University  of  Rochester. 

PHYSIOLOGY 

BRUST,  MANFRED,  Student,  New  York  University. 

COOK,  JOHN  ALFRED,  George  Washington  University. 

FERGUSON,  ALICE  HOWARD,  Graduate  Assistant,  Louisiana  State  University. 

FLINKER,  MARIE-LOUISE  M.,  Assistant  in  Physiology,  Vassar  College. 

FOGERSON,  VIRGINIA  LEE,  Student,  Drury  College. 

FOSTER,  ELIZABETH  JANE,  Student,  University  of  Illinois. 

GOLDSMITH,  YVETTE,  Perth  Amboy,  New  Jersey. 

HAJEK,  NORMA  MARY,  Cornell  University. 

HECHT,  LISELOTTE  ISABELLA,  Student,  University  of  Michigan. 

RESNICK,  OSCAR,  Resident  Scholar,  Harvard  University. 

WEISS,  MICHAEL  S.,  Student,  Washington  Square  College. 

WOLFF,  MARY  LYDA,  Instructor,  Cedar  Crest  College. 

WORKEN,  BARNEY,  3400  Wayne  Avenue,  New  York  City. 

ZOOLOGY 

ARONOWITZ,  OLGA,  New  York  University. 

BATES,  MARY  FLORENCE,  Student,  Vassar  College. 

BAYORS,  WINIFRED  M.,  Student  Seton  Hill  College. 

BEAL,  JUDITH  D.,  Vassar  College. 

BENJAMIN,  MRS.  REZSIN  C.,  Undergraduate  Student,  University  of  Rochester. 

BERNARD,  SISTER  MARIE,  Fordham  University. 

BERNIER,  GERMAINE,  Instructor,  University  of  Montreal. 

BEZILLA,  HELEN,  Student,  Seton  Hill  College. 

BRADIN,  JOHN  L.,  Northwestern  University. 

CALVERT,  JULIE  NEIL,  Student,  Wilson  College. 

CARLSON,  ALICE  MARIE,  Laboratory  Assistant,  University  of  Minnesota. 

CHAFFIN,  EVELYN  L.,  Student.  Drury  College. 

CLARK,  CARL  CYRUS,  Amherst  College. 

CUMMINGS,  REV.  GEORGE  W.,  Graduate  Student,  Catholic  University. 

DAILEY,  DOROTHY  HELEN,  Depamv  University. 

DAWSON,  MARY  JEAN,  Student,  Mt.  Holyoke  College. 

DEMPSEY,  ELLEN,  Oberlin  College. 

DICKASON,  MARY  ELIZABETH,  Student,  Smith  College. 

FARNHAM,  CAROL  JEAN,  Student,  Drury  College. 

FREITAG,  JANET  FAITH,  Student,  University  of  Connecticut. 

GOLDIS,  BERNICE  RUTH,  Graduate  Student,  University  of  Pennsylvania. 

HANLON,  REV.  JAMES  J.,  Graduate  Student,  Fordham  University. 

HILL,  SHIRLEY  B.,  Student,  Vassar  College. 

HINES,  EILEEN  BARBARA,  State  University  of  Iowa. 

JONES,  DOROTHY  B.,  Student,  University  of  Connecticut. 

JOSITA,  SISTER  M.,  Student,  Fordham  University. 

JULIER,  EDITH  VAILLANT,  Student,  Vassar  College. 


REPORT  OF  THE  DIRECTOR 


41 


KREKELER,  CARL  H.,  Student,  Washington  University. 

KUHN,  ALICE  ROBERTS,  Western-Maryland  College. 

LOWENS,  MARY  DOROTHY,  Student,  Swarthmore  College. 

McCLAiN,  MARYLOW,  Student,  Swarthmore  College. 

MCGREGOR,  ELIZABETH,  Instructor,  Mount  Holyoke  College. 

McVicKER,  SISTER  MAUREEN,  Teacher  of  Biology,  St.  Joseph's  College  for  Women 

MALLOCH,  JEAN,  Vassar  College. 

MEIHACK,  HELEN  LLOYD,  Student,  Oberlin  College. 

MINA,  FRANK  A.,  Laboratory  Instructor,  Fordham  University. 

OSBORN,  JOAN  A.,  Student,  Barnard  College. 

PETERS,  REV.  JOSEPH  J.,  Graduate  Student,  Fordham  University. 

RAYMOND,  BARBARA,  Student,  Swarthmore  College. 

RIGGS,  AUSTIN  F.,  Student,  Harvard  University. 

ROGERS,  HENRY  CRAMPTON,  Deerfield  Academy. 

SCHAEFER,  GERTRUDE,  Undergraduate,  Temple  University. 

SEAMAN,  ARLENE,  Zoology  Assistant,  Cornell  University. 

SNIPES,  ANNE,  Wheaton  College. 

STEES,  NANCY,  Teacher,  West  Chester  State  Teachers  College. 

SURRARRER,  THOMAS  C,  Professor  of  Biology,  Baldwin-Wallace  College. 

THORNTON,  DOROTHY  GOLDEN,  Assistant  in  Zoology  Dept.,  Wellesley  College. 

TUPPER,  LYLA,  Graduate  Student,  Northwestern  University. 

UBER,  VIRGINIA  M.,  Student,  Pennsylvania  College  for  Women. 

WAX,  FLORENCE  SIMA,  Student,  Oberlin  College. 

WHYTE,  MARJORIE  ANN,  Assistant,  Cornell  University. 

WILCOX,  BARBARA  L.,  Student,  Radcliffe  College. 

WILLIAMS,  OLWEN,  Teacher  of  Biology  and  Chemistry,  The  Putney  School. 

WILSON,  FAITH  EVELYN,  Johns  Hopkins  University. 

WILSON,  MARIE  ELLEN,  Student,  Western  Maryland  College. 


4.     TABULAR  VIEW  OF  ATTENDANCE 


1942      1943      1944      1945 


INVESTIGATORS — Total  337 

Independent  197 

Under  instruction  / 59 

Library  readers  31 

Research  assistants  50 

STUDENTS — Total  131 

Zoology  55 

Embryology  37 

Physiology  24 

Botany  15 

TOTAL  ATTENDANCE  468 

Less  persons  registered  as  both  students  and  investigators  7 


461 

INSTITUTIONS    REPRESENTED — Total     144 

By   investigators    102 

By    students    72 

SCHOOLS  AND  ACADEMIES  REPRESENTED   

By   investigators    5 

By    students    2 

FOREIGN  INSTITUTIONS  REPRESENTED  

By  investigators  3 

By  students  1 


201 

132 

16 

28 

25 

74 

36 

24 

6 

8 

275 
2 

273 

126 

83 

43 


160 
89 
19 
35 
17 
68 
47 
13 
8 

228 
6 

222 

116 

70 

41 

2 
1 


—     2 


193 

112 

11 

50 
20 

75 
37 
23 
10 

5 
276 

1 

275 

106 

74 

41 

1 
2 

2 
3 


212 

138 

10 

38 

26 

96 

55 

23 

13 

5 

308 


124 

100 

49 

2 
2 


42 


MARINE  BIOLOGICAL  LABORATORY 


5.     SUBSCRIBING  AND  COOPERATING  INSTITUTIONS 


1945 


Albany  Medical  College 

Amherst  College 

Biological  Institute,  Philadelphia,  Pennsylvania 

Bowdoin  College 

Bryn  Mawr  College 

Cathedral  College 

The  Catholic  University  of  America 

Columbia  University 

Cornell  University 

Cornell   University  Medical   College 

Duke  University 

Fish  and  Wild  Life  Service,  U.  S.  Dept.  of 

the  Interior 
Fordham  University 
Goucher  College 
Harvard  University 
Harvard  University  Medical  School 
Industrial  and  Engineering  Chemistry,  of  the 

American  Chemical  Society 
Johns  Hopkins  University 
Johns  Hopkins  Medical   School 
Lee  Foundation 
Eli  Lilly  &  Company 
Long  Island  University 
Macy  Foundation 

Massachusetts  Institute  of  Technology 
McGill  University 
Miami  University 
Mount  Holyoke  College 
New  York  University 

New  York  University  College  of  Medicine 
New  York  University  School  of  Dentistry 
New    York    University    Washington    Square 

College 


Oberlin  College 

Ohio  State  University 

Pennsylvania  College  for  Women 

Princeton  University 

Radcliffe  College 

Rockefeller  Institute  for  Medical  Research 

St.  Joseph  College  for  Women 

Smith  College 

State  University  of  Iowa 

Syracuse  University 

Syracuse   University   Medical   School 

Temple  University 

University  of  Chicago 

University  of  Connecticut 

University  of  Illinois 

University  of  Maryland  Medical  School 

University  of  Michigan 

University  of  Missouri 

University  of  Pennsylvania 

L'niversity  of  Pennsylvania  School  of  Medicine 

University  of  Rochester 

Vanderbilt  University  Medical  School 

Vassar  College 

Washington  University 

Wayne  University 

Wellesley  College 

Wesleyan  University 

Western  Maryland  College 

Western  Reserve  University 

Wheaton  College 

Williams  College 

Wilson  College 

Woods  Hole  Oceanographic  Institution 

Yale  University 


6.     EVENING  LECTURES,  1945 

Friday,  June  29 

PROF.  P.  W.  WHITING "The    Development    of    Hymenopteran    Ge- 
netics." 
Friday,  July  6 

DR.  R.  R.  GATES "Human    Heredity    in    Relation    to    Animal 

Genetics." 
Friday,  July  13 

DR.    I.   FANKUCHEN    "X-Ray  Diffraction  and  Protein  Structure." 

Friday,  July  20 

PROF.  S.  C.  BROOKS "Our  Interrelationships  with  South  Ameri- 
can Universities,  together  with  Illustrated 
Travel  Notes." 
Friday,  July  27 

PROF.  E.  G.  BUTLER "Problems  of  Differentiation  and  Dediffer- 

entiation   in   Amputated   Urodele   Limbs." 


REPORT  OF  THE  DIRECTOR  43 

Friday,  August  3 

DR.  BOSTWICK  H.  KETCHUM   "The  Prevention  of  Ship  Bottom  Fouling." 

Friday,  August  10 

DR.  DANIEL  MERRIMAN   "A  Study  in  Pure  and  Applied  Marine  Bi- 
ology.    The  Life   History  and  Economic 
Importance  of  the  Ocean  Pout." 
Friday,  August  17 

DR.  DETLEV  W.  BRONK  "Biological  Research  During  the  War  and 

Postwar   Periods." 
Friday,  August  24 

DR.  F.  L.  HISAVV  "Endocrines    and    the    Evolution    of    Vivi- 

parity  among  the  Vertebrates." 
Monday,  August  27 

GEORGE  G.  LOWER   "Local  Invertebrates." 

• 
Wednesday,  August  29 

DR.  PAUL  S.  GALTSOFF   "Impressions    of    a    Biologist    at    the    San 

Francisco  Conference." 
Thursday,  August  30 

MAJOR  A.  H.  NEUFELD "Medical  Research  Organization  in  the  Ca- 
nadian Army." 
Thursday,  August  30 

CAPT.  W.  R.  DURYEE "Medical  Military  Training." 

7.     SHORTER  SCIENTIFIC  PAPERS,  1945 

Tuesday,  July  24 

DR.  M.  M.  BROOKS "The  Redox  Potential  of  Penicillium  rota- 
turn  Medium  under  Some  Different  Con- 
ditions of  Growth." 

DR.  WILBUR  ROBBIE "The  Use  of  Cyanide  in  Manometric  Ex- 
perimentation." 

DR.  SEARS  CROWELL "The    Displacement   of   Terns   by   Gulls   at 

Weepecket  Island." 
Tuesday,  July  31 

DR.  P.  W.  WHITING  "The   Problem  of   Reversal   of   Male   Hap- 

loidy  by  Selection." 

DR.  BERTA  SCHARRER "Experimental  Tumors  after  Nerve  Section 

in  an   Insect." 

DR.  P.  S.  GALTSOFF "Reactions  of  Oysters  to  Free  Chlorine." 

Tuesday,  August  7 

DR.  T.  H.  BULLOCK  "Organization  of  Giant  Nerve  Fibers  in  cer- 
tain Polychaetes." 

DR.  ERNST  SCHARRER "The    Origin    of    Neurosecretory    Granules 

from   Basophil   Substances   in  the   Nerve 
Cells  of  Fishes." 

DR.  C.  H.  TAFT "The  Action  of  Quitenine  on  the  Livers  of 

Tautog  and  Toadfish." 

DR.  A.  M.  SHANES  "Evidence  of  a  Metabolic  Effect  by  Potas- 
sium in  Lowering  the  Injury  Potential  of 
Nerve." 


44  MARINE  BIOLOGICAL  LABORATORY 

Tuesday,  August  14 

DR.  R.  CHAMBERS    "Interrelations       between       Sperm-Nucleus, 

Egg-Nucleus  and  Cytoplasm  in  Asterias 
Egg." 

DR.  KURT  G.  STERN    "Physical-chemical  Studies  on  Chromosomal 

Nucleoproteins." 
Tuesday,  August  21 

DR.   DOROTHY  WRINCH    "Hemoglobin  and  other  Native  Proteins." 

DR.  E.  R.  WIT.KUS    "Endomitosis  in  Plants." 

DR.  C.  A.  BERGER "Recent  Cytological  Studies  in  Culex." 

Thursday,  August  23 

DR.  ETHEL  B.  HARVEY   "Development  of  Granule-free  Fractions  of 

Arbacia  eggs." 

DR.  ALEXANDER  SANDOW   "Studies  of  the  Muscle  Twitch  by  Methods 

of  Electronic  Recording." 

DR.  C.  D.  BEERS  "The  Role  of  Bacteria  in  the  Excystment  of 

the  Ciliate  Didinium." 
Monday,  August  27 

DR.  ANNA  R.  WHITING "Differences     in     Sensitivity,     Hatchability 

Curves  and  Cytological  Effects  between 
Eggs  X-rayed  in  First  Meiotic  Prophase 
and  Metaphase." 

DR.  W.  W.  WAINIO   "Aerobic    Oxidation    of    Simple    sugars    by 

Mammalian  Liver." 

DR.  DUGALD  E.  S.  BROWN "The  Role  of  Myosin  and  Myosin  Triphos- 

phatase  ;';;  Vitro  and  in  Muscle." 
Tuesday,  August  28 

DR.  LLOYD  M.  BERTHOLF "Accelerating  Metamorphosis  in  the  Tuni- 
cate Styela." 

DR.  ALFRED  FROELICH  "The  Influence  of  Drugs  on  Heat-narcosis." 

DR.  W.  MALCOLM  REID  "In  Vivo  and  in  Vitro  Glycogen  Utiliza- 
tion in  the  Avial  Nematode  Ascardia 
Galli." 

8.     MEMBERS  OF  THE  CORPORATION,  1945 

1.     LIFE  MEMBERS 

ALLIS,  MR.  E.  P.,  JR.,  Palais  Carnoles,  Menton,  France. 

BECKWITH,  DR.  CORA  J.,  Vassar  College,  Poughkeepsie,  New  York. 

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

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

COLE,  DR.  LEON  J.,  College  of  Agriculture,  Madison,  Wisconsin. 

CONKLIN,  PROF.  EDWIN  G.,  Princeton  University,  Princeton,  New  Jersey. 

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

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

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

KING,  MR.  CHAS.  A. 


REPORT  OF  THE  DIRECTOR  45 

KINGSBURY,  PROF.  B.  F.,  Cornell  University,  Ithaca,  New  York. 

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

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

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

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

MORGAN,  MRS.  T.  H.,  Pasadena,  California. 

MORGAN,  PROF.  T.  H.,  Director  of  Biological  Laboratory,  California  Institute  of 

Technology,  Pasadena,  California. 
NOYES,  Miss  EVA  J. 

PORTER,  DR.  H.  C.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 
SCOTT,  DR.  ERNEST  L.,  Columbia  University,  New  York  City,  New  York. 
SEARS,  DR.  HENRY  F.,  86  Beacon  Street,  Boston,  Massachusetts. 
SHEDD,  MR.  E.  A. 

STRONG,  DR.  O.  S.,  Columbia  University,  New  York  City,  New  York. 
WAITE,  PROF.  F.  C.,  144  Locust  Street,  Dover,  New  Hampshire. 
WALLACE,  LOUISE  B.,  359  Lytton  Avenue,  Palo  Alto,  California. 

2.     REGULAR  MEMBERS 

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

ADDISON,  DR.  W.  H.  F.,  University  of  Pennsylvania  Medical  School,  Philadelphia, 
Pennsylvania. 

ADOLPH,  DR.  EDWARD  F.,  University  of  Rochester  Medical  School,  Rochester,  New 
York. 

ALBAUM,  DR.  HARRY  G.,  Biology  Dept.,  Brooklyn  College,  Brooklyn,  N.  Y. 

ALBERT,  DR.  ALEXANDER,  383  Harvard  Street,  Cambridge,  Mass. 

ALLEE,  DR.  W.  C.,  The  University  of  Chicago,  Chicago,  Illinois. 

AMBERSON,  DR.  WILLIAM  R.,  Department  of  Physiology,  University  of  Maryland, 
School  of  Medicine,  Lombard  and  Greene  Streets,  Baltimore,  Maryland. 

ANDERSON,  DR.  RUBERT  S.,  University  of  Maryland  School  of  Medicine,  Depart- 
ment of  Physiology,  Baltimore,  Maryland. 

ANDERSON,  DR.  T.  F.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 

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

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

BAITSELL,  DR.  GEORGE  A.,  Yale  University,  New  Haven,  Connecticut. 

BAKER,  DR.  H.  B.,  Zoological  Laboratory,  University  of  Pennsylvania,  Philadelphia, 
Pennsylvania. 

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

BALLENTINE,  DR.  ROBERT,  Columbia  University,  Department  of  Zoology,  New  York 
City,  New  York. 

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

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

BARRON,  DR.  E.  S.  GUZMAN,  Department  of  Medicine,  The  University  of  Chicago, 
Chicago,  Illinois. 

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


46  MARINE  BIOLOGICAL  LABORATORY 

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

BEADLE,  DR.  G.  W.,  School  of  Biological  Sciences,  Stanford  University,  California. 

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

BECK,  DR.  L.  V.,  Hahnemann  Medical  College,  Philadelphia,  Pennsylvania. 

BEHRE,  DR.  ELINOR  H.,  Louisiana  State  University,  Baton  Rouge,  Louisiana. 

BERTHOLF,  DR.  LLOYD  M.,  Western  Maryland  College,  Westminster,  Maryland. 

BIGELOW,  DR.  H.  B.,  Museum  of  Comparative  Zoology,  Cambridge,  Massachusetts. 

BIGELOW,  PROF.  R.  P.,  Massachusetts  Institute  of  Technology,  Cambridge,  Massa- 
chusetts. 

BINFORD,  PROF.  RAYMOND,  Guilford  College,  North  Carolina. 

BISSONNETTE,  DR.  T.  HUME,  Trinity  College,  Hartford,  Connecticut. 

BLANCHARD,  PROF.  K.  C,  Johns  Hopkins  Medical  School,  Baltimore,  Maryland. 

BODINE,  DR.  J.  H.,  Department  of  Zoology,  State  University  of  Iowa,  Iowa  City, 
Iowa. 

BORING,  DR.  ALICE  M.,  Dickinson  House,  South  Haclley,  Massachusetts. 

BRADLEY,  PROF.  HAROLD  C.,  University  of  Wisconsin,  Madison,  Wisconsin. 

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

BRONFENBRENNER,  DR.  JACQUES  J.,  Department  of  Bacteriology,  Washington  Uni- 
versity Medical  School,  St.  Louis,  Missouri. 

BROOKS,  DR.  MATILDA  M.,  University  of  California,  Department  of  Zoology,  Berke- 
ley, California. 

BROOKS,  DR.  S.  C.,  University  of  California,  Berkeley,  California. 

BROWN,  DR.  DUGALD  E.  S.,  New  York  University,  College  of  Dentistry,  209  East 
23d  Street,  New  York  City,  New  York. 

BROWN,  DR.  FRANK  A.,  JR.,  Department  of  Zoology,  Northwestern  University, 
Evanston,  Illinois. 

BUCK,  DR.  JOHN  B.,  Industrial  Hygiene  Research  Lab.,  National  Institute  of 
Health,  Bethesda,  Maryland. 

BUCKINGHAM,  Miss  EDITH  N.,  Sudbury,  Massachusetts. 

BUDINGTON,  PROF.  R.  A.,  Winter  Park,  Florida. 

BULLINGTON,  DR.  W.  E.,  Randolph-Macon  College,  Ashland,  Virginia. 

BULLOCK,  DR.  T.  L.,  University  of  Missouri,  Columbia,  Missouri. 

BURBANCK,  DR.  WILLIAM  D.,  Department  of  Biology,  Drury  College,  Springfield, 
Missouri. 

BURKENROAD,  DR.  M.  D.,  Yale  University,  New  Haven,  Connecticut. 

BUTLER,  DR.  E.  G.,  Princeton  University,  Princeton,  N.  J. 

BYRNES,  DR.  ESTHER  F.,  1803  North  Camac  Street,  Philadelphia,  Pennsylvania. 

CAMERON,  DR.  J.  A.,  Baylor  College  of  Dentistry,  Dallas,  Texas. 

CANNAN,  PROF.  R.  K.,  New  York  University  College  of  Medicine,  477  First  Ave- 
nue, New  York  City,  New  York. 

CARLSON,  PROF.  A.  J.,  Department  of  Physiology,  The  University  of  Chicago,  Chi- 
cago, Illinois. 

CAROTHERS,  DR.  E.  ELEANOR,  134  Avenue  C.  East,  Kingman,  Kansas. 

CARPENTER,  DR.  RUSSELL  L.,  Tufts  College,  Tufts  College,  Massachusetts. 

CARROLL,  PROF.  MITCHELL,  Franklin  and  Marshall  College,  Lancaster,  Pennsyl- 
vania. 


REPORT  OF  THE  DIRECTOR  47 

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

CATTELL,  DR.  McKEEN,  Cornell  University  Medical  College.  1300  York  Avenue, 
New  York  City,  New  York. 

CATTELL,  MR.  WARE,  1621  Connecticut  Are.,  Washington,  D.  C. 

CHAMBERS,  DR.  ROBERT,  Washington  Square  College,  New  York  University,  Wash- 
ington Square,  New  York  City,  New  York. 

CHASE,  DR.  AURIN  M.,  Princeton  University,  Princeton,  New  Jersey. 

CHENEY,  DR.  RALPH  H.,  Biology  Department,  Long  Island  University,  Brooklyn, 
New  York. 

CHIDESTER,  PROF.  F.  E.,  Auburndale,  Massachusetts. 

CHILD,  PROF.  C.  M.,  Jordan  Hall,  Stanford  University,  California. 

CHURNEY,  DR.  LEON,  155  Powell  Lane,  Upper  Darby,  Pennsylvania. 

CLAFF,  MR.  C.  LLOYD,  Research  Fellow  in   Surgery,  Harvard   Medical   School, 
Boston,  Mass. 

CLARK,  PROF.  E.  R.,  University  of  Pennsylvania  Medical  School,  Philadelphia, 
Pennsylvania. 

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

CLARKE,  DR.  G.  L.,  Harvard  University  Biol.  Lab.,  16  Divinity  Ave.,  Cambridge 
38,  Mass. 

CLELAND,  PROF.  RALPH  E.,  Indiana  University,  Bloomington,  Indiana. 

CLOWES,  DR.  G.  H.  A.,  Eli  Lilly  and  Company,  Indianapolis,  Indiana. 

COE,  PROF.  W.  R.,  Yale  University,  New  Haven,  Connecticut. 

COHN,  DR.  EDWIN  J.,  183  Brattle  Street,  Cambridge,  Massachusetts. 

COLE,  DR.  ELBERT  C.,  Department  of  Biology,  Williams  College,  Williamstown, 
Massachusetts. 

COLE,  DR.  KENNETH  S.,  University  of  Chicago,  Chicago,  Illinois. 

COLLETT,  DR.  MARY  E.,  Western  Reserve  University,  Cleveland,  Ohio. 

COLTON,  PROF.  H.  S.,  Box  601,  Flagstaff,  Arizona. 

COOPER,  DR.  KENNETH  W.,  Department  of  Biology,  Princeton  University,  Prince- 
ton, New  Jersey. 

COPELAND,  PROF.  MANTON,  Bowdoin  College,  Brunswick,  Maine. 

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

COSTELLO,  DR.  HELEN  MILLER,  Department  of  Zoology,  University  of  North  Caro- 
lina, Chapel  Hill,  North  Carolina. 

CRAMPTON,  PROF.  H.  E.,  American  Museum  of  Natural  History,  New  York  City, 
New  York. 

CRANE,  JOHN  O.,  Woods  Hole,  Massachusetts. 

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

CROASDALE,  HANNAH  T.,  Dartmouth  College,  Hanover,  New  Hampshire. 

CROUSE,  DR.  HELEN  V.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 

CROWELL,  DR.  P.  S.,  JR.,  Department  of  Zoology,  Miami  University,  Oxford,  Ohio. 

CURTIS,  DR.  MAYNIE  R.,  377  Dexter  Trail,  Mason,  Michigan. 

CURTIS,  PROF.  W.  C.,  University  of  Missouri,  Columbia,  Missouri. 

DAN,  DR.  KATSUMA,  Misaki  Biological  Station,  Misaki,  Japan. 

DAVIS,  DR.  DONALD  W.,  College  of  William  and  Mary,  Williamsburg,  Virginia. 

DAWSON,  DR.  A.  B.,  Harvard  University,  Cambridge,  Massachusetts. 


48  MARINE  BIOLOGICAL  LABORATORY 

DAWSON,  DR.  J.  A.,  The  College  of  the  City  of  New  York,  New  York  City,  New 
York. 

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

DEMEREC,  DR.  M.,  Carnegie  Institution  of  Washington,  Cold  Spring  Harbor,  Long 
Island,  New  York. 

DILLER,  DR.  WILLIAM  F.,  1016  South  45th  Street,  Philadelphia,  Pennsylvania. 

DODDS,  PROF.  G.  S.,  Medical  School,  University  of  West  Virginia,  Morgantown 
West  Virginia. 

DOLLEY,  PROF.  WILLIAM  L.,  University  of  Buffalo,  Buffalo,  New  York. 

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

DuBois,  DR.  EUGENE  F.,  Cornell  University  Medical  College,  1300  York  Avenue, 
New  York  City,  New  York. 

DUGGAR,  DR.  BENJAMIN  M.,  c/o  Lederle  Laboratories  Inc.,  Pearl  River,  New 
York. 

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

DURYEE,  DR.  WILLIAM  R.,  Surgeon  General's  Office,  Washington,  D.  C. 

EDWARDS,  DR.  D.  J.,  Cornell  University  Medical  College,  1300  York  Avenue,  New 
York  City,  New  York. 

ELLIS,  DR.  F.  W.,  1175  Centre  Street,  Newton,  Massachusetts. 

EVANS,  DR.  TITUS  C.,  College  of  Physicians  and  Surgeons,  630  West  168th  Street, 
New  York  City,  New  York. 

FAILLA,  DR.  G.,  College  of  Physicians  and  Surgeons,  630  West  168th  Street,  New 
York  City,  New  York. 

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

FAUST,  DR.  ERNEST  C.,  Tulane  University  of  Louisiana,  New  Orleans,  Louisiana. 

FERGUSON,  DR.  JAMES  K.  W.,  Department  of  Pharmacology,  University  of  Toronto, 
Ontario,  Canada. 

FIGGE,  DR.  F.  H.  J.,  4636  Schenley  Road,  Baltimore,  Maryland. 

FISCHER,  DR.  ERNST,  Department  of  Physiology,  Medical  College  of  Virginia,  Rich- 
mond, Virginia. 

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

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

FORBES,  DR.  ALEXANDER,  Harvard  University  Medical  School,  Boston,  Massachu- 
setts. 

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

FURTH,  DR.  JACOB,  Cornell  University  Medical  College,  1300  York  Avenue,  New 
York  City,  New  York. 

GALTSOFF,  DR.  PAUL  S.,  420  Cumberland  Avenue,  Somerset,  Chevy  Chase,  Mary- 
land. 

GARREY,  PROF.  W.  E.,  Vanderbilt  University  Medical  School,  Nashville,  Tennessee. 

GATES,  DR.  REGINALD  R.,  Woods  Hole,  Massachusetts. 

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

GERARD,  PROF.  R.  W.,  The  University  of  Chicago,  Chicago,  Illinois. 

GLASER,  PROF.  O.  C.,  Amherst  College,  Amherst,  Massachusetts. 


REPORT  OF  THE  DIRECTOR  49 

GOLDFORB,  PROF.  A.  J.,  College  of  the  City  of  New  York,  Convent  Avenue  and  139th 
Street,  New  York  City,  New  York. 

GOODCHILD,  DR.  CHAUNCEY  G.,  State  Teachers  College,  Springfield,  Missouri. 

GOODRICH,  PROF.  H.  B.,  Wesleyan  University,  Middletown,  Connecticut. 

GOTTSCHALL,  DR.  GERTRUDE  Y.,  919  20th  Street,  Washington,  D.  C. 

GRAHAM,  DR.  J.  Y.,  Roberts,  Wisconsin. 

GRAND,  CONSTANTINE  G.,  Biology  Department,  Washington  Square  College,  New 
York  University,  Washington  Square,  New  York  City,  New  York. 

GRAVE,  PROF.  B.  H.,  DePauw  University,  Greencastle,  Indiana. 

GRAY,  PROF.  IRVING  E.,  Duke  University,  Durham,  North  Carolina. 

GREGORY,  DR.  LOUISE  H.,  Barnard  College,  Columbia  University,  New  York  City, 
New  York. 

GUDERNATSCH,  DR.  J.  FREDERICK,  New  York  University,  100  Washington  Square, 
New  York  City,  New  York. 

GUTHRIE,  DR.  MARY  J.,  University  of  Missouri,  Columbia,  Missouri. 

GUYER,  PROF.  M.  F.,  University  of  Wisconsin,  Madison,  Wisconsin. 

HAGUE,  DR.  FLORENCE,  Sweet  Briar  College,  Sweet  Briar,  Virginia. 

HALL,  PROF.  FRANK  G.,  Duke  University,  Durham,  North  Carolina. 

HAMBURGER,  DR.  VIKTOR,  Department  of  Zoology,  Washington  University,   St. 
Louis,  Missouri. 

HANCE,  DR.  ROBERT  T.,  The  Cincinnati  Milling  Machine  Co.,  Cincinnati  9,  Ohio. 

HARGITT,  PROF.  GEORGE  T.,  Department  of  Zoology,  Duke  University,  Durham, 
North  Carolina. 

HARMAN,  DR.  MARY  T.,  Kansas  State  Agricultural  College,  Manhattan,  Kansas. 

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

HARPER,  PROF.  R.  A.,  R.  No.  5,  Bedford,  Virginia. 

HARRISON,  PROF.  Ross  G.,  Yale  University,  New  Haven,  Connecticut. 

HARTLINE,  DR.  H.  KEFFER,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 

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

HARVEY,  DR.  E.  NEWTON,  Guyot  Hall,  Princeton  University,  Princeton,  New  Jer- 
sey. 

HARVEY,  DR.  ETHEL  BROWNE,  48  Cleveland  Lane,  Princeton,  New  Jersey. 

HAYDEN,  DR.  MARGARET  A.,  Wellesley  College,  Wellesley,  Massachusetts. 

HAYES,  DR.  FREDERICK  R.,  Zoological  Laboratory,  Dalhousie  University,  Halifax, 
Nova  Scotia. 

HAYWOOD,  DR.  CHARLOTTE,  Mount  Holyoke  College,  South  Hadley,  Massachusetts. 

HECHT,  DR.  SELIG,  Columbia  University,  New  York  City,  New  York. 

HEILBRUNN,  DR.  L.  V.,  Department  of  Zoology,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania. 

HENDEE,  DR.  ESTHER  CRISSEY,  Russell  Sage  College,  Troy,  New  York. 

HENSHAW,  DR.  PAUL  S.,  National  Cancer  Institute,  Bethesda,  Maryland. 

HESS,  PROF.  WALTER  N.,  Hamilton  College,  Clinton,  New  York. 

HIATT,  DR.  E.  P.,  Duke  University,  Durham,  North  Carolina. 

HIBBARD,  DR.  HOPE,  Department  of  Zoology,  Oberlin  College,  Oberlin,  Ohio. 

HILL,  DR.  SAMUEL  E.,  18  Collins  Avenue,  Troy,  New  York. 

HINRICHS,  DR.  MARIE,  Department  of  Physiology  and  Health  Education,  Southern 
Illinois  Normal  University,  Carbondale,  Illinois. 


50  MARINE  BIOLOGICAL  LABORATORY 

HISAW,  DR.  F.  L.,  Harvard  University,  Cambridge,  Massachusetts. 

HOADLEY,  DR.  LEIGH,  Harvard  University,  Cambridge,  Massachusetts. 

HOBER,  DR.  RUDOLF,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 

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

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

HOLLAENDER,  DR.  ALEXANDER,  c/o  National  Institute  of  Health,  Laboratory  of  In- 
dustrial Hygiene,  Bethesda,  Maryland. 

HOPKINS,  DR.  D.WIGHT  L.,  Mundelein  College,  6363  Sheridan  Road,  Chicago.  Illi- 
nois. 

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

HOWLAND,  DR.  RUTH  B.,  Washington  Square  College,  New  York  University, 
Washington  Square  East,  New  York  City,  New  York. 

HOYT,  DR.  WILLIAM  D.,  Washington  and  Lee  University,  Lexington,  Virginia. 

HYMAN,  DR.  LIBBIE  H.,  American  Museum  of  Natural  History,  New  York  City, 
New  York. 

IRVING,  LT.  COL.  LAURENCE,  Wright  Field,  Dayton,  Ohio. 

ISELIN,  MR.  COLUMBUS  O'D.,  Woods  Hole,  Massachusetts. 

JACOBS,  PROF.  MERKEL  H.,  School  of  Medicine,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania. 

JENKINS,  DR.  GEORGE  B.,  1336  Parkwood  Place,  N.W.,  Washington,  D.  C. 

JENNINGS,  PROF.  H.  S.,  Department  of  Zoology,  University  of  California,  Los  An- 
geles, California. 

JOHLIN,  DR.  J.  M.,  Vanderbilt  University  Medical  School,  Nashville,  Tennessee. 

JONES,  DR.  E.  RUFFIN,  JR.,  College  of  William  and  Mary,  Williamsburg,  Virginia. 

KAUFMANN,  PROF.  B.  P.,  Carnegie  Institution,  Cold  Spring  Harbor,  Long  Island, 
New  York. 

KEMPTON,  PROF.  RUDOLF  T.,  Vassar  College,  Poughkeepsie,  New  York. 

KIDDER,  DR.  GEORGE  W.,  Brown  University,  Providence,  Rhode  Island. 

KIDDER,  JEROME  F.,  Woods  Hole,  Massachusetts. 

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

KINDRED,  DR.  J.  E.,  University  of  Virginia,  Charlottesville,  Virginia. 

KING,  DR.  HELEN  D.,  Wistar  Institute  of  Anatomy  and  Biology,  36th  Street  and 
Woodland  Avenue,  Philadelphia,  Pennsylvania. 

KING,  DR.  ROBERT  L.,  State  University  of  Iowa,  Iowa  City,  Iowa. 

KNOWLTON,  PROF.  F.  P.,  Syracuse  University,  Syracuse,  New  York. 

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

KRAHL,  DR.  M.  E.,  College  of  Physicians  and  Surgeons,  630  West  168th  Street, 
New  York  32,  New  York. 

KRIEG,  DR.  WENDELL  J.  S.,  303  East  Chicago  Ave.,  Chicago,  Illinois. 

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

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

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

LANGE,  DR.  MATHILDE  M.,  Wheaton  College,  Norton,  Massachusetts. 


REPORT  OF  THE  DIRECTOR  51 

LAVIN,  DR.  GEORGE  I.,  Rockefeller  Institute,  66th  Street  and  York  Avenue,  New 
York  City,  New  York. 

LEWIS,  PROF.  I.  F.,  University  of  Virginia,  Charlottesville,  Virginia. 

LILLIE,  PROF.  FRANK  R.,  The  University  of  Chicago,  Chicago,  Illinois. 

LILLIE,  PROF.  RALPH  S.,  The  University  of  Chicago,  Chicago,  Illinois. 

LITTLE,  DR.  E.  P.,  Phillips  Exeter  Academy,  Exeter,  New  Hampshire. 

LOCHHEAD,  DR.  JOHN  H.,  Department  of  Zoology,  University  of  Vermont,  Bur- 
lington, Vermont. 

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

LOEB,  DR.  R.  F.,  180  Ft.  Washington  Avenue,  New  York  City,  New  York. 

LOEWI,  PROF.  OTTO,  155  East  93d  Street,  New  York  City,  New  York. 

LOWTHER,  MRS.  FLORENCE  DEL.,  Barnard  College,  Columbia  University,  New  York 
City,  New  York. 

LUCAS,  DR.  ALFRED  M.,  Regional  Poultry  Research  Laboratory,  East  Lansing, 
Michigan. 

LUCRE,  PROF.  BALDUIN,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 

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

LYNCH,  DR.  RUTH  STOCKING,  Dept.  of  Zoology,  University  of  California,  Los 
Angeles  24,  California. 

LYNN,  DR.  WILLIAM  G.,  Department  of  Biology,  The  Catholic  University  of  Amer- 
ica, Washington,  D.  C. 

MACDOUGALL,  DR.  MARY  S.,  Agnes  Scott  College,  Decatur,  Georgia. 

MACNAUGHT,  MR.  FRANK  M.,  Marine  Biological  Laboratory,  Woods  Hole,  Massa- 
chusetts. 

McCoucH,  DR.  MARGARET  SUMWALT,  University  of  Pennsylvania  Medical  School, 
Philadelphia,  Pa. 

MCGREGOR,  DR.  J.  H.,  Columbia  University,  New  York  City,  New  York. 

MACKLIN,  DR.  CHARLES  C.,  School  of  Medicine,  University  of  Western  Ontario, 
London,  Canada. 

MAGRUDER,  DR.  SAMUEL  R.,  Department  of  Anatomy,  Tufts  Medical  School,  Bos- 
ton, Massachusetts. 

MALONE,  PROF.  E.  F..  153  Cortland  Avenue,  Winter  Park,  Florida. 

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

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

MARTIN,  PROF.  E.  A.,  Department  of  Biology,  Brooklyn  College,  Bedford  Avenue 
and  Avenue  H,  Brooklyn,  New  York. 

MAST,  PROF.  S.  O.,  Johns  Hopkins  University,  Baltimore,  Maryland. 

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

MATTHEWS,  DR.  SAMUEL  A.,  Thompson  Biological  Laboratory,  Williams  College, 
Williamstown,  Massachusetts. 

MAYOR,  PROF.  JAMES  W.,  Union  College,  Schenectady,  New  York. 

MAZIA,  DR.  DANIEL,  Department  of  Zoology,  Gowen  Field,  Boise,  Idaho. 

MEDES,  DR.  GRACE,  Lankenau  Research  Institute,  Philadelphia,  Pennsylvania. 

MEIGS,  MRS.  E.  B.,  1736  M  Street,  N.W.,  Washington,  D.  C. 

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


52  MARINE  BIOLOGICAL  LABORATORY 

MENKIN,  DR.  VALY,  Duke  University,  School  of  Medicine,  Durham,  North  Caro- 
lina. 

METZ,  PROF.  CHARLES  W.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 

MICHAELIS,  DR.  LEONOR,  Rockefeller  Institute,  66th  Street  and  York  Avenue,  New 
York  City,  New  York. 

MILLER,  DR.  J.  A.,  Division  of  Anatomy,  College  of  Medicine,  University  of  Ten- 
nessee, Memphis,  Tennessee. 

MINNICH,  PROF.  D.  E.,  Department  of  Zoology,  University  of  Minnesota,  Minne- 
apolis, Minnesota. 

MITCHELL,  DR.  PHILIP  H.,  Brown  University,  Providence,  Rhode  Island. 

MOORE,  DR.  CARL  R.,  The  University  of  Chicago,  Chicago,  Illinois. 

MOORE,  DR.  J.  A.,  Barnard  College,  New  York  City,  New  York. 

MORGAN,  DR.  ISABEL  M.,  Poliomyelitis  Research  Center,  1901  E.  Madison  Street, 
Baltimore  5,  Maryland. 

MORRILL,  PROF.  C.  V.,  Cornell  University  Medical  College,  1300  York  Avenue, 
New  York  City,  New  York. 

MULLER,  PROF.  H.  J.,  Department  of  Zoology,  Indiana  University,  Bloomington, 
Indiana. 

NACHMANSOHN,  DR.  D.,  College  of  Physicians  and  Surgeons,  630  W.  168th  Street, 
New  York  City,  New  York. 

NAVEZ,  DR.  ALBERT  E.,  Department  of  Biology,  Milton  Academy,  Milton,  Massa- 
chusetts. 

NEWMAN,  PROF.  H.  H.,  173  Devon  Drive,  Clearwater,  Florida. 

NICHOLS,  DR.  M.  LOUISE,  Rosemont,  Pennsylvania. 

NONIDEZ,  DR.  JOSE  F.,  Cornell  University  Medical  College,  1300  York  Avenue, 
New  York  City,  New  York. 

NORTHROP,  DR.  JOHN  H.,  The  Rockefeller  Institute,  Princeton,  New  Jersey. 

OCHOA,  DR.  SEVERQ,  New  York  University,  College  of  Medicine,  477  First  Avenue, 
New  York  16,  New  York. 

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

OSBURN,  PROF.  R.  C.,  Ohio  State  University,  Columbus,  Ohio. 

OSTERHOUT,  PROF.  W.  J.  V.,  Rockefeller  Institute,  66th  Street  and  York  Avenue, 
New  York  City,  New  York. 

OSTERHOUT,  MRS.  MARIAN  IRWIN,  Rockefeller  Institute,  66th  Street  and  York 
Avenue,  New  York  City,  New  York. 

PACKARD,  DR.  CHARLES,  Marine  Biological  Laboratory,  Woods  Hole,  Massachu- 
setts. 

PAGE,  DR.  IRVINE  H.,  Cleveland  Clinic,  Cleveland,  Ohio. 

PAPPENHEIMER,  DR.  A.  M.,  5  Acacia  Street,  Cambridge,  Massachusetts. 

PARKER,  PROF.  G.  H.,  Harvard  University,  Cambridge,  Massachusetts. 

PARMENTER,  DR.  C.  L.,  Department  of  Zoology,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania. 

PARPART,  DR.  ARTHUR  K.,  Princeton  University,  Princeton,  New  Jersey. 

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

PAYNE,  PROF.  F.,  University  of  Indiana,  Bloomington,  Indiana. 

PEEBLES,  PROF.  FLORENCE,  Lewis  and  Clark  College,  Portland,  Oregon. 


REPORT  OF  THE  DIRECTOR  53 

PIERCE,  DR.  MADELENE  E.,  Vassaf  College,  Poughkeepsie,  New  York. 

PINNEY,  DR.  MARY  E.,  Milwaukee-Downer  College,  Milwaukee,  Wisconsin. 

PLOUGH,  PROF.  HAROLD  H.,  Amherst  College,  Amherst,  Massachusetts. 

POLLISTER,  DR.  A.  W.,  Columbia  University,  New  York  City,  New  York. 

POND,  DR.  SAMUEL  E.,  53  Alexander  Street,  Manchester.  Connecticut. 

PRATT,  DR.  FREDERICK  H.,  Wellesley  Hills  82,  Massachusetts. 

PROSSER,  DR.  C.  LADD,  University  of  Chicago,  Chicago,  Illinois. 

RAND,  DR.  HERBERT  W.,  Harvard  University,  Cambridge,  Massachusetts. 

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

REDFIELD,  DR.  ALFRED  C.,  Harvard  University,  Cambridge,  Massachusetts. 

REID,  DR.  W.  M.,  Monmouth  College,  Monmouth,  Illinois. 

RENN,  DR.  CHARLES  E.,  Harvard  University,  Cambridge,  Massachusetts. 

RENSHAW,  DR.  BIRDSEY,  Rockefeller  Institute  for  Medical  Research,  66th  Street 
and  York  Avenue,  New  York  City,  New  York. 

DERENYI,  DR.  GEORGE  S.,  Department  of  Anatomy,  University  of  Pennsylvania, 
Philadelphia,  Pennsylvania. 

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

RICE,  PROF.  EDWARD  L.,  Ohio  Wesleyan  University,  Delaware,  Ohio. 

RICHARDS,  PROF.  A.,  University  of  Oklahoma,  Norman,  Oklahoma. 

RICHARDS,  DR.  A.  GLENN,  Entomology  Department.  University  Farm,  Univ.  of 
Minnesota,  St.  Paul  8,  Minnesota. 

RICHARDS.  DR.  O.  W..  Research  Dept.  American  Optical  Co.,  19  Doat  Street, 
Buffalo,  New  York. 

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

ROGERS,  PROF.  CHARLES  G.,  Oberlin  College,  Oberlin,  Ohio. 

ROGICK,  DR.  MARY  D.,  College  of  New  Rochelle,  New  Rochelle,  New  York. 

ROMER,  DR.  ALFRED  S.,  Harvard  University,  Cambridge,  Massachusetts. 

ROOT,  DR.  R.  W.,  Department  of  Biology,  College  of  the  City  of  New  York,  Con- 
vent Avenue  and  139th  Street,  New  York  City,  New  York. 

ROOT,  DR.  W.  S.,  College  of  Physicians  and  Surgeons,  Department  of  Physiology, 
630  West  168th  Street,  New  York  City,  New  York. 

RUEBUSH,  DR.  T.  K.,  Dayton,  Virginia. 

RUGH,  DR.  ROBERTS,  Department  of  Biology,  Washington  Square  College,  New 
York  University,  New  York  City,  New  York. 

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

SASLOW,  DR.  GEORGE,  Washington  University  Medical  School,  St.  Louis,  Missouri. 

SAUNDERS,  LAWRENCE,  W.  B.  Saunders  Publishing  Company,  Philadelphia,  Penn- 
sylvania. 

SAYLES,  DR.  LEONARD  P.,  Department  of  Biology,  College  of  the  City  of  New  York, 
139th  Street  and  Convent  Avenue,  New  York  City,  New  York. 

SCHAEFFER,  DR.  ASA  A.,  Biology  Department,  Temple  University,  Philadelphia, 
Pennsylvania. 

SCHARRER,  DR.  ERNST  A.,  Western  Reserve  University,  School  of  Medicine,  2109 
Adelbert  Road,  Cleveland  6,  Ohio. 

SCHECHTER,  DR.  VICTOR,  College  of  the  City  of  New  York,  139th  Street  and  Con- 
vent Avenue,  New  York  City,  New  York. 


54  MARINE  BIOLOGICAL  LABORATORY 

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

SCHMITT,  PROF.  F.  O.,  Department  of  Biology,  Massachusetts  Institute  of  Tech- 
nology, Cambridge,  Massachusetts. 

SCHOTTE,  DR.  OSCAR  E.,  Department  of  Biology,  Amherst  College,  Amherst,  Massa- 
chusetts. 

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

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

SCHRAMM,  PROF.  J.  R.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 

SCOTT,  DR.  ALLAN  C.,  Union  College,  Schenectady,  New  York. 

SCOTT,  PROF.  WILLIAM  B.,  7  Cleveland  Lane,  Princeton,  New  Jersey. 

SCOTT,  SISTER  FLORENCE  MARIE,  Professor  of  Biology,  Seton  Hill  College,  Greens- 
burg,  Pennsylvania. 

SEMPLE,  MRS.  R.  BOWLING,  140  Columbia  Heights,  Brooklyn,  New  York. 

SEVERINGHAUS,  DR.  AURA  E.,  Department  of  Anatomy,  College  of  Physicians  and 
Surgeons,  630  West  168th  Street,  New  York  City,  New  York. 

SHANES,  DR.  ABRAHAM  M.,  New  York  University,  College  of  Dentistry,  New 
York. 

SHAPIRO,  DR.  HERBERT,  Radiation  Laboratory,  Massachusetts  Institute  of  Technol- 
ogy, Cambridge,  Massachusetts. 

SHELFORD,  PROF.  V.  E.,  Vivarium,  Wright  and  Healey  Streets,  Champaign,  Illinois. 

SHULL,  PROF.  A.  FRANKLIN,  University  of  Michigan,  Ann  Arbor,  Michigan. 

SHUMWAY,  DR.  WALDO,  University  of  Illinois,  Urbana,  Illinois. 

SICHEL,  DR.  FERDINAND  J.  M.,  University  of  Vermont,  Burlington,  Vermont. 

SICHEL,  MRS.  F.  J.  M.,  35  Henderson  Terrace,  Burlington,  Vermont. 

SINNOTT,  DR.  E.  W.,  Osborn  Botanical  Laboratory,  Yale  University,  New  Haven, 
Connecticut. 

SLIFER,  DR.  ELEANOR  H.,  Department  of  Zoology,  State  University  of  Iowa,  Iowa 
City,  Iowa. 

SMITH,  DR.  DIETRICH  CONRAD,  Department  of  Physiology,  University  of  Mary- 
land School  of  Medicine,  Lombard  and  Greene  Streets,  Baltimore,  Maryland. 

SNYDER,  PROF.  L.  H.,  Ohio  State  University,  Department  of  Zoology,  Columbus, 
Ohio. 

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

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

STARK,  DR.  MARY  B.,  1  East  105th  Street,  New  York  City,  New  York. 

STEINBACH,  DR.  H.  BURR,  Department  of  Zoology,  Washington  University,  St. 
Louis,  Missouri. 

STERN,  DR.  CURT,  Department  of  Zoology,  University  of  Rochester,  Rochester, 
New  York. 

STERN,  DR.  KURT  G.,  Polytechnic  Institute,  Department  of  Chemistry,  85  Living- 
ston Street,  Brooklyn,  New  York. 

STEWART,  DR.  DOROTHY  R.,  University  of  Pennsylvania  Medical  School,  Depart- 
ment of  Physiology,  Philadelphia  4,  Pennsylvania. 

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


REPORT  OF  THE  DIRECTOR  55 

STUNKARD,  DR.  HORACE  W.,   New  York  University,  University  Heights,   New 

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

California. 
SUMMERS,   DR.  FRANCIS  MARION,  Univ.   of  California,   College  of  Agriculture, 

Davis,  California. 

TAFT,  DR.  CHARLES  H.,  JR.,  University  of  Texas  Medical  School,  Galveston,  Texas. 
TASHIRO,  DR.  SHIRO,  Medical  College,  University  of  Cincinnati,  Cincinnati,  Ohio. 
TAYLOR,  DR.  C.  V.,  Leland  Stanford  University,  Leland  Stanford,  California. 
TAYLOR,  DR.  WILLIAM  R.,  University  of  Michigan,  Ann  Arbor,  Michigan. 
TE\VINKEL,  DR.   L.  E.,   Department  of   Zoology,   Smith   College,   Northampton, 

Massachusetts. 

TURNER,  DR.  ABBY  H.,  Mt.  Holyoke  College,  South  Hadley,  Massachusetts. 
TURNER,  PROF.  C.  L.,  Northwestern  University,  Evanston,  Illinois. 
TYLER,  DR.  ALBERT,  California  Institute  of  Technology,  Pasadena,  California. 
UHLENHUTH,  DR.  EDUARD,  University  of  Maryland,  School  of  Medicine,  Balti- 
more, Maryland. 

VISSCHER,  DR.  J.  PAUL,  Western  Reserve  University,  Cleveland,  Ohio. 
WAINIO,  DR.  W.  W.,  New  York  University,  College  of  Dentistry,  New  York  City. 
WALD,    DR.    GEORGE,    Biological    Laboratories,    Harvard    University,    Cambridge, 

Massachusetts. 

WARBASSE,  DR.  JAMES  P.,  Woods  Hole,  Massachusetts. 
WARD,  PROF.  HENRY  B.,  1201  W.  Nevada,  Urbana,  Illinois. 

WARREN,  DR.  HERBERT  S.,  1405  Greywall  Lane,  Overbrook  Hills,  Pennsylvania. 
WATERMAN,  DR.  ALLYN  J.,  Department  of  Biology,  Williams  College,  Williams- 
town,  Massachusetts. 
WEISS,  DR.  PAUL  A.,  Department  of  Zoology,  The  University  of  Chicago,  Chicago, 

Illinois. 

WENRICH,  DR.  D.  H.,  University  of  Pennsylvania,  Philadelphia,  Pennsylvania. 
WHEDON,  DR.  A.  D.,  North  Dakota  Agricultural  College,  Fargo,  North  Dakota. 
WHITAKER,  DR.  DOUGLAS  M.,  P.  O.  Box  2514,  Stanford  University,  California. 
WHITE,  DR.  E.  GRACE,  Wilson  College,  Chambersburg,  Pennsylvania. 
WHITING,  DR.  PHINEAS  W.,  Zoological  Laboratory,  University  of  Pennsylvania, 

Philadelphia,  Pennsylvania. 

WHITNEY,  DR.  DAVID  D.,  University  of  Nebraska,  Lincoln,  Nebraska. 
WICHTERMAN,  DR.  RALPH,  Biology  Department,  Temple  University,  Philadelphia, 

Pennsylvania. 

WIEMAN,  PROF.  H.  L.,  University  of  Cincinnati,  Cincinnati,  Ohio. 
WILLIER,  DR.  B.  H.,  Department  of  Biology,  Johns  Hopkins  University,  Baltimore, 

Maryland. 

WILSON,  DR.  J.  W.,  Brown  University,  Providence,  Rhode  Island. 
WITSCHI,  PROF.  EMIL,  Department  of  Zoology,  State  University  of  Iowa,  Iowa 

City,  Iowa. 
WOLF,    DR.    ERNST,    Biological    Laboratories,    Harvard    University,    Cambridge, 

Massachusetts. 

WOODRUFF,  PROF.  L.  L.,  Yale  University,  New  Haven,  Connecticut. 
WOODWARD,  DR.  ALVALYN  E.,  Zoology  Department,  University  of  Michigan,  Ann 

Arbor,  Michigan. 


56 


MARINE  BIOLOGICAL  LABORATORY 


WRINCH,  DR.  DOROTHY,  Smith  College,  Northampton,  Massachusetts. 

YNTEMA,  DR.  C.  L.,  Department  of  Anatomy,  Cornell  University  Medical  College, 

1300  York  Avenue,  New  York  City,  New  York. 
YOUNG,  DR.  B.  P.,  Cornell  University,  Ithaca,  New  York. 
YOU-NG,  DR.  D.  B.,  7128  Hampden  Lane,  Bethesda,  Maryland. 

9.     ASSOCIATES  OF  THE  MARINE  BIOLOGICAL  LABORATORY 


BARTOVV.  MRS.  FRANCIS  D. 

BEHNKE.  JOHN. 

BROWN,  MR.  AND  MRS.  THEODORE. 

CALKINS,  MRS.  GARY  N. 

COOPER,  CHARLES  P. 

CROSSLEY,  MR.  AND  MRS.  ARCHIBALD. 

CROWELL,  PRINCE. 

CURTIS,  DR.  WILLIAM  D. 

FAY,  MRS.  H.  H. 

FOSTER,  RICHARD  W. 

GARFIELD,  IRVIN  McD. 

GREEN,  GEORGE  S. 

GREEN,  Miss  GLADYS. 

HARRISON,  R.  G.,  JR. 

HUNT,  MRS.  REID. 

JANNEY,  MRS.  WALTER. 


KNOWER,  MRS.  H.  McE. 
LILLIE,  MRS.  F.  R. 

McMlTCHELL,   MRS.  J.   McC. 

MURPHY,  DR.  W.  J. 
NEWTON,  Miss  HELEN. 
NIMS,  MRS.  E.  D. 
NORMAN,  EDWARD  A. 
RIGGS,  MRS.  LAWRASON. 
RUDD,  MRS.  H.  W.  D. 
SAUNDERS,  MRS.  LAWRENCE. 
STOCKARD,  MRS.  C.  R. 
SWOPE,  MR.  AND  MRS.  GERARD. 
TEBBETS,  WALTER. 
WEBSTER,  MR.  AND  MRS.  E.  S. 
WICK,  MRS.  MYRON  T. 
WILSON,  MRS.  E.  B. 


^v 


C 


<*D 


LIBRA*\ 


J*4S* 

o        •* 

* 


THE  INFLUENCE  OF  TEXTURE  AND  COMPOSITION  OF 

SURFACE  ON  THE  ATTACHMENT  OF  SEDENTARY 

MARINE  ORGANISMS* 

C.  M.  POMEKAT  AND  C.  M.  WEISS 
Medical  Branch,  University  of  Tc.vas  and  the  IToods  Hole  Oceanographic  Institution  * 

Marine  installations  of  various  kinds  necessitate  exposure  of  construction  mate- 
rials under  sea  water.  Data  dealing  with  the  amount  of  fouling  accumulated  by 
such  materials  are  not  abundant.  Information  which  might  be  of  aid  to  the  scien- 
tist seeking  the  most  favorable  material  upon  which  to  collect  sedentary  organisms 
for  study  is  also  scanty.  The  present  study  was  undertaken  to  determine  the  effect 

TABLE  I 

Effect  of  surface  texture  of  glass  on  attachment  of  sedentary  organisms.  (Numbers  of  individuals 

on  each  surface  of  80  square  inches  of  plate) 


Surface  number 

Plain 
0 

Sand- 
blasted 
1 

Factrolite 
2 

Prestlitt- 
.? 

Ribbed 
4 

Pentecor 

5 

Series  No.  1,  Tahiti  Beach1 

39  days  (8/22/42-9/30/42) 

Hydroides  sp. 

143 

265 

152 

506 

349 

197 

Spirorbis  sp. 

85              1  88 

122 

90 

163 

110 

Barnacles 

1,948          1.072           1,162 

975 

1,674 

2,140 

Total 

2,176          1,525 

1  ,436 

1,571 

2,186 

2,447 

Average  pop. 

725.3          508.3 

478.7 

523.7    '       728.7 

815.7 

Average/square  inch 

9.1 

6.4 

6.0 

6.5 

9.1 

10.2 

Series  No.  2,  Miami  Beach- 

17  days  (8/22/42-9/8/42) 

Wet  weight  (grams) 

5  1  .0 

45.5 

50.0 

41.0 

50.0 

4  1  .0 

Drv  weight  (grams) 

8.5 

6.4 

7.9 

5.5 

7.5 

8.8 

Barnacles 

308 

227 

268 

213 

263 

331 

Series  No.  J,  Miami  Bcach- 

30  days  (9/15/42-10/15/42) 

Wet  weigh  1   (grams) 

164.5 

174.0 

149.0 

1  50.0 

126.0 

155.5 

1  )ry  weight  (grams) 

51.5 

50.0 

30.0 

24.0 

24.0 

33.0 

Barnacles 

642 

515 

554 

778 

798 

977 

1  Subtropical  testing  service. 


2  Beach  boat  slips. 


*  The  observations  described  here  were  made  while  the  authors  were  engaged  by  the  Woods 
Hole  Oceanographic  Institution  in  an  investigation  of  fouling,  under  contract  with  the  Bureau 
of  Ships,  Navy  Department,  which  has  given  permission  for  their  publication.  The  opinions 
presented  here  are  those  of  the  authors  and  do  not  necessarily  reflect  the  official  opinion  of  the 
Navy  Department  or  the  naval  service  at  large.  Contribution  No.  349  from  the  Woods  Hole 
Oceanographic  Institution. 

57 


58 


C.  M.  POMERAT  AND  C.  M.  WEISS 


of  surface  irregularities  and  of  substrate  composition  on  the  establishment  of  sessile 
populations.  The  experiments  were  conducted  in  Biscayne  Bay  at  Miami,  Florida, 
where  subtropical  conditions  favor  the  attachment  of  fouling  organisms  throughout 
the  year. 

Grateful  acknowledgment  is  made  to  Dr.  A.  C.  Redfield  and  Dr.  F.  G.  Walton 
Smith  for  many  helpful  suggestions. 


FIGURE  1.     Glass  surfaces  used  in  testing  the  relation  of  surface  irregularities  to  fouling.     0.  Plain. 
1.   Sandblasted.     2.  Factrolite.     3.  Prestlite.     4.  Ribbed.     5.  Pentecor. 

EFFECT  OF  SURFACE  IRREGULARITY 

Commercial  glasses,  manufactured  by  the  Pittsburgh  Glass  Company,  with 
various  surface  irregularities  were  used  for  this  study.  Six  8  X  10  inch  glass  plates 
were  assembled,  irregular  surface  down,  in  a  rack  suitable  for  floating  on  the  surface 
of  the  water.  The  floats  were  constructed  in  such  a  way  that  sea  water  could  move 
freely  on  both  sides  of  the  exposed  surface.  The  backs  of  the  panels  which  were 
all  relatively  smooth  were  placed  upward.  The  fouling  on  the  back  surfaces  was 


ATTACHMENT  OF  MARINE  ORGANISMS  59 

not  recorded.     The  surface  irregularities  of  the  panels  are  shown  in  Figure  1  and 
may  be  described  as  follows  : 

Surface  Number: 

0.  Plain  Smooth  glass,  polished. 

1 .  Sandblasted     Glass  sandblasted  on  lower  side. 

2.  Factrolite         Surface  consisted  of  pyramidal  depressions  of  which  there  were 

about  144  per  square  centimeter. 

3.  Prestlitc  Approximately  nine  pyramidal  depressions  per  square  centi- 

meter. 

4.  Ribbed  Surface  of  V-shaped  grooves,  nine  grooves  per  centimeter  of 

width. 

5.  Pcntecor  Approximately    three    V-shaped    grooves    per    centimeter    of 

width. 

Results  obtained  from  three  series  of  experiments  in  which  the  glass  surfaces 
were  exposed  are  shown  in  Table  1. 

The  Sessile  populations  which  grew  on  the  glass  plates  were  composed  pri- 
marily of  barnacles  and  tubeworms,  with  irregular,  perhaps  seasonal,  appearances 
of  tunicates  and  Anoinia  sp.  Barnacles  (B.  iinprorisns  and  B.  amphitritc  niveus  in 
order  of  relative  abundance)  were  numerous  in  both  locations,  but  those  at  Tahiti 
Beach  were  always  very  small  compared  to  those  at  Miami  Beach.  Many  more 
barnacles  attached  to  the  lower  (shaded)  surfaces  of  the  panels  than  to  the  upper 
surfaces  where  light  was  more  abundant.  This  is  in  agreement  with  the  experience 
of  Pomerat  and  Reiner  (  1(M2).  who  report  that  larger  numbers  of  barnacles  accu- 
mulate on  dark  surfaces  than  on  light  surfaces.  The  shaded  undersides  of  the  glass 
panels,  being  darker  than  the  upper  sides,  appear  to  attract  more  cyprids  and  hence 
show  a  greater  barnacle  accumulation. 

The  various  surface  textures  of  glass  had  little  influence  on  the  number  of  at- 
tached organisms.  In  these  experiments  barnacles  were  consistently  slightly  more 
numerous  on  Pentecor  than  on  smooth  glass.  This  behavior  was  confirmed  in  the 
experiment  reported  in  the  following  section  although  conditions  of  exposure  were 
not  exactly  parallel.  In  the  first  experiment  the  glass  panels  were  floated  at  the 
surface  in  a  shaded  location,  while  in  the  second  they  hung  vertically  below  low  tide 

TABLE  II 

Influence  of  substrate  on  fouling,  sixty  days'  exposure  at  the  beach  boat  slips,  September  25,  1942- 

November  25,  1942 

Weight  of  fouling  on   panel  area  of  264  sq.   in.,  that  of  wood 

panels  employed 
Substrate  Wet  weight  grams  Dry  wt.  grams 

1.  Dade  County  pine  675.1  346.5 

2.  Gum  1127.6  531.4 

3.  Magnolia  1165.4  446.4 

4.  White  pine  968.7  446.8 

5.  Cypress  954.8  392.0 

6.  Tile  980.1*  487.3 

7.  Cement  1033.0*  534.1 

8.  Glass  386.1  167.3 

*  Corrected  to  an  area,  264  sq.  in.,  equal  to  that  of  the  wood  panels. 


60 


C.  M.  POMERAT  AND  C.  M.  WElSS 


under  sun  exposure.  Counts  of  tubeworms  were  made  on  only  one  set  of  expo- 
sures. H \droidcs  sp.  was  most  abundant  on  Prestlite  and  Sf>irorbis  sp.  was  most 
abundant  on  sandblasted  gla.ss. 

COMPOSITION  OF  THE  SURFACE 

Unpainted  panels  of  wood  of  five  species,  clay  roofing  tiles,  cement  roofing  plates, 
and  a  glass  panel  were  exposed  for  60  days  at  the  Reach  Roat  Slips  in  Miami  Reach. 

TABLE  1 1 1 

Effect  of  substrate  on  fouling,  exposures  of  three  months  at  South  Dock,  Belle  Isle,  Miami  Beach, 
Florida,  January  9,  1943-April  9,  1943,  all  materials  applied  to,  or  mounted  on,  wood  unless 

otherwise  noted 


Composition  of  surfaces 

\Yet 
weight* 
(grams) 

Dry 

weight* 
(grams) 

Number* 
of  bar- 
nacles 

Notes 

Plastics 

1.  Celluloid 

3.8, 

2.2 

11 

Thin  coat  of  algae. 

2.   Plasticel 

24.3 

12.2 

124 

Barnacles'  bases  easily  removed. 

3.  Lucite 

5.6 

1.7 

41 

4.  Formica 

6.9 

3.2 

11 

5.   Isobutyl 

15.4 

7.2 

70 

Film  applied  to  glass  panel. 

Methacrylate 

Plastic  peels  intact  with  barnacles. 

Glass 

6.  Prestlite 

57.0 

25.2 

176 

Some  barnacles  12  mm.  across. 

7.  Pentecor 

46.0 

25.0 

148 

Some  barnacles  12  mm.  across. 

8.  Sandblasted 

23.6 

7.0 

46 

6  calcareous  tubeworms;  tunica  tes. 

9.  Smooth 

4.5 

1.7 

16 

Green  slime  may  have  caused  fish  to 

remove  young  barnacles. 

Paints  and  ingredients** 

- 

Coatings  applied  to  steel 

panels 

10.  Ester  gum  vehicle 

36.3 

8.1 

58 

Tunica  tes  and  bryozoa. 

1  1  .   Rosin  vehicle 

2.7 

0.4 

0 

Fish  spawn  both  sides. 

12.  Anticorrosive  paint  42-A 

2.7 

0.5 

9 

Baracles  very  small. 

13.  Vehicle  of  15RC 

6.1 

3.3 

43 

14.  Antifouling  paint  7C 

0.0 

0.0 

0 

Some  slime  film. 

15.  Antifouling  paint  8C 

0.6 

0.3 

14 

Small  barnacles  close  to  edge. 

Coatings  Applied  to  Wood 

16.   Ceraloid 

57.6 

38.5 

183 

17.   Paraffin 

11.3 

6.1 

59 

Lomnoria  active  in  breaking  paraffin. 

18.  Asphalt  u  m 

121.4 

34.3 

768 

Barnacles  onlv. 

19.  Asphaltum  varnish 

67.8 

13.8 

256 

Some  bryozoa. 

20.  Spar  varnish 

45.1 

7.0 

304 

2  1  .   Navy  grev 

41.6 

5.6 

150 

Algae. 

22.  Anti-corrosive  42-A 

48.2 

10.7 

156 

*  Corrected  to  an  area  of  144  square  inches. 

*  Anticorrosive  42A  is  a  standard  Navy  formula.  Vehicle  of  15RC  is  the  non-pigmented 
portion  of  a  standard  Navy  antifouling  paint.  Antifouling  paints  7C  and  8C  are  experimental 
modifications  of  a  standard  Navy  antifouling  paint  of  the  cold  plastic  type  in  which  the  toxic 
pigment  is  reduced  to  50  and  60  percent  of  the  normal  value. 


ATTACHMENT  OF  MARINE  ORGANISMS 


61 


TABI.K   I II  —  Continued 


Wet 

Dry 

Number* 

Composition  of  surfaces 

weight* 

weight* 

of  bar- 

Notes 

(grams) 

(grams) 

nacles 

Woods 

23.   Dade  County  pine 

395.2 

120.7 

748 

Bryozoa. 

(soaked  60  days) 

24.  Gum  (soaked  60  days) 

452.1 

133.4 

686 

25.   Hade  County  pine 

144.3 

27.3 

125 

Hydrozoa,  bryozoa. 

(unsoaked) 

26.  Gum  (unsoaked) 

249.8 

43.5 

222 

27.  Soft  pine 

57.6 

11.5 

184 

28.  Teak 

143.8 

88.7 

306 

Large  barnacles. 

29.   Maderia 

173.7 

84.2 

358 

Manv  lish  eggs. 

30.  Greenheart 

77.0 

40.8 

342 

31.   Balsa 

2.9 

1.6 

5 

Wood  verv  soft. 

Metals 

32.  Steel 

224.4 

42.8 

88 

33.  Galvanized  iron 

2.6 

0.7 

6 

Barnacles  easily  removed. 

34.  Zinc 

1.0 

0.2 

0 

Active  corrosion. 

35.  Lead 

30.6 

50.9 

396 

Large  barnacles. 

36.   Monel 

1.6 

0.5 

6 

Manv  fish  eggs. 

37.   Nickel 

43.2 

10.7 

126 

38.  Galvanized  iron  pipe 

4.7 

3.0 

27 

Barnacle  on  rusted  threads  and  dam- 

aged edges. 

Miscellaneous 

39.   Linoleum 

79.7 

23.0 

193 

40.   Deck  canvas  no.  10 

5.1 

2.3 

7 

Sagging-algae  eaten  by  fish. 

41  .  Sole  leather 

32.4 

12.4 

66 

42.   Masonite,  heat  tempered 

138.6 

31.8 

594 

Brown  tunicates. 

43.  Asbestos 

284.2 

65.9 

980 

Bryozoa,    Anoniia,    hydrozoa,    calcar- 

eous tubeworm. 

All  panels  were  suspended  in  a  vertical  position  approximately  two  feet  beneath  the 
mean  low  water  mark.  The  site  was  well  shaded  by  a  protecting  roof.  The  results 
obtained  are  presented  in  Table  II. 

The  weights  of  the  populations  (barnacles,  tubeworms,  tunicates,  bryozoa,  and 
algae)  which  accumulated  on  the  woods,  tile,  and  cement  were  of  the  same  order  of 
magnitude,  though  variations  as  great  as  59  per  cent  were  observed.  The  weight 
of  organisms  accumulated  on  glass  was  approximately  30  per  cent  of  that  collected 
from  other  substrate  materials. 

A  much  larger  number  of  materials  were  tested  in  a  second  experiment,  the  re- 
sults of  which  are  given  in  Table  III.  Exposure  was  made  for  three  months  at 
South  Dock,  Belle  Isle,  in  Miami  Beach,  where  conditions  of  bright  sunlight,  active 
current  movement,  and  moderate  fouling  incidence  were  found.  Growth  on  the 
panels  consisted  primarily  of  barnacles  (B.  hnprovisus  and  B.  amphitrite  niveus) 
with  occasional  tufts  of  hydrozoa  and  patches  of  colonial  tunicates.  A  blanket  of 
algae  having  very  short  filaments  grew  on  panels  of  light  color  or  shaded  back- 
ground. Large  sets  of  fish  eggs  were  found  on  the  rosin  vehicle  and  Monel.  Bor- 
ings of  Liuuwria  sp.  were  everywhere  evident  in  unprotected  wood. 


62 


C.  M.  POMERAT  AND  C.  M.  WEISS 


The  substrates  accumulating  heaviest  populations  were  asbestos,  asphaltum, 
Dade  County  pine  (pre-soaked  60  days),  gum  wood  (pre-soaked  60  days)  and 
Masonite.  Asbestos  shingle,  commonly  used  as  clapboarding,  yielded  the  richest 
harvest  as  measured  by  the  number  of  barnacles.  A  comparison  of  asbestos  and 
Masonite,  two  of  the  best  collectors,  is  shown  in  Figure  2.  The  asphaltum  used 
was  of  the  type  employed  as  aquarium  cement.  It  accumulated  barnacles  only. 

Panels  of  gum  and  Dade  County  pine,  which  had  been  exposed  for  60  days  in 
the  earlier  test  reported  in  Table  II,  were  included  for  comparison  with  unsoaked 
specimens  of  these  woods.  The  unsoaked  woods  developed  much  less  fouling. 


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2.     Accumulation  oi  fouling  organisms  on  masonite  and  asbestos  after  90  days'  exposure 

at  Belle  Isle,  Miami  Beach,  Florida. 


Intact  galvanizing  on  iron  was  very  resistant  to  marine  life.  No  barnacles  were 
obtained  on  zinc,  on  the  experimental  antifouling  paint  7C  or  on  rosin  vehicle,  a 
common  paint  component. 

Materials  with  hard  non-porous  and  non-fibrous  surfaces  were  in  general  rather 
poor  collectors  of  fouling.  The  best  accumulation  of  sedentary  populations  was 
found  on  surfaces  which  were  porous  and/or  fibrous.  Surface  of  paints,  paint  in- 
gredients and  linoleum  are  in  general  non-porous  and  non-fibrous.  Compared  to 
the  size  and  strength  of  the  barnacle  cyprid  they  are  also  smooth  and  hard.  The 
histogram  (Fig.  3)  summarizes  the  collective  efficiency  of  the  substrates. 

Some  results  were  undoubtedly  spurious,  and  these  should  be  noted.  Fouling 
on  the  antifouling  paint  SC  which  occurred  along  the  edges  of  the  panel  was  probably 


ATTACHMENT  OF  MARINE  ORGANISMS 


63 


Grams  Wet  Weight  of  Foulini 


lOO 


200 


400 


300 


PLASTICS 
— —      GJ.ASSZS 


PA/HTS  AND  PAINT 


BALSA 


MSTALS  (except  Steel)  -  STEEL 

•CAHVAS-LEATHIK.-LIMOLEUM  -    MASOMIT£  -  ASBESTOS 


WOODS 


FICUKE  3.     Relative  amounts  of  foulins;  on  various  classes  ot  materials  used  as  test  panels. 


FIGURE  4.     Bases   of  barnacles   grown   on   various   substrates.     A.    Navy   grey.     B.   Antifouling 
paint  15RC.     C.  Ester  gum.     D.  Anticorrosive  paint  42A. 


64 


C.  M.  POMERAT  AND  C.  M.  WEISS 


due  to  imperfections  of  the  paint  surface.  In  contrast,  7C  which  contained  less 
copper  was  not  fouled.  Deck  canvas  and  smooth  glass  both  supported  a  culture  of 
green  algae  which  evidently  served  as  food  for  fish.  Active  feeding  on  these  panels 
unquestionably  disturbed  other  fouling  organisms.  Balsa  wood  was  apparently 
sloughing  its  surface  and  thus  loosening  attached  forms.  In  spite  of  these  minor 
qualifications,  the  results  involve  a  range  of  population  numbers  sufficiently  wide 
to  indicate  the  relative  merits  of  the  substrates  used. 

One  of  the  most  interesting  of  the  results  was  observed  when  barnacles  were 
removed  from  the  various  substrates.  Some  of  the  substrates  bore  barnacles  with 
deeply  scalloped  margins  (Fig.  4)  instead  of  the  typical  smooth  edges.  These 
margins  suggested  that  localized  irregular  marginal  growth  interruptions  had  taken 
place.  Such  barnacles  were  collected  from  : 

Spar  varnish 

Linoleum 

Navy  grey  topside  paint  (P— 50) 

Antifouling  paint  vehicle   15RC 

Ester  gum  paint  vehicle 

Anticorrosive  paint  42-. \ 


"' 


ft*,  c? 


FIGURE  5.     Bases  of  barnacles  grown  on  various  substrates.     A.  Isobutyl  methacrylate. 
B.  Plasticel.     C.  Soft  paraffin.     D.  Ceraloid. 


ATTACHMENT  OF  MARINE  ORGANISMS  65 

Barnacles  growing  on  soft  paraffin  had  distinctly  concave  bases.  Mosaics  of 
bases  witb  angular  margins  were  typical  of  barnacles  attached  to  lead  but  were  also 
found  on  other  overcrowded  substrates.  It  was  possible  to  remove  barnacles  with 
intact  bases  very  easily  from  several  materials,  including  plasticel,  ceraloid,  and  iso- 
butyl  methacrylate  (Fig.  5).  This  finding  might  prove  useful  in  designing  experi- 
ments in  which  the  minute  anatomy  of  basal  structures  was  to  be  studied. 

SUMMARY 

1.  Submerged  samples  of  40  different  construction  materials  were  used  as  sub- 
strates for  the  collection  of  sedentary  populations.     The  barnacle  counts  in  the  popu- 
lations ranged  from  980  on  asbestos  shingles  to  zero  on  zinc  and  on  two  paint  coat- 
ings, after  three  months'  immersion  in  Biscayne  Bay  at  Miami  Beach,  Florida. 

2.  Various  surface  textures  of  glass  plates  were  found  to  exert  no  significant 
influence  on  the  accumulation  and  growth  of  sedentary  marine  organisms,  although 
smooth  clear  glass  accumulated  smaller  populations  in  the  comparatively  short  expo- 
sure periods,  1—3  months  . 

3.  The  results  suggest  that  efficiency  of  a  substrate  as  a  fouling  collector  is  in 
general   correlated   with   porosity   of   surface   or   with   fibrous   nature   of   surface. 
Smooth,  non-porous,  non-fibrous  surfaces,  especially  if  also  hard,  seem  to  be  poor 
accumulators  of  sedentary  organisms. 

4.  Further  testing  of  substrates  is  greatly  to  be  desired  in  this  connection. 

REFERENCES 

POMERAT,  C.  M.,  AND  E.  R.  REINER,  1942.     The  influence  of  surface  angle  and  of  light  on  the 
attachment  of  barnacles  and  other  sedentary  organisms.     Biol  Bull.,  82    (1)  :   14. 


THE  DEVELOPMENTAL  HISTORY  OF  AMAROECIUM  CONSTEL- 
LATUM.     II.  ORGANOGENESIS  OF  THE  LARVAL 

ACTION  SYSTEM 

SISTER  FLORENCE  MARIE  SCOTT 

The  Marine  Biological  Laboratory,  Woods  Hole,  Mass..  and  the  Biology  Department,  Scion  Hill 

College.  Greensburg,  Pennsylvania 

INTRODUCTION 

The  early  development  of  the  embryo  of  A-maroecium  constellation  has  been  pre- 
sented in  a  previous  paper  (Scott,  1945).  The  accumulation  of  yolk  modifies  the 
pattern  of  mosaic  development  characteristic  of  Tunicates  to  the  extent  that  gastru- 
lation  is  accomplished  in  an  atypical  manner.  Convergence  of  the  cells  of  the  lateral 
margins  of  the  posterior  blastoporal  lip  is  accomplished  to  the  left  of  the  mid-line. 
The  neural  plate  elongates  posteriorly  at  the  place  where  the  lateral  blastoporal  lips 
meet  and  close.  The  chordal  cells  are  inflected  at  the  anterior  lip  and  lie  in  the 
median  axis.  The  potential  muscle  cells  of  the  morphological  right  side  lie  dorsal 
to  the  notochord  as  a  result  of  their  growth  across  the  mid-dorsal  plane,  the  muscle 
cells  of  the  morphological  left  side  lie  below  the  level  of  the  notochord  on  the  curved 
left  side  of  the  embryo.  The  two  groups  of  muscle  cells  are  separated  by  the  poste- 
rior extension  of  the  neural  plate. 

MATERIALS  AND  METHODS 

Amaroecium  constellation  is  abundant  along  the  eastern  coast  of  the  United 
States.  The  breeding  season  lasts  throughout  the  summer  months.  Material  for 
this  study  was  collected  at  Woods  Hole,  Massachusetts.  The  embryos,  squeezed 
from  adult  colonies,  were  selected  and  arranged  into  a  progressive  series  of  stages 
for  study.  They  were  fixed  in  Bouin's  fluid.  Some  were  stained  by  Conklin's 
modification  of  Delafield's  haematoxylin,  others  with  borax-carmine,  then  cleared 
according  to  the  benzyl-benzoate  method  described  in  Romeis'  "Taschenbuch  der 
Mikroscopischen  Technik."  Corresponding  stages  were  sectioned  serially,  stained 
in  Mayer's  or  Gallagher's  or  iron  haematoxylin,  and  counterstained  with  eosin  or 
triosin.  All  drawings  were  made  with  the  aid  of  a  camera  lucida.  The  photo- 
micrographs were  made  with  a  Leitz  "Macca"  camera  using  Zeiss  apochromat,  20  X , 
and  fluorite  oil  immersion,  100  X,  objectives  with  a  Zeiss  microscope. 

Later  embryonic  development 

It  seems  advisable  to  present  a  descriptive  series  of  developmental  stages  that 
may  be  used  as  points  of  reference  for  structures  differentiating  during  the  organ- 
forming  period.  For  convenience  the  developmental  period  following  gastrulation 
is  divided  into  four  stages;  1)  the  tail  bud  stage,  2)  early  tadpole  stage,  3)  pre- 
hatching  stage,  and  4)  the  free-swimming  tadpole  stage.  The  free-swimming  larva 

66 


AMAROECIUM  CONSTELLATUM.    II 


67 


or  tadpole  has  been  described  thoroughly  by  Grave  (1921)  and  shall  be  presented 
here  in  brief  summary  since  reference  to  it  is  necessary.  A  short  description  of  the 
external  appearance  of  these  stages  will  be  given  first  and  referred  to  in  subsequent 
treatment  of  organogenesis  as  Stages  I,  II,  III,  and  IV.  The  terms,  larval  action 
system  and  adult  action  system,  used  by  Grave  (1935,  1944)  will  be  adopted  for 
the  structures  functioning  during  larval  life  and  those  functioning  during  adult  life 
respectively. 

The  tail  bud  stage 

By  the  end  of  gastrulation  the  embryo  is  approximately  spherical  except  for  a 
shallow  postero-ventral  invagination  of  the  ectoderm  constricting  tail  from  trunk 
region.  The  furrow  appearing  first  on  the  right  side  is  deeper  there,  and  less  deep 
as  it  extends  to  the  left  side.  The  tail  bud  is  short  and  rounded,  curving  immedi- 
ately toward  the  ventral  side  of  the  trunk.  Through  the  thin  epidermis  quadruple 
rows  of  large  muscle  cells  can  be  seen  lying  dorsal  and  ventral  to  the  notochord. 
The  neural  plate  is  elevated  at  the  periphery  to  form  the  neural  groove,  enclosing; 
anteriorly  a  wide  depression,  the  presumptive  brain  region,  posteriorly  a  narrow, 
trough-like  depression  lying  to  the  left  of  the  notochord,  the  presumptive  neural 
tube  (Fig.  I  A). 


h.v 


FIGURE  1.  A.  Stage  I,  embryo  before  neural  folds  close.  160  X.  B.  Stage  II,  early  tad- 
pole; beginning  of  differentiation  of  digestive  and  nervous  systems.  160  X.  b.  v.,  brain  vesicle; 
d.  ph.,  dorsal  diverticulum  of  pharynx;  m.  bd.,  muscle  band;  n.  t.,  neural  tube;  ph.,  pharynx; 
y.  m.,  yolk  mass. 

A  transverse  section  through  the  tail  bud  stage  discloses  that  the  embryo  is  solid. 
A  single  layer  of  definitive  endoderm  lies  under  the  concave  neural  plate  (Fig.  5£). 
This  layer  of  cells  develops  from  the  cells  that  form  the  superficial  "pseudo- 
invagination"  cavity  of  gastrulation.  The  depression  closes  by  a  reversal  in  change 
of  shape  of  the  cells  involved  rather  than  by  approximation  of  the  lips  of  the  blasto- 
pore  thus  producing  a  solid  archenteron  (Scott,  1945).  The  endodermal  cells 
spread  under  and  anterior  to  the  neural  plate.  Ventral  to  them  is  located  the  mass 
of  heavily  yolk-laden  cells  derived  from  the  macromeres.  Wedged  between  the 


68  FLORENCE  MARIE  SCOTT 

thin  ectoderm  and  the  solid  endoderm  on  either  side  is  a  mass  of  mesenchyme,  small, 
polygonal  cells  with  prominent  nuclei  (Fig.  5£). 

Posteriorly  the  definitive  endoderm  lies  adjacent  to  the  chordal  cells  which  are 
beginning  to  interdigitate  in  the  base  of  the  tail  bud.  The  mesenchyme  terminates 
abruptly  in  this  region  against  the  muscle  cells  of  the  tail. 

Early  tadpole  stage 

The  embryo  increases  in  size  and  acquires  the  shape  that  justifies  its  being  called 
"tadpole."  The  trunk  region  elongates  slightly  in  the  antero-posterior  axis  remain- 
ing curved  at  the  anterior  end.  The  tail  encircles  the  body  meridionally  as  it  grows 
in  length.  The  embryo  is  still  opaque. 

The  neural  folds  are  closed,  the  position  of  the  sensory  vesicle  being  marked  by 
aggregations  of  black  pigment  which  show  through  the  surface  of  the  body.  The 
neural  tube  is  faintly  visible  along  the  side  of  the  tail.  More  conspicuous  are  the 
large  muscle  cells  dorsal  and  ventral  to  the  prominent  notochord  which  forms  the 
axis  of  the  tail  throughout  its  length.  Dorsally,  on  either  side  of  the  sensory  vesicle 
there  is  a  slight  ectodermal  invagination,  rudiments  of  the  atrial  chambers.  The 
embryo  is  confined  within  a  test  the  cells  of  which  are  arranged  in  a  compact  layer 
(Fig.  IB). 

Pre-hatching  stage 

Changes  in  the  external  appearance  of  the  later  embryo  depend  on  the  develop- 
ment of  siphons  and  adhesive  papillae  and  the  secretion  of  a  tunic.  As  body  growth 
continues  and  organs  of  the  larval  action  system  differentiate,  the  body  becomes 
transparent  except  where  the  mass  of  yolk  is  lodged  in  the  pharynx. 

The  trunk  region  continues  to  elongate  antero-posteriorly  becoming  elliptical  in 
shape.  Posteriorly  the  body  narrows  to  the  base  of  the  tail ;  anteriorly  it  flares  in 
the  dorso-ventral  axis  in  relation  to  the  vertical  position  of  the  adhesive  papillae. 
Laterally  the  body  is  compressed.  A  thickening  layer  of  tunic  invests  the  entire 
trunk.  It  is  indented  at  the  junction  of  trunk  and  tail  and  continues  over  the  sur- 
face of  the  tail.  The  tail  encircles  the  body  meridionally  being  pressed  into  a  groove 
in  the  tunic.  The  tunic  of  the  tail  projects  laterally  into  fins. 

The  sensory  vesicle  occupies  a  dorsal  position  at  the  posterior  end  of  the  trunk. 
Two  masses  of  pigment  project  into  its  cavity.  Immediately  in  front  of  it  lies  the 
elevation  of  the  oral  siphon ;  behind  it  and  on  the  posterior  curve  of  the  body  lies  the 
atrial  siphon.  Much  of  the  internal  structure  is  visible  through  the  tunic  and 
mantle.  The  incipient  adhesive  papillae  appear  as  three  disc-like  projections  in 
verticle  series  at  the  rounded  anterior  end  (Fig.  2). 

The  free-swimming  tadpole  stage 

The  trunk  of  the  tadpole  of  Amaroecium  at  its  release  measures  about  600  micra 
in  length;  it  measures  about  270  micra  in  depth.  The  tubular  atria  with  their  triple 
rows  of  gill  slits  are  pressed  into  the  dorsal  pharynx  through  half  of  its  length 
posteriorly.  An  obvious  structure  in  the  pharynx  is  the  dorsal,  heavily  ridged  endo- 
style  which  seems  to  rest  on  the  lateral  masses  of  yolk  that  form  the  wall  of  the 
pharynx.  The  transparent  pericardium  occupies  a  large  space  below  the  yolk  ante- 


AMAROECIUM  CONSTELLATUM.     II 


69 


riorly  in  front  of  the  loop  of  alimentary  tract.  On  the  right  side  of  the  body  the 
stomach  extends  along  the  posterior  and  ventral  curvature  of  the  yolk.  On  the  left 
the  narrow  intestine  curves  along  the  side  of  the  stomach  up  to  the  left  atrium  where 
it  terminates.  The  root  of  the  tail  lies  in  the  posterior  third  of  the  length  of  the 
body. 

Anteriorly,  the  adhesive  papillae  project  into  the  tunic  in  a  vertical  row  slightly 
to  the  right  of  the  median  plane.  The  test  vesicles  lie  loosely  within  the  tunic  or 
many  of  them,  even  at  the  time  of  hatching,  retain  a  slender  connection  with  the 
cone  or  ridge  from  which  they  originate.  Where  the  tail  is  continuous  with  the 
trunk  the  tunic  dips  clown  into  an  abrupt  pocket.  The  epidermis  secretes  a  thin 
sheath  of  tunic  about  the  tail.  Laterally  it  expands  into  wide  sail-like  fins  (Fig.  3). 


,5.V. 


ad. 


ps~ 


FIGURE  2.  Stage  III,  lateral  view  of  tadpole  with  incipient  adhesive  papillae.  About  120  X. 
ad.  p.,  adhesive  papillae;  end.,  endostyle ;  ep.,  epidermis;  /.  a.,  left  atrium;  >i.  t.,  neural  tube;  oes., 
oesophagus;  p.  c.,  pericardia!  cavity;  ph.,  pharynx;  st.,  stomach-intestine  rudiment;  s.  v.,  sensory 
vesicle ;  tu.,  tunic ;  y.  »i.,  yolk  mass. 

ORGANOGENESIS  OF  THE  LARVAL  ACTION  SYSTEM 
Digestive  system 

The  pharyngeal  cavity  develops  in  Stage  II  by  delamination  between  the  layer 
of  definitive  endoderm  and  the  mass  of  yolk-laden  cells,  appearing  first  below  the 
brain  and  spreading  from  that  point  (Fig.  IB,  5F,  6E}.  It  extends  back  to  the 
base  of  the  notochord  as  an  upwardly  directed  diverticulum.  Ventral  to  the  base 
of  this  projection  a  second  invagination  appears,  the  rudiment  of  the  stomach  and 
intestine  located  a  little  to  the  right  of  the  median  plane  on  the  inner  side  of  the 
visceral  ganglion  (Fig.  2,  6F). 

The  pharynx  deepens  in  Stage  III  encroaching  upon  the  mass  of  yolk  cells. 
Gradually  thin  septa  of  epithelium  divide  the  yolk  mass  into  four  compact  longi- 
tudinal columns,  the  two  on  each  side  being  continuous  at  the  bottom.  The  central 


70 


FLORENCE  MARIE  SCOTT 


two  are  lower  than  the  outer  two,  thus  providing  greater  depth  for  the  limited 
pharyngeal  cavity  (Fig.  6A,  F).  This  supply  of  nutritive  material  in  the  pharynx 
remains  to  be  digested  during  the  active  life  of  the  larva  and  throughout  the  critical 
period  of  metamorphosis.  All  other  tissues  lose  their  meager  supply  of  yolk  almost 
entirely,  leaving  their  cytoplasm  clear. 

Along  the  roof  of  the  pharynx,  anterior  to  the  place  of  origin  of  the  oral  siphon, 
the  epithelium  rises  up  into  a  double  fold  enclosing  the  endostyle,  restricted  to  the 
dorsal  side  above  and  between  the  lateral  masses  of  yolk  and  passing  to  the  anterior 
end  of  the  yolk  mass  (Fig.  2,  6A}.  Before  the  tadpole  is  released  from  its  test,  the 
cells  in  the  floor  of  the  groove  develop  long  cilia.  The  pharynx  grows  out  above 
and  below  the  atrial  sacs,  bringing  the  mesial  atrial  and  lateral  pharyngeal  walls  into 
intimate  contact  (Fig.  65). 

Due  to  the  combined  activity  of  atrial  and  pharyngeal  epithelia,  three  horizontal 
rows  of  gill  slits  are  formed,  each  consisting  of  seven  or  eight  perforations.  The 


oral  Siphon 


FIGURE  3.  Stage  IV,  tadpole  at  hatching.  About  120  X.  end.,  endostyle;  cp.,  epidermis: 
int.,  intestine;  p.  c.,  pericardia!  cavity;  st.,  stomach;  s.  v.,  sensory  vesicle;  te.  v.,  test  vesicles: 
tit.,  tunic ;  y.  m.,  yolk  mass. 

bordering  cells  of  each  gill  develop  a  heavy  brush  of  cilia,  precocious  equipment  from 
the  functional  point  of  view.  Even  though  the  mouth  breaks  through  to  the  bran- 
chial chamber,  the  tunic  fills  up  the  oral  and  atrial  siphons  until  metamorphosis  is 
completed. 

The  rudiment  of  the  oesophagus  grows  forward  along  the  curvature  of  the  yolk 
and  dilates  to  form  the  stomach.  The  diverticulum  extends  to  the  midventral  region 
of  yolk  where  it  turns  sharply  upon  itself  and  continues  backward  as  the  slender 
intestine.  With  a  gradual  slope  upward  the  intestine  retraces  the  course  of  the 
stomach  on  its  left  side  terminating  ventral  to  the  posterior  end  of  the  left  atrium 
(Fig.  3,  6B).  Later  the  anus  opens  into  the  atrium  here. 

There  are  no  cilia  evident  in  the  intestine  or  stomach  during  this  period  of  de- 
velopment. The  wall  of  the  stomach  is  thicker  than  the  wall  of  the  remaining  parts 
of  the  digestive  tract  although  the  alimentary  epithelium,  throughout  its  length,  con- 
sists of  a  single  layer  of  cells. 


AMAROECIUM  CONSTELLATUM.     II  71 

With  rapid  general  growth  of  the  body,  the  loop  of  intestine  and  stomach  in- 
creases in  length  anteriorly,  extending  through  the  posterior  half  of  the  body  cavity 
below  and  behind  the  yolk-laden  pharynx  (Fig.  3).  The  pericardium  lies  directly 
in  front  of  it.  Between  the  arms  of  the  loop  posteriorly  are  lodged  the  bases  of 
the  axial  organs  of  the  tail. 

Atrium — During  Stage  II  the  atrium  or  peribranchial  sac  appears  as  a 
pair  of  ectodermal  invaginations,  one  on  either  side  of  the  sensory  vesicle  (Fig. 
6£).  At  the  place  of  its  origin  the  neck  of  each  depression  constricts  and  separates 
from  the  surface. 

In  the  transition  from  Stage  II  to  Stage  III,  the  atria,  in  contact  with  the  lateral 
endodermal  wall  of  the  pharynx,  grow  in  an  anterior  direction  only,  with  the  result 
that  the  atrial  chambers  are  horizontal  capsular  cavities  located  dorsally,  one  on 
either  side  of  the  pharynx  (Fig.  6B).  They  extend  through  the  posterior  two- 
thirds  of  the  trunk,  curving  gently  upward  posteriorly  where  they  grow  towards 
each  other  and  unite  behind  the  pharynx  (Fig.  3).  The  atrial  siphon  opens 
through  the  dorsal  wall  of  this  connecting  canal  between  the  two  cavities. 

The  atrial  walls  are  characteristically  thin  and  the  cells  lose  their  intercell  mem- 
branes. Occasional  yolk  granules  are  scattered  through  the  cytoplasm.  During 
Stage  III  the  gill  slits  perforate  the  walls  in  three  horizontal  rows  on  the  inner  side 
in  direct  contact  with  the  wall  of  the  pharynx.  The  lowermost  row  develops  first, 
the  atrial  and  pharyngeal  fusing  first  in  these  regions.  The  slits  number  between 
seven  and  nine  in  each  row.  Later  in  the  free-swimming  period  the  cells  bordering 
the  gill  aperture  produce  long  cilia.  The  endoderm  has  no  part  in  atrial  formation 
except  insofar  as  the  gill  slits  are  the  product  of  joint  activity  of  atrial  and  pharyn- 
geal walls  (Caullery,  1895). 

Oral  and  Atrial  Siphons — Late  in  Stage  III  the  dorsal  ectoderm  in  front 
of  the  sensory  vesicle  thickens  and  invaginates,  pushing  the  endoderm  of  the  pharynx 
before  it.  The  circle  of  epidermis  around  the  invaginated  area  becomes  elevated, 
giving  the  oral  siphon  a  crater-like  appearance  (Fig.  68} .  The  floor  of  the  invagi- 
nation  thins  out  in  a  flat  layer  against  the  pharyngeal  roof  with  which  it  is  in  con- 
tact. The  lower  part  of  the  cavity  projects  outward  from  the  center  and  produces 
a  ring-shaped  extension  on  the  mouth  opening.  The  oral  cavity  assumes  the  shape 
of  a  flask  with  a  long  neck  and  a  flattened  base  (Fig.  3).  Into  this  ectodermal 
cavity,  or  stomodaeum,  the  hypophysial  duct  opens,  just  before  hatching  of  the  tad- 
pole. Although  the  oral  plate  breaks  through  late  in  the  tadpole's  development, 
the  tunic  fills  up  the  stomodaeal  portion  and  prevents  the  passage  of  both  food  and 
water  during  larval  life. 

The  atrial  siphon,  like  the  oral,  is  formed  by  ectodermal  invagination.  The 
thickened  mantle  is  elevated,  raising  the  siphon  above  the  level  of  the  rest  of  the 
mantle  in  knob-like  fashion  (Fig.  6C).  The  floor  of  the  invagination  fuses  with 
the  dorsal  wall  of  the  connecting  arm  of  the  atrium.  The  atrial  siphon  is  situated 
on  the  downward  curve  of  the  dorsal  surface  just  posterior  to  the  sensory  vesicle 
and  anterior  to  the  insertion  of  the  tail  (Fig.  3,  6G,  H).  The  epithelial  lining  of 
the  oral  and  atrial  siphons  projects  into  each  opening  at  several  points  forming 
small  tentacles.  The  mesenchymatous  muscles  in  the  mantle  in  this  region  provide 
the  contractile  elements  that  control  the  apertures  when  the  siphons  begin  to  function. 


FLORENCE  MARIE  SCOTT 

Heart  and  pericardium 

Towards  the  end  of  Stage  III,  the  endodermal  cells  extend  completely  around 
the  yolk  mass  as  a  definite  epithelium.  Mid-ventrally  it  evaginates  into  the  body 
space  and  constricts  off  from  the  yolk  epithelium.  The  bladder-like  vesicle  is  the 
pericardium  which  invaginates  mid-dorsally  into  an  inner  enclosed  vesicle,  the  heart. 
The  cells  lose  their  inter-cell  membranes  and  the  nuclei  bulge  irregularly  in  both 
cardial  and  pericardial  walls  (Fig.  6 A).  The  heart  does  not  develop  beyond  this 
point  at  present,  the  circulatory  system  not  functioning  during  larval  life. 

The  nervous  system 

The  neural  folds  of  Stage  I  close  in  the  early  phase  of  Stage  II  thus  forming  the 
hollow  nervous  system  typical  of  chordates  except  in  one  point,  the  curving  of  the 
neural  tube  through  90°  to  the  left  of  the  brain  region.  The  anterior  portion  of  the 
nervous  system  produces  the  sensory  vesicle  with  its  sensory  organs,  the  hypophysis, 
definitive  ganglion,  and  the  so-called  subneural  gland.  The  intermediate  part  in- 
cluding the  origin  of  curvature  and  a  small  contribution  from  the  brain  region  gives 
rise  to  the  visceral  ganglion  and  the  spinal  enlargement,  the  posterior  part  becomes 
the  neural  tube. 

The  cavity  of  the  brain  region  is  slightly  dilated  and  its  wall  uniformly  thick. 
The  neural  tube  consists,  in  section,  of  four  cuboidal  cells  surrounding  a  small  lumen 
(Fig.  4C).  Cell  membranes  in  both  regions  are  distinct  at  this  stage,  the  nuclei 
are  large  and  contain  heavily  staining  nucleoli.  The  cytoplasm  is  reticular  in  ap- 
pearance and  has  occasional  yolk  granules. 

During  Stage  III  the  brain  vesicle  differentiates  into  two  structures,  the  sensory 
vesicle  in  the  entire  right  side  and  the  rudiment  of  the  hypophysis  on  the  left  poste- 
rior side  (Fig.  4A,  5 A}.  The  vesicle  expands,  its  walls  becoming  thin;  the  rudi- 
ment of  the  hypophysis  remains  small  with  thick  walls.  This  secondary  cavity  is 
separated  completely  from  the  sensory  vesicle  at  the  region  of  evagination  but  their 
walls  remain  attached  throughout  subsequent  development  (Fig.  5B,  C). 

The  sensory  vesicle — Two  sensory  structures  develop  in  the  sensory  vesi- 
cle, the  statolith  and  the  eye.  The  left  posterior  wall  of  the  vesicle  thickens,  the 
right  wall  expands  dorsally  and  laterally ;  all  the  cells  lose  their  inter-cell  mem- 
branes. The  left  wall  of  the  cavity  remains  thick  and  constitutes  the  sensory 
ganglion  of  the  brain.  One  cell  on  the  ventro-anterior  wall  projects  into  the  cavity 
and  large  pigment  granules  are  deposited  in  its  cytoplasm ;  these  coalesce  to  form 
the  statolith  (Fig.  4B,  SB).  In  Stage  IV  the  statolith  is  a  spherical  mass  of  pig- 
ment confined  within  the  cell  membrane  and  attached  to  the  ganglionic  wall  by  a 
stout  stalk,  the  remaining  part  of  the  cell  (Fig.  4D,  SC,  D}. 

A  group  of  cells  situated  dorso-laterally  at  the  left  posterior  limit  of  the  vesicle 
initiates  the  development  of  the  eye  by  the  deposition  of  pigment  granules  of  much 
smaller  size  than  those  that  form  the  statolith.  Absence  of  cell  membranes  makes 
it  difficult  to  ascertain  the  number  of  cells  that  participate  in  this  activity.  The  pig- 
ment is  deposited  in  the  shape  of  a  cup,  its  concavity  facing  dorso-laterally  and  to 
the  right  within  the  vesicle.  Three  ganglionic  cells  which  retain  their  membranes 
fill  up  the  concavity  in  series.  They  secrete  globules  of  liquid  which  increase  in  size 
both  by  the  gradual  addition  of  the  secretion  and  by  the  fusion  of  globules.  The 
globules  of  liquid  form  the  so-called  lens  cell  (Fig.  4A,  B,  D,  5D).  The  nuclei 


AMAROECIUM  CONSTELLATUM.     II 


73 


..ret 


FIGURE  4.  .4.  Transverse  section  through  brain  after  the  neural  folds  close,  Stage  II.  750  X. 
B.  Longitudinal  section  through  brain  of  same  stage.  750  X.  C.  Cross  section  through  tail  of 
tadpole,  Stage  III.  300  X.  D.  Section  through  brain  of  tadpole  just  before  hatching,  oblique  to 
include  both  sensory  organs.  750  X.  E.  Longitudinal  section  through  tail  of  tadpole  in  Stage 
III.  300  X.  F.  Section  through  epidermis  and  test  of  Stage  III.  750  X.  G.  Reconstruction 
of  brain  and  related  structures  of  Stage  III,  viewed  from  left  side.  300  X.  b.  v.,  brain  vesicle; 
con.  fib.,  contractile  fibrils;  dcf.  g.,  definitive  ganglion;  cp.,  epidermis;  hyp.,  hypophysis;  /.  c., 
lens  cell;  m.  bd.,  muscle  band;  n.  c.,  neural  canal;  nch.,  notochord ;  n.  t.,  neural  tube;  ret.,  retinal 
cells  of  eye;  s.  gn.,  sensory  ganglion;  sn.  gl.,  subneural  gland;  stat.,  statolith ;  .?.  c.,  sensory  cell; 
s.  e.,  spinal  enlargement ;  .y.  in.,  smooth  muscle  cells  of  mantle ;  j.  p.,  sensory  pigment ;  t.  c.,  test 
cells ;  tu.,  tunic ;  v.  g.,  visceral  ganglion ;  y.  g.,  yolk  granules. 


74  FLORENCE  MARIE  SCOTT 

which  at  first  occupy  a  central  position  in  the  cells  are  pushed  to  the  periphery  as 
the  lenses,  increasing  in  size,  come  eventually  to  monopolize  the  entire  cell. 

The  pigment  granules  of  the  eye  always  remain  discrete,  not  coalescing  as  do 
those  of  the  otolith.  Extending  through  the  concentrated  pigment  are  small  rods 
of  clear  cytoplasm.  They  run  from  the  hase  of  the  cup  back  towards  the  ganglion. 
Seven  or  eight  of  them  may  be  seen  in  embryos  of  Stage  IV  that  are  mounted  in  a 
mixture  of  benzyl-benzoate  and  oil  of  wintergreen. 

The  hypophysis — The  rudiment  of  the  hypophysis  early  in  Stage  III  ap- 
pears as  an  extension  or  small  evagination  of  the  brain  cavity  (Fig.  4A,  5 A).  The 
cells  retain  their  membranes,  their  nuclei  are  smaller  than  those  of  the  adjoining 
part  of  the  brain.  Histologically  they  present  the  appearance  of  epithelial  tissue. 
Upon  its  separation  from  the  primary  cavity  during  Stage  III  it  elongates  antero- 
posteriorly  along  the  left  side  of  the  sensory  ganglion  (Fig.  4G).  In  Stage  IV  it 
ends  blindly  at  the  posterior  wall  of  the  oral  siphon.  Later  these  walls  fuse  and  the 
hypophysis  communicates  with  the  posterior  region  of  the  stomodaeum,  extending 
along  the  side  of  the  ganglion  with  a  gentle  slope  upward  as  far  as  the  atrial  siphon 
where  it  terminates  blindly.  The  floor  of  the  duct,  corresponding  in  position  to 
the  region  of  the  eye,  deepens  abruptly  (Fig.  4D,  G).  The  ventral  wall  of  the 
pocket  becomes  slightly  thicker,  the  indentation  with  its  thickened  floor  constituting 
the  subneural  gland.  Hjort  (1896)  reviews  the  opposing  views  concerning  this 
structure  in  the  early  works  on  Tunicates. 

The  definitive  ganglion — By  a  proliferation  of  cells  in  the  mid-region  of 
its  roof  in  Stage  II  the  hypophysial  duct  produces  an  oval  mass  containing  small 
nuclei  similar  to  those  in  the  hypophysial  duct  itself.  The  cell  membranes  dis- 
appear and  the  nuclei  wander  out  toward  the  periphery  where  they  collect  in  several 
rows  with  the  granular  cytoplasm  concentrated  in  the  center  (Fig.  4D,  4G,  SC). 
This  part  of  the  nervous  system,  the  definitive  ganglion,  persists  through  meta- 
morphosis and  together  with  the  hypophysis  gives  rise  to  the  permanent  nervous 
system  of  the  adult. 

Visceral  ganglion — The  visceral  ganglion  originates  in  that  part  of  the 
neural  plate  that  curves  toward  the  left  in  Stage  I.  The  lumen  is  obliterated,  the 
large  nuclei  migrate  to  the  periphery  leaving  the  medulla  mass  of  interlacing  fibrils 
and  granules  (Fig.  4D).  The  visceral  ganglion  lies  posterior  to  and  ventral  to  the 
sensory  vesicle.  Dorsally  where  it  merges  with  the  sensory  ganglion,  it  exceeds  the 
sensory  vesicle  in  diameter  but  it  gradually  diminishes  in  diameter  towards  the  base 
of  the  tail  where  it  is  continuous  with  the  neural  tube.  At  the  junction  there  is  a 
slight  enlargement  called  the  spinal  enlargement  (Fig.  4G).  The  neural  tube  re- 
tains its  lumen.  It  runs  through  the  length  of  the  tail  to  the  left  of  the  notochord. 
In  Stage  IV  a  single  nerve  emerges  from  the  visceral  ganglion  on  its  right  side  just 
below  the  hypophysis.  It  runs  anteriorly  and  sends  out  branches  to  the  smooth 
musculature  of  the  mantle. 

THE  NOTOCHORD 

At  the  end  of  the  gastrulation  period  the  chordal  cells  lie  under  the  posterior  part 
of  the  neural  plate.  Anteriorly  adjacent  to  them  are  endodermal  cells;  dorsally,  the 
potential  muscle  cells  of  the  right  lateral  margin  of  the  blastopore ;  ventro-laterally, 
the  potential  muscle  cells  of  the  left  lateral  margin  of  the  blastopore.  Posteriorly 
the  chordal  cells  extend  into  the  rudiment  of  the  tail. 


AMAROECIUM  CONSTELLATUM.     II 


75 


.          Vi 

•       .,   •          "I 


* 


FIGURE  5.  A.  Transverse  section  corresponding  to  Figure  4  A.  225  X.  B.  Transverse  sec- 
tion through  brain  of  Stage  III,  hypophysis  separated  from  brain  vesicle.  650  X.  C,  D.  Sec- 
lions  through  sensory  vesicle  and  definitive  ganglion  of  Stage  IV;  oblique,  thus  including  both 
sensory  organs.  650  X.  E.  Transverse  section  through  Stage  I;  anterior  region.  150  X.  F. 
Transverse  section  through  early  Stage  II;  neural  folds  closed.  150  X.  G.  Longitudinal  sec- 
tion through  adhesive  papilla  of  Stage  III.  650  X.  d.  c.,  definitive  endoderm  ;  dcf.  <;.,  definitive 
ganglion;  <//.  c.,  gland  cells;  //y/1.,  hypophysis;  inch.,  mesenchyme  cells;  11.  p.,  neural  plate;  stal., 
statolith. 


Some  of  the  endodermal  cells  of  the  yolk  mass  lie  along  the  right  side  of  the 
chordal  cells  and  when  the  tail  is  constricted  from  the  trunk  region  these  cells  form 
the  loose  column  of  caudal  endoderm.  In  Stage  IV  little  of  it  remains  (Fig.  4C, 
65). 

In  Stage  I  the  notochordal  cells  begin  to  shift  in  position.  They  interdigitate 
into  a  row  of  disc-shaped  cells  occupying  the  central  axis  of  the  short  tail.  The  cells 


76  FLORENCE  MARIE  SCOTT 

resemble  the  endodermal  cells  of  the  yolk  mass  in  possessing  delicate  membranes, 
nuclei  smaller  than  those  of  adjoining  muscle  cells,  and  yolk  granules. 

During  Stages  II  and  III  the  notochord  elongates  as  the  tail  lengthens.  The 
chordal  cells  lengthen ;  the  inter-cell  membranes  separate  from  each  other  converting 
them  into  hour-glass  shaped  cells  with  the  nucleus  resting  in  the  constricted  neck 
between  the  peripheral  masses  of  protoplasm  (Fig.  4E). 

In  Stage  IV  the  cell  halves  separate  completely  giving  the  chord  the  appearance 
of  a  tube  with  a  scalloped  lining.  The  proximal  end  retains  its  relationship  with 
the  hinder  end  of  the  pear-shaped  mass  of  yolk  between  the  atrial  cavities  and  the 
arms  of  the  digestive  tract  (Fig.  6C,  H).  Distally  it  corresponds  in  length  to  the 
neural  tube  and  tail  muscles. 

Muscle  cells  of  the  tail 

Mesoderm  differentiates  into  three  structures  of  the  larva,  one  of  which  is  re- 
stricted to  the  larval  action  system,  two  of  which  function  in  both  the  larval  and  adult 
action  systems.  The  former  includes  the  muscles  of  the  tail,  the  latter  the  muscles 
of  the  mantle  and  mesenchymatous  connective  tissue  in  the  body  cavity.  The  asym- 
metry of  the  posterior  lip  of  the  blastopore  at  the  end  of  gastrulation  (Stage  I) 
places  the  muscle  cells  of  the  right  side  dorsal  to  the  chordal  cells  and  to  the  right 
of  the  neural  plate  at  its  posterior  end,  the  muscle  cells  of  the  left  side  to  the  left  of 
the  posterior  neural  plate  but  ventral  to  the  notochord  (Fig.  4C).  Each  band  is 
made  up  of  four  cells  in  fairly  regular  rows. 

In  Stage  II  the  myoblasts  are  the  most  prominent  cells  in  the  body  because  of 
their  size  and  heavy  membranes.  Each  cell  contains  a  large  faintly  reticular  nucleus 
with  a  conspicuous  nucleolus.  The  deeper  cytoplasm  is  grossly  reticular  and  retains 
an  occasional  yolk  granule  (Fig.  6D). 

In  Stage  III  the  peripheral  cytoplasm  elaborates  in  its  cortex,  in  a  slightly  spiral 
direction,  along  the  longitudinal  axis  rows  of  contractile  fibrillae  composed  of  minute 
granules  so  distributed  that  they  resemble  the  individual  myofibrillae  of  striated 
muscle  of  the  higher  chordates  (Fig.  4E,  6D).  The  myofibrillae  are  continuous 
from  one  cell  to  another  throughout  the  length  of  the  muscle  bands.  Grave  (1921) 
has  described  this  in  the  free-swimming  tadpole  of  Amaroecium.  The  bases  of  the 
muscle  bands,  like  that  of  the  notochord  in  Stage  IV,  are  located  well  within  the 
posterior  part  of  the  trunk  just  behind  the  mass  of  yolk  (Fig.  6H). 

Muscles  of  the  mantle 

In  the  late  embryonic  period  (Stage  III)  many  of  the  mesenchyme  cells  located 
directly  under  the  ectoderm  unite  end  to  end  to  form  the  smooth  fibres  of  the  mantle 
(Fig.  4F).  One  set  of  such  muscle  fibres  radiates  from  each  of  the  siphons.  The 
other  set  encircles  the  trunk  obliquely  from  the  dorsal  to  the  ventral  side. 

Mcscnclivinc  of  the  body  cavity 

In  Stage  I  two  compact  lateral  masses  of  mesenchyme  cells  lie  pressed  tightly 
between  the  nutritive  endoderm  and  shallow'  ectodermal  cells.  The  one  on  the  right 
side  is  disposed  more  dorsally  than  the  one  on  the  left  side  (Fig.  5E).  They  ex- 
tend from  the  posterior  muscle  cells  towards  the  anterior  end  of  the  body. 


AMAROECIUM  CONSTELLATUM.     II 


77 


w 


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*} 


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Ph    *VA  KF-  ^V 

'it   7Kf5    f    ^4  i 

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PC— V-^  /       /•* 

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H 


FIGURE  6.  A,  B,  C.  Transverse  sections  through  tadpoles  of  Stage  IV;  A,  anterior.  B.  In 
region  of  oral  siphon.  C.  In  region  of  atrial  siphon.  About  200  X.  D.  Section  through  part 
of  muscle  hand ;  middle  cell  through  center  of  muscle  cell,  lateral  cell  through  peripheral  cyto- 
plasm where  myonbrillae  are  formed.  About  850  X.  E.  Transverse  section  through  Stage  II  to 
show  atrial  invaginations.  300  X.  /;.  Longitudinal  section  through  Stage  III.  About  150  X. 
G.  Tadpole  just  before  hatching,  chorion  not  ruptured.  About  250  X.  H.  Tadpole  at  hatching. 
Note  insertion  of  notochonl  at  posterior  end  of  trunk.  About  250  X.  ad.  p.,  adhesive  papilla; 
at.,  atrium;  at.  in.,  atrial  invagination ;  at.  .?.,  atrial  siphon;  end.,  endostyle ;  int.,  intestine;  i)i.  c., 
muscle  cell;  ncli..  notochonl;  or.  .v..  oral  siphon;  ph..  pharynx;  p.  c.,  pericardial  cavity;  st.. 
stomach ;  ^.  v.,  sensor}-  vesicle ;  v,  y.,  visceral  ganglion. 


78  FLORENCE  MARIE  SCOTT 

In  Stage  II  both  masses  of  cells  multiply  and  spread  out  under  the  ectoderm  in 
all  directions  except  posteriorly.  A  small  amount  of  mesenchyme  is  found  in  the 
tail,  probably  derived  from  the  cells  in  the  mid-region  of  the  posterior  lip  of  the 
blastopore.  Being  crowded  together  the  cells  appear  angular  in  section.  The  nu- 
clei are  relatively  large  (Fig.  4F,  5(7). 

During  the  transition  from  Stage  II  to  Stage  III  growth  of  the  body  and  absorp- 
tion of  the  yolk  effect  a  separation  between  the  epithelial  cells  of  the  epidermis  and 
the  endodermal  cells  (Fig.  SF,  6F).  As  the  body  cavity  enlarges  the  cells  round 
up,  separate  from  each  other,  and  wander  freely  about,  dividing  frequently  and  even- 
tually filling  up  all  available  space  except  in  the  area  around  the  base  of  the  tail  ( Fig. 
6A,B,Q. 

Other  mesenchyme  cells  assume  stellate  shape  and  send  out  long  slender  strain  IN 
of  protoplasm  by  means  of  which  they  form  a  reticulum  of  mesenchymatous  tissue. 
This  is  the  nearest  approach  to  a  coelomic  epithelium  that  is  found  in  Tunicates  with 
the  possible  exception  of  the  perivisceral  cavity  of  Ciona. 

The  mantle  and  tunic 

The  epidermis  in  Stage  I  is  a  layer  of  thin  cells  small  in  surface  view  dorsally 
where  they  adjoin  the  neural  plate,  larger  towards  the  ventral  body  region.  In 
Stage  II  the  cells  are  of  uniformly  small  size  and  cuboidal  in  section  except  where 
they  invaginate  to  form  the  atrium  and  are  columnar  in  shape.  In  surface  view  all 
present  the  characteristic  polygonal  arrangement  of  epithelial  tissue. 

During  Stage  III  the  protoplasm  becomes  vacuolated  medially,  the  nuclei  being 
pushed  to  the  periphery  where  the  cytoplasm  is  more  granular  (  Fig.  5B,  6E).  The 
epidermal  cells  grow  thinner  as  development  progresses  and  the  inter-cell  mem- 
branes disappear.  \Yhen  the  epidermis  has  assumed  the  characteristic  appearance 
of  the  Tunicate  mantle  in  Stage  III  it  secretes  a  thick  layer  of  structureless  tunic. 
Occasional  cells  of  the  test  of  the  ovum  are  trapped  in  the  clear  tunicin,  the  greater 
number,  however,  being  pushed  with  the  test  ahead  of  the  tunic  (Fig.  6C).  The 
tunic  is  grooved  where  it  is  secreted  about  the  tail  and  when  the  tail  is  released,  with 
the  disappearance  of  the  test,  the  groove  remains  in  evidence  marking  the  embryonic 
position  of  the  tail. 

Dcrh'ati'i'cs  <>j  tJie  epidermis 

Adhesive  papillae — A  conspicuous  feature  of  the  Amaroecium  larva  is  a 
vertical  row  of  three  adhesive  papillae  at  the  anterior  end  (Fig.  3,  6/;,  6",  H).  Each 
papilla  first  appears  early  in  Stage  III  as  a  local  thickening  of  ectoderm  forming  a 
pad  of  columnar  cells.  The  cells  at  the  periphery  of  the  thickened  pad  form  a  stem 
which  increases  in  length  as  the  tunic  thickens,  the  whole  organ  becoming  goblet- 
shaped.  It  retains  its  connection  with  the  body  cavity  through  its  slender  hollow 
stem  (Fig.  6G,  H).  The  papillae  extend  through  the  thickness  of  the  tunic  and 
are  exposed  at  its  surface.  Cells  that  constitute  the  functional  portion  become  vacu- 
olated and  reticular  proximally  and  toward  the  center  of  the  cup,  where  the  long 
cells  converge,  they  produce  secretion  granules  which  lodge  in  the  concavity  of  the 
papilla  (Fig.  56").  The  bordering  epidermis  surrounds  the  disc  forming  a  thin 
layer  over  the  cup-shaped  depression.  During  the  free-swimming  life  of  the  larva 


AMAROECIUM  CONSTELLATUM.     II  79 

the  secretion  granules  are  converted  into  a  viscid  substance  by  means  of  which  the 
tadpole  becomes  attached.  The  entire  glandular  structure  is  of  ectodermal  origin. 
Grave  (1921)  from  his  study  of  the  fully  formed  tadpole  supposed  that  mesenchyme 
cells  gave  rise  to  the  glandular  portion  of  the  papilla.  Mesenchyme  cells  wander 
from  the  body  cavity  into  the  hollow  stalk  but  they  are  not  incorporated  into  its 
structure.  The  tail  encircling  the  body  crowds  the  papillae  a  little  to  the  right  of 
the  sagittal  plane  thus  adding  to  the  asymmetry  of  the  larva.  The  three  papillae 
cannot  be  homologized  with  the  tactile  papillae  of  Botryllus,  which  are  integral  parts 
of  the  peripheral  nervous  system.  Here  they  serve  only  as  gross  organs  of 
attachment. 

Test  vesicles — During  Stage  III,  when  the  adhesive  papillae  are  differ- 
entiating, the  test  vesicles  originate  as  numerous  small  ectodermal  evaginations  in 
four  distinct  regions  at  the  anterior  end  of  the  trunk.  Two  groups,  separated  from 
each  other  by  the  median  papilla,  are  directed  forward.  The  dorsal  group  is  de- 
rived from  a  short  ridge  extending  in  the  direction  of  the  oral  siphon.  The  ventral 
group,  below  the  ventral  papilla,  is  derived  from  a  long  ridge  extending  posteriorly 
through  about  a  third  of  the  length  of  the  trunk  (Fig.  5,  6G,  H).  The  vesicles 
themselves  originate  as  independent  hollow  slender  projections  of  the  ectoderm. 
The  attached  end  of  each  evagination  becomes  narrow,  finally  constricting  off  and 
severing  its  connection  at  the  base.  Frequently  this  separation  is  not  effected  by  the 
time  of  hatching  of  the  vesicle  still  being  attached  to  the  epidermis  by  their  stalk-like 
bases.  When  detached  the  slightly  pear-shaped  vesicle  rounds  up  and  becomes  a 
sphere  consisting  of  a  single  layer  of  cells  which  lose  their  definition  on  the  proximal 
side  where  they  are  extremely  thin. 

The  use  of  the  word  "test"  in  connection  with  these  vesicles  is  unfortunate.  The 
chorion  of  the  egg  of  Tunicates  is  called  the  test  and  the  cells  that  either  lie  freely 
in  the  enclosed  liquid  or  are  resolved  into  pavement  epithelium  are  called  the  test 
cells.  The  tunic  of  the  tadpole  is  a  purely  ectodermal  derivative.  The  tunic  of  the 
adult  colonies  being  the  product  of  secretory  activity  of  these  vesicles,  the  vesicles 
should,  with  greater  accuracy,  be  called  the  "tunic  vesicles." 

SUMMARY 

1.  The  digestive  system  of  Amaroecium  lacks  an  open  archenteron  at  the  end  of 
gastrulation.     The  pharynx  appears  as  a  narrow  incision  with  a  thin  roof  and  heavy 
floor.     An  oesophageal  evagination  differentiates  into  stomach  and  intestine. 

2.  Heart  and  pericardium  originate  from  the  floor  of  the  pharynx. 

3.  Atrium  and  siphons  are  ectodermal  structures  that  become  associated  with 
the  digestive  system. 

4.  The  nervous  system  consists  of  a  sensory  vesicle  enclosing  two  sensory  masses 
of  pigment,  a  hypophysis  lying  beside  two  sensory  ganglion,  a  visceral  ganglion  de- 
scending laterally  to  the  neural  tube  which  lies  to  the  left  of  the  notochord  through- 
out the  length  of  the  tail. 

5.  The  notocord  is  derived  from  chordal  cells  invaginated  at  gastrulation.     Its 
cells  become  vacuolated.     The  notochord  is  confined  to  the  tail  and  posteriormost 
region  of  the  trunk. 

6.  Muscle  cells  differentiate  from  mesodermal  cells  of  the  blastoporal  margins. 
Asymmetry  of  the  blastopore  places  the  cells  of  its  right  margin  dorsal  to  the  noto- 


80  FLORENCE  MARIE  SCOTT 

chord,  the  cells  of  the  left  margin  ventral  to  the  notochord.  Each  band  of  muscle 
cells  consists  of  four  longitudinal  rows.  Cells  separate  from  the  two  lateral  masses 
of  mesenchyme  and  move  into  the  body  space  of  the  developing  tadpole.  They  give 
rise  to  muscles  of  the  mantle. 

LITERATURE  CITED 

CAULLERY,  M.,  1895.     Contribution  1'fitude  des  Ascidies  Composees.     Bull,  dc  la  France  ct  de  la 

Belglquc,  27:  1-158. 
CONKLIN,  E.  G.,   1905.     Organization  and  cell  lineage  of  the  Ascidian  egg.     Jour.  Acad.  Nat. 

Sci.  Phild.,  13:  1-119. 
GRAVE,  C.,  1920.     The  origin,  function  and  fate  of  the  test  vesicles  of  Amaroucium  constellatum. 

Anat.  Rcc.,  17 :  350. 
GRAVE,    C.,    1921.     Amaroucium   constellatum — The    structure   and   organization    of   the   tadpole 

larva.     Jour.  Morfh.,  36:  71-101. 
GRAVE,  C.,   1935.     Metamorphosis  of  Ascidian  larvae.     Papers  from   the   Tortugas  Laboratory, 

29 :  209-292. 
GRAVE,  C.,  1944.     The  larva  of  Styela   (Cynthia)   partita:   Structure,  activities  and  duration  of 

life.    Jour.  Morph.,  75  :  173-190. 

HJORT,  J.,  1896.     Germ  layer  studies  based  upon  the  development  of  Ascidians.     Cliristiania. 
MAURICE,  C.  ET  M.  SCHULGIN,  1884.     Embryogenie  de  1'Amaroecium  proliferum   (Ascidie  com- 

posee).     Ann.  Sci.  Nat.,  (6)   17:  I^t6. 
SCOTT,  SISTER  FLORENCE  M.,  1945.     The  developmental  history  of  Amaroecium  constellatum.     I. 

Early  embryonic  development.     Biol.  Bull.,  88:  126-138. 


COMPARATIVE  SENSITIVITY  OF  SPERM  AND  EGGS  TO 
ULTRAVIOLET  RADIATIONS 

ARTHUR  C.  GIESE * 

Marine  Biological  Laboratory,   Woods  Hole,  Mass,  and  Hopkins  Marine  Station, 
Pacific  Grove,  Calif,  and  Stanford  University,  California 

The  sperm  of  the  sea  urchin  are  more  sensitive  to  ultraviolet  radiations  than 
the  eggs  when  the  effectiveness  of  the  rays  is  compared  by  the  retardation  of  cleav- 
age of  unexposed  eggs  fertilized  with  irradiated  sperm  on  the  one  hand  and  of  ir- 
radiated eggs  fertilized  with  unexposed  sperm  on  the  other  (Giese,  1939c).  It 
would  be  interesting  to  know  whether  sperm  are  generally  more  susceptible  to  these 
radiations  than  eggs ;  therefore,  the  experiments  were  repeated  on  a  number  of 
marine  forms.  It  is  also  desirable  to  find  an  explanation  for  this  differential  sus- 
ceptibility in  those  cases  where  it  occurs.  Insight  might  be  gained  by  determining 
action  spectra  for  the  sperm  and  egg,  therefore  the  relative  efficiency  of  action  of 
different  wave-lengths  of  ultraviolet  light  in  retarding  cleavage  of  irradiated  eggs 
and  of  eggs  fertilized  with  irradiated  sperm  was  determined  as  described  below. 

MATERIALS  AND  METHODS 

Arbacia  punctulata,  Nereis  limbata,  Chaetoptcrus  pcrgamcntaccus,  and  Mactra 
sp.  were  studied  at  Woods  Hole,  Mass.  Strongylocentrotus  franciscaims  and  S. 
purpuratus,  collected  at  Moss  Beach,  and  Urcchis  caiipo  collected  at  Bolinas  Bay, 
California,  were  used  at  Stanford  University,  and  Dendrastcr  e.vccntricns  and  Pa- 
tcria  miniata  were  studied  at  the  Hopkins  Marine  Station,  each  type  of  egg  being 
used  during  the  active  breeding  season. 

The  methods  for  studying  the  eggs  were  similar  to  those  previously  described 
(Giese,  1938).  Except  for  the  work  on  the  action  spectrum,  the  mercury  argon 
discharge  tube  which  emits  about  85  per  cent  of  its  light  at  A  2537  A  was  used  and 
the  light  intensity  was  measured  with  a  Hanovia  Ultraviolet  Meter  (No.  949).  The 
dishes  were  kept  in  running  sea  water  to  attain  a  lower  temperature  than  that  of  the 
room.  The  work  on  the  action  spectrum  was  done  with  a  mercury  arc  and  a  natural 
quartz  monochromator  and  the  intensity  of  the  light  was  measured  with  a  thermopile 
as  in  previous  studies  (Giese,  1938).  The  eggs  were  kept  in  dishes  in  moist  cham- 
bers and  in  a  constant  temperature  room  at  15°  C. 

Sperm  were  used  in  dilutions  of  between  1  :  200  and  1  :  1 .000  of  the  spawn.  Such 
dilution  is  necessary  because  in  denser  suspensions  ultraviolet  light  is  completely 
removed  by  the  sperm  first  reached.  Irradiated  sperm  lose  their  fertilizing  power 
rapidly,  therefore  they  must  be  used  soon  after  exposure  (see  Hinrichs,  1927,  for 
studies  on  inactivation). 

1  This  work  was  supported  in  part  by  grants  from  the  Rockefeller  Foundation.  The  writer 
is  indebted  to  Dr.  C.  Packard,  Director  of  the  Marine  Biological  Laboratory,  and  to  Dr.  L.  R. 
Blinks,  Director  of  the  Hopkins  Marine  Station,  for  making  available  the  facilities  and  for  the 
many  kindnesses  extended  to  the  author  during  the  course  of  this  work. 

81 


82 


ARTHUR  C.  GIESE 


EXPERIMENTAL 


Comparison  of  various  eggs 


A  summary  of  the  general  results  obtained  with  all  the  eggs  studied  will  be  found 
in  Table  I.  Not  all  the  eggs  respond  to  ultraviolet  radiations  in  the  same  way. 
Thus  cleavage  of  eggs  of  Arbacia  and  Strongylocentrotus  is  merely  slowed  up  but 
remains  normal  after  small  and  medium  dosages  so  that  comparisons  of  the  effects 
of  various  dosages  and  wave-lengths  is  relatively  easy.  Abnormalities  only  appear 
after  larger  dosages.  In  Urechis,  Nereis,  and  some  of  the  other  eggs  the  threshold 
for  abnormal  development  is  relatively  low.  While  per  cent  of  abnormal  develop- 
ment could  be  used  for  analysis  of  effects  of  radiations,  it  would  be  much  more 
difficult. 

It  is  readily  apparent  that  with  regard  to  ultraviolet  susceptibility,  there  are  two 
types  of  sperm :  in  the  Echinoderms,  especially  Arbacia  and  Strongylocentrotus,  the 

TABLE  I 

Comparative  action  of  ultraviolet  radiation2  on  eggs  and  sperm  of  various  marine  animals 


Species 


Effects  on  eggs 


Effects  on  sperm 


Strongylocentrotus 
purpuratus 


Arbacia  punctulata 


Dendr aster  excen- 
tricus 

Urechis  caupo 


Chaetopterus  perga- 
mentaceus 


Nereis  limbata 


Mactra  sp. 


Delay  just  noticeable  after  about 
100  ergs/mm.2;  will  develop  even 
after  4,000;  after  8,000  ergs/mm.2 
become  quite  abnormal. 

Noticeable  delay  after  200  ergs/ 
mm.2  but  even  after  2,000  ergs/ 
mm.2  plutei,  normal  but  smaller 
than  controls,  develop  from  eggs. 
After  4,000-8,000  ergs/mm.2  eggs 
are  quite  abnormal. 

Slight  delay  after  1,600  ergs/mm.2; 
strong  after  6,400;  quite  abnormal 
after  25,000  ergs/mm.2 

Some  delay  after  200  ergs/mm.2 
Marked  injury  with  abnormal 
cleavage  after  5,000  ergs/mm.2 

Slight  delay  only  after  about  4,000 
ergs/mm.2;  after  16,000  ergs/mm.2 
still  cleave  but  much  delay  and 
many  cytolize. 

Even  after  4,000  ergs/mm.2  de- 
velop with  little  delay  to  the 
trochophore  stage;  after  8,000  ergs 
show  delayed  cleavage. 

Very  slight  delay  after  500  ergs/ 
mm.2;  striking  after  4,000-8,000. 


Noticeable  delay3  even  after  10- 
20  ergs/mm.2  Marked  retardation 
as  dosage  above  this  is  used. 

Noticeable  delay  even  after  less 
than  50  ergs/mm.2  After  250  ergs 
still  develop  larvae  but  after  500 
quite  abnormal.  Even  after  4,000 
ergs/mm.2  sperm  activate  eggs. 

Slight  delay  after  200  ergs/mm.2; 
abnormal  after  800  ergs/mm2. 

Marked  abnormalities  after  200 
ergs/mm.2 

Slight  delay  after  2,000  ergs/mm.2; 
killed  after  about  8,000-16,000 
ergs/mm2. 

Slight  delay  between  4,000-8,000 
ergs/mm.2  8,000  kills  most  sperm. 


Delay    after    250    ergs/mm.2    and 
progressive  delay  thereafter. 


2  The  measurements  with  the  Hanovia  meter  are  accurate  to  about  10  per  cent  as  checked  by 
thermopile  measurements  in  one  instance. 

3  Amounting  to  15-30  minutes  delay  at  the  third  cleavage  of  eggs  fertilized  with  such  irradiated 
sperm.     Marked  delay  means  a  delay  of  an  hour  or  more. 


SENSITIVITY  OF  SPERM  AND  EGGS 


83 


sperm  are  much  more  sensitive  than  the  eggs ;  in  the  worms  such  as  Urechis, 
Nereis,  and  Chaetopterus  the  sperm  is  slightly  if  at  all  more  sensitive  than  the  egg, 
as  judged  by  cleavage  delay. 

Such  a  lack  of  differences  in  susceptibility  of  the  gametes  might  be  more  ap- 
parent than  real.  It  is  possible  that  when  there  is  little  or  no  cleavage  delay  follow- 
ing fertilization  of  an  egg  with  an  irradiated  sperm,  the  sperm  may  be  serving  only 
to  activate  the  egg  to  haploid  parthenogenesis.  Eggs  of  Arbacia  and  of  Chaetop- 
terus were,  therefore,  fertilized  with  sperm  treated  either  to  a  small  dosage  or  to  a 
medium  dosage  of  radiations  and  at  appropriate  intervals  samples  were  fixed  in 
Benin's  fluid  and  stained  with  iron  hematoxylin.  Although  the  preparations  were 

k 

ACTION  SPECTRUM  FOR  RETARDATION  OF   CLEAVAGE 


100 


80 

\ 
I6" 


40 


20 


SPERM^ 


A 

X.  S 


EGGS      D 

SETTING  ACTION  AT  2804 


N 


\          EQUAL   TO  100 
\ 

\ 
\ 
\ 
\ 
\ 
\ 


P 


EGGS  COMPARED  TO  SPERM 
ON  AN  ABSOLUTE  BASIS 


2400 


2600  280O 

WAVELENGTH  IN  A 


3000 


320O 


FIGURE  1.  Action  spectra  for  retardation  of  cleavage  of  eggs  fertilized  with  irradiated  sperm 
at  A  and  for  irradiated  eggs  at  C.  At  B  the  data  for  the  eggs  are  compared  on  a  relative  basis 
setting  the  value  at  \  2,804  A  as  100  per  cent  efficient.  See  text  below. 

not  entirely  satisfactory,  evidence  for  pronuclear  fusion  was  observed  in  both  cases. 
No  lagging  or  disintegrating  sperm  were  observed  in  the  cytoplasm  of  either  egg. 
Since  neither  cytological  nor  physiological  evidence  suggests  parthenogenesis,  it 
seems  likely  that  for  the  dosage  ranges  tested  the  delayed  cleavage  follows  fusion 
of  the  gametic  nuclei.  The  difference  between  the  two  types  of  sperm  must  lie  in 
some  other  factor.  Possible  explanations  will  be  considered  in  the  discussion. 

The  data  in  Table  I  show  that  the  threshold  for  effects  on  cleavage  is  quite  dif- 
ferent for  eggs  of  different  species.  Thus  Strongylocentrotus,  Arbacia,  Mactra, 
and  Urechis  eggs  are  retarded  after  brief  exposures  to  ultraviolet  as  compared  to 


84 


ARTHUR  C.  GIESE 


Nereis,  Chaetopterus,  and  Dendraster.  Whether  this  is  due  to  mere  physical 
screening  by  some  inert  materials  in  the  egg  or  to  differences  in  concentration  of 
some  light  sensitive  materials  is  not  known. 

Action  spectra  for  egg  and  sperm 

If  irradiation  of  the  nucleus  alone  causes  retardation  of  division  of  the  cell,  the 
same  action  spectrum  should  be  found  for  egg  and  sperm ;  that  is,  there  should  be 
no  qualitative  difference  in  effectiveness  of  different  wave-lengths  even  though  the 
general  susceptibility  of  the  sperm  is  greater.  If  elements  in  the  cell  other  than  the 

ACTION   SPECTRA   FOR   SPERM  AND  EGG  AND  PROTEIN  ABSORPTION 


100  i- 


i 
I 


I 


i 


\          ABSORPTION  BY 


SERUM  ALBUMIN 


'      SPERM 
ACTION 
SPECTRUM 


\ 
ABSORPTION 

BY  NUCLEIC  ACID 


EGG  ACT/ON 
SPECTRUM 


20    - 


£300 


2500 


270O 


2900 


3100 


WAVELENGTH   IN  ANGSTROM    UNITS 


FIGURE  2.  Comparison  of  the  action  spectra  of  Figure  1  with  absorption  spectra  of  nucleic 
acid  and  serum  albumin.  Data  for  nuclei  acid  from  Caspersson  (1938),  for  proteins  from 
Smith  (1929).  Note  that  the  action  spectrum  for  the  egg  is  quite  different  from  the  absorption 
spectrum  for  albumin  at  both  ends. 

nucleus  are  involved  the  egg  may  show  an  action  spectrum  different  from  that  of 
the  sperm. 

The  methods  employed  for  the  studies  at  different  wave-lengths  are  similar  to 
those  already  described  elsewhere  (Giese,  1938,  1939c),  therefore,  only  the  briefest 
mention  need  be  made  of  them.  The  irradiated  eggs  are  fertilized  with  normal 
sperm.  The  rate  of  division  is  then  determined  by  observing  for  percentage  of 
cleavage  every  15  minutes.  The  times  at  which  the  eggs  reach  the  2,  4,  8,  16,  and 
32  cell  stages  are  recorded  and  the  number  of  cleavages  is  plotted  against  the  time 
after  fertilization  and  compared  with  the  control.  The  increase  in  time  required  to 
reach  the  third  cleavage  is  taken  as  a  measure  of  the  retarding  action  of  the  radi- 


SENSITIVITY  OF  SPERM  AND  EGGS  85 

ations.  The  retardation  is  then  plotted  against  dosage.  From  such  curves  for  each 
of  the  wave-lengths  the  dosage  required  to  bring  about  a  given  retardation  can  be 
determined.  For  Figures  1  and  2  the  reciprocals  of  the  relative  amounts  of  energy 
at  different  wave-lengths  required  to  produce  a  retardation  of  division  by  1.5  hours 
were  determined.  In  Figure  1  at  A  and  C  the  sperm  and  egg  are  compared  on 
this  basis  and  a  great  difference  in  susceptibility  between  the  gametes  is  evident. 
In  B  the  data  for  the  eggs  are  compared  amongst  themselves  on  a  relative  basis 
setting  the  action  at  A  2,804  A  as  100  per  cent  efficient. 

The  shape  of  the  curves  indicates  that  different  materials  are  being  affected  in 
the  two  cases,  since  the  action  spectrum  is  considered  to  be  a  measure  of  the  ab- 
sorption by  the  active  constituent.  To  see  if  the  absorbing  materials  can  be  identi- 
fied the  absorption  spectra  for  serum  albumin  and  nucleic  acid  are  given  in  Figure 
2.  It  is  apparent  that  the  action  spectrum  for  sperm  matches  the  absorption  spec- 
trum for  nucleic  acid  better  than  the  absorption  spectrum  for  albumin ;  the  reverse 
is  true  for  the  action  spectrum  of  the  egg.  Since  the  simple  proteins  and  nucleo- 
proteins  are  the  major  structural  constituents  of  the  cell  and  none  of  the  other  or- 
ganic or  inorganic  constituents  have  very  specific  absorption,  the  resemblances  while 
imperfect  are  indicative  of  absorption  by  these  two  classes  of  compounds  in  the 
action  of  ultraviolet  radiations  on  the  gametes. 

DISCUSSION 

The  occurrence  of  a  differential  susceptibility  of  gametes  with  the  sperm  more 
sensitive  to  ultraviolet  light  than  the  egg  as  first  found  in  the  sea  urchin,  Strongylo- 
centrotus  pwpuratus,  was  verified  on  Arbacia  and  Dendraster  and  in  preliminary 
trials  on  Pateria  and  6\  franciscanus  but  not  on  Urechis,  Chaetopterus,  and  Nereis. 
In  the  latter  forms  the  sperm  appears  to  be  only  slightly  more  sensitive  than  the  egg 
(Table  I).  The  former  group  of  species  belongs  embryologically  to  the  radially- 
cleaving,  indeterminate  egg  type,  the  latter  group  to  the  spiral  determinate  type. 
In  addition,  the  radial  eggs  used  here  are  mature  or  nearly  so  at  the  time  of  shedding 
whereas  the  spiral  eggs  are  generally  immature.  An  illustration  of  the  difference 
in  response  to  ultraviolet  light,  depending  on  this  difference  in  organization  is  seen 
in  the  local  "burns"  occurring  in  the  spiral  eggs.  Thus  a  Nereis  egg  given  a  uni- 
lateral dosage  of  between  8,000-16,000  ergs/mm.2  may  develop  apparently  normally 
except  on  the  burned  surface  which  appears  blistered.  A  Strongylocentrotus  egg 
on  the  other  hand  unless  given  a  large  dosage  of  light  will  show  general  effects  dis- 
tributed throughout  the  retarded  egg.  However,  it  is  not  possible  to  say  which 
features  of  the  organization  account  for  the  difference  in  sensitivity  of  the  eggs  and 
sperm  of  the  two  groups. 

One  might  envisage  that  in  eggs  the  retarding  effects  of  radiation  on  cleavage  are 
due  to  the  inactivation,  by  substances  formed  during  irradiation,  of  some  catalyst 
which  is  necessary  for  the  reactions  involved  in  cleavage.  In  one  group  of  eggs 
perhaps  the  catalyst  is  present  in  excess  of  that  necessary  for  a  characteristic  rate 
of  cleavage,  the  rate  being  controlled  by  some  other  limiting  factor,  in  the  other  it  is 
present  in  just  adequate  concentration  and  itself  constitutes  the  limiting  factor. 
Even  a  considerable  dosage  of  radiations  will  not  reduce  the  concentration  of  cata- 
lyst below  the  critical  level  in  the  first  case  but  will  readily  do  so  in  the  second.  In 
the  first  case  no  cleavage  delay  would  be  expected  until  very  large  dosages  of  radi- 


86  ARTHUR  C.  GIESE 

ations  had  been  administered,  in  the  second  the  cleavage  should  be  affected  after 
very  small  dosages.  One  would  have  to  assume  that  irradiated  sperm  on  penetrat- 
ing unirradiated  eggs  introduce  similar  cleavage-inhibiting  substances  acting  on  the 
catalyst  as  those  formed  in  the  irradiated  egg.  In  this  case  also  the  effect  on  cleav- 
age should  depend  upon  the  amount  of  catalyst  present  in  the  egg — if  in  excess,  the 
cleavage  should  not  be  easily  inhibited,  if  limiting,  the  reverse  should  be  true.  \Ye 
should  expect  both  sperm  and  egg  to  be  relatively  insensitive  to  the  radiations  in 
the  former  and  this  is  found  in  most  spirally  cleaving  eggs. 

Against  the  above  postulation  is  the  fact  that  the  action  spectrum  for  sperm  re- 
sembles nucleoprotein  absorption  while  for  the  egg  it  resembles  simple  protein  ab- 
sorption indicating  two  different  ultraviolet  absorbing  materials  in  the  gametes  by 
which  the  cleavage-retarding  effect  is  produced.  It  is  possible  that  absorption  by 
both  of  these  types  of  proteins  leads  to  the  formation  of  toxic  photoproducts  which 
inhibit  the  same  catalyst.  It  is  also  possible  that  the  toxic  substance  is  much  more 
rapidly  formed  by  the  nucleoproteins,  but  the  necessary  assumptions  strain  the 
imagination. 

It  should  be  pointed  out  that  the  retardation  of  the  early  cleavage-  is  only  the 
initial  effect  of  the  radiation.  If  the  delayed  effect  could  be  studied  we  might  find 
that  the  recovery  from  injury  to  the  egg  would  resemble  absorption  by  nucleo- 
protein, indicating  a  more  lasting  injury  to  the  nucleus  than  to  the  cytoplasm,  as  is 
the  case  for  division  of  Paramecium  (Giese,  1945a).  Because  the  number  of  cells 
cannot  be  satisfactorily  determined  in  the  later  cleavages  such  experiments  were  not 
attempted  with  eggs. 

The  action  spectrum  obtained  for  the  egg  is  similar  to  that  observed  for  "cyto- 
plasmic"  effects  such  as  increased  time  of  ciliary  reversal,  retardation  of  excystment, 
immobilization  of  cilia,  and  prevention  of  hatching  of  eggs.  The  action  spectrum 
for  the  sperm  resembles  that  for  "nuclear"  effects  such  as  recovery  of  paramecia 
from  sublethal  effects,  bactericidal  and  fungicidal  effects  and  the  production  of  muta- 
tions (see  Giese,  1945b,  for  references).  It  is  interesting  to  note  the  difference  be- 
tween the  action  spectrum  for  retardation  of  cleavage  of  the  egg  and  for  activation 
studied  by  Hollaender  (1938).  In  the  latter  case  no  action  was  found  until  about 
A  2,650  A  and  the  effectiveness  of  the  light  increased  as  the  wave-length  decreased. 
The  mechanism  of  action  of  the  ligbi  must  be  different  in  these  two  instances.  The 
action  spectrum  data  thus  lay  the  foundation  for  further  analysis  of  the  effect  of 
these  radiations  upon  gametes. 

SUMMARY 

1.  The  action  spectrum  for  the  retardation  of  division  of  eggs  fertilized  with  ir- 
radiated sperm  resembles  the  absorption  of  ultraviolet  light  by  nucleoproteins. 

2.  The  action  spectrum  for  retardation  of  division  of  irradiated  eggs  of  the  sea 
urchin  resembles  absorption  by  simple  proteins  like  albumin  except  that  at  the  short 
wave-length  end  there  is  no  increase  in  action  at  A  2,483  A  where  absorption  shows 
a  definite  upswing. 

3.  The  absolute  amount  of  energy  required  to  affect  division  to  the  same  extent 
by  affecting  the  sperm  is  very  much  less  than  that  required  to  affect  eggs. 

4.  Other  Echinoderms  tested  show  a  similar  difference  in  susceptibility  of  eggs 
and  sperm :  6\  jranciscanus,  Arbacia  punctnlata,  Dendraster  ex  centric  us,  and  Pa- 
ter la  miniata 


SENSITIVITY  OF  SPERM  AND  EGGS  87 

5.  Animals  other  than  Echinoderms  tested  did  not  show  as  striking  a  difference 
between  susceptibility  of  eggs  and  sperm  :  Urechis  caupo,  Mactra  sp.,  Chactoptcrus 
pcrgamentaceus,  and  Nereis  limb  at  a. 

6.  In  the  eggs  listed  in  paragraph  5,  determinations  are  made  more  difficult  by 
the  tendency  for  the  eggs  to  show  irregular  cleavage  rather  than  retarded  cleavage 
as  the  dosage  increases.     Such  irregular  cleavage  occurs  in  Echinoderm  eggs  as  well 
but  the  threshold  is  higher. 

7.  If  both  eggs  and  sperm  of  the  sea  urchin  are  irradiated  the  effect  on  the  rate 
of  division  is  less  than  the  sum  of  the  effects  which  would  be  expected  on  each  of  the 
gametes  alone.     However,  the  percentage  of  abnormal  cleavage  greatly  increases. 

LITERATURE  CITED 

CASPKRSSON,  T.,  1936.     Uher  den  chemischen  Aufbau  des  Strukturen  des  Zellkernes.     Skandinav. 

arch.  f.  physiol.  Suppl,  8  to  Vol.  73,  1-151. 
GIESE,  A.  C.,  1938.     The  effects  of  ultraviolet  radiations  of  various  wavelengths  upon  cleavage 

of  sea  urchin  eggs.     Biol.  Bull.,  65 :  238-247. 
GIESE,  A.  C.,  1939a.     Retardation  of  early  cleavage  of  Urechis  by  ultraviolet  light.     PhvsioJ. 

'  Zool.,  12  :  319-327. 
GIESE,  A.  C.,   1939b.     Ultraviolet  light  and  cell  division.     Effects  of  x  2654  and  2804A  upon 

Paramecium  caudatum.     /.  Cell.  Conip.  Physiol.,  13:   139-150. 

GIESE,  A.  C.,  1939c.     Ultraviolet  radiation  and  cell  division.     Nuclear  sensitivity  :  effect  of  ir- 
radiation of  sea  urchin  sperm.     /.  Cell.  Conip.  Physiol.,  14:  371-382. 
GIESE,  A.  C.,  1945a.     The  ultraviolet  action  spectrum  for  retardation  of  division  of  Paramecium. 

/.  Cell.  Comp.  Physiol.,  26 :  47-55. 

GIESE,  A.  C.,  1945b.     Ultraviolet  radiations  and  life.     Physiol.  Zool.,  18 :  223-250. 
HINKICHS,   M.   A.,    1927.     Ultraviolet   radiation   and   the   fertilizing   power   of   Arbacia   sperm. 

Biol.  Bull.,  53:  416-437. 
HOI.LAENDER,  A.,  1938.     Monochromatic  ultraviolet  radiation  as  an  activating  agent  for  the  eggs 

of  Arbacia  punctulata.     Biol.  Bull.,  75 :  248-257. 
SMITH,  F.  C.,  1929.     The  ultraviolet  absorption  spectra  of  certain  aromatic  ainino  acids  and  of 

proteins.     Proc.  Roy.  Soc.  London  B,  104:  198-205. 


OBSERVATIONS  ON  THE  FUNCTIONING  OF  THE  ALIMENTARY 
SYSTEM  OF  THE  SNAIL  LYMNAEA  STAGNALIS 

APPRESSA  SAY 

MELBOURNE  ROMAINE  CARRIKER 
Zoological  Laboratory  of  the   University  of   Wisconsin,  Madison 

INTRODUCTION 

Although  records  exist  of  functional  studies  on  the  alimentary  system  of  Basom- 
matophora  as  far  back  as  the  early  eighteen  hundreds,  the  detailed  story  of  the 
course  and  ultimate  fate  of  food  in  the  alimentary  tract  and  the  simultaneous  move- 
ments of  the  tract  is  thinly  scattered  and  far  from  complete.  In  the  more  recent 
emphasis  placed  on  some  gastropods  because  of  their  importance  as  vectors  of  para- 
sites of  man,  domestic  animals,  wild  game,  and  fish,  it  is  vitally  important  that  the 
normal  physiology  of  the  system  most  frequented  by  these  parasites  be  better  known. 

It  is  the  purpose  of  this  paper  to  integrate  the  previous  work  on  the  physiology 
of  the  alimentary  system  of  Lymnaea  st  agnails  and  allied  forms  (suborder  Basom- 
matophora,  order  Pulmonata)  with  original  research  on  the  same  system  in  L.  s. 
apprcssa  Say.  The  basic  morphological  (Carriker,  1945)  and  histological  (Car- 
riker  and  Bilstad,  1946)  studies  on  this  system  in  L.  s.  apprcssa  have  been  com- 
pleted and  are  in  press.  All  terms  used  in  this  research  have  been  described  in  these 
two  papers. 

L.  s.  apprcssa  has  been  selected  for  this  research  because  it  is  a  representative 
vector  and  because  of  its  excellent  response  to  laboratory  culture,  its  relatively 
large  size  (maximum  shell  length,  62.5  mm.)  as  compared  with  other  fresh  water 
pulmonates,  its  short  life  cycle,  and  its  relatively  thin  semitransparent  shell  and  semi- 
transparent  tissues.  Snails  used  in  the  research  were  cultured  entirely  in  the  labo- 
ratory. They  were  grown  through  many  generations  in  large  battery  jars  and  fed 
on  lettuce  and  cooked  "cream  of  wheat"  cereal.  The  water  in  the  jars  was  aerated 
by  means  of  a  small  Marco  air  pump  (Noland  and  Carriker,  1946).  The  original 
snails  were  collected  in  Fox  Lake,  Wisconsin,  in  1939.  Parasite-free  cultures 
(especially  of  trematodes)  from  the  original  snails  were  obtained  by  the  isolation 
of  the  egg  mass  soon  after  oviposition  in  separate  aquaria.  Each  new  culture  was 
started  in  this  way  rendering  transmission  of  infection  very  improbable.  Detailed 
examination  of  succeeding  generations  has  not  disclosed  parasites. 

This  work  was  carried  out  at  the  University  of  Wisconsin  (1939-1943)  under 
the  stimulating  guidance  of  Prof.  L.  E.  Noland,  whose  advice,  encouragement,  and 
friendly  cooperation  were  much  appreciated. 

HISTORICAL  REVIEW 

Scanty  observations  on  the  function  of  the  anterior  part  of  the  alimentary  tract 
of  Lymnaea  were  given  by  Semper  (1857),  Geddes  (1879)  and  Moquin-Tandon 
(1885)  ;  more  detailed  information  was  given  by  Amaudrut  (1898),  Pieron  (1908) 

88 


ALIMENTARY  SYSTEM  OF  LYMNAEA  89 

and  by  Baecker  (1932).  The  stomach  region  was  investigated  by  Gartenauer 
(1875),  Moquin-Tandon  (1885),  Colton  (1908)  and  Heidermanns  (1924).  These 
experimental  contributions  of  Colton  and  of  Heidermanns,  particularly  of  the 
latter,  are  noteworthy.  The  liver  has  been  the  object  of  most  of  the  physiologi- 
cal work  although  the  research  has  usually  been  incidental  to  that  on  the  stylom- 
matophoran  Helix:  Barfurth  (1880,  1881,  1883a  and  b),  Frenzel  (1886),  Cuenot 
(1892),  Enriques  (1901,  1902),  Faust  (1920),  Peczenik  (1925)  and  Krijgsman 
(1928).  Only  the  investigation  of  Peczenik  is  exclusively  on  L.  stagnalis. 

EXPERIMENTAL  METHODS  AND  RESULTS 
Lymnaea  physiological  salt  solution 

The  study  of  the  living  system  has  required  the  development  of  a  physiological 
salt  solution  which  will  approximate  the  ionic  and  osmotic  balances  of  the  blood  of 
Lymnaea  more  closely  than  do  such  commonly  used  solutions  as  Ringer's.  On  the 
basis  of  incomplete  data  given  by  Duval  (1928)  on  Lymnaea  and  by  Bernard  and 
Bonnet  (1930)  on  Helix  on  the  molecular  concentration  of  blood,  the  following 
solution  was  developed  for  L.  s.  appressa: 

NaCl 2.0  gms.  per  liter 

NaHCO., 2.0     "       "      " 

KH2PO4 0.1     "       "      " 

MgCl2 0.3     "       "       " 

CaCl2 0.3     "       "      " 

This  solution  consists  of  0.47  per  cent  salts  and  gives  a  pH  of  approximately  7.8. 
After  about  a  week  considerable  precipitation  of  CaCO.,  occurs,  although  this 
seems  to  have  no  noticeable  effect  on  the  isolated  organs.  The  vas  deferens  was 
used  in  testing  the  solution  and  was  found  superior  to  the  heart  for  this  purpose. 
The  vas  deferens,  terminal  preputium  and  prostate  gland  were  removed  under  the 
physiological  salt  solution  from  the  cephalic  hemocoel  without  bruising.  This  por- 
tion of  the  reproductive  tract  is  in  part  a  strong  muscular  tube  which  is  easily  ex- 
cised and  maintains  a  continuous  squirming  motion  as  long  as  the  tissues  are  alive. 
It  continued  squirming  for  about  66  hours  in  the  solution  described  above.  A 
Ringer's  solution  of  0.7  per  cent  salts  keeps  it  moving  for  about  12  hours,  although 
at  a  much  reduced  rate. 

Hydrogen  ion  concentration 

The  first  work  on  the  estimation  of  the  pH  of  the  alimentary  tract  of  a  fresh 
water  snail  seems  to  be  that  done  by  A.  H.  Rosenbloom  on  L.  s.  appressa  in  his 
bachelor's  thesis  in  1942  (unpublished)  in  this  laboratory.  He  has  kindly  con- 
sented to  the  incorporation  of  his  results  in  this  paper.  His  method  was  essentially 
the  colorimetric  one  employed  by  Yonge  (1925)  :  fluids  from  the  various  lumina  of 
the  alimentary  tract  of  the  snails  under  variable  feeding  conditions  were  pressed 
out  onto  paraffined  plates  and  thoroughly  mixed  with  indicators  (brom-thymol 
blue,  neutral  red,  and  methyl  red).  The  colors  were  compared  with  those  of  indi- 
cators freshly  prepared  in  buffered  solutions  checked  on  a  Coleman  pH  electrome- 
ter. The  results  are  given  in  Table  I : 


90 


MELBOURNE  ROMAINE  CARRIKER 


TABLE  I 

pH  of  the  contents  of  various  lumina  of  the  alimentary  tract  of  L.  s.  appressa 


Organ 

Average  pH 

Maximum  and 
minimum  pH 

Number  of  tests 

Number  of  snails 

Buccal  cavitv 

Same  as  water  in 

external  medium. 

Proesophagus 

6.9 

7.2-6.3 

10 

10 

Postesophagus  

7.2 

7.6-6.6 

10 

10 

Gizzard  and  crop  . 

6.4 

7.2-6.3 

12 

12 

Pylorus  .... 

6.6 

7.0-5.8 

10 

10 

Intestine  

7.1 

7.8-6.2 

35 

15 

Enzymes 

Preliminary  tests  were  made  for  non-purified  cathepsin,  pepsin,  trypsin,  and 
amylase.  The  tests  for  the  proteinases  were  made  according  to  the  methods  of 
Anson  (1938),  Bradley  (1938)  and  Folin  and  Ciocalteu  (1927)  ;  those  on  amylase, 
by  the  iodine  test  of  Hawk  and  Bergeim  (1937).  Semi-micro  technics  were  ap- 
plied to  large  numbers  of  the  excised  organs. 

Maximum  catheptic  activity  (at  pH  3)  over  a  ten-day  period  was  found  in  the 
liver.  That  occurring  in  the  buccal  mass  and  gizzard  and  other  portions  of  the 
alimentary  system  was  not  significant  as  compared  to  that  in  the  liver.  In  an  effort 
to  determine  to  what  extent  cathepsin  might  be  secreted  from  the  liver,  gut  fluid 
from  which  the  amebocytes  had  been  centrifuged  was  tested.  Under  the  conditions 
of  the  experiment,  at  least,  no  cathepsin  was  found  in  the  gut  juice.  In  some  tests 
tryptic  activity  was  found  in  the  salivary  glands.  A  very  active  amylase,  optimum 
pH  7,  was  present  in  the  salivary  glands  and  in  the  liver. 

The  only  investigation  of  the  hydrolytic  enzymes  of  the  alimentary  system  of  the 
Basommatophora  reported  in  the  literature  is  that  by  Heidermanns  (1924).  He 
described  a  positive  test  for  cellulase  present  in  the  digestive  juice  of  the  stomach 
organs  (crop,  gizzard,  and  pylorus)  of  L.  stagnalis. 

Ciliatlon 

Ciliary  currents  were  studied  by  the  injection  of  fine  carmine  suspensions  in 
Lymnaea  physiological  salt  solution  through  various  portions  of  the  exposed  tract, 
by  application  of  carmine  particles  to  the  epithelium  of  the  opened  tract  and  by 
placing  small  bits  of  gut  wall  in  a  carmine  suspension  on  an  uncovered  microscopic 
slide  under  high  magnification.  In  some  dissections  the  undisturbed  food  particles 
were  seen  passing  through  various  portions  of  the  excised  gut  on  the  natural  ciliary 
currents. 

No  work  has  been  performed  previously  on  the  ciliation  of  the  alimentary  system 
of  the  Basommatophora.  Merton  (1923)  in  his  research  on  the  external  ciliation 
of  pulmonates  included  a  brief  study  of  the  ciliation  of  the  hepatic  ducts  of  Helix. 

The  entire  alimentary  system  of  L.  s.  apprcssa,  with  the  exception  of  the  gizzard 
and  portions  of  the  buccal  cavity,  is  ciliated  (see  later  in  this  paper),  Figures  3,  9, 
and  11. 


ALIMENTARY  SYSTEM  OF  LYMNAEA  91 

Muscular  activity 

The  activity  of  the  alimentary  system  was  observed  under  binoculars  through  the 
transparent  walls  of  normal  living  young  snails  and  in  adult  unanesthetized  snails 
opened  under  Lymnaea  physiological  salt  solution.  The  independent  activity  of 
the  radula  over  the  odontophore  was  clearly  observed  and  conclusively  verified  by 
watching  snails  under  the  binocular  under  the  following  conditions :  snails  deprived 
of  food  for  a  day  were  placed  in  a  finger  bowl  of  well  aerated  water  to  which  had 
been  added  strips  of  lettuce  (1-2  mm.  wide).  A  Petri  dish  was  floated  over  the 
lettuce  and  the  water.  As  the  snails  crawled  upside  down  under  the  glass,  feeding 
on  the  lettuce,  the  action  of  the  radula  and  mouth  parts  was  clearly  visible  under  a 
strong  beam  of  light. 

Sand  in  the  gizzard 

In  order  to  check  the  experiments  of  Heidermanns  (1924)  and  to  add  additional 
information  on  the  role  of  sand  in  the  comminution  of  food  by  the  gizzard  of  L.  s. 
apprcssa,  the  following  experiments  were  devised. 

Sixteen  adult  snails  were  placed  in  each  of  four  aerated  aquaria  containing  a 
one-half  inch  mesh  wire  platform  over  the  bottom.  By  means  of  this  contrivance 
the  feces  were  removed  from  the  vicinity  of  the  snails  soon  after  defecation.  To 
three  of  the  aquaria  the  following  foods  were  added  respectively :  ( 1 )  cooked  "cream 
of  wheat,"  (2)  filter  paper,  and  (3)  lettuce.  (4)  No  food  was  added  to  the  fourth 
tank.  (5)  A  fifth  tank  was  assembled  as  a  control  without  the  wire  platform  and 
with  lettuce  and  sand.  One  snail  from  each  aquarium  was  killed  daily  and  opened 
immediately.  After  ten  days  the  following  was  disclosed  :  eight  of  the  forty-three 
experimental  pulmonates  contained  no  sand  in  the  tract,  thereby  showing  that  it  is 
possible  to  rid  completely  the  tracts  of  a  few  of  the  snails  of  sand ;  however,  there 
was  extensive  variation  in  the  ability  of  the  different  snails  to  retain  sand.  As  the 
quantity  of  sand  in  the  gut  decreased,  the  snails  consumed  less  food,  until  in  the 
absence  of  sand  in  the  tract,  no  food  was  ingested  and  the  guts  became  void  of  food 
material  and  feces.  The  different  diets  indicated  no  significant  difference  in  their 
respective  values  as  sand  eliminators.  Sand  was  found  most  abundantly  in  the  giz- 
zard lumen,  then  in  decreasing  amounts  in  the  crop  and  retrocurrent  passage  of  the 
pylorus  '(anatomical  terminology  has  been  described  elsewhere,  Carriker,  1945). 
After  the  quantity  of  sand  in  the  lumen  of  the  gizzard  reached  a  certain  low  level, 
it  was  retained  with  surprising  tenacity  for  many  days.  The  material  in  the  fecal 
pellets  of  the  control  snails,  particularly  of  the  gizzard  residues,  was  markedly 
brown  and  more  thoroughly  triturated  than  those  of  snails  with  sand-free  diets. 

In  a  second  set  of  experiments  snails  approximately  10  mm.  in  length  were 
placed  in  a  one-quarter  inch  mesh  wire  basket  suspended  in  a  large  laboratory  snail 
stock  tank.  The  feces,  propelled  by  the  sluggish  circulation  of  the  water  in  the 
tank,  passed  out  of  the  basket.  All  lettuce  placed  in  the  basket  was  carefully  washed 
to  remove  sand.  The  experiment  was  continued  for  several  months.  In  spite  of 
precautions,  small  quantities  of  fine  sand  were  always  present  in  the  tracts  of  some 
of  the  animals ;  however,  this  did  not  seem  to  be  enough  for  proper  trituration  as 
many  of  the  snails  died  abnormally  at  an  early  age  and  none  reached  the  normal 
adult  size  of  the  control  snails  in  the  tank  outside  the  experimental  basket.  There 


MELBOURNE  ROMAINE  CARRIKER 

is  unquestionably  a  vital  need  for  the  presence  of  at  least  a  limited  quantity  of  sand 
in  the  gizzard  of  these  snails  for  sufficient  breaking  down  of  the  food. 

These  results  are  in  agreement  with  the  findings  of  Heidermanns  (1924)  and  of 
Colton  (1908).  Heidermanns  accidently  discovered  that  the  only  way  to  entirely 
remove  the  sand  from  a  live  snail  was  to  cause  it  to  hibernate,  in  which  state  it 
emitted  the  total  contents  of  the  tract.  Colton  noted  that  in  the  presence  of  sand 
the  plant  food  was  cut  to  pieces  by  L.  colnniclla,  but  that  in  the  absence  of  sand  it 
went  unmolested. 

Digestive  cell  ingestiou 

By  the  use  of  a  method  patterned  after  that  of  Peczenik  (1925)  the  ingestion  of 
participate  food  by  the  digestive  cells  was  investigated.  White  of  egg  was  strained 
through  cheese  cloth.  Carbon  (lamp  black)  was  ground  into  the  egg  albumen  and 
the  mixture  was  thoroughly  beaten.  This  was  steamed  to  a  stiff  mass  and  fed  to 
snails  starved  for  a  few  days.  After  feeding  commenced,  the  snails  were  opened 
every  other  day.  Indigestible  residues  within  vacuoles  in  the  digestive  cells  as  well 
as  similar  residues  in  the  fecal  pellets  showed  the  presence  of  very  minute  particles 
of  carbon,  particles  not  present  in  the  control  snails.  The  indigestible  residues  in 
the  digestive  cells  appeared  very  similar  to  the  albumen  passing  down  the  intestine 
in  the  gizzard  residues. 

Fecal  rhythms 

Some  information  was  gathered  on  the  rhythms  of  the  liver  and  of  the  gizzard 
by  a  study  of  the  rate  and  extent  of  passage  of  the  various  fecal  strings.  The  fecal 
pellets  of  a  40  mm.  snail  were  observed  daily  for  twenty-four  days.  The  animal 
was  isolated  in  a  two-liter  glass  jar  over  the  bottom  of  which  was  placed  a  paraf- 
fined one-half  inch  mesh  galvanized  metal  screen,  so  that  all  fecal  pellets  fell  to  the 
bottom  of  the  jar  and  could  not  be  reconsumed.  The  mollusc  was  fed  lettuce  on 
which  was  sufficient  sand  for  the  needs  of  the  stomach  region.  Three  egg  masses 
were  oviposited  by  the  snail,  and  it  added  2  mm.  of  shell  during  the  twenty-four 
day  period.  Upon  dissection  at  the  end  of  the  experiment  the  animal  appeared  nor- 
mal in  all  respects.  For  the  first  ten  days  the  pellets  were  collected  and  examined 
microscopically  every  few  hours  during  the  day ;  during  the  latter  part  of  the  ex- 
periment they  were  collected  every  twelve  hours.  Numerous  examinations  were 
made  of  fecal  pellets  from  the  stock  snail  tanks  to  corroborate  the  findings  on  the 
experimental  snail. 

PHYSIOLOGY  OF  THE  ALIMENTARY  TRACT 
Bitccal  mass  and  esophagus 

L.  s.  appressa  is  primarily  an  herbivore.  In  the  laboratory  it  may  complete  its 
life  cycle  on  lettuce  alone  and  in  its  natural  state  feeds  on  the  aquatic  vegetation  of 
its  surroundings.  Specialization  of  the  alimentary  system  (Carriker,  1945)  has 
been  in  keeping  with  a  plant  diet.  However,  animal  food  is  also  consumed  as  has 
been  observed  by  Walter  (  1906)  and  by  seven  other  authors  cited  by  him.  Repeat- 
edly in  this  laboratory  L.  s.  appressa  has  been  observed  to  eat  the  bodies  out  of  the 


ALIMENTARY  SYSTEM  OF  LYMNAEA  93 

shells  of  dead  snails  in  the  aquaria.     Biochemical  tests  disclose  the  presence  of  some 
tryptic  activity  in  the  secretion  of  the  salivary  glands. 

Pieron  (1908)  has  found  in  L.  auricularia  and  L.  stagnalis  that  there  is  a  total 
absence  of  food  discrimination  in  the  buccal  mass  and  that  their  feeding  is  a  reflex 
which  keeps  the  radula  working  most  of  the  time.  The  only  portion  of  the  body 
showing  any  discrimination  is  the  anterior  surface  of  the  foot  which  contains  faintly 
sensitive  chemoreceptors.  In  aquaria  in  this  laboratory  L.  s.  appressa  rasps  much 
of  the  time,  whether  on  lettuce  or  over  the  newly  cleaned  glass  surface  of  its  tank. 
However  it  does  also  pass  through  regular  "resting"  periods  in  which  no  rasping 
occurs.  In  the  rasping  stroke  the  radula  passes  first  to  one  side  and  then  to  the 
other  describing  a  broad  feeding  track. 

Feeding  can  be  followed  clearly  in  normal  immature  "albino"  L.  s.  appressa  (a 
strain  with  very  little  dark  pigment)  feeding  on  a  "cream  of  wheat"  food  mixture 
blackened  with  lamp  black.  This  can  lie  seen  to  pass  as  far  as  the  stomach  region. 
On  the  protractor  stroke  the  radula  cups  to  an  elongated  spoon-shaped  trowel  about 
one-half  the  width  of  the  upper  mandible,  and  working  against  this,  cuts  out  long 
narrow  bits  of  food.  Each  denticle  is  sharp  so  the  concerted  action  of  the  numerous 
denticles  on  the  radula,  sliding  independently  over  the  odontophore,  provides  an 
effective  cutting-rasping  apparatus.  The  food  bits  are  pushed  back  through  the 
dorsal  food  channel  to  the  rear  of  the  buccal  cavity  which  dilates  to  receive  them. 
The  tip  of  the  radula  closely  appresses  to  the  dorsal  wall  of  the  buccal  cavity  in  its 
rearward  passage,  as  attested  by  the  jagged  pattern  of  the  dorsal  chitinous  surface. 
The  buccal  aperture  constricts  strongly  and  rapidly  after  the  receding  radula.  Some 
bits  of  food  are  dropped  and  remain  in  the  dorsal  food  channel  for  the  next  rear- 
ward swing  of  the  food-laden  radula.  Several  food  bits  clump  in  the  rear  of  the 
buccal  cavity  prior  to  being  forced  down  the  esophagus.  The  radula  functions 
principally  in  cutting  pieces  of  food  of  suitably  small  dimensions  for  convenient 
transport  through  the  anterior  portion  of  the  alimentary  tract ;  it  does  not  triturate 
the  food  to  any  considerable  degree. 

Only  the  posterior  third  of  the  buccal  cavity  is  ciliated.  These  cilia  and  those 
in  the  densely  ciliated  esophagus  beat  strongly  posteriorly,  bearing  food  bits  from 
the  rear  of  the  buccal  cavity  to  the  crop. 

In  connection  with  the  functioning  of  the  buccal  mass,  refer  to  a  previous  paper 
(Carriker,  1945)  for  the  names,  origin,  and  insertion  and  relations  of  the  muscles 
and  parts  of  the  mass.  The  muscular  activity  of  the  buccal  mass  is  divisible  into 
four  major  synchronous  movements:  (1)  opening  and  closing  of  the  oral  aperture 
and  consequent  spreading  and  approximation  of  the  mandibles  and  lips,  as  well  as 
dilation  and  contraction  of  the  circular  muscles  about  the  anterior  portion  of  the 
buccal  cavity,  (2)  backward-forward  and  simultaneous  elevator-depressor  move- 
ments of  the  odontophore,  with  some  slight  turning  of  the  odontophore  on  its  longi- 
tudinal axis  and  some  movement  to  the  right  and  to  the  left,  (3)  movement  of  the 
radula  and  radular  sac  over  the  cartilage,  and  (4)  backward -forward  and  simul- 
taneous elevator-depressor  movements  of  the  entire  buccal  mass.  Consequently 
there  exist  in  the  buccal  mass  three  intrinsic  focal  points  about  which  the  ma- 
jority of  the  muscles  radiate:  (1)  the  oral  aperture,  (2)  the  odontophoral  cartilage, 
and  (3)  the  radula  and  the  radular  sac. 

The  activity  of  the  odontophore  with  respect  to  the  remainder  of  the  buccal  mass 
may  be  arbitrarily  divided  into  four  phases,  and  described  as  follows:  (1)  the  quies- 


94  MELBOURNE  ROMAINE  CARRIKER 

cent  stage  in  which  the  odontophore  lies  at  rest  in  the  rear  of  the  huccal  cavity  with 
its  longitudinal  axis  in  a  dorsoventral  position.  (2)  The  protracting  stroke  in 
which  the  proximal  end  of  the  odontophore  swings  in  an  arc  of  about  130°  from  its 
basal  position  to  a  point  where  it  lies  above  the  plane  of  the  distal  end,  which  then 
is  in  a  position  to  pass  partly  out  of  the  buccal  cavity,  bringing  the  radula  against 
the  substratum.  At  the  beginning  of  this  stroke  the  odontophore  assumes  a  hori- 
zontal position  as  a  result  of  the  lowering  of  the  distal  end  by  contraction  of  the 
dorsal  odontophoral  flexor  muscle,  and  a  simultaneous  raising  of  the  proximal  end 
by  strong  contraction  of  the  posterior  jugalis  muscle.  The  oral  aperture  and  the 
anterior  portions  of  the  buccal  mass  dilate  to  permit  partial  protrusion  of  the  odon- 
tophore through  the  mouth ;  the  labial  retractors,  suboral  dilators  and  dorsomandibu- 
lar  dilators  spread  the  mouth.  The  extrinsic  postventral  levators  and  posterior 
jugalis  further  raise  the  rear  of  the  buccal  mass  so  that  the  distal  tip  of  the  odon- 
tophore is  directed  towards  the  oral  aperture,  to  which  it  seems  to  be  guided  prin- 
cipally by  the  action  of  the  dorsal  odontophoral  flexor  muscles.  The  inframedian 
radular  tensors  draw  the  radula  over  the  distal  end  of  the  cartilage  to  the  point 
where  most  of  the  radula  outside  the  radular  sac  lies  on  the  under  side  of  the  hori- 
zontally inclined  cartilage,  and  the  collostylar  hood  lies  just  behind  the  distal  crest 
of  the  cartilage.  The  combined  action  of  the  radular  sac  and  cartilage  tensors  holds 
the  radula  tautly  drawn  over  the  cartilage  in  readiness  for  the  rasping  stroke.  Con- 
traction of  the  intracartilage  tensors  adds  considerably  to  the  rigidity  of  the  cushion 
under  the  radula.  As  Woodward  (1895)  points  out  for  Natalina  caffra,  the  fibers 
of  the  cartilage  act  in  much  the  same  way  as  the  intrinsic  muscles  of  the  human 
tongue  and  in  contraction  cause  an  elongation  and  consequent  slight  protrusion  of 
the  radula.  The  pressure  of  the  blood  in  the  odontophoral  sinus  probably  provides 
further  turgidity.  Contraction  of  the  extrinsic  as  well  as  of  the  intrinsic  protractor 
muscles  brings  the  odontophore  to  the  substratum.  (3)  In  the  rasping  stroke  the 
distal  tip  of  the  odontophore  is  drawn  over  the  substrate  in  a  licking  motion.  The 
radula,  independent  of  the  principal  motion  of  the  cartilage  under  it,  is  itself  simul- 
taneously slid  quickly  backward  most  of  its  length  over  the  cartilage  by  the  action 
of  the  heavy  supralateral  and  supramedian  radular  tensor  muscles.  The  odonto- 
phore is  aided  by  contraction  of  the  extrinsic  preventral  levator  muscles  which  pull 
the  anteroventral  floor  of  the  buccal  cavity  forward  and  upward.  As  the  mouth 
opens  during  the  previous  stroke,  the  cutting  distal  margin  of  the  dorsal  mandible 
is  turned  partly  forward  by  contraction  of  the  dorsomandibular  dilators  and 
possibly  the  posterior  jugals.  Thus  as  the  radula  rasps  forward  it  makes 
connection  with  and  scrapes  past  the  inner  side  of  the  dorsal  mandible,  much 
as  two  jaws  would  come  together,  so  that  the  snail  when  feeding  on  thin  por- 
tions of  lettuce  actually  "bites"  off  pieces  with  each  rasping  stroke.  It  is  only  when 
feeding  on  thicker  foods  that  true  "rasping"  comes  into  play.  The  dorsal  mandible 
is  governed  by  the  dorsomandibular  approximator  muscle.  The  lateral  mandibles 
afford  mechanical  protection  to  the  sides  of  the  mouth,  and  close  in  medially  after 
the  radula  and  under  and  behind  the  dorsal  mandible.  (4)  The  retractor  stroke 
returns  the  odontophore  to  the  resting  condition,  and  completes  the  cycle,  by  action 
of  the  extrinsic  retractor  muscles  and  the  supralateral  and  supramedian  radular  ten- 
sors and  relaxation  of  the  protractors.  The  oral  aperture  is  closed  after  the  reced- 
ing odontophore  by  action  of  the  labial  sphincter  and  the  mandibular  approximator 
muscles ;  the  buccal  cavity,  by  a  contraction  of  the  buccal  sphincter  and  related  mus- 


ALIMENTARY  SYSTEM  OF  LYMNAEA  95 

cles  of  the  walls.  In  assuming  the  resting-  position,  the  raclular  sac  is  depressed 
behind  the  cartilage  and  the  radula  rests  principally  behind  the  vertically  arranged 
cartilage  so  that  the  ventral  tip  of  the  sac  projects  slightly  below  the  level  of  the 
buccal  mass.  As  observed  by  Amaudrut  (1898)  for  Lyiniiaca,  the  ventral  wall  of 
the  buccal  cavity  between  the  esophageal  ledge  and  the  collostylar  hood  is  also  de- 
pressed, forming  a  slight  dilation  in  front  of  the  esophageal  opening.  As  both  the 
oral  aperture  and  the  proesophagus  are  closed  during  the  retractive  stroke  of  the 
radula,  it  is  likely  that  this  dilation  is  instrumental  in  creating  a  slight  vacuum  in 
front  of  the  esophageal  opening  which  aids  in  disengaging  food  particles  from  the 
radula.  The  dilation  is  caused  principally  by  depression  of  the  radular  sac  and 
possibly  by  contraction  of  the  superior  suspensor  muscle  of  the  radular  sac  and  the 
hood  tensor  muscles. 

The  proesophagus  is  limited  in  its  muscular  activity  to  slight  peristaltic  waves 
proceeding  towards  the  postesophagus ;  while  the  latter  undergoes  pronounced  peri- 
staltic activity  in  either  a  forward  or  a  backward  direction,  dilating  broadly  and 
contracting  its  entire  length.  In  dilation  it  may  become  so  large  as  to  fill  much  of 
the  cephalic  hemocoel  of  the  expanded  mollusc.  In  expansion  it  is  filled  with  a 
reddish  fluid  from  the  stomach  region  and  food  particles. 

In  the  buccal  cavity  the  food  receives  generous  quantities  of  fluid  from  the  buccal 
gland  cells,  a  fluid  which  is  probably  mostly  mucoid  in  nature,  judging  from  the 
positive  mucicarmine  stain  and  from  negative  tests  for  amylase  and  trypsin.  This 
does  not  however  preclude  the  possibility  of  the  presence  of  other  enzymes  which 
were  not  tested  for.  As  food  passes  under  the  openings  of  the  salivary  ducts  it 
receives  mucus,  amylase,  trypsin,  and  possibly  other  enzymes  from  the  salivary 
glands. 

The  proesophagus  adds  more  secretion  from  buccal  glands  and  mucous  cells. 
The  postesophagus  functions  as  a  temporary  reservoir  for  the  retention  of  food 
when  the  crop  is  full.  Being  capable  of  considerable  distension,  it  may  retain 
larger  quantities  of  food  than  the  crop.  Digestion  begins  in  the  postesophagus 
because  of  enzymatic  secretions  received  from  the  salivary  glands. 

Stomach  region 

Comminution  of  food  particles  is  completed  in  the  crop,  gizzard,  and  anterior 
portions  of  the  retrocurrent  passage  of  the  pylorus.  These  three  organs  act  as  a 
unit  comparable  to  a  grist-mill.  The  kneading  motion  of  the  anterior  and  posterior 
gizzard  constrictor  muscles  and  the  gizzard  lobes  over  the  sand  in  the  lumen  pro- 
vides the  grinding  action.  Food  bits  forced  between  the  sand  are  soon  crushed  to 
minute  particles  upon  which  the  digestive  enzymes  may  act  more  efficiently.  Two 
synchronized  movements  are  present  in  the  gizzard.  In  the  first  the  anterior  and 
posterior  gizzard  constrictor  muscles  alternate  smoothly  in  mild  contraction,  thus 
mixing  and  forcing  the  contents  of  the  gizzard  slowly  back  and  forth  ;  in  the  second, 
not  as  frequent  as  the  first,  the  bulk  of  the  gizzard  compressor  muscles  contract 
suddenly  and  strongly,  bringing  pressure  to  bear  on  the  contents  of  the  gizzard. 
The  presence  of  gritty  material  in  the  gizzard  of  the  Lymnaeidae  has  been  noted  by 
many:  Cuvier  (1817),  Wetherby  (1879),  Whitfield  (1882),  Moquin-Tandon 
(1885),  Colton  (1908),  F.  C.  Baker  (1900,  1911),  and  Heidermanns  (1924). 

In  the  crop,  all  ciliary  currents  lead  to  the  anterior  margin  of  the  right  gizzard 


96  MELBOURNE  ROMAINE  CARRIKER 

pad,  those  on  the  left  side  beating  ventrad  and  over  to  the  right  (Fig.  3).  Thus 
fine  food  material  accumulates  on  the  right  side  of  the  crop  at  the  anterior  edge  of 
the  right  gizzard  lobe.  The  crop  receives  food  from  the  postesophagus  and  forces 
it  into  the  gizzard  lumen.  When  ample  sand  is  accessible  to  the  animal,  the  crop 
and  anterior  portions  of  the  retrocurrent  pyloric  passage  are  both  filled  with  it. 
The  walls  of  these  organs  act  as  mechanical  obstructions  to  the  open  ends  of  the 
gizzard  lumen  and  concentrate  the  pressure  of  the  gizzard  musculature  upon  the 
contents.  They  also  cooperate  in  the  muscular  activity  of  the  gizzard  in  a  unified 
kneading  and  a  slow  rotation  of  the  gritty  contents.  The  retrocurrent  passage  re- 
turns to  the  crop  those  particles  which  have  been  dislodged  from  the  gizzard  con- 
tents by  muscular  movements  of  the  stomach  region.  In  this  fashion  the  contents 
of  the  gizzard  undergo  thorough  comminution  and  partial  digestion  before  the  resi- 
dues are  shunted  down  the  procurrent  passage  to  the  prointestine. 

The  epithelium  of  the  stomach  region  bears  a  complicated  system  of  ciliary  cur- 
rents (Figs.  1,  2,  3,  9).  Cilia  in  the  procurrent  passage  direct  fine  particles  from 
the  right  ventral  side  of  the  gizzard  cavity  to  the  prointestine.  Those  in  the  retro- 
current  passage  are  directed  anteriad  towards  the  left  side  of  the  gizzard  cavity. 
The  dorsal  passage  bears  what  in  fixed  sections  appears  to  be  nothing  more  than  a 
brush  border.  Even  in  carmine  suspensions  under  high  magnification  no  distinct 
current  could  be  detected  in  it.  The  cilia  on  the  ventral  fold  are  divided  into  two 
distinct  functional  areas :  those  on  the  right  half  of  the  fold  beat  obliquely  posteriad 
and  laterally  in  the  direction  of  the  currents  in  the  procurrent  passage ;  those  on  the 
left  half,  obliquely  anterolaterad  in  the  direction  of  the  gizzard  and  the  currents  in 
the  retrocurrent  passage.  The  currents  on  the  minor  fold  whip  obliquely  antero- 
laterad;  those  on  the  medial  half  of  the  major  fold  pass  obliquely  anterolaterad; 
while  those  on  the  lateral  half  of  the  major  fold  and  those  on  the  medial  half  of  the 
fold  adjacent  the  hepatic  vestibule  reach  posterolaterad.  The  ciliary  currents  in  the 
retrocurrent  passage  are  noticeably  faster  than  those  in  the  procurrent  passage. 
Currents  on  the  atrial  corrugations  run  into  the  incurrent  tubule  of  the  cecum. 
Thus  the  pylorus  in  cross  section  (Fig.  2)  is  composed  of  three  channels,  each  with 
distinct  ciliary  currents  and  of  three  folds  which  almost  meet  centrally  and  whose 

EXPLANATION  OF  PLATE  I 
(All  figures  concern  L.  s.  appressa) 

FIGURE  1.  Stereogram  of  the  pylorus,  hepatic  vestibule,  atrium,  cecum,  anterior  portion  of 
prointestine,  and  liver  lobes.  The  vascularization  is  stressed.  (Small  arrows  indicate  the  flow 
of  blood  in  the  arteries ;  large  arrows,  the  direction  of  movement  of  the  contents  of  this  part  of 
the  tract.)  X  6. 

FIGURE  2.  Stereogram  of  cross-section  of  the  pylorus,  taken  midway  between  the  gizzard 
and  the  hepatic  vestibule.  The  stippled  surfaces  are  heavily  ciliated.  (The  small  arrows  in- 
dicate the  direction  of  the  ciliary  beat;  the  large  arrows,  the  direction  of  passage  of  material  in 
the  pylorus.  The  arrows  with  broken  stems  designate  the  direction  of  ciliary  beat  on  surfaces 
behind  the  folds.)  X  25. 

ABBREVIATIONS 

AT,  atrial  artery;  CC,  cecal  artery;  d.p.p.,  dorsal  pyloric  passage;  GD,  dorsogastric  artery; 
HN,  minor  hepatic  artery;  HP,  prohepatic  artery;  IP,  prointestinal  artery;  »;./>./.,  major  pyloric 
fold;  n.p.j.,  minor  pyloric  fold;  pc.p.,  procurrent  pyloric  passage;  PM,  major  pyloric  artery; 
PN,  minor  pyloric  artery;  PP,  propyloric  artery;  PV ,  ventropyloric  artery;  re. p.,  retrocurrent 
pyloric  passage ;  v.p.j.,  ventropyloric  fold ;  VT,  vestibular  vascular  arborescence. 


ALIMENTARY  SYSTEM  OF  LYMNAEA 


97 


PLATE  I 


PI/ 


or  /ode  of  //t/er 


typhlosote 
-pm/f)fest/ne 


ftepot/c  vestibule 
Aepafic  ducts 


/ode 


c/orsa/  passage 
minor- 


major  fo/d 


uentra/  fo/d 


ct/iatecf  str/a 


ci/ia 


98  MELBOURNE  ROMAINE  CARRIKER 

ciliary  currents  pass  out  of  the  dorsal  into  both  the  procurrent  and  the  retrocurrent 
passages.  The  major  fold  in  addition  bears  a  thin  longitudinal  strip  of  long  cilia 
at  its  boundary  with  the  dorsal  passage.  The  major  and  minor  folds  in  the  living 
animal  nearly  always  touch  along  their  crests,  so  that  the  fluid  contents  of  the  dorsal 
passage  may  pass  into  the  two  ventral  passages  but  coarse  material  from  the  ventral 
passage  may  not  pass  into  the  dorsal  passage.  The  juxtaposition  of  the  two  folds 
is  continued  under  the  hepatic  vestibule,  where  the  folds  provide  a  ventral  floor  to 
this  chamber.  At  this  point  the  cilia  on  the  folds  direct  a  powerful  current  out  and 
away  from  the  vestibule,  again  preventing  the  entrance  of  coarse  material  into  the 
hepatic  ducts  and  liver. 

As  discovered  for  Helix  by  Merton  (1923),  the  corrugations  of  the  larger  proxi- 
mal portions  of  the  hepatic  ducts  of  L.  s.  apprcssa  bear  two  ciliary  countercurrents 
(Fig.  11)  :  the  cilia  on  the  crests  of  the  corrugations  are  long  and  beat  into  the 
liver,  those  in  the  grooves  are  shorter  and  pass  particles  in  the  direction  of  the  he- 
patic vestibule  and  into  the  incurrent  tubule  of  the  cecum.  The  particles  in  the 
grooves  are  quickly  entrapped  in  mucus  secreted  there  and  formed  into  delicate 
strings.  The  currents  directed  into  the  liver  could  be  traced  with  certainty  only  in 
the  large  hepatic  ducts,  although  cilia  were  observed  as  far  as  the  peripheral  folli- 
cles in  isolated  bits  of  living  liver  tissue.  Yonge  (1936)  states  that  in  Mollusca 
where  food  passes  into  the  liver  and  waste  material  out,  the  ducts  are  ciliated  in 
such  a  way  that  an  inward  passage  exists  on  one  side  and  an  outward  one,  on  the 
other.  Such  counter  currents  could  not  be  determined  in  L.  s.  appressa. 

In  the  cecum  the  cilia  on  the  cecal  folds  beat  off  the  folds  into  the  tubules  (Fig. 
9)  ;  those  in  the  incurrent  tubule  pass  carmine  particles  directly  to  the  distal  end 
and  around  this  into  the  excurrent  tubule.  Here  the  cilia  beat  circumferentially, 
rotating  the  contents  of  the  tubule  along  the  longitudinal  axis.  In  the  continuation 
of  the  excurrent  tubule  across  the  pyloric  wall  the  ciliary  stream  is  directed  towards 
the  prointestine. 

The  crop,  pylorus,  liver,  and  hepatic  ducts  are  as  active  as  the  postesophagus. 
Besides  the  usual  peristaltic  movements,  they  undergo  a  series  of  violent  alternating 
pulsations,  here  designated  pulsatory  movements,  in  which  the  crop,  pylorus,  hepatic 
ducts,  and  liver  pulsate  successively,  forcing  the  fluid  contents  slowly  back  and 
forth  in  swirling  currents.  In  the  pylorus  the  pulsations  commence  at  a  point  be- 
tween the  typhlosole  and  the  atrium  and  pass  towards  the  gizzard.  They  are  of 
two  types :  ( 1 )  very  strong  pulsations  in  which  the  entire  structure  contracts  and 
(2)  minor  pulsations  running  over  restricted  portions  of  the  pylorus.  In  the  liver 
the  pulsations  pass  as  far  as  the  terminal  follicles.  This  marked  movement  is  most 
vividly  observed  in  bits  of  living  liver  tissue  under  high  magnification.  Individual 
cells  are  seen  to  move  against  each  other  by  contraction  of  the  thin  muscular  con- 
nective sheet  enveloping  each  follicle.  The  pylorus  undergoes  the  most  pronounced 
movements  and  appears  to  lead  the  other  organs  in  activity.  The  incurrent  tubule 
of  the  cecum  is  relatively  thin-walled  and  does  not  appear  to  undergo  peristaltic 
activity.  The  excurrent  tubule  is  thicker-walled  and  has  definite  peristaltic  move- 
ment in  the  direction  of  the  outlet. 

It  follows  then  that  one  of  the  important  functions  of  the  pylorus  is  that  of  a 
filter  chamber,  separating  the  digested  and  the  fine,  partly  digested  food  particles 
from  the  gross  material  and  sand.  This  is  the  conclusion  which  Heidermanns 
(1924)  also  reached  when  he  stated  that  most  of  the  time  sand  and  gross  material 


ALIMENTARY  SYSTEM  OF  LYMNAEA  99 

are  kept  from  passing  into  the  liver  by  the  pyloric  folds.  The  major  and  minor 
folds  remain  in  close  approximation  along  their  crests,  leaving  a  narrow  slit  be- 
tween the  dorsal  and  the  ventral  passages  which  may  be  called  the  pyloric  filter. 
The  cilia  on  the  folds  are  well  developed  and  beat  away  from  the  dorsal  passage. 
During  the  pulsatory  movements  of  the  stomach  region  only  the  finest  particles  and 
fluid  material  are  permitted  ingress  to  the  liver  through  this  filter.  The  pulsatory 
currents,  as  these  in  the  gut  lumen  may  be  named,  are  relatively  strong  and  in  their 
streaming  between  the  sand  particles  and  foot  bits  in  the  gizzard  cavity  dislodge 
large  particles  of  food.  Those  which  are  carried  into  the  pro-  and  retrocurrent 
passages  and  which  are  too  large  to  pass  through  the  pyloric  filter,  become  entangled 
in  the  ciliary  currents  of  the  folds  and  are  carried  quickly  back  to  the  left  side  of 
the  gizzard  lumen  by  way  of  the  retrocurrent  passage.  The  particles  carried  into 
the  crop  on  the  forward  streaming  of  the  contents  are  soon  entangled  in  the  ciliary 
currents  of  the  crop  and  conveyed  to  the  right  side  of  the  gizzard  lumen.  Here, 
then,  is  a  delicate  adjustment  by  which  the  larger  particles  dislodged  from  the  giz- 
zard contents  are  equally  redistributed  for  further  grinding  within  the  gizzard. 

At  certain  intervals  during  the  day  the  pulsatory  movements  appear  to  cease  and 
a  portion  of  the  residual  material  and  sand  in  the  gizzard  pass  out  through  the  pro- 
current  passage  to  the  prointestine.  The  propulsion  of  gizzard  strings  (Fig.  10), 
as  these  residues  may  be  named,  through  the  procurrent  passage  is  very  slow  and 
mostly  by  cilia  supplemented  by  slight  peristalsis.  Cilia  were  found  active  through- 
out all  portions  of  the  alimentary  tract  whenever  opened ;  no  cessation  of  ciliary 
activity  (as  occurs  in  some  lamellibranchs  during  increase  of  CO2  concentration) 
or  reversal  of  beating  was  observed.  During  emission  of  the  gizzard  string,  the 
large  portion  of  the  ventral  pyloric  fold  which  partly  occludes  the  gizzard  lumen 
flattens  to  enlarge  the  opening.  As  suggested  by  Howells  (1942)  for  Aplysia,  it 
appears  that  the  shape  and  position  of  the  pyloric  folds  in  L.  s.  appressa  are  partly 
maintained  by  blood  pressure  in  the  sinuses. 

To  what  extent  digestion  does  occur  in  the  postesophagus,  crop,  gizzard,  and 
pylorus  is  questionable.  As  amylase  from  the  liver  and  from  the  salivary  glands, 
trypsin  from  the  salivary  glands  and  cellulase,  at  least,  are  present  in  the  gut  con- 
tents, some  food  may  be  partly  hydrolized.  Part  of  the  remaining  available  food 
is  reduced  mechanically  to  particles  small  enough  for  ingestion  by  the  digestive 
cells  of  the  liver.  The  amebocytes  of  the  gut  also  appear  to  aid  in  digestion.  Ac- 
cording to  Heidermanns  (1924)  fats  and  carbohydrates  are  absorbed  in  the  pylorus 
by  the  ciliated  cells. 

The  pyloric  filter  permits  only  minute  food  particles  to  pass  into  the  liver.  Most 
of  the  radular  teeth  which  are  discarded  continuously  from  the  radula  throughout  the 
life  of  the  snail  (Carriker,  1943a)  and  grains  of  sand  as  large  as  90^.,  by  reason  of 
the  fact  that  they  are  considerably  heavier  than  the  lighter  food  particles  of  the 
same  dimensions,  are  carried  past  the  cilia  by  the  force  of  the  pulsatory  currents. 
The  larger  free  food  particles,  especially  of  lettuce,  are  very  light  and  are  readily 
barred  by  the  cilia  of  the  filter.  In  the  proximal  portions  of  the  hepatic  ducts,  be- 
cause of  counter  ciliary  currents,  only  the  finer  particles  that  fall  into  the  grooves  of 
the  corrugations  can  be  carried  towards  the  cecum ;  thus  teeth  and  larger  sand 
grains  are  held  at  this  point  by  the  ciliary  currents  of  the  crests  of  the  corrugations 
until  sufficient  fecal  material  passes  out  of  the  liver  to  carry  them  with  it. 

Ciliation  of  the  crests  of  the  corrugations  may  play  a  small  role  in  the  conduction 


100  MELBOURNE  ROMAINE  CARRIKER 

of  food  material  into  the  follicles  of  the  liver,  but  probably  the  principal  conveyers 
are  the  pulsatory  currents.  Food  in  solution  and  in  suspension  is  thus  brought  to 
all  the  internal  surfaces  of  the  liver  follicles.  Larger  particles  finding  entrance 
through  the  filter  and  too  large  to  remain  readily  in  suspension  appear  to  fall  to 
the  ductal  epithelium.  The  smaller  of  these  are  soon  propelled  into  the  grooves  of 
the  corrugations.  Liberal  quantities  of  mucus  are  secreted  there,  trapping  the  par- 
ticles in  mucous  strings  which  pass  towards  the  cecum,  coalescing  as  they  advance 
into  the  larger  grooves  (Fig.  11).  From  the  incurrent  cecal  tubule  the  mucoid 
strings  pass  around  the  distal  end  of  the  cecum  into  the  excurrent  tubule.  There 
the  material  receives  a  further  transparent  layer  of  mucoid  and  cementing  material 
and  is  rotated  into  a  smooth  cylindrical  continuous  string,  here  designated  the  cccal 
string  (Fig.  10).  This,  partly  by  ciliary  action  and  partly  by  peristalsis,  then  passes 
on  into  the  prointestine  across  the  atrium.  In  snails  feeding  on  green  lettuce  the 
strings  are  a  vivid  green  because  of  a  heavy  accumulation  of  bits  of  chlorophyll 
bearing  bodies  which  become  entangled  in  mucous  strings  in  the  hepatic  ducts. 
In  gastropods  fed  on  a  food  containing  carbon,  the  cecal  strings  are  a  dense  black. 
In  animals  on  a  starvation  diet,  the  cecum  continues  to  pass  out  cecal  strings,  just 
as  in  the  feeding  animal,  but  the  strings  are  a  mucoid,  transparent,  milky-white  color 
and  much  reduced  in  diameter.  It  thus  would  seem  that  the  function  of  the  grooves 
in  the  hepatic  corrugations  and  of  the  cecum  is  to  collect  and  eliminate  those  fine 
particles  which  pass  through  the  pyloric  filter  but  which  are  too  large  to  be  engulfed 
by  the  digestive  cells  and  which  are  thus  mechanically  eliminated  by  a  "supple- 
mentary filter."  Cecal  strings  pass  out  continuously,  apparently  at  the  same  uni- 
form rate  and  without  apparent  interruption.  They  provide  a  kind  of  "time  clock" 
by  which  the  rate  of  passage  of  the  gizzard  strings  and  the  residues  from  the  liver 
can  be  compared  (Fig.  10). 

EXPLANATION  01-  PLATE  11 
(All  figures  concern  L.  s.  appressa ) 

FIGURE  3.  Ciliation  currents  of  the  postesophagus,  crop,  gizzard,  pylorus,  hepatic  vestibule, 
atrium,  and  anterior  portion  of  prointestine.  The  tract  has  been  slit  ventrally  and  spread.  X  6. 

FIGURE  4.  Irregular  blue-green  excretion  bodies  (in  vacuoles)  taken  from  the  liver  string. 
X500. 

FIGURE  5.  Smooth  blue-green,  or  brown,  excretion  bodies  (in  vacuoles)  taken  from  the 
liver  string.  X  500. 

FIGURE  6.     "Signet"  excretion  body   (in  vacuole)  appearing  in  the  liver  strings.     X  500. 

FIGURE  7.  Clear  nodules  found  in  the  liver  strings  which  when  pressed  out  under  the  cover 
slip  display  their  crystalline  nature.  They  dissolve  in  dilute  HC1  and  seem  very  similar  to  the 
calciferous  concretions  of  the  vesicular  cells  of  the  connective  tissue.  X  500. 

FIGURE  8.  Indigestible  residues  from  digestive  cell  (in  vacuole),  found  abundantly  in  liver 
strings.  X  500. 

FIGURE  9.  Ciliation  currents  of  the  cecum,  which  has  been  opened  along  the  incurrent  cecal 
tubule  and  spread  flat.  X  6. 

FIGURE  10.  Typical  fecal  pellet,  showing  the  gizzard,  liver  and  cecal  strings,  and  the  im- 
pression of  the  typhlosole  in  the  pellet.  X  6. 

FIGURE  11.  Portion  of  the  corrugated  epithelium  of  the  hepatic  duct,  taken  at  the  opening  of 
the  duct  into  the  hepatic  vestibule.  (Large  arrows  indicate  the  direction  of  the  ciliary  currents 
in  the  grooves ;  the  small  arrows,  that  on  the  crests  of  the  corrugations. )  X  50. 

ABBREVIATIONS 

c.s.,  cecal  string;  cxcur.  tubule,  excurrent  tubule;  g.s.,  gizzard  string;  inc.  tubule,  incurrent 
tubule;  l.s.,  liver  string;  s.,  sand;  t.i.,  impression  of  typhlosole  in  fecal  pellet. 


PLATE  II 


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wnm^M'm 


procarren  t 
pa-ssoge 


^wm/'-m^r^---- 
:•  0:py'  -mr< -*-f  -•/-• 
Hl^f^Hii  -x//  ••. /.» 


passage 
'or  fo/d 
/ni/jor  fo/d 


.  fa  date 
ceco/  fo/o? 
ezcur.     fa  bate 


w 


1  «^.  ;V 

H^-  &-':.'• '.    '  •  .      .^ 

•'-•>   sJ^'r.iX'..    •:•  • . 


102  MELBOURNE  ROMAINE  CARRIKER 

The  excretory  bodies  and  indigestible  residues  in  the  liver  are  voided  periodi- 
cally. These  are  passed  simultaneously  in  minute  mucous  strings  from  all  parts  of 
the  liver  towards  the  central  hepatic  ducts,  there  converging  into  larger  strings 
which  pass  in  the  direction  of  the  hepatic  vestibule.  At  the  proximal  end  of  the 
hepatic  ducts  this  material  fills  most  of  the  main  duct.  The  combined  currents  in 
the  grooves  of  the  corrugations  appear  to  exert  a  stronger  force  than  those  on  the 
crests,  so  forcing  the  waste  material  directly  into  the  hepatic  vestibule  (Fig.  11). 
There  it  is  caught  by  the  outward  flowing  ciliary  currents  on  the  major  and  minor 
pyloric  folds  and  passed  rapidly  into  the  prointestine.  The  excretory  bodies  and 
indigestible  residues  passing  from  both  lobes  of  the  liver  are  compressed  in  the 
hepatic  vestibule  into  one  bulky  string  which  is  distinct  from  the  cecal  and  from 
the  gizzard  string  and  may  be  called  the  liver  string  (Fig.  10).  It  is  drawn  out 
of  the  liver  at  the  same  rate  as  the  cecal  string  passes  out  of  the  ceum.  Both  strings 
are  usually  found  parallel  to  each  other  and  uncoiled  in  the  fecal  pellets.  The  giz- 
zard string,  on  the  other  hand,  passes  out  much  more  slowly  so  that  the  cecal  string 
occurs  loosely  and  abundantly  coiled  therein  (Fig.  10).  A  lapse  of  time  seems  to 
occur  between  the  exit  of  the  gizzard  string  and  that  of  the  liver  string,  as  indicated 
by  a  conspicuous  coiling  of  the  cecal  string  between  the  last  portion  of  the  gizzard 
string  and  the  forward  end  of  the  liver  string.  The  gizzard  string  follows  the  liver 
string  immediately,  as  indicated  by  no  noticeable  coiling  of  the  cecal  string  between 
the  two.  There  is  also  some  evidence  that,  as  the  liver  string  is  drawn  from  the 
liver,  the  pulsations  of  the  stomach  region  cease.  In  animals  opened  for  physiologi- 
cal observation  of  the  tract,  the  stomach  region  was  never  pulsating  when  the  liver 
strings  were  passing  out  of  the  liver.  This  is  desirable  to  prevent  the  dismember- 
ment of  the  strings  and  their  mixing  with  food  material  brought  into  the  liver  by 
the  pulsatory  currents.  The  merger  of  the  strings  in  the  prointestine  produces  the 
fecal  pellets. 

The  pylorus  is  composed  of  a  complicated  system  of  folds  and  passages,  it  is  in- 
nervated by  a  pair  of  complex  nerve  plexuses  and  a  nerve  net,  and  all  of  the  parts 
are  exceptionally  well  vascularized.  Functionally  there  is  present  in  this  portion 
of  the  tract  an  intricate  system  of  counter  ciliary  currents  and  synchronized  mus- 
cular movements,  as  well  as  partial  vascular  control  of  the  folds.  The  pylorus  is 
thus  well  equipped  to  convey  digestive  fluids  from  the  liver  to  the  gizzard  and  crop, 
to  bear  digested  and  semi-digested  particles  into  the  liver  from  the  gizzard,  to  ex- 
clude large  sand  and  other  large  particulate  matter  from  the  liver  and  transfer  such 
residues  to  the  prointestine,  to  receive  waste  material  from  the  liver  and  transport 
it  to  the  intestine,  to  act  in  conjunction  with  the  cecum,  liver,  and  hepatic  ducts  in 
shunting  a  continuous  string  of  residual  particles  from  the  walls  of  these  organs 
into  the  prointestine,  to  secrete  fluids  (of  unknown  nature)  and  finally  to  absorb 
fats  and  carbohydrates. 

Liver 

The  liver  is  probably  the  most  important  organ  of  digestion  in  the  alimentary 
system  of  the  gastropods.  Peczenik  (1925)  shows,  as  has  been  indicated  in  this 
work  also  in  feeding  experiments,  that  such  proteins  as  egg  albumen  are  engulfed 
and  digested  intracellularly  in  the  digestive  cells,  and  the  indigestible  residues  are 
cast  out  in  vacuoles.  Krijgsman  (1928)  believes  that  digestive  cells  in  Lymnaea 


ALIMENTARY  SYSTEM  OF  LYMNAEA  103 

are  also  secretory  as  well  as  absorptive,  as  he  has  often  observed  numerous  typical 
secretion  granules  in  the  liver  cells  of  starved  snails.  Biochemical  tests  indicate 
that  the  greatest  catheptic  activity  of  the  snail  body  is  localized  in  the  liver,  yet  none 
of  this  activity  has  been  found  in  the  fluid  of  the  gut.  This  is  in  keeping  with  cath- 
eptic systems  in  other  animals  in  which  the  enzyme  has  been  shown  to  exist  entirely 
as  an  intracellular  protease.  Hurst  (1927)  writes  that  in  PJiysa  fat  and  glycogen 
are  stored  in  the  digestive  cells.  Fat  was  also  found  in  the  lime  cells  of  Helix  by 
Griinbauin  (1913).  The  problem  of  what  size  of  food  particle  is  engulfed  through 
the  distal  membrane  of  the  digestive  cells  is  still  an  open  question.  It  is  likely,  as 
indicated  by  the  work  of  Krijgsman  (1925.  1928)  on  Hcli.r.  that  the  lime  cells 
function  in  storing  and  in  periodically  secreting  a  buffering  agent  which  adjusts 
the  pH  of  the  gut  juice ;  this  point  has  not  been  investigated  in  L.  s.  appressa.  The 
mucous  cells  of  the  liver  provide  the  mucus  utilized  in  the  binding  of  the  indi- 
gestible residues  and  the  excretory  bodies  into  the  liver  strings. 

Amebocytes  were  found  in  varying  numbers  in  the  contents  of  the  lumina  of  the 
liver,  postesophagus,  gizzard,  and  pylorus.  These  were  similar  to  those  seen  in 
the  blood.  In  some  instances  those  in  the  gut  contained  fecal  vacuoles  so  large  as 
to  force  the  cell  into  a  peripheral  lobate  ring. 

Rhythmic  activity  of  the  liver  is  suggested  by  inspection  of  sectioned  liver  tissue, 
of  fecal  pellets  and  of  the  living  organ  in  various  phases  of  its  activity.  Pulsatory 
movements  of  the  stomach  region  are  apparently  interrupted  only  during  the  pas- 
sage of  liver  strings  and  of  gizzard  strings.  This  may  explain  why  smaller  hepatic 
excretory  bodies  occur  in  the  upper  pylorus,  gizzard,  crop,  and  postesophagus  in 
such  insignificant  numbers.  If  the  pulsatory  currents  persisted  during  the  elimina- 
tion of  the  liver  residues  one  would  expect  to  find  liver  string  detritus  scattered 
over  the  gut  in  as  great  profusion  as  in  the  liver,  along  with  the  reddish  colored  se- 
cretions from  the  liver. 

The  inclusion  bodies  of  the  digestive  cells  of  L.  s.  appressa  have  been  studied 
in  detail  in  the  living  cells  of  normally  feeding  snails,  starved  snails,  snails  fed  on 
special  diets  and  in  preserved  tissue  sections.  The  egested  bodies  have  been  fol- 
lowed  in  the  fecal  pellets  over  a  period  of  weeks.  The  results  of  the  study  clearly 
indicate  the  presence  in  the  digestive  cells  of  excretion  bodies,  of  indigestible  resi- 
dues and  of  secretion  in  separate  vacuoles. 

Figure  8  illustrates  a  vacuole  from  the  digestive  cells  which  is  filled  with  indi- 
gestible particles.  These  vacuoles  measure  12  to  25  /j.  in  diameter.  In  snails  feed- 
ing on  lettuce  the  contents  are  colored  a  greenish  brown  to  dark  brown  and  are 
composed  of  minute  irregular  particles,  some  of  the  larger  ones  of  which  measure 
about  3  ju,  in  diameter.  In  the  digestive  cells  they  occur  one  per  cell  and  in  varying 
stages  of  particulate  concentration.  These  constitute  the  bulk  of  the  liver  strings 
and  retain  their  identity  in  fecal  pellets  which  have  been  voided  for  several  days. 

The  secretion  granules  are  clearly  evident  in  preserved  histological  sections 
stained  with  iron  hematoxylin,  especially  grouped  towards  the  distal  area  of  the  cell. 
Larger  granules  measure  as  much  as  4  ^  in  diameter. 

The  excretion  vacuoles  (Figs.  4,  5,  6)  when  in  the  cells  may  measure  as  much 
as  25  p.  in  diameter,  but  in  the  fecal  pellets  have  shrunk  somewhat.  In  the  living 
cells  excretion  bodies  are  found  in  variable  form  and  color  and  are  best  observed 
when  the  cells  are  slowly  pressed  out  under  a  cover  slip  as  the  fluids  evaporate. 
The  cell  contents  then  pass  rolling  and  turning  from  the  ruptured  cells,  exposing  the 


104  MELBOURNE  ROMAINE  CARRIKER 

different  surfaces  of  the  inclusions.  There  is  one  series  in  which  the  vacuoles  range 
from  small  to  large  vacuoles  containing  variable  numbers  and  sizes  of  minute  blue- 
green,  translucent,  many-angled  particles.  The  smaller  particles  are  in  constant 
Brownian  movement,  dancing  around  like  a  swarm  of  bees,  and  indicating  the  low 
viscosity  of  the  fluids  in  the  vacuoles  (Fig.  4).  In  a  second  series  the  same  vari- 
ation in  size  of  the  vacuole  is  encountered  but  the  blue-green  bodies  are  present  in 
groups  of  only  one  to  four  per  vacuole  and  are  spherical  and  smooth  ( Fig.  5 ) .  In 
a  third  series  the  vacuoles  and  bodies  are  identical  in  form  to  the  second  series,  but 
the  color  of  the  bodies  varies  from  a  light  brown  to  a  dark  solid  brown.  The  largest 
of  these  bodies  are  sometimes  found  free  of  the  vacuoles.  When  compressed  under 
a  cover  slip  they  spread  with  a  flowing  viscous  movement,  much  as  a  drop  of  heavy 
molasses  spreads  when  pressed  between  two  smooth  surfaces.  In  the  fecal  pellets 
these  vacuoles  are  usually  found  varying  in  diameter  from  3  to  15  ju,  and  the  vacuole 
membrane  presses  closely  around  the  excretion  body.  A  fourth  type  of  excretion 
body  is  found  which  varies  in  diameter  from  12  to  18  /*,  is  colored  a  dark  brown 
with  a  smooth  center  and  possesses  a  periphery  of  irregular  markings,  such  that  the 
body  resembles  a  signet  ring  (Fig.  6).  The  excretion  bodies  described  above  are 
present  principally  in  the  liver  strings,  and  only  in  negligible  numbers  in  the  cecal 
strings.  The  "browns"  and  "signets,"  particularly,  stain  with  methylene  blue  and 
neutral  red  and  do  not  dissolve  in  strong  HC1.  The  different  types  described  are 
not  all  present  in  any  liver  string  in  equal  abundance  at  any  one  time,  but  vary 
independently,  in  a  sequence  which  did  not  seem  significant.  Because  of  the  transi- 
tional stages  between  some  of  these  excretion  bodies  it  is  probable  that  they  are  all 
different  phases  of  the  same  type  of  metabolic  excretion ;  but  the  method  of  their 
formation  is  still  a  puzzle. 

Intestine  and  rectum 

Cilia  on  the  typhlosole  beat  towards  the  lateral  sides  of  the  typhlosole  (Fig.  3)  ; 
those  over  the  prointestine  around  the  typhlosole  beat  circumferentially  and  some- 
what obliquely  from  the  dorsal  to  the  ventral  sides  in  a  symmetrical  pattern.  The 
division  of  the  currents  occurs  along  the  dorsal  line  of  the  prointestine.  Over  the 
pellet-compressor  the  cilia  beat  transversely  across  the  intestine.  The  raphe  bears 
a  strong  current  which  streams  directly  posteriad.  Thus  in  the  pellet-forming  re- 
gion, through  ciliation  and  muscular  movement,  loose  particles  are  gathered,  rolled 
inward  about  the  typhlosole  and  folded  into  a  compact  pellet.  Strong  ciliary  cur- 
rents in  the  remainder  of  the  intestine  and  rectum  are  limited  almost  entirely  to 
the  costae,  raphe,  and  pseudoraphe ;  cilia  of  the  intercostal  surfaces  are  relatively 
short  and  weak.  Peristaltic  activity  is  evident  throughout  the  intestine  and  rectum, 
being  noticeably  strongest  in  the  early  portions  of  the  prointestine,  just  behind  the 
pellet-compressor. 

Abundant  vascularization  of  the  prointestine,  in  contrast  to  the  relatively  poor 
vascularization  of  the  esophagus,  suggests  that  this  region  of  the  intestine  may  also 
function  in  the  absorption  of  food  and  water. 

Consolidation  of  the  cecal  and  liver  strings  occurs  at  the  hepatic  vestibule ;  of  the 
gizzard  residues  and  cecal  string,  in  the  pellet-forming  region.  The  cecal  string  as 
it  is  moulded  in  the  cecum  is  already  a  smooth  well  cemented  string  and  undergoes 
no  further  change  as  it  is  forced  continuously  across  the  outer  margin  of  the  atrium. 


ALIMENTARY  SYSTEM  OF  LYMNAEA 


105 


The  liver  string,  characterized  hy  a  fine  dark  brown  mottling  and  almost  as  well 
concentrated  as  the  cecal  string,  receives  a  final  transparent  envelope  of  cementing 
fluid  which  binds  the  cecal  string  to  it  (Fig.  10). 

The  chief  function  of  the  pellet-forming  region  is  that  of  consolidating  and  ce- 
menting the  loose  straggling  gizzard  residues  which  constitute  by  far  the  greatest 
bulk  of  the  fecal  pellet.  The  large  numbers  of  mucous  cells,  basophilic  flask  cells 
and  basal  secreting  cells  about  the  pellet-forming  region  are  indicative  of  the  large 
quantities  of  cementing  substance  secreted  during  the  moulding  of  the  pellets.  By 
means  of  ciliary  streams  and  constriction  of  the  tube  at  the  pellet-forming  region 
the  gizzard  residues  are  pressed  into  pellets,  and  the  cecal  strings,  lying  loosely 


40 


30 


20 


10 


length 

/engfn  fiver  •str/fryi 

number  t/cer  strings 


10 


15 


20 


25 


Days 


FIGURE  1.  Length  in  millimeters  of  the  liver  and  gizzard  strings  and  number  of  liver  strings 
of  the  fecal  pellets,  calculated  on  a  twenty-four  hour  basis.  These  were  voided  in  a  period  of 
twenty-four  days  by  a  forty  millimeter  L.  s.  afiprcssa.  The  vertical  arrows  indicate  the  time 
at  which  egg  masses  were  oviposited. 

coiled  in  these  residues,  are  simultaneously  incorporated  in  the  pellets.  These  are 
then  forced  out  of  the  pellet-forming  region  by  ciliary  activity  and  by  strong  peri- 
staltic movements  which  are  noticeably  stronger  immediately  behind  the  pellet- 
compressor.  Peristaltic  activity  gradually  diminishes  in  the  direction  of  the  anus. 
The  conspicuous  impression  of  the  typhlosole  remains  in  the  fecal  pellet,  particu- 
larly in  the  gizzard  string  portion,  as  long  after  defecation  as  the  pellet  retains  its 
form.  Moore  (1931)  has  found  variable  patterns  in  the  fecal  pellets  of  different 
Gastropoda  and  points  out  the  importance  of  identification  of  animals  by  means  of 
their  pellets.  A  most  striking  fact  about  fecal  pellet  formation  is  the  extreme  com- 


106  MELBOURNE  ROMAINE  CARRIKER 

pleteness  with  which  fecal  material  is  compressed  and  cemented.  This  presumably 
prevents  fouling  of  any  portion  of  the  tract. 

For  any  given  snail  the  diameter  of  the  gizzard  string  portion  of  the  fecal  pellet 
is  constant,  varying  principally  with  the  size  of  the  snail.  The  liver  string  varies 
in  diameter  from  that  of  the  gizzard  string  to  that  of  the  fecal  string.  Figure  1 
indicates  for  a  forty  millimeter  snail  over  a  period  of  twenty-four  days  the  rate  and 
extent  of  voidance  of  fecal  pellets.  For  the  tabulation  of  this  data  the  fecal  pellets 
were  collected  daily  and  arranged  end  to  end  under  the  binoculars  and  measured  to 
the  nearest  millimeter.  The  measurements  given  indicate  only  the  lengths  of  the 
gizzard  and  liver  strings,  as  the  cecal  string  generally  occurs  embedded  in  the  first 
two  strings.  The  diameter  of  the  gizzard  string  is  reliably  constant ;  that  of  the 
liver,  less  so. 

Most  conspicuous  is  the  fact  that  the  quantity  of  fecal  pellets  voided  daily  is 
quite  variable  from  day  to  day.  The  quantity  of  gizzard  strings  fluctuates  far  more 
erratically  than  does  that  of  the  liver  strings,  indicating  that  the  volume  of  material 
utilized  by  the  liver  is  more  constant  than  that  which  may  pass  through  the  gizzard. 
The  number  of  liver  strings  is  a  more  conservative  indicator  than  the  length  of 
strings,  and  is  probably  not  as  accurate.  Passage  of  food  through  the  gizzard,  and 
thus  food  consumption,  seems  to  diminish  during  oviposition. 

As  indicated  by  the  following  data,  feces  were  voided  in  about  equal  quantity 
day  and  night,  with  just  a  slight  daily  increase,  over  a  period  of  twenty  days  (9  P.M. 
to  9  A.M.,  and  9  A.M.  to  9  P.M.,  respectively)  : 

Pellets  Night  Day 

Total  length  of  pellets,  mm 1,987  2,134 

Total  length  of  liver  strings,  mm 530  588 

Total  number  of  liver  strings 110  113 

The  total  length  of  fecal  pellets  passed  in  the  twenty-four  days  was  5,645  mm. ; 
and  the  total  length  of  liver  strings,  1,491  mm.,  was  passed  in  289  liver  strings,  giv- 
ing an  average  length  of  5.1  mm.  per  liver  string.  Actually  the  liver  strings  varied 
in  length  from  one  to  10  mm.  The  average  calculated  length  of  fecal  pellets  passed 
in  twenty-four  hours  was  235  mm. ;  of  liver  strings,  62  mm.  In  a  normally  feeding 
snail  the  sequence  of  the  liver  strings  with  the  gizzard  strings  was  always  one  of 
alternation.  Liver  strings  do  not  generally  mix  with  the  gizzard  strings.  Gizzard 
strings  as  long  as  52  mm.  were  found  connecting  liver  strings.  Three  typical  series 
of  fecal  pellets  taken  from  days  one,  two,  and  three  on  Figure  1  are  given  below. 
The  liver  and  gizzard  strings  are  represented  by  the  lengths  in  millimeters  of  the 
strings  in  the  order  of  their  elimination ;  the  figures  for  lengths  of  the  gizzard  strings 
are  italicized.  The  total  time  for  elimination  of  the  pellets  is  given  to  the  right  in 
parenthesis : 

(1)  640  7  33  7  11  644  5  (5  hrs.  15  rnins.) 

(2)  48  7  52  8  13  4  50  5  38  6     (10  hrs.  15  mins.) 

(3)  207    87226299243     (10  hrs.  30  mins.) 

As  indicated  by  the  curve  for  total  fecal  pellets  in  Figure  1  and  by  the  lengths  of 
the  gizzard  strings  in  the  series  above,  consumption  of  food  appeared  to  follow  an 
alternating  heavy  and  light  cycle. 


ALIMENTARY  SYSTEM  OF  LYMNAEA  107 

In  snails  deprived  of  food  the  elimination  of  the  gizzard  strings  ceased  and  liver 
strings  then  became  connected  only  by  slender  lengths  of  cecal  strings.  When 
starvation  had  continued  for  ten  or  more  days  nothing  but  delicate  white  cecal 
strings  and  a  few  much  reduced  liver  strings  containing  metabolic  excretion  bodies 
were  found  in  the  intestine. 

A.  H.  Rosenbloom  (unpublished  bachelor's  thesis,  1942)  by  feeding  colored 
food  to  L.  s.  appressa  at  different  times  through  a  period  of  a  month  found  that  in 
normally  feeding  snails  of  approximately  forty  millimeters  shell  length  the  minimum 
time  for  the  passage  of  food  from  the  mouth  to  the  anus  was  two  hours  and  twenty 
minutes ;  in  snails  previously  starved  for  a  week,  five  hours  and  fifty  minutes.  He 
found  also  that  previously  starved  snails  feed  for  a  longer  consecutive  time  than 
do  normally  feeding  snails.  The  present  investigation  shows  clearly  that  the  ali- 
mentary system  becomes  completely  emptied  of  food  a  few  days  after  starvation 
commences.  Considerably  more  food  and  a  longer  time  are  required  for  a  starved 
animal  to  fill  the  alimentary  tract  with  food  to  the  point  where  fecal  material  is 
voided  than  for  a  normally  feeding  snail. 

The  rhythm  of  passage  of  liver  strings  is  in  keeping  with  the  rhythm  of  the  liver 
itself  in  which  all  digestive  cells  appear  to  assimilate  food  together  and  discharge 
indigestible  residues  simultaneously.  This  cycle,  as  indicated  by  the  passage  of 
liver  strings,  is  not  completely  unvarying,  because  the  number  of  liver  strings  dis- 
charged daily  varied  approximately  from  eight  to  nineteen.  Thus  the  interval  be- 
tween the  discharge  of  liver  residues,  probably  the  time  during  which  the  liver  was 
digesting  food,  varied  in  this  experiment  from  seventy-five  minutes  to  three  hours. 
It  is  possible  that  oviposition  ( Fig.  1 )  may  account  for  some  of  the  variability. 

There  seems  to  be  nothing  in  the  literature  concerning  fecal  cycles  in  the  Gas- 
tropoda. Some  few  scattered  observations  are  reported  on  the  length  of  the  fecal 
pellets.  For  example,  Heidermanns  (1924)  writes  that  a  48  mm.  L.  stagnalis 
with  a  90  mm.  intestine,  eliminated  120  mm.  of  feces  in  24  hours. 

The  long  intestine  is  characteristic  of  the  herbivorous  snail  nutrition  of  L.  s. 
appressa.  One  of  the  most  striking  facts  about  the  functioning  of  the  alimentary 
system  is  the  meticulous  care  with  which  all  loose  particles  are  collected  and  properly 
disposed  of,  in  this  way  serving  as  a  highly  efficient  sanitation  system.  The  fecal 
pellets  receive  additional  external  layers  of  cementing  material  as  they  pass  down 
the  length  of  the  intestine  and  rectum.  The  pH  of  the  intestine  is  slightly  more 
alkaline  than  that  in  the  stomach  region.  As  pointed  out  by  Yonge  (1935)  mucus 
is  an  amphoteric  protein  whose  viscosity  is  augmented  by  higher  pH,  thus  more 
efficient  consolidation  of  the  feces  occurs.  Elimination  of  the  fecal  pellets  through 
the  anus  is  a  fairly  rapid  and  uniform  process.  The  strong  anal  sphincter  muscle 
remains  tightly  contracted  except  during  defecation.  Fecal  pellets,  being  slightly 
heavier  than  water,  settle  slowly  to  the  bottom  of  the  aquaria.  The  marked  effi- 
ciency of  the  mucoid  coating  over  the  feces  is  indicated  by  the  extended  period  after 
defecation  that  pellets  retain  their  identity.  Thus  it  would  seem  that  the  alimentary 
system  has  not  only  become  specialized  in  the  maintenance  of  hygienic  conditions 
within  the  system,  but  also  in  furthering  a  healthy  external  environment. 

Fecal  pellets  are  ingested  by  snails  even  in  the  presence  of  fresh  food  and  the 
animals  appear  to  derive  some  nourishment  from  them.  It  is  to  be  recalled  that 
the  gizzard  is  not  a  thoroughly  efficient  grinding  mechanism  and  in  many  cases, 


108  MELBOURNE  ROMAINE  CARRIKER 

particularly  in  the  absence  of  sufficient  fine   sand,  considerable  unused   available 
food  passes  out  in  the  gizzard  strings. 

DISCUSSION 

The  question  as  to  whether  the  radula  slides  over  the  cartilage  independent  of 
cartilage  activity  has  been  a  favorite  point  of  academic  controversy  with  certain 
malacologists  for  some  time  (in  Lymnaeidae  see  Geddes,  1879;  Amaudrut,  1898; 
and  Pelseneer,  1935;  in  the  Stenoglossa,  a  review:  Carriker,  1943b).  In  L.  s. 
appressa  (and  possibly  in  the  majority  of  snails  carefully  investigated)  there  is  no 
question  but  that  the  principal  activity  of  the  radula  is  that  effected  by  the  action 
of  the  cartilage  and  muscles  under  it,  and  a  sliding  of  the  total  radula  over  the 
cartilage  independent  of  the  movement  of  the  cartilage. 

A  study  of  the  movements  of  the  gut  in  L.  s.  apprcssa  suggests  that  rather  than 
the  presence  of  different  pH  in  the  different  portions  of  the  gut,  the  pH  may  vary 
with  the  rhythms  and  secretions  of  the  liver,  the  secretions  of  the  salivary  glands, 
the  secretions  of  the  unicellular  glands  of  the  gut  wall  and  with  feeding.  It  is  quite 
unlikely  that  with  the  constant  mixing  of  the  gut  contents  as  a  result  of  the  pulsatory 
movements  at  certain  periods,  the  pH  would  vary  markedly  in  the  different  lumina 
of  the  tract  at  any  time.  The  wide  range  obtained  between  the  maximum  and  the 
minimum  pH's  and  the  insignificant  variation  of  the  maximum  and  of  the  minimum 
pH's  is  in  keeping  with  this  suggestion.  The  partial  isolation  of  the  intestine  from 
the  movements  of  the  stomach  region  is  in  keeping  with  the  slightly  higher  pH  found 
in  the  intestinal  lumen. 

The  complexity  and  abundance  of  nervous  tissues  about  the  stomach  region  sug- 
gests a  possible  nervous  control  of  the  movements  of  the  stomach  region  and  of  the 
liver.  In  its  muscular  structure  there  is  no  doubt  that  the  buccal  mass  is  the  most 
complex  organ  in  the  alimentary  system ;  functionally  it  appears  that  the  region  in 
and  about  the  pylorus  is  the  most  intricate.  The  dense  ramifications  of  blood  ves- 
sels, the  presence  of  two  nerve  plexuses,  the  intricate  series  of  folds  and  the  compli- 
cated ciliary  streams  in  this  region  lend  credence  to  this  postulation. 

Heidermanns  (1924)  has  opened  the  question  of  the  function  of  sand  in  the 
basommatophoran  gizzard  in  his  comparative  study  of  Ancylus,  Planorbis,  Physa, 
Lymnaea  and  certain  stylommatophorans.  He  points  out  that  in  land  pulmonates 
the  flaring  portion  of  the  esophagus  is  called  the  stomach,  whereas  in  the  aquatic 
pulmonates  the  esophagus  is  normal  and  the  stomach  has  become  differentiated  into 
the  crop,  gizzard  and  pylorus.  Thus  the  Stylommatophora  have  no  organs  that 
could  properly  be  homologized  with  the  stomach  of  the  Basommatophora.  The  giz- 
zard and,  with  few  exceptions,  sand  in  the  tract  are  absent  from  the  land  pulmo- 
nates. The  gizzard,  he  states,  reaches  its  peak  of  specialization  in  L.  stagnalis  and 
probably  rose  by  reason  of  the  ingestion  of  sand  with  food.  He  observed  that  in  all 
Basommatophora  the  gizzard  originates  in  front  of  the  first  flexure  of  the  gut,  appar- 
ently as  a  muscular  band  whose  primitive  function  was  to  dispose  of  sand  masses 
tending  to  congest  there.  This  primitive  type  of. gizzard  is  exemplified  by  that  of 
Ancylus  and  the  intermediate  type  by  that  of  Planorbis.  Heidermanns  in  support 
of  his  theory  of  the  origin  of  the  gizzard  through  a  specialization  of  a  primordial 
portion  of  undifferentiated  gut,  attempted  to  show  modification  of  the  gizzard  in 
one  snail  generation  by  the  use  of  various  diets.  As  might  be  anticipated,  he  got 
no  significant  structural  changes. 


ALIMENTARY  SYSTEM  OF  LYMNAEA  109 

The  fact  that  Lymnaca  possesses  the  gizzard  grinding  mill  may  explain  the  ob- 
servation stressed  by  Heidermanns  that  the  cellulase  of  this  snail  is  less  active  than 
that  of  Helix  which  has  not  developed  a  gizzard  and  consequently  needs  a  strong 
cellulase  for  the  hydrolysis  of  the  cell  walls  of  plant  food  consumed. 

There  is  striking  similarity  in  the  functioning  of  the  alimentary  tract  of  the 
herbivore  Onchidella  ccltica,  ably  presented  by  Fretter  (1943)  in  a  recent  paper, 
and  that  of  L.  s.  apprcssa.  Perhaps  this  similarity  is  not  to  be  wondered  at  when, 
as  Fretter  writes,  "Many  of  the  features  which  the  Onchidiidae  share  with  the  pul- 
monates  may  be  attributed  to  the  close  origin  of  the  two  groups,  the  similarity  of 
their  diet  and  their  air-breathing  habit." 

SUMMARY 

1.  A  balanced  physiological  salt  solution  was  developed  which  maintains  con- 
tractions of  the  vas  deferens  for  approximately  66  hours. 

2.  Cathepsin  was  found  in  greatest  concentration  in  the  liver  and  no  activity 
could  be  ascertained  in  the  gut  fluids.     Some  trypsin  was  indicated  in  the  salivary 
glands.     Amylase  showed  greatest  activity  in  the  salivary  glands  and  the  liver. 

3.  Muscular  activity  of  the  alimentary  system  involves  the  manipulation  of  the 
mouth  parts  in  the  buccal  mass,  peristalsis  in  the  remainder  of  the  tract,  marked 
pulsatory  movements  of  the  postesophagus,  crop,  pylorus  and  liver,  and  a  kneading 
motion  of  the  gizzard.     The  radula  is  moved  principally  by  the  action  of  the  odonto- 
phore  but  also  operates  independently  of  it. 

4.  The  entire  alimentary  system,  with  the  exception  of  the  gizzard  and  parts  of 
the  buccal  cavity,  is  ciliated.     The  cilia  show  definite  directional   streams  which 
function  in  propelling  food  particles,  in  sorting  food  and  in  consolidating  fine  refuse 
particles  with  the  aid  of  mucoid  substances. 

5.  Sand   is  consumed   normally  by   the   snail   and   is  necessary  for  the  proper 
functioning  of  the  gizzard  in  the  crushing  of  food  particles.     Very  little  trituration 
is  performed  by  the  mouth  parts. 

6.  The  pylorus  is  composed  of  a  complicated  system  of  folds  and  passages  and 
counter  ciliary  currents  and  functions  as  a  filter  which  permits  only  the  soluble  and 
the  finer  food  particles  to  pass  into  the  liver.     It  shunts  the  undigested  residues 
from  the  gizzard  into  the  prointestine. 

7.  In  the  liver  the  digestive  cells  function  in  secretion,  assimilation-,  intracellular 
digestion  and  excretion.     The  indigestible  foods  and  the  excretory  products,  as  vari- 
ably shaped  and  colored  inclusion  bodies,  are  eliminated  in  vacuoles. 

8.  The  cecum  functions  in  collecting  the  finer  residues  from  the  liver  and  forces 
them  in  a  continuous  string  into  the  prointestine. 

9.  The  residual  material  coming  from  the  gizzard,  liver  and  cecum  is  charac- 
teristic for  each  organ  and  is  readily  identified  as  distinct  in  the  fecal  pellet. 

10.  The  prointestine  is  specialized  in  the  final  consolidation  of  gizzard,  liver  and 
cecal  strings  with  the  aid  of  cementing  substances  secreted  by  the  basophilic  flask 
cells  and  the  basal  cells. 

11.  The  rhythmic  nature  of  the  liver  is  disclosed  principally  by  a  study  of  the 
fecal  pellets. 

12.  L.  s.  apprcssa  is  an  herbivore.     Food  bits  are  cut  away  by  the  radula  and 
swallowed.     In  the  buccal  cavity  the  food  receives  mucus  from  the  buccal  gland 


110  MELBOURNE  ROMAINE  CARRIKER 

cells,  mucous  cells  and  the  salivaries  and  enzymes  from  the  latter.  Temporary  stor- 
age and  initial  digestion  occur  in  the  postesophagus.  Digestive  fluids  pass  up  from 
the  liver  in  the  pulsatory  movements  of  the  stomach  region  which  keep  the  fluid  gut 
contents  in  constant  circulation.  The  crop,  gizzard  and  anterior  portion  of  the 
retrocurrent  passage  of  the  pylorus  comminute  the  food.  Amebocytes  present  in  the 
gut  contents  appear  to  aid  in  digestion.  Soluble  and  fine  particles  of  food  pass 
through  the  pyloric  filter  into  the  liver  where  it  is  assimilated  by  the  digestive  cells. 
Assimilation  also  occurs  in  the  pylorus  and  absorption  possibly  in  the  intestine. 
There  is  some  evidence  that  the  pulsatory  movements  of  the  stomach  region  cease 
during  the  passage  of  the  gizzard  and  the  liver  strings. 

LITERATURE  CITED 

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Jour.  Gen.  PhysioL,  22  :  79. 
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Chicago  Acad.  Sci.,  2:  191-211. 
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502. 
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Zool.  Anz.,  .4  :  20-23. 
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435-439. 
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TEMPORARY  PAIR  FORMATION  IN  PARAMECIUM 

BURSARIA  1 


TZE-TUAN  CHEN 

Department  of  Zoology,  University  of  California,  Los  Angeles 

In  Parame'cium  bursaria,  the  two  members  of  a  conjugating  pair  normally  re- 
main united  twenty  to  thirty-eight  or  more  hours.  During  this  time  various  nu- 
clear processes  take  place,  including  three  pregamic  divisions,  exchange  and  fusion 
of  pronuclei,  and  three  post-zygotic  divisions.  Clones  that  are  capable  of  under- 
going normal  conjugation  as  described  above  are  called  "normal  clones." 

But  there  are  some  clones  of  this  species  which  are  abnormal  -  in  that  when  they 
are  mixed  with  normal  clones  the  pairs  formed  are  not  lasting  but  separate  within 
a  few  hours.  An  examination  was  made  of  such  temporary  pairs  in  order  to  dis- 
cover what  nuclear  or  other  changes  occur  in  them. 

Fourteen  clones  of  Parameciion  bursaria,  all  belonging  to  Variety  I,  have  been 
used  in  the  present  study.  These  clones,  all  of  which  were  collected  in  nature,  are 
listed  in  Table  I  with  data  on  each  clone  including  (1)  the  mating  type  to  which 
it  belongs  and  (2)  the  locality  where  it  was  collected. 

TABLE  I 

Clones  of  Paramecium  bursaria  employed  in  study  of  temporary  pair  formation 


Clone  number 

Original  designation 
of  clone 

Mating  type 

Locality  collected 

1 

SAaS 

A 

Santa  Ana  River,  Cal. 

2 

Or3 

A 

Vicinity  of  Capistrano,  Cal. 

3 

SGa3 

A 

San  Gabriel  River,  Cal. 

4 

BG35 

A 

Los  Angeles,  Cal. 

5 

La3 

B 

Laguna  Canyon,  Cal. 

6 

SAa7 

B 

Santa  Ana  River,  Cal. 

7 

UC13 

B 

Los  Angeles,  Cal. 

8 

BH2 

B 

Beverly  Hills,  Cal. 

9 

SAa4 

D 

Santa  Ana  River,  Cal. 

10 

LP10 

D 

Lone  Pine,  Cal. 

11 

UC14 

D 

Los  Angeles,  Cal. 

12 

SAal 

C 

Santa  Ana  River,  Cal. 

13 

BH7 

C 

Beverly  Hills,  Cal. 

14 

BH101 

C 

Beverly  Hills,  Cal. 

Clones  1-11  are  normal  clones  in  that  they  are  capable  of  undergoing  normal  conjugation. 
Clones  12-14  are  abnormal  clones  in  that  when  they  are  mixed  with  normal  clones  the  pairs  formed 
are  not  lasting  but  separate  within  a  few  hours. 

1  This  work  was  aided  by  grants  from  the  Committee  for  Research  in  Problems  of  Sex, 
National  Research  Council ;  and  from  the  Joseph  Henry  Fund  of  the  National  Academy  of 
Sciences. 

-  These  clones  are  considered  abnormal  here  only  because  they  are  incapable  of  taking  part 
in  the  formation  of  lasting  pairs  when  they  are  mixed  with  other,  normal  clones. 

112 


TEMPORARY  PAIR  FORMATION  IN  PARAMECIUM  113 

The  animals  were  cultured  in  essentially  the  manner  described  by  Jennings 
(1939).  For  cytological  study  the  animals  were  fixed  in  Schaudinn's  fluid  con- 
taining glacial  acetic  acid,  stained  in  iron  hematoxylin,  and  destained  in  saturated 
aqueous  solution  of  picric  acid,  following  the  technique  the  writer  has  described 
(Chen,  1944). 

EXPERIMENTAL  STUDIES 

Most  of  the  present  work  was  done  with  the  two  abnormal  clones  (BH7,  SAal), 
although  some  study  was  also  made  on  a  third  abnormal  clone  (BH101).  All  of 
these  three  abnormal  clones  belong  to  mating  type  C  of  Variety  I.  These  abnormal 
clones  were  mixed  with  normal  clones.  Eleven  such  normal  clones  (all  belonging 
to  Variety  I)  were  used.  Four  of  these  normal  clones  belong  to  mating  type  A; 
four  to  type  B  ;  and  three  to  type  D  (see  Table  I). 

As  an  example  of  the  phenomenon  of  temporary  pair  formation,  the  reaction  be- 
tween the  abnormal  clone  SAal  and  the  normal  clone  UC13  will  be  described.  On 
November  27.  1940,  a  large  number  of  animals  belonging  to  each  of  these  two  clones 
were  mixed  at  about  eleven  o'clock  in  the  morning.  Strong  agglutinative  mating 
reaction  occurred  almost  immediately.  Half  an  hour  after  mixture,  pairs  were 
being  formed.  An  hour  after  mixture  (about  noon)  many  pairs  were  formed. 
But  in  the  early  afternoon  the  pairs  broke  apart  into  single  animals.  By  five  o'clock 
all  but  a  few  pairs  had  separated.  By  evening  all  had  separated. 

Such  temporary  pair  formation  was  also  observed  when  the  abnormal  clone 
SAal  was  mixed  with  the  following  normal  clones:  Or3,  SGa3,  SAaS,  La3,  SAa7, 
BH2,  SAa4,  LP10.  and  UC14;  or  when  the  abnormal  clone  BH7  was  mixed  with 
the  normal  clone  LP10;  or  when  the  abnormal  clone  BH101  was  mixed  with  the 
normal  clone  BG35. 

If  such  a  mixture  was  placed  in  a  moist  chamber  and  kept  from  drying  (with 
occasional  replacement  of  the  fluid  that  evaporated),  the  typical  agglutinative  mating 
reaction  and  temporary  pair  formation  recurred  the  following  day  and  almost  daily 
over  a  period  of  many  days.  Some  such  mixtures  were  kept  under  daily  observa- 
tion over  a  period  of  nineteen  days.  The  following  is  the  characteristic  daily  be- 
havior of  the  animals  in  such  a  mixture.  The  agglutinative  mating  reaction  occurs 
in  the  late  morning.  By  noon,  many  pairs  are  formed.  These  pairs  persist  for  a 
few  hours.  Between  four  and  six  o'clock  in  the  afternoon  only  a  few  pairs  are 
found.  In  the  early  evening  one  or  two  pairs  may  remain ;  none  can  be  found  after 
nine  o'clock  in  the  evening. 

CYTOLOGICAL  STUDIES 
Nuclear  conditions  in  the  clones  that  jorm  only  temporary  pairs 

The  writer  has  made  a  cytological  study  of  twenty-one  abnormal  clones,  includ- 
ing the  two  clones  BH7  and  SAal,  and  nineteen  of  the  twenty-two  such  clones  de- 
scribed by  Jennings  (1944).  It  was  found  that  fifteen  of  these  clones  possess 
micronuclei,  while  six  appear  to  be  amicronucleate. 

Thus  the  amicronucleate  condition  is  not  the  general  cause  of  the  peculiar  be- 
havior of  these  abnormal  clones.  It  is  probable  that  the  persistence  of  the  amicro- 
nucleate condition  is  a  result  of  the  inability  to  conjugate  and  acquire  a  micronucleus, 
rather  than  the  cause  of  it.  Apparently  there  are  conjugating  and  non-conjugating 


114  TZE-TUAN  CHEN 

races  of  amicronucleate  ciliates.  In  nature  those  that  can  conjugate  do  so  and  ac- 
quire a  micronucleus,  leaving  in  the  amicronucleate  condition  only  those  incapable 
of  conjugation.  In  my  experience  with  P.  bursaria,  which  includes  a  study  of  the 
nuclei  and  chromosomes  of  many  clones  (collected  from  different  parts  of  the  United 
States.  Canada,  Russia,  England,  Ireland,  and  Czechoslovakia),  the  only  amicro- 
nucleate animals  found  in  nature  are  those  which  cannot  conjugate.  Since  they  can- 
not conjugate,  it  is  likely  that  such  clones  will  be  permanently  amicronucleate.  In 
nature  any  amicronucleate  animal  that  can  conjugate  would  not  remain  amicro- 
nucleate for  long,  since  it  would  become  micronucleate  after  mating  with  a  normal 
animal  from  whom  it  receives  a  pronucleus  as  a  result  of  conjugation  (Chen,  1940). 
Amicronucleate  animals  that  can  conjugate  have  been  found  in  P.  bnrsaria  (Chen, 
1940)  3  and  in  Euplotcs  patella  (Kimball,  1941).  They  arose  spontaneously  in  labo- 
ratory cultures. 

Nuclear  changes  in  temporary  pair  jormation 

To  determine  whether  nuclear  changes  occur  in  temporary  pair  formation,  a  series 
of  preparations  were  made,  in  December,  1940,  of  temporary  pairs  (abnormal  clone 
SAal  X  normal  clone  UC13)  4  and  a  number  of  separated  animals  belonging  to  the 
latter  clone.  The  material  included  pairs  5  to  6  hours  after  onset  of  temporary 
mating,  separated  animals  a  few  hours  after  separation,  13  to  17  hours  after  separa- 
tion, and  21  hours  after  separation.  The  micronuclei  in  these  temporary  pairs  and 
separated  animals  were  compared  with  the  micronuclei  of  vegetative  animals  of 
clone  UC13  (not  mixed  with  any  other  clone).  It  was  found  that  micronuclei  in 
the  majority  of  the  temporary  pairs  and  of  the  separated  animals  were  slightly 
swollen.  In  some,  no  nuclear  changes  were  apparent. 

In  June,  1943,  a  series  of  preparations  were  made  of  temporary  pairs  (abnormal 
clone  BH101  X  normal  clone  BG35)  5  and  a  number  of  separated  animals  belonging 

3  The  writer  has  recently  found  some  additional  cases  of  conjugation  between  amicronucleate 
and  normal  animals  in  Pafaincciinn  bnrsaria,  in  Variety  III.      (Normal  nuclear  changes  occur 
in  the  conjugants  having  the  micronuclei.)      These  amicronucleate  animals  arose  spontaneously 
in  laboratory  cultures. 

Schwartz  (1939)  in  a  brief  preliminary  paper  reported  "conjugation"  in  Paramecium 
bursaria  between  amicronucleate  and  normal  animals  and  between  two  amicronucleate  animals. 
In  view  of  the  lack  of  details  in  this  report,  it  is  impossible  to  tell  whether  temporary  or  lasting 
pair  formation  took  place. 

4  Clone  SAal  appears  to  be  amicronucleate;  clone  UC13  has  a  deeply  staining  micronucleus. 

5  Clone  BH101  has  a  small,  lightly  staining  micronucleus ;  clone  BG35  has  a  relatively  large, 
deeply  staining  micronucleus. 

EXPLANATION  OF  FIGURES 

FIGURES  1-34.  Micronuclei  of  animals  belonging  to  clone  UC13  before,  during,  and  after 
temporary  pairing  with  animals  of  clone  BH7  (drawn  by  Mr.  Earl  Nielsen).  All  drawings 
were  made  with  a  camera  lucida.  X  3,300. 

FIGURES  1-5.     Resting  micronuclei  of  vegetative  animals. 

FIGURES  6-10.  Micronuclei  in  the  members  of  temporary  pairs  4  hours  after  onset  of  pair- 
ing. 

FIGURES  11-16.     Micronuclei  in  the  separated  animals  18  hours  after  separation. 

FIGURES  17-22.     Micronuclei  in  the  separated  animals  30  hours  after  separation. 

FIGURES  23-28.     Micronuclei  in  the  separated  animals  42  hours  after  separation. 

FIGURES  29-34.     Micronuclei  in  the  separated  animals  51  hours  after  separation. 


TEMPORARY  PAIR  FORMATION  IN  PARAMECIUM 


115 


6 


1  •         29 


17 


c 

& 

v£- 


II 


12 


,'    f 

'•  r 


1 


25 


31 

FIGURES  1-34. 


13 


J'i 


14 


116  TZE-TUAN  CHEN 

to  the  latter  clone.  The  material  included  pairs  3  to  4  hours  after  onset  of  temporary 
pairing,  and  separated  animals  2  hours  after  separation,  and  a  day  after  separation. 
It  was  found  that  the  micronuclei  in  the  majority  or  most  of  the  temporary  pairs 
and  separated  animals  were  slightly  swollen.  In  others  no  nuclear  changes  were 
apparent. 

In  October,  1944,  a  series  of  preparations  were  made  of  the  temporary  pairs  (ab- 
normal clone  BH7  X  normal  clone  UC13)  6  and  separated  animals  belonging  to  the 
latter  clone.  The  material  included  pairs  4  hours  after  onset  of  temporary  mating, 
and  separated  animals  7  hours  after  separation,  18  hours  after  separation,  30  hours 
after  separation;  42  hours  after  separation,  51  hours  after  separation.  A  series  of 
preparations  of  vegetative  animals  of  clone  UC13  (not  mixed  with  any  other  clone) 
were  used  as  controls  (Figs.  1-5).  It  was  found  that  the  micronuclei  in  nearly  all 
of  the  temporary  pairs  and  separated  animals  were  slightly  but  noticeably  swollen 
(Figs.  6-34).  This  is  true  even  of  the  separated  animals  51  hours  after  separation 
(Figs.  29-34),  indicating  that  the  physiological  effect  of  the  contact  between  the 
animals  in  temporary  pairing  (as  shown  by  the  swelling  of  the  micronucleus)  is  of 
long  duration. 

GENERAL  RELATIONS 

The  temporary  pair  formation  described  in  the  present  paper  is  similar  to  that 
reported  by  Sonneborn  (1942)  in  P.  aurclia  and  by  Jennings  (1944)  in  P.  bursaria. 
Sonneborn  (1942)  concluded  from  his  data  that  cell  adhesion  occurring  in  the  initial 
stage  of  the  mating  reaction  and  cell  fusion  occurring  during  subsequent  conjugation 
are  due  to  two  different  mechanisms. 

SUMMARY  AND  CONCLUSIONS 

1.  In  normal  conjugation  of  Paramecium  bursaria,  the  two  members  of  each  pair 
remain  united  for  20  to  38  or  more  hours,  during  which  time  various  nuclear  proc- 
esses take  place  including  three  pregamic  divisions,  exchange  and  fusion  of  pro- 
nuclei,  and  three  post-zygotic  divisions.     Clones  that  are  capable  of  undergoing  nor- 
mal conjugation  as  described  above  are  called  "normal  clones." 

2.  Some  clones  of  this  species  are  abnormal  in  that  when  they  are  mixed  with 
normal  clones  the  pairs  formed  are  not  lasting  but  separate  within  a  few  hours. 

3.  In  temporary  pair  formation,  the  animals  of  diverse  mating  types  when  mixed 
exhibit  the  typical  agglutinative  mating  reaction.     Within  an  hour  many  pairs  are 
formed  but  in  a  few  hours  these  pairs  break  apart  into  single  animals. 

4.  If  such  a  mixture  is  placed  in  a  moist  chamber  and  kept  from  drying  (with 
occasional  replacement  of  fluid  that  evaporates)  such  agglutinative  mating  reaction 
and  temporary  pair  formation  will  recur  daily  over  a  period  of  many  days. 

5.  Cytological  study  of  21  such  abnormal  clones  shows  that  most  of  these  clones 
have  micronuclei ;  some  appear  to  be  amicronucleate.     Therefore,  amicronucleate 
condition  cannot  explain  the  incapacity  for  taking  part  in  the  formation  of  lasting 
pairs.     It  is  probable  that  the  persistence  of  the  amicronucleate  condition  is  a  result 
of  the  inability  to  conjugate  and  acquire  a  micronucleus  rather  than  the  cause  of  it. 

6.  In  temporary  pair  formation,  there  are  no  conspicuous  nuclear  changes  either 
in  the  pairs  or  in  the  animals  after  their  separation.     In  the  majority  of  the  tempo- 

G  Clofie  BH7  appears  to  be  amicronucleate. 


TEMPORARY  PAIR  FORMATION  IN  PARAMECIUM  117 

rary  pairs  and  separated  animals,  there  is,  however,  a  slight  swelling  of  the  micro- 
nuclei.  This  swelling  persists  for  a  considerable  length  of  time  after  the  separation 
of  the  animals,  indicating  that  the  physiological  effect  of  the  contact  between  the  ani- 
mals in  temporary  mating  (as  shown  by  the  swelling  of  the  micronuclei)  is  of  long 
duration. 

LITERATURE  CITED 

CHEN,  T.  T.,  1940.     Conjugation  in  Paramecium  bursaria  between  animals  with  diverse  nuclear 

constitutions.     Jour.  Hercd.,  31 :   185-196. 

CHEN,  T.  T.,  1944.     Staining  nuclei  and  chromosomes  in  Protozoa.     Stain  Tcchn.,  19 :  83-90. 
JENNINGS,  H.  S.,  1939.     Genetics  of  Paramecium  bursaria.     I.  Mating  types  and  groups,  their 

interrelations  and  distribution  ;   mating  behavior  and  self-sterility.     Genetics,  24 :   202- 

233. 
JENNINGS,  H.  S.,  1944.     Paramecium  bursaria:  life  history.     I.  Immaturity,  maturity  and  age. 

Bio!.  Bull.,  86:  131-145. 
KIMBALL,   R.  F.,   1941.     Double  animals  and  amicronucleate  animals   in  Euplotes   patella  with 

particular  reference  to  their  conjugation.     Jour.  Exp.  Zool.,  86 :   1-32. 

SCHWARTZ,  V.,   1939.     Konjugation  micronucleusloser   Paramaecien.     Natumnss.,  27:   724. 
SONNEBORN,   T.   M.,    1942.     Evidence   for   two   distinct   mechanisms    in   the   mating   reaction   of 

Paramecium  aurelia.     Anat.  Rec.,  84 :  542-543. 


Vol.  91,  No.  2  October,  1946 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED  BY  THE  MARINE  BIOLOGICAL  LABORATORY 


THE  SPACE-TIME  PATTERN  OF  SEGMENT  FORMATION 

IN  ARTEMIA  SALINA 

PAUL  B.  WEISZ 

Department  of  Zoology,  McGill  University,  Montreal,  Canada 

i 

INTRODUCTION 

The  present  work  was  carried  out  in  an  attempt  to  arrive  at  a  primary  under- 
standing of  the  regularities  and  laws  in  the  phenomenon  of  metameric  segmentation, 
as  related  to  the  shape  and  size  of  animals.  To  date  this  phenomenon,  although  of 
widespread  occurrence  amongst  the  higher  animal  phyla  and  thus  probably  an  in- 
tegral part  in  the  more  complex  patterns  of  evolutionary  organization,  was  never- 
theless surprisingly  rarely,  if  at  all,  subjected  to  analytical  inquiry.  The  reason 
for  this  can  probably  be  found  in  an  essential  lack  in  the  past  of  well-defined  con- 
cepts about  the  interrelations  between  mass,  shape,  growth,  and  degree  of  develop- 
ment of  living  organisms.  The  problem  of  segment  formation  in  relation  to  size 
and  shape  is  primarily  one  involving  a  clear  appreciation  of  the  dynamic  geometry 
of  living  matter,  and  initial  insight  into  the  problem  can  therefore  only  emerge  from 
rigorous  observation  on  a  quantitative  level,  followed  preferably  by  geometrical 
and  mathematical  analysis.  Such  a  method  has  been  employed  in  the  present 
work,  and  the  results  gained  are  conclusive  enough  not  only  to  point  the  way  for 
further  study  of  the  problem  at  hand,  but  also  to  promise  reasonable  success  in  the 
application  of  the  quantitative,  geometrical  method  to  questions  of  biological  space- 
time  pattern  in  general. 

The  choice  of  Artemia  has  proven  particularly  fortunate  for  a  study  of  meta- 
meric segmentation.  The  animal,  held  to  be  amongst  the  most  primitive  of  living 
Crustaceans  (Lockhead,  1941),  develops  few,  if  any,  specialized  structural  features 
which  would  ordinarily  tend  to  obscure  the  fundamental  processes  of  morphogenesis. 
Moreover,  the  development  of  as  many  as  nineteen  body  segments,  a  further  primi- 
tive trait,  is  of  obvious  advantage  in  the  investigation  of  the  underlying  principles 
of  formation.  Also,  Artemia  is  easily  obtained  and  can  be  reared  in  the  laboratory 
without  difficulty. 

METHODS  AND  MATERIALS 

Larvae  of  Artemia  salina  were  obtained  from  commercial,  air-dried  egg  cysts. 
Since  excystment  is  retarded  or  inhibited  in  water  above  a  certain  salinity  (Jennings 
and  Whitaker,  1941),  water  of  a  specific  gravity  of  1.020  was  used  throughout  as 
the  initial  medium.  The  egg  shells  cracked  open  usually  12  to  18  hours  after 
contact  with  the  water,  and  emergence  of  the  larvae  (Whitaker,  1940)  took  place 

119 


120  PAUL  B.  WEISZ 

between  18  and  24  hours.  Portions  of  five  stock  solutions  of  brines  with  different 
salt  concentration  were  employed  as  further  media.  The  solutions  were  obtained 
from  the  original  sea  water  by  either  diluting  with  doubly  glass  distilled  water  or 
concentrating  with  NaCl  to  specific  gravities  of  1.022,  1.033,  1.047,  1.066,  and 
1.085,  respectively.  All  solutions  were  vigorously  aerated  daily,  and  possible 
deviations  from  the  proper  specific  gravity  were  adjusted  in  weekly  intervals.  All 
work  was  carried  out  at  room  temperature,  corresponding  to  an  average  water 
temperature  of  21-22°  C.  As  soon  as  the  embryos  had  emerged,  still  enclosed 
within  the  fine  hatching  membrane,  they  were  transferred  to  water  from  either  of 
the  five  stock  solutions.  The  moment  of  hatching,  occurring  within  24  to  30  hours 
after  the  cysts  had  first  made  water  contact,  was  taken  as  zero  time  for  all  further 
determinations. 

For  observation  the  larvae  were  reared  singly,  in  heavy  crystal  watch  glasses. 
In  the  course  of  several  observational  series,  a  total  of  up  to  100  individuals  were 
observed,  at  least  for  certain  periods  of  their  development;  of  the  100,  about  25 
individuals,  evenly  distributed  among  the  solutions,  were  reared  from  hatching  to 
the  adult  stage.  The  presence  or  absence  of  a  molted  shell,  the  time,  the  tempera- 
ture, the  stage  of  development  reached,  and  a  series  of  measurements  on  bodily 
proportion  were  recorded  for  each  individual  twice  daily  in  the  earlier  stages  and 
daily  for  later  stages.  The  animals  were  fed  once  every  two  days  on  a  sea-water- 
yeast  suspension.  Each  watch  glass  containing  an  animal  was  covered  so  that 
evaporation  was  nearly  abolished,  but  a  minimum  of  air  circulation  was  always 
allowed  for  to  equilibrize  the  CO2  released  by  the  yeast  and  the  animal.  The  water 
was  changed  at  two-day  intervals  for  the  younger  stages  and  daily  for  older  ones. 

Larval  body  measurements  were  taken  under  the  microscope  with  the  help  -of 
a  hemocytometer  slide  whose  grid  allows  the  direct  reading  off  of  lengths  of  50 
micra  and  consistent  estimations  of  lengths  of  10,  20,  and  30  micra.  If  the  larva 
is  placed  on  a  coverslip  with  a  minimum  of  water,  the  whole  can  be  adjusted  in 
relation  to  the  grid  ;  evaporation  is  sufficiently  slow  to  allow  five  or  six  measure- 
ments at  a  time.  The  error  inherent  in  this  method,  viz.,  the  parallax  due  to  the 
thickness  of  the  coverslip,  is  small  enough  to  be  negligible  ;  also,  since  all  measure- 
ments were  taken  in  this  way,  the  relative  values  are  consistent. 

PRELIMINARY  OBSERVATIONS 

Barigozzi  (1939)  and  Rugh  (1941)  observed  the  total  developmental  time  from 
hatching  to  the  adult  stage  of  Artemia  to  be  3  to  4  weeks.  This  is  true  as  a  broad 
generalization,  but  with  the  egg  cysts  in  the  various  salinity  media  here  employed, 
certain  statistically  preferred  tendencies  become  apparent,  expressed  empirically  by 


r)_r).  o 

*->  x  —  •k'O    ^ 


-e.s 


where  Dx  is  the  time,  in  days,  for  complete  development  from  hatching  in  a  solution 
of  salt  concentration  x\  D0,  the  (hypothetical)  time,  similarly,  for  development  in 
distilled  water  (the  algebraic  value  turned  out  to  be  36.55)  ;  and  Sx,  the  specific 
gravity  of  the  solution  of  concentration  x.  This  relation  holds  good  only  in  a 
statistical  sense,  within  a  specific  gravity  range  of  1.020  and  1.1  ;  it  indicates  that 
with  higher  salt  concentrations  the  rate  of  development  tends  to  be  greater  (Fig.  1). 
Irrespective  of  concentration  a  sigmoid  curve  of  growth  is  always  obtained. 


ARTEMIA  SEGMENTATION  PATTERN 


121 


Morphologically,  different  salinities  have  no  differential  effect  on  relative  body 
proportions,  a  result  to  be  expected  in  view  of  the  conclusions  of  Bond  (1932). 
An  inverse  relation  between  total  size  and  salinity,  observed  by  Bond,  Heath 
(1924),  and  Warren  (1938)  for  larvae  from  non-excysted  eggs  in  the  natural 
habitat,  however,  could  not  be  observed  for  the  excysted  larvae  here  used;  in  the 
latter,  total  sizes  are  identical  at  equivalent  stages  of  development,  irrespective  of 
salinity. 

The  number  of  molts  between  hatching  and  sexual  maturity  is  not  constant. 
Even  when  reared  in  the  same  medium,  slight  differences  in  molting  frequency 
between  several  larvae  may  occur.  Moreover,  there  exists  a  rough  statistical 
relation  between  salinity  and  the  total  number  of  molts,  approximating  closely  the 


0    I/  S4  5  fi  7    8    9     10      II   12   13    14        15  IS          17 


05 


FIGURE  1.  The  effect  of  salinity  on  developmental  time,  from  hatching  to  sexual  maturity; 
absolute  growth  curves.  Abscissa:  total  larval  length;  Ordinate:  time  in  days.  A-E,  media 
of  salt  water,  specific  gravities  from  1.022-1.085  respectively;  numbers  1-19  above  abscissa  refer 
to  number  of  body  segments  present. 

above  relation  between  salinity  and  the  time  required  for  complete  development; 
in  general,  however,  the  number  of  molts  for  a  given  salinity  is  somewhat  lower  than 
the  number  of  days  required  for  development.  In  larvae  from  excysted  eggs  and 
under  artificial  food  conditions,  a  range  of  12  to  16  molts  was  observed  between 
hatching  and  maturity,  at  a  specific  gravity  of  1 .085 ;  this  compares  with  25  to  29 
molts  at  a  specific  gravity  of  1.022,  and  gradually  decreasing  molting  frequencies 
for  the  intermediate  salinity  ranges.  A  staging  of  larval  development  according 
to  molts,  as  Heath  has  done  for  non-excysted  individuals,  would  therefore  not  be 
possible  in  the  present  case.  Heath's  13  stages  would  hold  for  excysted  larvae 
only  when  reared  in  brine  of  a  specific  gravity  of  1 .085 ;  even  then  certain  definite 
differences  in  the  degree  of  development  of  equivalent  molting  stages  can  be 


122  PAUL  B.  WEISZ 

observed,  as  comparison  of  Heath's  descriptions  with  those  below  makes  apparent. 
With  increasing  developmental  age  the  duration  of  instars  increases;  a  12  to 
24  hour  interval  between  molts  in  the  younger  stages  compares  with  24  to  30 
hour  intervals  in  older  ones.  The  two  factors  of  salinity  and  developmental  age 
also  determine  the  size  increase  between  molts ;  for  higher  salinities,  as  well  as  for 
older  larvae,  the  size  increase  is  greater.  There  is  no  observable  relation  however 
between  the  time  at  which  a  molt  occurs  and  the  size  or  the  developmental  stage 
attained,  irrespective  of  whether  test  larvae  are  reared  in  the  same  or  at  different 
salinities.  Molting  is  also  greatly  influenced  by  the  food  supply.  Starving  animals 
do  not  molt ;  after  3-5  days  an  abortive  attempt  at  molting  is  made  which  usually 
results  in  the  death  of  the  animal.  Conversely,  overfed  larvae  may  molt  twice  in 
rapid  succession  without  undue  increase  in  size. 

ANALYSIS  OF  SEGMENT  FORMATION 
Observations  and  definitions 

The  larval  development  of  Artemia  can  best  be  dealt  with  in  terms  of  the 
number  of  body  segments  present.  The  first  three  segments  become  visible  almost 
simultaneously  at  a  total  larval  size  of  0.745  mm.  (stage  3),  after  the  embryonic 
yolk  has  been  digested  away,  and  the  termination  of  the  hatching,  nauplius,  and 
metanauplius  stages  can  therefore  be  represented  as  the  termination  of  stages  0,  1, 
and  2,  respectively ;  at  the  end  of  any  following  stage  the  stage  number  will  thus  indi- 
cate directly  the  number  of  body  segments  present.  It  will  be  convenient  to  distin- 
guish between  a  thoracic  period  of  development,  comprising  the  first  11  stages,  and 
an  abdominal  period,  including  stages  12  to  19;  the  latter  can  again  be  divided  into 
a  genital  period  (stages  12  and  13)  and  a  post-abdominal 'one  (stages  14  to  19). 

Except  for  the  first  three,  each  individual  segment  is  initially  recognizable  as  a 
transverse  ring  of  thickened  mesoderm,  the  segment  rudiment,  immediately  under- 
neath the  otherwise  smooth  epidermal  layers  (segmental  stage  a).  Later,  partial 
transverse  constrictions  appear  externally  in  the  epidermis  and  the  chitin,  in  a 
plane  just  posterior  to  that  of  the  segment  rudiment  (segmental  stage  b}.  Even- 
tually, the  constrictions  become  complete  and  deepen,  with  a  concomitant  bulging 
out  of  the  body  wall  in  the  region  of  the  segment  rudiment  (segmental  stage  c). 
At  this  stage,  the  segment  can  be  considered  "laid  down,"  its  shape  resembling 
more  or  less  a  short  cylinder.  In  thoracic  segments,  appendage  buds  appear  in 
stage  c  ventro-laterally,  on  either  side.  The  segments  are  considered  mature  when 
their  pairs  of  swimming  appendages  first  become  independently  motile.  Stages  a 
to  c  of  the  first  and  second,  and  stages  a  and  b  of  the  third  segment  can  never  be 
clearly  seen;  the  first  stages  of  these  segments  are  attained  prior  to  hatching  and 
during  the  nauplius  and  metanauplius  phases,  when  the  presence  of  dense  yolk 
conceals  details  of  structure.  As  these  segments  become  plainly  visible  in  the 
third  stage  of  the  thoracic  period,  segment  3  is  in  stage  c,  but  segments  1  and  2 
are  already  correspondingly  ahead,  both  in  size  and  the  degree  of  their  development. 

At  the  end  of  the  thoracic  period  the  llth  segment  has  reached  stage  c  and  the 
first  five  segments  have  become  mature.  The  llth  segment  attains  maturity  at 
the  end  of  the  abdominal  phase  of  development  (stage  19).  Appendage  buds 
similar  to  those  on  more  anterior  segments  also  develop  on  segments  12  and  13. 
But  instead  of  developing  into  swimming  appendages  the  buds  on  either  side  of 


ARTEMIA  SEGMENTATION  PATTERN  123 

both  segments  enlarge,  and  in  the  female  fuse  into  a  sac  in  stage  18,  forming  the 
left  and  right  brood  pouch;  no  male  larvae  were  investigated.  The  remaining  six 
segments  develop  similarly  from  segment  rudiments,  but  no  appendage  buds  are 
ever  formed  and  stage  c  represents  the  first  stage  of  maturity.  At  the  end  of  the 
abdominal  period  the  19th  and  last  segment  has  become  mature.  In  the  head,  the 
ocellus  becomes  pigmented  in  stage  2  and  the  compound  eyes  in  stage  4.  The 
maxillae  and  maxillulae  also  form  in  stage  2.  The  end  of  stage  19  marks  the  time 
when  the  gnathobase  and  the  setae  have  been  lost  entirely  from  the  second  antenna. 
After  stage  19  an  arbitrary  number  of  non-segmental  stages  ensue  before  sexual 
maturity  is  reached. 

When  individuals  in  identical  stages  of  development  from  the  same  or  from 
different  salinity  media  are  compared,  it  is  strikingly  apparent  that  total  lengths 
and  body  proportions  in  general  fall  within  well-defined  size-classes ;  the  deviations 
from  the  underlying  averages  in  no  case  exceed  ±  3  per  cent.  In  Table  I  the 
averages  of  a  variety  of  body  measurements  are  shown,  from  the  25  individuals 
watched  throughout  development,  with  the  stage  number  as  the  basis  of  calculation ; 
these  values,  within  ±  3  per  cent,  are  true  for  individuals  from  any  of  the  salinities 
here  examined.  A  schematic  diagram  of  an  Artemia  larva  indicates,  in  Figure  2, 
how  the  various  entities  have  been  defined.  Head  length  is  understood  to  include 
maxillar  and  maxillular  segments.  The  length  of  a  segment  refers  to  axial  and 
the  width  to  its  lateral  extent.  Total  abdominal  length  is  the  length  of  the  seg- 
mental  portion,  whether  actually  cut  up  into  segments  or  not,  plus  the  length  of  a 
terminating  anal  piece;  the  segmental  portion  is  the  pygidium  of  annelid  forms, 
and  in  Artemia  is  readily  distinguished  from  the  anal  piece,  or  urosome,  by  a  con- 
striction. During  the  abdominal  period  of  development,  the  segmental  abdomen 
contains  a  genital  region  composed  of  segments  12  and  13,  as  well  as  a  post- 
abdomen  (presumptive  segments  14  to  19)  with  segmented  and  non-segmented 
portions. 

From  observation  and  from  examination  of  the  data  in  Table  I  the  following 
facts  concerning  the  formation  of  segments  in  relation  to  larval  shape  and  size  are 
consistently  found  to  occur : 

1.  Every  thoracic  segment  when  newly  formed   (stage  c)   has  a  fixed  length 
of  0.03  mm.  and  a  fixed  width  of  0.144  mm. 

2.  Every  time  a  new  thoracic  segment  is  laid  down  in  stage  c,  preceding  seg- 
ments increase  in  length  and  in  width. 

3.  Throughout  the  thoracic  period,  the  segmental  part  of  the  abdomen  has  a 
constant  average  length  of  0.249  mm.;  its  anterior  width,  being  slightly  smaller 
than  the  width  of  the  newest  segment,  is  also  constant  (C  =  0.142  mm.). 

4.  During  the  thoracic  period,  the  lateral  contour-lines  of  the  thorax  are  straight 
lines  converging  posteriorly;  the  lateral  abdominal  contour-lines  are  also  straight, 
but  generally  they  converge  with  a  greater  degree  of  taper  than  the  thoracic  contours. 

5.  Appendages  are  longer  the  more  anterior  they  are;  the  line  joining  the  tips 
of  the  appendages  on  one  side  of  the  body  is  more  or  less  a  straight  line. 

6.  As  the  llth  segment  appears  in  stage  c,  the  5th  segment  has  matured  and 
the  19th  segment  has  appeared  in  stage  a. 

7.  Between  stage  a  and  stage  c  of  the  thoracic  segments,  4  stages  of  larval 
development  intervene ;  an  interval  of  more  than  4  developmental  stages  is  neces- 
sary for  an  abdominal  segment  to  reach  stage  c  from  stage  a. 


124 


PAUL  B.  WEISZ 


TO. 
"t* 

§ 


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ARTEMIA  SEGMENTATION  PATTERN 


125 


8.  Between  stage  c  and  maturity  in  the  first  5  segments,  6  stages  of  develop- 
ment intervene;  8  stages  intervene  before  maturity  of  segments  6  to  11. 

9.  The  anterior  width  of  the  non-segmented  portion  of  the  abdomen  during  the 
abdominal  phase  is  of  a  fixed  and  constant  magnitude  and  identical  to  the  constant 
anterior  width  of  the  abdomen  during  the  thoracic  phase ;  thus  at  the  end  of  stage 


wu 


FIGURE  2.  Schematic  diagram  of  a  larva  of  Artcmia  salina.  A,  larva  in  the  thoracic 
period ;  B,  larva  in  the  post-abdominal  period  of  development.  A,  length  of  segmental  abdomen 
(pygidium)  ;  a,  angle  of  thoracic  taper;  /3,  angle  of  abdominal  taper;  C,  Wo,  WA0,  constant 
anterior  width  of  segmental  abdomen;  GL,  length  of  genital  region;  H,  head  length;  NSPA, 
non-segmented  post-abdomen ;  P,  length  of  post-abdomen ;  PS,  PSm,  length  of  a  post-abdominal 
segment;  SPAL,  length  of  segmented  post-abdomen;  SR,  segment  rudiment;  T,  Tn,  Tm,  length 
of  thorax ;  TAL,  total  abdominal  length ;  TS,  TSn,  length  of  first  thoracic  segment ;  TSL,  Ti, 
length  of  last  (newest)  thoracic  segment:  Tot.L,  total  larval  length;  U,  urosomal  length;  W, 
Wn,  width  of  first  thoracic  segment;  WTSL,  Wi,  width  of  last  thoracic  segment;  WA,  WAr, 
width  of  first  abdominal  (12th)  segment;  WASL,  width  of  last  (newest)  abdominal  segment; 
WU,  anterior  width  of  urosome. 


126  PAUL  B.  WEISZ 

19,  the  width  of  the  urosome  is  the  same  as  the  posterior  width  of  the  head  in 
stage  0  when  segmental  development  started.  A  constant  width  has  seemingly 
travelled  down  the  larva. 

10.  At  the  beginning  of  stage  12,  the  segmental  abdomen  starts  to  grow  in 
length  at  a  fast  rate,  having  retained  a  constant  length  in  the  thoracic  period. 

.11.  Segments  12  and  13  are  of  equal  length  at  any  time  after  their  formation; 
they  are  individually  always  somewhat  longer  than  the  llth  thoracic  segment  and 
become  progressively  shorter,  relatively,  than  the  14th  segment.  At  stage  18, 
6  developmental  stages  after  segment  12  has  reached  stage  c,  segment  12  and  13 
fuse  to  form  the  brood  pouch  in  the  female  and  can  then  be  considered  matured. 
The  interval  for  attainment  of  maturity  is  thus  equal  to  the  similar  interval  in  the 
first  5  thoracic  segments. 

12.  Segments  14  to  19  are  not  of  equal  length  when  formed ;  more  posterior 
segments,  when  formed,  are  longer  than  more  anterior  ones  when  formed.     Also, 
any  one  of  these  segments  has  always  one-sixth  of  the  length  of  the  post  abdomen, 
and  at  a  given  time  post-abdominal  segments  are  of  equal  length. 

13.  When  segment  19  reaches  stage  c,  the  llth  segment  attains  maturity. 

14.  During  the  abdominal  phase,  the  thorax  changes  shape  in  the  following 
way:  the  2nd  segment  becomes  longer  and  wider  than  the  1st,  then  the  3rd  larger 
than  the  2nd,  etc.,  and  the  5th  becomes  the  largest,  coincident  with  the  end  of 
stage  19.     As  a  result,  the  lateral  thoracic  contours  become  curved,  the  widest  part 
of  the  thorax  being  at  segment  2  in  stage  16,  at  segment  3  in  stage  17,  etc.,  and  at 
segment  5  in  stage  19. 

15.  Similar  differential  increases  take  place  in  the  appendages;  at  stage  19, 
the  5th  pair  of  swimming  appendages  is  longest  and  appendageal  length  regularly 
decreases  towards  the  1st  and  the  llth  pair.     The  line  joining  the  appendage  tips 
on  one  side  of  the  thorax  is  now  also  curved. 

16.  During  the  abdominal  phase,  and  paralleling  the  differential  increases  in 
the  thoracic  segments,  a  progressive  dorsal  thoracic  curvature  develops,  with  an 
analogous  shift  backwards  of  the  maximal  flexure ;  the  latter  arrives  similarly  at 
segment  5  at  the  end  of  stage  19.     Due  to  this  flexure  the  head  now  appears  bent 
ventrad. 

17.  The  lateral  contours  of  the  abdomen  remain  straight  lines  throughout  the 
abdominal  phase,  with  a  definite  taper  directed  backwards. 

18.  At  the  end  of  stage  19  segmental  development  is  completed ;  further  devel- 
opment is  still  to  take  place  in  the  head.     The  essential  overall  shape  of  the  animal 
as  now  established,  i.e.,  the  possession  of  a  barrel-shaped  thorax  and  a  straight 
tapering  abdomen,  is  carried  through  to  sexual  maturity,  although  changes  of  detail 
do  still  occur. 

These  observations  are  now  to  be  interpreted  and  integrated  analytically. 

The  thoracic  phase  of  development 

The  lengths  of  the  first  thoracic  segment,  in  successive  stages,  are  0.03,  0.04, 
0.05,  0.058,  0.065,  0.076  mm.,  etc.  (Table  I).  The  differences  between  these 
values,  taken  for  all  11  thoracic  stages,  are  very  close  to  an  average  difference  of 
0.0097  mm.  The  length  of  the  first  thoracic  segment  in  successive  stages  can 


ARTEMIA  SEGMENTATION  PATTERN  127 

therefore  be  expressed  as  an  arithmetical  series 

TSn  =  (TSi  --  TS0)  +  (n--  l)-Aj  (1) 


where  TSn  refers  to  the  length  of  the  first  thoracic  segment  at  stage  n;  n,  to  the 
successive  stage  numbers  from  1  to  11  (and  thus  to  the  number  of  segments 
present  at  the  time)  ;  (TS\  —  TSo),  to  the  initial  length  of  the  first  segment  at 
the  end  of  stage  1  ;  and  As,  to  the  increase  of  segmental  length  per  stage  (0.0097 
mm.).  The  expression  would  mean  that  the  first  segment  grows  in  length  by  a 
constant  amount  As  during  each  stage. 

Since  every  other  thoracic  segment  is  known  to  start  off  with  an  identical  value 
for  (TSi  —  TSo),  viz.,  0.03  mm.,  it  could  be  possible  that  other  thoracic  segments 
also  increase  a  constant  amount  As  during  each  stage.  If  that  were  true,  then  the 
newest  segment,  at  any  given  stage,  would  have  a  length  of  (TSi  -•  TSQ},  the 
segment  immediately  preceding  it  a  length  of  (TSi  --  TSo)  +  As,  the  third  but 
last  a  length  of  (TSi  -•  TS0)  +  2As,  .  .  .  etc.,  and  the  first  segment  again  a 
length  of  [(7\S"i  --  TSo)  +  (n  --  l)-As].  In  other  words  the  length  of  the  entire 
thorax,  being  the  sum  of  individual  segments,  should  be  the  sum  of  an  arithmetical 
series  whose  first  term  is  (TSi  -•  TS0)  and  whose  last  term  is  [(TSi  —  T5"0) 
+  (n  --  l)-As],  This  can  be  put  as 


Tn  =  n-  (TS,  -  TS0)  +  -As  (2) 

where  Tn  is  the  total  thoracic  length  at  stage  n;  and  (TSi  -•  TSQ),  the  constant 
length  of  the  newest  segment  (or  the  length  of  the  first  segment  when  in  stage  c). 
Taking  As  as  0.0097  mm.  and  (TSi  -•  TS0)  as  0.03  mm.,  Tn  for  successive  values 
of  n  can  be  calculated.  These  calculated  values  are  compared  with  the  observed 
values  for  thoracic  length  in  Table  II  ;  the  largest  discrepancy  is  only  approximately 
5  per  cent,  and  the  original  suggestion  is  thus  shown  to  be  fact,  i.e.,  every  thoracic 
segment  grows  in  length  for  a  constant  amount  As,  in  each  stage  of  the  thoracic 
period. 

A  similar  approach  can  be  employed  to  analyze  thoracic  changes  in  width. 
While  in  stage  0  thoracic  length  Tn  is  also  0,  the  anterior  width  of  the  presumptive 
thorax  is  already  0.142  mm.  (C).  In  stage  1,  the  width  of  the  first  segment  is 
0.144  mm.  (Table  I),  and  the  initial  increase  (W\--C),  analogous  to  (TS\ 
•  TSo)  in  equations  1  and  2,  is  therefore  0.002  mm.  ;  the  anterior  width  of  the 
presumptive  second  segment  is  again  C  =  0.142  mm.  (each  segment  after  stage  c 
being  regarded  as  a  short  cylinder).  In  succeeding  stages,  the  width  of  the  first 
segment  increases  0.002,  0.004,  0.005  mm.  .  .  .  etc.  (Table  I).  The  increments 
per  stage  are  then  not  constant,  as  they  were  for  segmental  length,  but  the  figures 
suggest  that  the  increases  of  the  increments  per  stage  might  be  constant.  If  the 
increment  in  stage  2  were  0.003  instead  of  0.002  mm.,  the  increase  Azy  over  the 
initial  increment  (W\  —  C)  would  be  0.001,  and  (W\  --  C)  +  Aw  would  repre- 
sent the  increase  in  width  during  stage  2.  Similarly  (W\  —  C)  +  2Aw  and 
(W\  —  C)  +  3  Aw  would  indicate  the  increases  during  stages  3  and  4  respectively. 
In  general, 


(Wn  -  Wn^  =  (Wi  -  C)  +  (n  -  1)  -Aw  (3) 


128 


PAUL  B.  WEISZ 


would  be  true,  where  (Wn~  Wn-i)  represents  the  increase  in  width  of  the  first 
segment  during  stage  n.  The  total  width  increase  of  the  first  segment  during  the 
first  n  stages  would  then  be  the  sum  of  an  arithmetical  series  whose  first  term  is 
(Wi  —  C)  and  whose  last  term  is  [(Wi  --  C)  +  (n  --  1)  -Aw],  for  similar  reasons 
as  in  thoracic  length ;  or 


and 


n(n  --  1) 


n(n  -•  1) 


Aw 


(4) 


(5) 


TABLE  II 

Calculated  and  observed  magnitudes  of  certain  larval  body  regions,  in  millimeters 


Thoracic  length 

Width  of  1st  thoracic  segment 

Observed 

Calculated 

Observed 

Calculated 

.    1 

0.030 

0.030 

0.144 

0.144 

2 

0.070 

0.069 

0.146 

0.147 

3 

0.122 

0.119 

0.150 

0.151 

4 

0.185 

0.178 

0.155 

,  0.156 

5 

0.242 

0.247 

0.162 

0.162 

6 

0.311 

0.325 

0.167 

0.169 

7 

0.390 

0.413 

0.179 

0.177 

8 

0.480 

0.511 

0.191 

0.186 

9 

0.600 

0.619 

0.209 

0.196 

10 

0.733 

0.736 

0.225 

0.207 

11 

0.861 

0.863 

0.245 

0.220 

Length  of  post-abdomen 

Length  of  a  post-abdominal 
segment 

Width  of  12th  segment 

Observed 

Calculated 

Observed 

Calculated 

Observed 

Calculated 

12 

(0.29) 

(0.28) 

(0.041) 

(0.04) 

0.150 

0.147 

13 

(0.325) 

(0.30) 

(0.054) 

(0.05) 

0.160 

0.154 

14 

0.38 

0.36 

0.06 

0.06 

0.170 

0.163 

15 

0.45 

0.45 

0.08 

0.075 

0.185 

0.174 

16 

0.58 

0.57 

0.09 

0.095 

0.190 

0.187 

17 

0.73 

0.72 

0.12 

0.12 

0.200 

0.202 

18 

0.91 

0.90 

0.15 

0.15 

0.210 

0.219 

19 

1.13 

1.1-1- 

0.18 

0.185 

0.230 

0.238 

Taking  for  (Wi  —  C)  and  Aw  the  values  0.002  and  0.001  mm.  respectively,  Wn 
has  been  calculated  for  successive  values  of  n,  and  the  comparison  with  the  observed 
values  is  shown  in  Table  II.  The  percentage  discrepancies  are  greater  than  those 
observed  for  thoracic  length,  but  nevertheless  insignificant  in  view  of  the  greater 
difficulty  of  taking  accurate  measurements  of  entities  of  so  much  smaller  magni- 
tude. It  is  to  be  concluded  that  the  width  of  the  first  thoracic  segment  grows 
similarly  as  the  length  of  the  thorax,  i.e.,  by  adding,  in  each  stage,  another  term 


ARTEMIA  SEGMENTATION  PATTERN  129 

of  an  arithmetical  series  in  which  consecutive  terms  differ  by  a  constant  amount  Aw. 
It  must  now  be  shown  that  other  thoracic  segments  also  increase  in  width 
according  to  equations  4  and  5  ;  actual  measurements  for  these  segments  have  not 
been  taken,  but  the  proof  can  be  arrived  at  indirectly.  It  is  known  from  observa- 
tion that  the  lateral  thoracic  contours  are  straight  lines  converging  posteriorly.  The 
angle  of  taper  a  (Fig.  2)  is  always  expressed  by 

Wn-C 

tan  a  =        ?T  (6) 

LL  „ 

and  this  angle,  on  calculation,  is  seen  to  be  very  nearly  constant  for  successive 
values  of  n.  For  n—\  and  n  —  11,  tan  a  equals  0.033  and  0.045  respectively; 
the  average  from  all  eleven  values  is  0.039,  corresponding  to  an  angle  of  2°  18', 
±  15'.  Since  the  contours  are  then  straight  lines,  with  a  constant  taper  in  all 
thoracic  stages,  the  taper  of  individual  segments  must  also  be  constant  and  identical, 
i.e.,  (IV  n  --  Wn-\)/2TSn  .',  as  the  length  TSV  of  a  given  segment  in  a  given  stage 
can  be  shown  to  be  equal  to  the  length,  in  the  preceding  stage,  of  the  segment 
immediately  anterior  to  it,  an  analogous  equality  must  obtain  for  the  width  of  a 
segment,  for  the  taper  in  each  case  must  be  identical.  In  other  words,  when  the 
width  of  the  first  segment  is  Wn,  the  width  of  the  succeeding  segment  is  Wn_\, 
in  the  same  stage;  this  proves  however,  by  extension,  that  all  thoracic  segments 
must  increase  in  a  manner  identical  to  the  first,  since  Wn  and  Wn-\  represent  sums 
of  the  same  arithmetical  series  as  that  in  equation  5,  Wn  containing  one  term  more 
than  Wn-i- 

The  segmental  abdomen  during  the  thoracic  phase  maintains  a  constant  length 
(A  =  0.249  mm.)  and  a  constant  anterior  width  (C  =  0.142  mm.).  The  posterior 
width  W  U,  identical  to  the  "width  of  the  urosome,"  however  increases  (Table  I). 
The  angle  of  taper  ft,  therefore,  expressed  by 

C    - 

~~ 


2A       '- 

decreases.  Stated  in  other  words,  the  convergence  of  the  abdominal  contour-lines 
gradually  diminishes.  A  stage  will  eventually  be  reached  at  which  the  thoracic 
and  abdominal  contours  will  form  continuous  straight  lines,  the  thoracic  contours 
having  a  constant  taper  (equation  6)  ;  at  this  time 


tan  a  =  tan  ft 

and 

Wn-C      C  -  WUn 


(8) 


2Tn  2A 

from  which  WUn  can  be  calculated,  all  other  terms  being  known.  WUn  from 
equation  (8)  is  0.123  mm.;  the  value  of  WUn  closest  to  this  in  Table  I  is  0.125 
mm.  in  stage  11.  It  follows  therefore  that  the  thoracic  and  abdominal  contours 
become  continuous  straight  lines  as  the  end  of  the  thoracic  period  of  development 
is  reached. 

For  analytical  purposes  thoracic  shape  during  the  thoracic  period  can  be  re- 
garded as  a  regular  cone  from  which  the  tip  was  cut  off  (frustrum  of  a  cone). 
Dorso-ventral  extent  at  any  level  would  be  very  nearly  equal  to  the  lateral  width 


130  PAUL  B.  WEISZ 

at  that  level.  The  diameters  of  the  end  faces  of  the  frustrum  can  thus  be  assumed 
to  be  Wn  and  W-L  respectively,  and  since  the  length  of  the  frustrum  is  always  given 
by  Tn,  the  volume  and  the  surface  area  of  the  thorax  can  be  approximated  by  the 
use  of  known  geometrical  formulae.  If  the  volume  V\  of  the  first  segment  is 
known  the  total  thoracic  volume  Vn  at  any  stage  can  also  be  calculated  from  a 
sum-of-a-series  equation,  of  the  general  form 


which  must  obtain,  since  both  length  and  width  changes  are  governed  by  such 
equations.  Furthermore,  As  and  Aw  are  obviously  related  mathematically  to 
Av.  In  sum,  if  the  initial  size  and  shape  of  the  thorax  (n—  1),  and  the  .values 
As  and  Aw  are  known,  the  size  and  shape  of  the  thorax  at  any  further  thoracic 
stage  can  be  predicted. 

The  abdominal  phase  of  development 

Abdominal  growth.  —  At  the  beginning  of  the  abdominal  period,  the  segmental 
abdomen  starts  to  grow  in  length,  having  been  constant  before.  During  stages  12 
and  13  the  abdominal  increases  are  0.08  mm.  per  stage,  or  almost  exactly  8  X  As 
(Table  I)  ;  since  the  initial  abdominal  length  at  the  beginning  of  stage  12  (or  at 
the  end  of  stage  11)  is  0.249  mm.  or  approximately  8  X  0.03  m.,  it  follows  that  during 
the  genital  period  each  0.03  mm.  portion  of  the  segmental  abdomen  grows  an 
amount  As  per  stage.  In  other  words,  the  segmental  abdomen  behaves  as  though 
it  were  already  cut  up  into  its  eight  segments,  and  each  of  these  hypothetical  seg- 
ments has  the  same  antero-posterior  growth  potential  as  thoracic  segments  when 
first  laid  down,  viz.,  increasing  As  per  stage  after  having  a  length  of  0.03  mm.  If 
the  12th  segment  were  laid  down  in  the  manner  in  which  thoracic  segments  are 
formed,  it  would  reach  stage  c  at  a  length  of  0.03  mm.  But  after  stage  11  the 
entire  segmental  abdomen  has  already  started  to  grow,  at  a  rate  of  As  per  stage 
per  0.03  mm.  Thus  at  the  end  of  stage  12  when  the  12th  segment  reaches  stage  c, 
it  will  be  0.03  +  As,  or  0.04  mm.  instead  of  0.03  mm.  long  ;  the  entire  segmental 
abdomen  should  then  be  eight  times  0.04,  or  0.32  mm.,  and  the  post-abdomen  0.28 
mm.  long.  Analogously  during  stage  13,  each  0.04  mm.  portion  of  the  segmental 
abdomen  will  now  add  an  amount  As,  so  that  segment  13  when  in  stage  c  will  be 
0.05  mm.  and  the  entire  segmental  abdomen  eight  times  0.05,  or  0.40  mm.  long. 
At  this  point  the  genital  region  should  be  0.10  (2  X  0.05)  mm.  and  the  post-abdomen 
0.30  mm.  long.  Actual  figures  in  Table  I  support  such  an  interpretation  rather 
well,  and  the  conclusion  is  justified  that  during  the  genital  period  the  segmental 
tissue  of  the  abdomen  acquires  the  same  growth  potential  in  length  as  that  of 
equivalent  amounts  of  thoracic  tissue  during  the  thoracic  period. 

The  genital  region  continues  to  grow  in  length  at  the  indicated  rate,  as  the  data 
in  Table  I  tend  to  show.  The  post-abdomen  would  similarly  do  so,  were  it  not 
for  the  fact  that  another  change  in  the  mode  of  growth  occurred  at  the  end  of  stage 
13.  Successive  post-abdominal  lengths  Pm  from  stage  13  on  are  0.325,  0.38,  0.45, 
0.58  mm.  etc.,  in  other  words  the  increments  are  increasing.  A  sum-of-a-series 
expression,  similar  to  that  for  thoracic  width  changes,  fits  these  figures  very 


ARTEMIA  SEGMENTATION  PATTERN  131 

closely,  i.e., 

Pm  -  Fo  =  (Pi  -  Fo)  -m  +  m(m~  1}  •  A/>  (         (10) 

and 


where  Pm  represents  the  total  post-abdominal  length  for  stages  14  to  19  ;  PQ,  the 
initial  length  at  the  end  of  stage  13;  PI,  the  length  at  the  end  of  stage  14;  m,  the 
successive  integers  from  1  to  6;  and  A/>,  the  increments  per  stage  over  the  initial 
increase  (Pi  —  P0).  The  theoretical  value  for  P0  was  previously  seen  to  be  0.30 
mm.,  and  with  0.06  and  0.03  for  (Px  —  P0)  and  A/?  respectively,  the  calculated 
values  for  Pm  compare  well  with  the  observed  ones  (Table  II). 

If  equation  (11)  is  divided  by  six,  the  growth  formula  for  individual  segments 
is  obtained,  since  each  of  these  segments  is  one-sixth  of  the  entire  post-abdomen; 


+  (PS,  -  PS0)  -m  +  •  A(/>)  (12) 

PSo,  PSi,  and  A(/>)  are  0.05,  0.06  and  0.005  mm.  respectively,  and  (PSl  -  PS0) 
is  therefore  0.01,  or  very  closely  As;  thus  the  initial  increase  of  the  presumptive 
segments  14  to  19,  at  the  beginning  of  the  post-abdominal  period,  is  identical  to 
the  increase  of  these  tissues  during  stages  12  and  13,  and  this  increment  is  then 
augmented  by  a  constant  amount  A(/>)  in  each  subsequent  stage.  What  is  re- 
sponsible for  this  change  in  the  mode  of  growth  of  post-abdominal  segments?  It 
is  more  than  likely  that  non-formation  of  appendages  is  related  to  this,  inasmuch 
as  newly  formed  tissue  will  not  be  diverted  for  the  establishment  and  subsequent 
growth  of  appendage  buds  ;  augmented  growth  of  the  segments  would  therefore 
be  facilitated.  It  can  now  be  stated  in  general,  that  while  body  segments  are 
formed,  length  increments  per  stage  for  all  segments  are  constant,  but  the  incre- 
ments may  be  added  to  an  initial  length  as  in  thoracic  and  genital  segments,  or  to 
an  initial  increase  of  length,  as  in  post-abdominal  segments. 

As  in  thoracic  segmentation,  the  anterior  width  of  the  segmental  abdomen  has 
the  constant  value  C  =  0.142  mm.,  during  the  abdominal  period.  This  value  is 
the  anterior  abdominal  width  at  the  end  of  stage  11,  and  the  anterior  width  of  the 
presumptive  13th  segment  at  the  end  of  stage  12.  The  12th  segment,  by  this  time, 
has  attained  a  width  of  0.15  mm.  (Table  I),  and  in  succeeding  stages  this  width 
increases  to  0.16,  0.17,  0.185  mm.  .  .  .  etc.  As  for  thoracic  width  the  increases 
are  found  not  to  be  uniformly  constant,  and  a  sum-of-a-series  expression  again 
approaches  the  data  best,  i.e., 


WAr-C  --r-(WAi-C}  +2        "Awa  (13) 

and 

J^r  =  C  +  r(0Mi  -  C)  +  r(r7  n-A7C'a  (14) 


where  WAr  represents  the  width  of  the  12th  segment  at  a  stage  r  of  the  abdominal 
period;  (WA\  —  C),  the  initial  increase  in  width  during  stage  12;  Awa,  the  in- 
crease in  width,  per  stage,  over  the  increment  during  the  preceding  stage;  and  r, 


132  PAUL  B.  WEISZ 


the  successive  integers  from  1  to  8.  If  for  (IVAi  —  C)  and  Awa  0.005  and 
0.002  mm.  respectively  are  taken,  the  calculated  values  for  WAr  compare  well 
with  the  observed  ones  (Table  II). 

Other  abdominal  segments  can  be  shown  to  follow  a  similar  mode  of  growth 
in  width.  The  lateral  contours  being  straight  lines,  the  angle  of  taper  /?  is 
expressed  by 

WAr  -   WUr 


where  Ar  is  the  length  of  the  entire  segmental  abdomen,  i.e.,  genital  plus  post- 
abdominal  lengths,  and  other  values  as  before.  Tan  /?,  when  calculated  from 
Table  I  for  successive  values  of  r,  centers  about  the  average  of  0.036  ±  0.004  ;  in 
other  words,  the  abdominal  taper  does  not  only  remain  constant  during  the  ab- 
dominal period,  but  this  taper  is  also  practically  identical  with  that  reached  by  the 
segmental  abdomen  at  the  end  of  stage  11  (cf.  above,  equation  8). 

Unlike  thoracic  segments,  which  start  development  at  stage  c  with  the  same 
length  as  that  of  more  anterior  segments  at  stage  c,  the  abdominal  segments  begin 
development  at  a  length  identical  with  that  of  more  anterior  segments  at  the  same 
time.  In  maintaining  a  constant  taper,  the  initial  increase  of  any  presumptive 
thoracic  segment  over  the  width  C  is  always  expressed  by  the  first  term  of  the 
series  applying  to  thoracic  width  (equations  3,  4,  and  5),  and  the  later  a  segment 
arises  the  fewer  terms  of  the  series  can  it  add  to  its  width  during  the  thoracic  period. 
Since  abdominal  segments  have  now  also  been  shown  to  maintain  a  constant  taper, 
and  since  their  lengths  at  stage  c  are  equal  to  those  of  more  anterior  segments 
already  beyond  stage  c,  an  analogous  relation  must  similarly  exist  for  segmental 
width  ;  namely,  the  initial  increase  of  a  presumptive  abdominal  segment  over  the 
width  C  must  be  identical  to  the  width  increase  experienced  by  other  abdominal 
segments  at  the  same  time.  If  (WA]_  --  C)  in  equation  (13)  represents  the  initial 
increase  of  segment  12,  then  (WA2  —  WA\)  would  do  similarly  for  segment  13. 
In  other  words,  the  width  of  both  segments  follow  the  same  series,  but  the  second 
term  for  segment  12  becomes  the  first  term  for  segment  13;  the  third  term  for 
segment  12,  similarly,  becomes  the  first  term  for  segment  14,  etc.,  and  the  eighth 
and  last  term  for  segment  12  is  the  first  and  last  term  for  segment  19.  Thus  as 
with  thoracic  segments,  the  later  an  abdominal  segment  arises  the  fewer  terms  are 
added  to  its  width,  but  while  the  width  increases  of  thoracic  segments  start  with 
the  same  and  end  with  consecutive  terms,  those  of  abdominal  segments  start  with 
consecutive  and  end  with  the  same  terms  of  the  series. 

It  should  be  observed  parenthetically  that  equation  (13)  may  have  a  slightly 
different  constant  Awa  for  the  genital  and  post-abdominal  segments  respectively, 
reflecting  the  different  modes  of  growth  in  length  of  these  two  groups  of  segments  ; 
or,  if  the  constant  is  actually  identical  the  lateral  abdominal  contour  would  theo- 
retically not  be  an  exact  continuous  straight  line,  but  rather  two  straight  lines  with 
slightly  different  taper,  joined  between  segments  13  and  14.  In  either  case,  the 
difference  would  be  so  small  as  to  be  unnoticeable  in  practice  ;  with  the  present 
techniques  of  observation  and  measurement,  a  single  series  relation  holds  for  both 
groups  of  segments,  and  even  if  two  separate  series  could  be  established  with  finer 
means,  the  principle  of  growth  in  width  as  outlined  above  would  nevertheless  hold. 

As  for  segmental  growth  in  length,  a  general  conclusion  can  now  be  stated  for 


ARTEMIA  SEGMENTATION  PATTERN  133 

growth  in  width,  viz.,  width  increments  per  stage  for  all  body  segments  are  con- 
stant, and  the  increments  are  always  added  to  an  initial  increase  in  width.  The 
combined  generalization  is  also  true,  that  total  segmental  mass  increments  per  stage 
are  constant,  and  the  increments  are  added  either  to  initial  masses  or  to  initial 
increases  of  mass. 

Thoracic  growth. — One  of  four  possible  reasons  could  a  priori  be  advanced 
in  an  attempt  to  account  for  the  differential  size  changes  in  the  thorax,  such  that 
the  5th  segment  ultimately  becomes  largest,  during  the  abdominal  period:  i.e., 
either  the  segment  rudiments  in  stage  a  differ  in  initial  size  but  follow  the  same 
growth  curves ;  or  the  analogous  converse ;  or  either  the  rudiments  have  both  equal 
initial  size  and  identical  growth  curves ;  or  the  analogous  negative.  Since  for  all 
thoracic  segments  four  stage-intervals  elapse  between  stage  a  and  stage  c,  length 
and  width  magnitudes  at  stage  c  are  identical,  and  the  increments  per  stage,  no 
matter  at  which  segmental  stage,  are  identical  (i.e.,  As),  only  the  conclusion  is 
admissible  that  thoracic  segment  rudiments  have  equal  initial  sizes  and  follow 
growth  curves  of  the  same  shape.  Under  such  conditions  there  are  two  factors 
which  must  be  held  responsible  for  the  observed  growth  of  thoracic  segments,  i.e., 
the  time  lag  in  the  formation  of  consecutive  segments,  and  segmental  age.  The 
time  lag  fully  accounts  for  the  regular  gradation  of  segmental  sizes  at  the  end  of 
the  thoracic  period  and  for  the  constant  taper  of  the  thorax ;  as  will  be  demon- 
strated below,  the  influence  of  this  original  time  lag  carries  over  importantly  into 
the  abdominal  phase,  and  this,  together  with  the  factor  of  segmental  age,  can  indeed 
be  made  the  basis  for  a  consistent  interpretation  of  the  manner  of  thoracic  growth. 

Data  in  Table  I  show  that  thoracic  length  remains  constant  during  the  genital 
phase.  Hereafter  the  values  for  length  fit  the  equation 

r/-r-i  /  T->  T      \  1%  \  1H  1  /       i  f  1  S-  \ 

m-:  Tm_!  +  (7\  -  •  T0)m  -          -^-    -  As  (16) 

where  T0  and  7\  represent  thoracic  length  at  the  end  of  stage  11  and  stage  14 
respectively,  and  m,  as  before,  the  integers  from  1  to  6.  With  0.86  and  0.92  mm. 
for  TO  and  7\,  and  As  as  before,  successive  calculated  values  for  T,,,  are  0.92,  1.03, 
1.18,  1.36,  1.56,  and  1.77  mm.,  significantly  close  to  the  observed  data;  the  in- 
creases per  stage  are  therefore  0.06,  0.11,  0.15,  0.18,  0.20,  and  0.21  mm.,  and  the 
differences  between  the  increases  are  seen  to  diminish  in  a  regular  manner. 

The  scheme  in  Table  III  will  account  for  such  a  series  of  increases.  The 
figures  in  this  table  represent  multiples  of  As  and  they  show  the  length  increase 
of  the  indicated  segment  during  the  indicated  stage.  Sums  of  figures  in  vertical 
rows,  multiplied  by  As,  indicate  the  increases  of  the  entire  thorax  during  the  given 
stages,  and  successive  sums  are  seen  to  be  equal  to  the  values  for  the  increases  per 
stage  as  calculated  from  equation  (16).  Horizontal  sums,  multiplied  by  As,  give 
the  total  increments  of  any  thoracic  segment  during  the  post-abdominal  period. 
This  scheme  is  reproduced  somewhat  differently  in  Table  IV,  in  which  the  figures, 
multiplied  by  As,  indicate  directly  the  size  of  any  of  the  19  body  segments  at  any 
of  the  19  developmental  stages ;  vertical  sums  have  meanings  analogous  to  equiva  • 
lent  sums  in  Table  III. 

It  will  be  observed  that  all  formulae  previously  deduced  in  connection  with 
length  increases  are  inherent  in  the  figures  in  Table  IV ;  observational  data  are 


134 


PAUL  B.  WEISZ 


also  incorporated.  For  example,  the  first  segment  when  reaching  maturity  in 
stage  7  has  a  length  of  0.09  mm.  (cf.  Tahle  I).  Succeeding  thoracic  segments 
must  also  mature  at  this  size,  in  consequence  to  the  equality  of  their  growth  curves ; 
thus  segment  5  is  shown  to  mature  in  stage  11  when  the  19th  segment  appears  in 
stage  a,  and  segment  11  in  stage  19,  in  conformity  to  the  observational  data  in 
Table  I.  The  scheme  in  Table  IV  also  shows  well  the  successive  segmental  pro- 
portions in  the  thorax  during  the  abdominal  phase.  In  stage  16,  segments  1  and  2 
are  longest,  in  stage  17  similarly  segments  2  and  3,  etc. ;  maximal  segmental  length 
thus  shifts  caudad,  fully  corroborating  observation. 

Segmental -growth  of  the  thorax  as  indicated  in  the  table  can  be  interpreted 
provided  two  assumptions  are  postulated,  i.e.,  (a)  a  segment  can  no  longer  grow 
by  regularly  increasing  amounts  after  having  passed  through  14  segmental  stages, 

TABLE  III 

Scheme  of  segmental  increments,  in  multiples  of  As,  in  the  thorax  during  the  post-abdominal 

phase  of  development 


Stage 

Total 

14 

15 

16 

17 

18 

19 

increases 

Segment 

1 

0 

1 

1 

1 

1 

1 

5 

2 

0 

1 

2 

2 

2 

2 

9 

3 

0 

1 

2 

3 

3 

3 

12 

4 

0 

1 

2 

3 

4 

4 

14 

5 

0 

1 

2 

3 

4 

5 

15 

6 

1 

1 

1 

1 

1 

1 

6 

7 

1 

1 

1 

1 

1 

1 

6 

8 

1 

1 

1 

1 

1 

1 

6 

9 

1 

1 

1 

1 

1 

1 

6 

10 

1 

1 

1 

1 

1 

1 

6 

11 

1 

1 

1 

1 

1 

1 

6 

Total 

thoracic 

increases 

6 

11 

15 

18 

20 

21 

counted  from  stage  c,  and  (b)  a  segment,  in  order  to  grow  by  increasing  amounts 
at  all,  must  have  matured  within  the  first  6  segmental  stages  of  its  existence, 
counted  from  stage  r.  These  two  provisions  constitute  the  limiting  conditions  of 
segmental  age. 

Table  IV  reveals  that  only  the  first  5  segments  fulfill  the  second  condition ; 
segments  6  to  1 1  would  also  have  matured  in  6  stages  of  their  individual  existence, 
were  it  not  for  the  fact  that  no  thoracic  growth  takes  place  during  the  genital  period, 
and  maturation  of  the  posterior  thoracic  segments  is  therefore  delayed  by  two 
stages.  Thus  only  the  first  five  segments  would  be  able  to  grow  by  increasing 
amounts,  whenever  such  growth  was  made  possible.  It  has  been  shown  previously 
that  at  the  beginning  of  the  post-abdominal  phase,  the  post-abdomen  ceases  to  grow 
by  constant  increments  and  begins  growth  by  increasing  increments,  with  an  initial 


ARTEMIA  SEGMENTATION  PATTERN 


135 


increase  during  stage  14  equal  to  that  of  stage  13.  Apparently  the  phenomenon  of 
increasing  increments  at  this  time  is  not  confined  to  the  post-abdomen  but  also 
affects  thoracic  segments,  subject  to  the  limiting  provisions  stated  above.  Thus 
the  first  five  segments  have  an  initial  increase  equal  to  the  increment  during  stage 
13,  viz.,  0;  segments  6  to  11,  not  fulfilling  condition  (b),  simply  continue  at  their 
former  constant  rates,  viz.,  As  per  stage  (cf.  data  in  Table  III,  under  increases 
during  stage  14).  From  here  on,  the  first  five  segments  augment  their  increases 
by  As  in  every  stage,  until  their  14th  segmental  stage  is  passed ;  then,  by  assump- 
tion, the  increment  of  the  14th  segmental  stage  can  no  longer  be  augmented,  but 

TABLE  IV 

Scheme  of  growth  of  body  segments,  in  multiples  of  A.v 
(a  refers  to  segmental  stage  a  of  any  given  segment) 


Stage 

• 

Thoracic  phase 

Genital 
phase 

Post-abdominal 

phase 

1234567 

8 

9 

10 

11            12       13 

14 

15 

16 

17 

18 

19 

Seg- 

ment 

1 

3456789 

10 

11 

12 

13 

13 

14 

15 

16 

17 

18 

2 

345678 

9 

10 

11 

12 

12 

13 

15 

17 

19 

21 

3 

34567 

8 

9 

10 

11 

11 

12 

14 

17 

20 

23 

4 

3456 

7 

8 

9 

10 

10 

11 

13 

16 

20 

24 

5 

a                        345 

6 

7 

8 

9 

9 

10 

12 

15 

19 

24 

6 

a                        34 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

7 

a                        3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

8 

a 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

9 

a 

3 

4.       5 

6 

7 

8 

9 

10 

11 

10 

a 

3 

4 

5 

6 

7 

8 

9 

10 

11 

a 

3 

4 

5 

6 

7 

8 

9 

12 

(a) 

4       5 

6 

7 

8 

9 

10 

11 

13 

a 

5 

6 

7 

8 

9 

10 

11 

14 

(a) 

6 

7.5 

9.5 

12 

15 

18.5 

15 

a 

7.5 

9.5 

12 

15 

18.5 

16 

(a) 

9.5 

12 

15 

18.5 

17 

a 

12 

15 

18.5 

18 

(a) 

15 

18.5 

19 

a 

18.5 

is  retained  as  a  constant  increment  till  growth  stops  altogether.  Thus  segment 
one  has  increased  its  increment  of  zero  by  As  at  the  end  of  stage  15  (Tables  III 
and  IV)  ;  but  at  this  point  its  14th  segmental  stage  has  already  been  passed  and 
hereafter  only  a-constant  increment  of  As  per  stage  is  possible.  Segment  2  on  the 
other  hand  is  younger  than  segment  one,  being  laid  down  in  stage  c  with  a  time 
lag  of  one  developmental  stage.  By  the  end  of  stage  15,  therefore,  when  segment 
one  has  just  passed  its  14th  segmental  stage,  segment  2  has  only  passed  its  13th 
segmental  stage  and  its  increment  of  As  during  stage  15  can  be  augmented  once 
more  by  As;  when  the  2nd  segment  has  passed  its  14th  segmental  stage,  its  in- 


136  PAUL  B.  WEISZ 

creases  in  subsequent  stages  will  therefore  be  2  As  per  stage.  Similarly,  segments 
3,  4,  and  5,  each  being  one  stage  younger  than  the  preceding  segment,  are  able  to 
augment  their  increments  by  As  3,  4,  and  5  times  respectively,  before  they  com- 
plete the  14th  segmental  stage.  Segment  5  in  consequence  is  as  long  as  segment 
4  at  the  end  of  stage  19,  but  the  former  will  continue  to  grow  at  a  rate  of  5  As 
per  stage,  while  the  rate  of  the  latter  can  only  be  4  As  per  stage ;  at  any  time  after 
stage  19  therefore  the  fifth  segment  will  be  longest. 

Analogous  changes  occur  with  regard  to  thoracic  growth  in  width,  and  the 
thoracic  cone-frustrum  of  stage  11  gradually  assumes  the  shape  of  a  barrel,  with 
the  "waist"  at  segment  5  after  stage  19.  The  dorsal  thoracic  curvature  of  the 
animal,  arising  similarly  after  stage  11,  can  also  be  interpreted  as  a  result  of 
differential  segmental  increases  in  a  dorsal  direction,  according  to  a  scheme  resem- 
bling that  in  Table  III. 

The  genital  segments  have  been  noted  to  mature,  i.e.,  to  form  a  broodpouch,  in 
stage  18.  Table  IV  reveals  that  at  the  end  of  this  stage,  segment  12  has  just  com- 
pleted its  6th,  and  segment  13,  its  5th  stage  of  segmental  development,  counted 
from  stage  c.  Thus  both  segments  fulfill  one  of  the  two  age  conditions  assumed 
for  thoracic  segments ;  the  fulfillment  of  the  other  might  be  expected.  Observa- 
tion proves  that  this  is  actually  so.  Genital  segments  of  older  larvae  are  known 
to  bulge  considerably  beyond  the  general  abdominal  contour,  giving  them  a  knobby 
appearance.  This  could  not  be  possible  if  the  constant  increases  observed  up  to 
stage  18  were  maintained  any  further;  rather,  after  stage  18  the  initial  increment 
of  an  increasing  rate  will  again  be  equal  to  the  increase  during  the  stage  just 
passed,  viz.,  As,  and  during  a  20th  stage  this  increment  will  be  augmented  by  a 
given  amount,  during  a  21st  stage  by  twice  this  amount,  etc.,  till  the  14th  seg- 
mental stage  is  passed. 

The  post-abdominal  segments  have  previously  been  shown  to  grow  by  regu- 
larly augmented  increases  as  soon  as  they  afe  laid  down.  But  since  these  segments 
bear  no  appendages,  stage  c  for  them  is  equivalent  to  attainment  of  maturity,  as 
already  observed  above.  Maturity  thus  proves  to  be  an  important  temporal 
threshold  for  all  body  segments,  and  the  statement  that  augmented  growth  will 
occur  in  any  mature  segment,  provided  maturity  was  reached  in  a  definite  time, 
has  general  application ;  the  concept  of  segmental  maturity  is  apparently  not  only 
a  working  hypothesis,  as  has  been  assumed  at  the  start,  but  seems  to  have  real 
biological  meaning. 

There  is  no  doubt  that  the  scheme  of  growth  here  presented  describes  correctly 
the  actual  events  of  later  thoracic  development ;  but  the  assumptions,  while  justified 
by  the  interpretations  they  allow,  still  remain  to  be  explained.  Only  tissue  culture 
studies  will  be  able  to  reveal  why  segments  not  matured  in  the  first  6  stages  of 
existence  are  at  too  early  a  stage  of  development,  and  why  segments  after  14  stages 
of  existence  are  at  too  advanced  a  stage  to  do  more  than  keep  up  a  constant  rate. 

Growth  of  appendages;  integration  oj  segmental  development 

In  the  preceding  section  it  has  been  reasoned  that  segment  rudiments  in  the 
thorax  have  equal  initial  size  and  identical  growth  curves;  observation  tends  to 
confirm  not  only  this  but  also  that  equal-sized  rudiments  develop  for  all  body 
segments.  It  can  be  assumed  that  in  these  rudiments  certain  tissue  masses  (ap- 


ARTEMIA  SEGMENTATION  PATTERN  137 

pendage  rudiments),  initially  also  of  equal  size  and  of  equal  growth  capacity  in 
equal  times,  differentiate  independently  towards  the  establishment  of  appendage 
buds.  Such  buds  however  never  appear  in  the  post-abdomen,  and  when  they 
appear  in  other  regions  they  may  develop  into  swimming  appendages  or  into  a 
broodpouch.  In  the  evidence  presented  in  Tables  I  and  IV,  an  important  clue 
can  be  found  to  at  least  one  of  the  factors  preventing  serial  analogy  despite  the 
observed  serial  homology  in  appendageal  development. 

Every  thoracic  appendage  rudiment  reaches  the  bud  stage  after  an  interval  of 
four  developmental  stages.  The  segment  as  a  whole  is  at  stage  c  at  this  point, 
and  the  appendage  buds  of  any  thoracic  segment  must  be  of  identical  size,  due  to 
the  identity  of  initial  size  and  of  growth  capacity  for  all  appendage  rudiments,  and 
of  identical  shape,  since  every  thoracic  segment  at  stage  c  has  identical  propor- 
tions. Enough  appendageal  tissue  has  apparently  been  manufactured,  during  the 
four  preceding  stages,  to  initiate  the  development  of  a  swimming  appendage. 

When  a  genital  segment  reaches  stage  c,  4  +  and  5  developmental  stages  have 
elapsed  since  stage  a.  The  appendage  rudiments  therefore  have  time  to  manu- 
facture proportionately  more  appendageal  tissue,  at  the  same  intensity  as  that  of 
thoracic  rudiments.  If  the  genital  segments  in  stage  c  had  larger  sizes,  propor- 
tionate to  the  longer  time  interval  available,  the  appendage  buds  of  genital  segments 
would  have  the  same  size  and  shape  as  those  of  thoracic  segments.  However,  both 
the  length  and  the  width  of  genital  segments  are  greater  in  stage  c  than  the  size 
which  would  be  proportionate  to  the  longer  time  of  formation.  The  length  of 
any  thoracic  segment  when  laid  down  is  0.03  mm.,  for  example,  and  four  develop- 
mental stages  have  elapsed  since  stage  a ;  the  length/time  ratio  is  thus  0.03/4.  In 
genital  segments  this  ratio  is  larger,  viz.,  0.04/4  +  and  0.05/5,  and  analogously  for 
width.  Appendageal  tissue  in  genital  segments  can  therefore  not  be  developed  in 
sufficient  quantity,  in  proportion  to  segmental  size,  to  produce  appendage  buds  of 
dimensions  equal  to  those  of  thoracic  buds,  even  though  more  time  is  available. 
Genital  buds  will  thus  be  relatively  smaller  and  flatter,  and  the  amount  of  appenda- 
geal tissue  manufactured  will  be  spread  more  thinly  over  the  presumptive  appen- 
dage region ;  the  quantity  of  tissue  present  per  unit  area  is  apparently  already 
below  the  threshold  necessary  for  the  formation  of  comparatively  specialized 
swimming  appendages,  and  only  enough  tissue  is  available  to  initiate  the  formation 
of  a  relatively  simple  sac. 

In  post-abdominal  segments  at  stage  c  the  size/time  ratio  becomes  progressively 
larger  still,  and  appendageal  tissue  consequently  cannot  even  accumulate  in  quanti- 
ties sufficient  to  form  a  bud. 

After  the  appendage  buds  are  laid  down,  an  appendage  retains  a  definite  size- 
proportionality  to  the  segment  bearing  it.  When,  for  example,  the  thoracic  con- 
tour is  a  straight  line,  during  the  thoracic  phase  of  development,  the  line  joining 
the  tips  of  the  appendages  on  one  side  is  also  a  straight  line,  and,  as  with  the 
segments  themselves,  the  time  lag  in  bud  formation  accounts  for  the  taper.  Simi- 
larly, as  the  thorax  gradually  becomes  barrel-shaped  in  the  abdominal  phase,  the 
transformation  is  reflected  in  differential  length  increases  in  the  appendages,  and 
when  the  appendageal  tips  on  one  side  of  the  body  are  joined  by  a  line,  the  result 
is  an  analogously  barrel-shaped  contour. 

From  the  above  analyses,  the  following  integrated  sequence  of  events  becomes 
apparent  with  regard  to  segmental  development. 


138  PAUL  B.  WEISZ 

Shortly  before  hatching  segmental  rudiments  of  equal  size  begin  to  be  formed, 
at  a  rate  of  one  per  developmental  stage ;  with  a  time  lag  of  four  stages,  segments 
are  constricted  off  in  posterior  succession,  all  with  constant  initial  sizes  and  in- 
creasing by  a  constant  amount  during  each  stage.  As  the  first  segment  reaches 
maturity,  the  rate  of  segment  rudiment  formation  increases  to  two  per  stage. 
Rudiments  are  laid  down  at  this  rate  till  the  newest  rudiment  appears  at  the 
posterior  end  of  the  segmental  abdomen  which  latter  had  so  'far  maintained  a 
constant  length.  The  last  formed  rudiment  happens  to  be  the  19th  and  by  this 
time,  11  segments  have  been  constricted  off,  five  of  which  have  already  matured. 

The  process  of  rudiment  deposition  and  segment  constriction  could  be  assumed 
to  go  on  at  length,  were  it  not  for  the  fact  that  the  "end"  of  the  animal  has  been 
reached.  This  is  apparently  the  cue  for  a  general  change  in  the  mode  of  growth. 
The  entire  segmental  abdomen  begins  growth,  increasing  as  yet  equal  amounts 
per  stage,  and  the  thorax  ceases  to  grow.  After  two  genital  segments  of  equal 
size  are  formed  another  general  change  occurs*  to  the  effect  that  hereafter  any 
segment  maturing  within  a  definite  time  may  grow  by  augmented  increases,  as 
described  in  detail  above.  This  type  of  growth  is  maintained,  in  each  segment  in 
which  it  takes  place  until  the  14th  segmental  stage  is  passed,  whereupon  the  total 
increment  of  the  14th  stage  is  reproduced  without  further  increase  in  each  suc- 
ceeding stage.  Segments  not  matured  within  the  required  time  continue  to  grow 
by  constant  increments.  The  eventual  result  of  this  varied  manner  of  growth, 
maintained  up  to  sexual  maturity,  is  the  barrel-shape  of  the  thorax,  the  presence 
of  a  dorsal  thoracic  curvature,  the  knobby  appearance  of  the  broodpouch  seg- 
ments, etc. 

DISCUSSION 

Throughout  the  present  analysis  of  metamerism  in  Artemia  salina,  the  time 
scale  employed  was  that  of  developmental  stages,  defined  as  the  number  of  body 
segments  present.  It  must  be  eminently  realized  that  this  is  a  scale  of  relative, 
biological  time.  Events  in  nature  take  place  in  a  space-time  continuum,  and  to 
Artemia  equivalent  happenings  in  space,  i.e.,  the  establishment  of  segments,  must 
be  correlated  to  the  passage  of  equivalent  units  of  (relative)  time,  i.e.,  what  here 
had  been  called  "stages."  In  hours  and  minutes,  segment  formation  occurs  of 
course  not  in  equivalent  times,  since  the  phenomenon  is  dependent  on  the  environ- 
ment on  the  one  hand,  and  on  changes  in  growth  rates  with  age  on  the  other. 
Artemia  and  other  similarly  primitive  forms  are  particularly  suited  for  a  ready 
identification  of  relative  time,  but  in  segmented  animals  of  greater  complexity,  as 
well  as  in  non-segmented  groups,  "equivalent  happenings  in  space"  cannot  be  picked 
out  with  comparative  ease,  and  it  will  be  more  difficult  to  tell  what  the  relative  time 
scale  actually  is ;  but  that  it  is  intrinsically  present  in  biological  phenomena  has 
already  been  acknowledged  by  others.  Thus  Needham  (1942),  after  briefly  re- 
viewing the  pertinent  literature,  states : 

"Mouse  time  must  bear  the  same,  or  a  similar,  relation  to  elephant  time  as 
mouse  spatial  magnitudes  to  elephant  spatial  magnitudes.  Indeed,  unless  the 
time  factor  is  brought  into  account,  we  may  understand  morphological  similarity, 
but  we  can  never  hope  to  understand  physiological,  still  less  embryological, 
similarity." 


ARTEMIA  SEGMENTATION  PATTERN  139 

t 

Measurements  on  Artemia  in  absolute  time  would  never  have  brought  to  light 
the  truly  amazing  simplicity  of  the  laws  of  segment  formation,  as  given  by  the  series 
and  the  sum-of-series  formulae,  and  in  terms  of  relative  time  these  formulae  as- 
sume a  simple  biological  meaning,  viz.,  (a)  that  equivalent  spatial  events  take 
place  during  equivalent  relative  times,  and  also  (b)  that  equivalent  spatial  events 
take  place  in  tissues  of  equivalent  relative  age.  For  illustration,  the  thorax  during 
the  thoracic  period  of  development  may  be  considered,  where  the  increments  per 
stage  of  (TSi  --  TS0)  and  (W\  --  C)  (equations  1  and  3)  are  indeed  equivalent 
and  constant,  and  where  every  other  segment  grows  similarly  in  this  same  manner ; 
summation  of  the  increments  must  then  result  in  the  sum-of-series  expression. 
Analogous  interpretations,  based  on  the  idea  of  spatial  and  temporal  equivalence 
can  be  adduced  in  every  other  case  in  which  the  formulae  hold,  i.e.,  virtually  for 
the  entire  period  of  segmental  development.  Before  and  after  this  period,  relative 
time  is  of  course  still  operative,  but  its  expression  is  latent,  inasmuch  as  its  passage 
is  not  paralleled  by  morphological  events  clearly  identified  as  equivalent.  The 
same  would  be  true  for  the  majority  of  living  organisms,  but  it  can  be  asserted 
with  a  fair  amount  of  logical  conviction,  that  if  and  when  it  will  be  possible  to 
make  explicit  the  relative  time  scales  of  living  organisms  as  a  whole,  size  incre- 
ments in  relative  time  units  will  be  found  to  be  equivalent,  and  series  formulae  of 
linear,  quadratic,  and  perhaps  even  of  higher  degree  will  be  found  to  hold. 

It  should  in  general  be  useful  to  have  a  specific  term  to  distinguish  relative 
biological  time  from  absolute  duration ;  the  concept  as  a  whole  might  be  called 
"biochronism,"  and  the  relative  time  scale  could  be  said  to  have  one  "biochron" 
as  its  unit.  Also,  in  order  to  transcend  the  usual  connotations  of  "growth  rate," 
"biochronal  rate"  could  be  substituted.  Whenever  in  the  text  above  "increase  per 
developmental  stage"  was  mentioned,  "increase  per  biochron"  was  really  implied. 
In  this  connection,  the  type  of  analysis  in  the  present  report  is  clearly  different 
from  "allometric,"  "heterauxetic,"  or  "heterogonic"  inquiries.  The  term  "mor- 
phometry"  is  suggested  to  indicate  generally  any  quantitative  appreciation  of  or- 
ganic size,  shape,  and  time  as  an  integrated  dynamic  pattern.  While  it  is  realized 
that  apologies  are  in  order,  more  or  less  categorically,  for  the  introduction  of  any 
new  term  into  present-day  biology,  it  should  be  kept  in  mind  that  new  terms  become 
unavoidable  as  different  methods  of  inquiry  and  fresh  fields  of  study  appear. 

Two  immediate  issues  have  not  been  touched  on  at  all  in  the  present  analysis. 
First,  what  determines  the  changes  in  the  mode  of  segmental  growth  at  the  end 
of  both  the  thoracic  and  the  genital  periods?  That  the  changes  occur  is  fairly 
definitely  established,  and  this  would  support  the  view  that  division  of  segmental 
development  into  periods  is  real,  i.e.,  physiological  as  well  as  morphological.  But 
beyond  that,  speculation  into  the  nature  and  history  of  the  changes  would  be  futile, 
for  lack  of  direct  evidence.  Secondly,  and  this  is  the  fundamental  question  in  the 
study  of  metamerism,  why  are  segments  formed  at  all?  It  will  readily  be  ad- 
mitted that  even  an  attempt  to  answer  this  problem  can  only  be  made  after  a  great 
deal  more  is  known  about  segmentation  phenomena  as  a  whole. 

Excepting  these  two  questions  however,  the  final  size  and  shape  of  Artemia 
nevertheless  has  here  been  accounted  for  in  terms  of  initial  body  proportions  much 
as  Berrill  (1941)  has  done  for  the  ascidian,  Botryllus.  When  copepods,  crayfish, 
and  other  diverse  crustacean  forms  oE  higher  evolutionary  rank  are  considered, 
similarly  in  possession  of  a  barrel-shaped  thorax  and  a  straight  tapering  abdomen, 


140  PAUL  B.  WEISZ 

it  is  perhaps  justifiable  to  reflect  that  Crustacea  as  a  group  might  have  evolved 
with  a  single  and  basic  geometrical  pattern  of  growth. 

SUMMARY 

1.  Growth  and  the  dynamic  pattern  of  segment  formation  in  excysted  larvae  of 
Artemia  salina  have  been  quantitatively  studied.     The  final  shape  of  Artemia  at 
sexual  maturity  can  be  accounted  for  in  terms  of  initial  shape  at  hatching. 

2.  In   analyzing   the  pattern   of   metamerism,   the   stages   of   development  are 
gauged  by  the  number  of  body  segments  present.     Growth  during  the  entire  period 
of  segment  formation  is  found  to  be  governed  by  arithmetical  series  and  sum-of- 
series  relations,  implying  that  growth  increments  per  stage  over  either  initial  sizes 
or  initial  increases  are  constant  and  identical  for  thoracic,  genital,  and  abdominal 
segments,   respectively.     Later  transformations  of  larval   shape,   resulting   in  the 
barrel-shape  of  the  thorax,  the  presence  of  a  dorsal  thoracic  curvature,  the  knobby 
appearance  of  the  genital  segments,  and  the  presence  of  a  straight  tapering  ab- 
domen, are  accounted  for  analytically  on  the  basis  of  concepts  concerning  the  age 
of  segments  and  the  time  lag  involved  in  segment  formation. 

3.  The  presence,  absence,  and  the  difference  of  structure  of  appendages  are 
shown  to  be  determined,  at  least  in  part,  by  the  size  of  segments  when  first  laid 
down,  and  by  the  time  available  for  appendage  rudiments  to  form  appendageal 
tissues.. 

4.  The  time  scale  employed   in   the  analysis   of  the   segmentation   pattern  in 
Artemia  is  interpreted  to  be  a  relative,  biological  one,  and  the  meaning  of  the  series 
formulae  with  regard  to  this   relative  scale  is   illustrated.     The  notion   of  "bio- 
chronism"  is  introduced,   as  a  general   concept  applying  to  biological   events   in 
relative  time. 

LITERATURE  CITED 

BARIGOZZI,  C,   1939.     La  biologia  di  Artemia  salina  Leach   studiata  aquario.    Atti  Soc.  Ital. 

Sci.  Nat.,  78 :  137-160. 

BERRILL,  N.  J.,  1941.     Size  and  morphogenesis  in  the  bud  of  Botryllus.     Biol.  Bull,  80:  185-193. 
BOND,  R.  M.,  1932.     Observations  on  Artemia  "franciscana"  Kellogg,  especially  on  the  relation 

of  environment  to  morphology.  Int.  Rev.  dcr  gcs.  Hydrobiol.  u.  Hydrogr.,  28:  117-125. 
HEATH,  H.,  1924.  The  external  development  of  certain  phyllopods.  Jour.  Morph.,  38 :  453-483. 
JENNINGS,  R.  H.  AND  D.  M.  WHITAKER,  1941.  The  effect  of  salinity  upon  the  rate  of  excyst- 

ment  of  Artemia.     Biol.  Bull,  80:  194-201. 

LOCKHEAD,  J.  H.,  1941.     Artemia,  the  brine  shrimp.     Turtox  'News,  19:  41—45. 
NEEDHAM,  J.,  1942.    Biochemistry  and  morphogenesis.     Cambr.  Univ.  Press,  Cambridge,  1942 

(p.  561). 

RUGH,  R.,  1941.    Experimental  embryology.     New  York  Univ.  Press,  N.  Y.,  1941    (p.  206). 
WARREN,  H.  S.,  1938.     The  segmental  excretory  glands  of  Artemia  salina  Linn.  var.  principalis 

Simon   (the  brine  shrimp).    Jour.  Morph.,  62:  263-289. 
WHITAKER,  D.  M.,  1940.     The  tolerance  of  Artemia  cysts  for  cold  and  high  vacuum.     Jour. 

E.rp.  Zoo/.  83 :  391-399. 


ELECTRON  MICROSCOPE  OBSERVATIONS  OF  THE 
TRICHOCYSTS  AND  CILIA  OF  PARAMECIUM 

M.  A.  JAKUS  AND  C.  E.  HALL 

/ V/></r///;<'»/  /if  Binliiiiy.  Massachusetts  Institute  of   Tcclui/>!o//y,   Cambridge,  .Massachusetts 

In  previous  publications,  electron  micrographs  have  been  shown  of  trichocysts 
(Jakus,  1945)  and  of  cilia  (Schmitt,  Hall,  and  Jakus,  1943).  Recently  we  have 
re-examined  both  these  organelles  using  the  shadow-casting  technique  of  Williams 
and  Wyckoff  (1945).  The  new  technique  shows  structural  detail  with  improved 
clarity  and  reveals  some  features  not  previously  visible  in  specimens  prepared  in 
the  conventional  manner. 

TUNGSTEN    FILAMENT 
WITH    METAL 


METAL  DEPOSIT 

^    COLLODION    FILM 
"SHADOW" 

FIGUKE  1.     Diagram  uf  shadow-casting  technique. 

The  shadow-casting  technique  is  illustrated  diagrammatically  in  Figure  1.  A 
specimen  is  placed  in  a  vacuum  bell- jar  containing  a  conical  tungsten  filament  in 
which  are  placed  some  small  pieces  of  a  suitable  metal  such  as  chromium.  When 
the  filament  is  raised  to  a  high  temperature  by  the  passage  of  an  electric  current 
the  metal  evaporates,  travelling  in  straight  lines  and  depositing  on  the  specimen  as 
indicated.  Structures  projecting  above  the  surface  of  the  supporting  film  cast  per- 
manent shadows  to  the  "leeward"  and  intercept  metal  to  the  "windward."  Speci- 
mens are  then  examined  in  the  electron  microscope  in  the  usual  manner.  In  posi- 
tive prints  the  shadows  appear  bright  because  they  represent  relatively  transparent 
regions  in  the  object.  It  is  customary,  therefore,  to  prepare  micrographs  as  nega- 
tive prints  so  that  the  shadows  will  appear  darker  than  the  background. 

TRICHOCYSTS 

The  structure  and  properties  of  the  trichocysts  of  Paramecium  have  been  de- 
scribed in  a  previous  paper  (Jakus,  1945).  In  electron  micrographs,  the  discharged 
trichocyst  consists  of  a  sharply-pointed  tip  and  an  elongated,  cross-striated  shaft 
with  a  periodicity  of  about  550  A.  The  cross-striated  structure  appears  to  be  a 

141 


142  M.  A.  JAKUS  AND  C.  E.  HALL 

thin  membrane  formed  by  the  lateral  aggregation  of  fine  fibrils.  The  tip,  in  con- 
trast to  the  shaft,  is  quite  opaque.  The  reason  for  this  opacity  was  not  obvious. 

Further  information  about  the  morphology  of  the  dried  extruded  trichocyst  is 
obtained  from  electron  micrographs  of  shadowed  specimens  (Fig.  2).  The  tip  is  seen 
to  be  a  compact  structure  which  stands  up  from  the  film  and  is  not  flattened  to  any 
great  extent  as  a  result  of  dehydration.  The  contour  of  its  shadow  indicates  that 
it  is  shaped  somewhat  like  a  golf  tee.  In  contrast  to  the  tip,  the  dried  shaft  is  very 
flat,  as  is  evident  from  the  short  shadow  it  casts.  The  cross  striations  previously 
observed  are  enhanced  by  the  metal,  indicating  that  the  surface  has  a  regularly  cor- 
rugated contour.  The  elevated  regions  correspond  to  the  darker  bands  in  both  un- 
treated trichocysts  and  those  stained  with  phosphotungstic  acid.  Other  details  of 
structure  observed  previously  may  also  be  found  in  some  shadowed  trichocysts. 
These  are  the  fine  longitudinal  striations  of  the  shaft  membrane  and  the  larger 
periodicity  (2.200  A)  frequently  noted  along  the  shaft.  The  latter  may  appear 
simply  as  a  slight  further  intensification  of  every  fourth  dark  band,  suggesting  that 
these  ridges  have  a  somewhat  higher  elevation  than  do  the  others. 

In  some  specimens  the  pointed  tip  appears  regularly  cross-striated,  if  the  amount 
of  metal  deposited  has  not  been  excessive  and  the  orientation  of  the  tip  is  approxi- 
mately parallel  to  the  direction  of  deposition.  This  banding  has  not  been  seen  in 
either  stained  or  unstained  specimens  and,  while  it  is  readily  visible  in  the  original 
micrographs  of  shadowed  tips,  it  is  not  considered  to  be  of  sufficient  clarity  for  re- 
production. Although  relatively  constant  in  any  one  tip,  the  spacing  varied  from 
280  to  365  A  in  the  different  tips  measured  and  had  an  average  value  of  about  300  A. 
This  is  to  be  compared  with  the  average  period  of  about  550  A  in  the  trichocyst 
shaft. 

>- 

CILIA 

The  cilia  of  Paramecium  are  shed  quite  readily  if  the  cell  is  injured  and  both 
intact  cilia  and  fragments  are  observed  frequently  in  preparations  of  trichocysts. 
Each  cilium  consists  of  a  bundle  of  fibrils  (about  eleven  in  number),  extending  the 
full  length  of  the  cilium  (Fig.  3).  The  diameter  of  the  dried  fibrils  lies  between 
300  and  500  A.  It  may  be  of  significance  that  both  the  number  of  fibrils  and  their 
diameter  are  in  close  agreement  with  the  corresponding  values  observed  in  the  sperm 
tails  of  numerous  animal  forms  ( Schmitt,  Hall,  and  Jakus.  1943). 

In  fixed  preparations  (for  example,  with  OsO4),  the  component  fibrils  usually 
adhere  to  form  a  compact  bundle,  while  in  unfixed  cilia  they  separate  to  a  greater 
or  lesser  extent.  They  are  clearly  defined  in  shadowed  specimens.  Usually  the 
separation  of  fibrils  is  not  complete  and  they  remain  in  close  contact  near  the  end  of 
the  cilium  which  was  attached  to  the  cell.  Here  they  appear  sometimes  to  be  joined 
into  two  closely  adjacent  bundles. 

It  is  not  evident  what  holds  the  fibrils  together  in  the  living  cilium.  No  spiral 
sheath  similar  to  that  observed  in  mammalian  sperm  tails  (Schmitt.  Hall,  and  Jakus, 
1943)  or  in  Euglena  flagella  (Brown.  1945)  has  been  seen.  If  a  sheath  does  exist, 
it  must  be  very  fragile  and  easily  ruptured.  In  some  cilia,  a  rather  poorly-defined 
cross-striation  has  been  noted,  particularly  in  two  or  more  adjacent  fibrils.  This 
striation  appears  to  be  unlike  that  of  clearly  cross-striated  proteins  and,  if  it  is  not 
an  inherent  periodicity  in  the  fibril,  it  may  represent  the  remnants  of  some  binding 
or  enveloping  structure. 


TRICHOCYSTS  AND  CILIA 


143 


FIGURE  2.     Trichocysts  from  Paramecium,  shadow-cast  with  chromium.     X  16,000. 
FIGURE  3.     Ciliuni  from  Paramecium,  shadow-cast  with  chromium.     X  11,000. 


144  M.  A.  JAKUS  AND  C.  E.  tJALL 

SUMMARY 

Electron  micrographs  of  shadow-cast  trichocysts  of  Paramecium  show  that  the 
dried  trichocyst  shaft  is  flattened  on  the  supporting  film,  while  the  pointed  tip  is 
apparently  more  resistant  to  collapse  on  dehydration.  Accentuation,  by  the  metal, 
of  the  cross  striation  previously  observed  in  the  shaft  indicates  that  the  periodicity 
is  accompanied  by  corrugation  of  the  dried  surface.  A  cross  striation  in  the  tip  is 
also  visible  in  some  micrographs  of  shadow-cast  specimens.  In  the  few  cases  where 
the  periodicity  could  be  measured,  the  average  spacing  was  about  300  A,  as  com- 
pared to  about  550  A  for  the  well-defined  shaft  striation. 

In  electron  micrographs  of  shadow-cast  specimens  of  Paramecium  cilia,  the 
component  fibrils  are  seen  with  greatly  increased  clarity. 

LITERATURE  CITED 

BROWN,  H.  P.,  1945.  On  the  structure  and  mechanics  of  the  protozoan  flagellum.  Ohio  Jour. 
Science.  45:  247-301. 

JAKUS,  M.  A.,  1945.  The  structure  and  properties  of  the  trichocysts  of  Paramecium.  Jour. 
E.r/>.  ZooL.  100:  457-485. 

SCHMITT,  F.  O.,  C.  E.  HALL,  AND  M.  A.  JAKUS,  1943.  The  ultrastructure  of  protoplasmic 
fibrils.  Biol.  Symp..  10:  261-276. 

WILLIAMS,  R.  C.,  AND  R.  W.  G.  WYCKOFF,  1945.  Electron  shadow-micrography  of  virus  par- 
ticles. Proc.  Soc.  E.\-p.  Hiol.  Mcd.,  58:  265-270. 


HYDROSTATIC  PRESSURE  EFFECTS  UPON  THE  SPINDLE  FIGURE 

AND  CHROMOSOME  MOVEMENT.     11.    EXPERIMENTS  ON 

THE  MEIOTIC  DIVISIONS  OF  TRADESCANTIA 

POLLEN  MOTHER  CELLS 

DANIEL  C.  PEASE 

Department  of  Anatomy,  the  Medical  School,  the  University  of  Southern  California, 

Los  Angeles,  California 

INTRODUCTION 

Hydrostatic  pressure  increments  are  known  to  reduce  progressively  the  rigidity 
of  plasmagels  and  the  viscosity  of  plasmasols.  Eventually  complete  solation  results. 
Marsland  (1939  and  1942)  has  been  able  to  formulate  what  appears  to  be  a  gen- 
eral quantitative  law  on  the  basis  of  a  considerable  volume  of  work  with  very  di- 
verse material.  He  has  found  that  with  each  increment  of  1,000  lbs./in.2  hydro- 
static pressure,  the  relative  rigidity  or  viscosity  decreased  to  76  per  cent  of  the 
initial  value.  This  applied  no  matter  whether  the  cytoplasm  of  amoebae,  Arbacia 
eggs,  or  Elodca  was  being  studied.  Furthermore,  these  direct  effects  have  always 
proved  very  rapidly  reversible  when  the  pressure  was  released.  The  subsequent 
pattern  of  cell  events,  however,  has  sometimes  been  found  to  have  been  changed  by 
new  reorganization  patterns  (cf.,  Pease,  1940,  1941). 

In  the  first  paper  of  this  series  (Pease,  1941),  experiments  w7ere  reported  in 
which  advantage  was  taken  of  these  known  effects  of  hydrostatic  pressure  to  study 
the  first  cleavage  division  spindle  apparatus  in  Urechis  eggs.  The  material  was  not 
well  suited  for  this  sort  of  work,  and  some  interpretations  were  open  to  question. 
However,  the  following  facts  were  clear  and  significant.  1 )  Pressure  could  so 
affect  the  cell  that  no  trace  of  the  spindle  figure  appeared  in  the  fixed  preparations, 
and  presumably  the  spindle  had  been  completely  liquified.  2)  The  pressures  de- 
stroying the  spindle  blocked  all  anaphase  movement.  3)  The  chromosomes  ag- 
gregated in  clumps  (originally  thought  to  be  vesicles)  under  lower  pressures  than 
were  required  to  block  anaphase  movement.  4)  Numerous  cytasters  appeared  in 
material  given  a  brief  recovery  period  before  fixation.  5)  Peculiar  "half-spindles" 
developed  de  novo  within  cytasters  whenever  the  latter  came  in  contact  with  nuclear 
material.  6)  By  their  very  nature,  the  half-spindles  lacked  "continuous  fibers" 
since  only  one  pole  was  involved,  and  also  there  were  no  "interzonal  fibers."  7) 
Yet  there  was  ample  evidence  that  such  half-spindles  were  functional  in  moving 
chromosomes,  and  even  recently-formed  nuclei  with  membranes  were  at  least  de- 
formed, and*  probably  moved,  by  them.  The  role  of  cytoplasmic  components  in  the 
spindle  was  stressed  (perhaps  unduly),  and  the  role  of  the  "traction  fibers"  mini- 
mized (perhaps  incorrectly  as  will  be  seen  later). 

To  find  out  whether  or  not  nuclear  gels  behaved  in  the  same  manner  as  cyto- 
plasmic gels  when  hydrostatic  pressures  were  applied,  the  extraordinary  equational 
meiotic  division  in  Stcatococcus  spermatocytes  has  been  studied  in  unpublished  work 
by  the  author.  In  these  cells  the  spindle  is  formed  inside  the  nuclear  membrane, 

145 


146  DANIEL  C.  PEASE 

and  the  anaphase  movement  nearly  completed,  before  the  nuclear  membrane  dis- 
integrates. In  this  case,  there  can  be  no  question  but  that  the  whole  spindle  appa- 
ratus is  of  nuclear  derivation.  It  was  found  that  sufficiently  high  pressures  de- 
stroyed it  by  liquefaction,  and  anaphase  movement  was  blocked.  The  spindle 
re-formed  once  more  when  the  pressure  was  removed  and  the  cells  allowed  a  short 
recovery  period.  Thus  the  physiological  action  of  hydrostatic  pressure  appears  to 
be  qualitatively  identical  in  gels  of  nuclear  and  cytoplasmic  origin. 

For  the  present  work,  Tradcscentia  pollen  mother  cells  (PMC)  were  selected 
as  material  for  several  reasons.  The  spindle  is  characterized  by  relatively  enor- 
mous "traction  fibers"  going  to  the  poles  from  comparatively  large  and  easily  visible 
kinetochores.  The  cells  have  the  advantage  of  a  small  number  of  chromosomes 
which  are  relatively  large.  The  only  important  disadvantages  are  the  impossibility 
of  getting  controls  which  necessarily  divide  at  the  same  time  as  the  experimental 
material,  and  the  extreme  difficulties  (which  proved  insuperable  with  pressure  tech- 
niques) of  actually  observing  the  divisions  in  vivo  (cf.,  Shimakura,  1934). 

The  material  was  collected  and  prepared  at  Stanford  University,  and  the  author 
is  indebted  to  Dr.  Reed  Rollins  of  that  institution's  botany  department  for  technical 
advice  on  handling  procedures  and  for  the  plants  which  were  used.  The  material 
was  studied  mostly  at  Columbia  University  before  the  war  interrupted  the  work. 
Dr.  F.  Schrader,  Dr.  S.  Hughes-Schrader,  and  Dr.  H.  Ris  followed  its  course  with 
interest,  enthusiasm,  and  valuable  suggestions.  Dr.  C.  W.  Metz  of  the  University 
of  Pennsylvania  also  contributed  excellent  comments  on  an  early  draft  of  the 
manuscript. 

MATERIAL  AND  METHODS 

The  half  dozen  Tradcscantia  paludosa  plants  used  in  these  experiments  pos- 
sessed six  pairs  of  chromosomes.  They  were  derived  from  a  common  stock.  The 
anthers  were  prepared  by  separating  the  connective  which  joins  the  two  lobes.  One 
lobe  was  then  fixed  as  a  control  just  at  the  time  of  pressure  application  to  the  other 
lobe.  The  bisection  of  the  anthers  with  a  small  lance  could  be  accomplished  easily 
without  rupturing  the  anther  lobe  walls.  The  lobes  were  handled  and  finally 
mounted  in  a  7.4  gm./lOO  ml.  saccharose  (Merck,  C.  P.)  solution  which  Shimakura 
(1934)  has  found  to  be  isotonic  with  Tradcscantia  pollen  mother  cells. 

The  pressure  bomb  used  in  these  experiments  held  a  half  dram  homeopathic  vial, 
and  was  so  designed  that  it  could  be  opened  very  rapidly.  After  filling  with  sugar 
solution  and  a  few  anther  lobes,  the  vial  was  sealed  with  "Parafilm"  wax  sheet  held 
in  place  with  a  rubber  band.  The  experimental  material  was  always  kept  under  the 
desired  hydrostatic  pressure  for  a  one  hour  period.  In  a  few  experiments  the  mate- 
rial was  fixed  30  minutes  after  the  release  of  pressure  which  allowed  time  for  some 
recovery.  But  in  most  of  the  experiments,  the  pressure  was  released,  the  bomb 
opened,  and  the  fixative  added  within  one  minute.  Preliminary  experiments  had 
shown  that  there  was  no  appreciable  reorganization  within  that  short  time  limit. 

Experiments  were  performed  using  1,000  Ib.  pressure  increments  from  1,000  to 
6,000  lbs./in.2,  and  with  8,000,  10,000,  and  15,000  lbs./in.2  Control  experiments 
were  performed  giving  identical  treatment,  but  at  atmospheric  pressure,  and  at  the 
relatively  low  pressure  of  150  lbs./in.2 

Bouin's  fixative,  to  which  3  per  cent  urea  was  added,  was  used  throughout. 
For  study,  eight  micra  sections  were  prepared,  and  stained  by  Heidenhain's  hema- 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.    II  147 

toxylin  method.  Both  mordanting  and  staining  were  prolonged  (never  less  than 
5  hours  each),  and  the  sections  were  destained  in  saturated  picric  acid  in  such  a 
fashion  that  considerable  stain  remained  in  the  cytoplasm.  There  was  a  good  deal 
of  shrinkage,  but  the  cytoplasmic  differentiation  (particularly  of  the  spindle)  was 
good. 

RESULTS 
Effects  upon  the  first  division  spindle 

The  first  division  spindle  was  particularly  sensitive  to  a  critical  hydrostatic  pres- 
sure that  was  found  to  be  between  4,000  and  5,000  lbs./in.2  Even  after  4,000  Ibs. 
had  been  applied,  the  spindle  figures  looked  essentially  normal.  There  was  no  re- 
duction in  the  length  or  diameter  of  "traction  fibers"  (compare  Fig.  28  with  Figs. 
25  and  26).  However,  many  of  the  "continuous  fibers"  had  apparently  been  lost 
for  the  net  effect  was  a  more  diffuse  looking  spindle  mass  with  fewer  and  less  con- 
spicuous continuous  fibers.  The  abnormalities  of  chromosome  movement  under 
even  the  lower  pressures  prevented  any  adequate  study  of  "interzonal  connections," 
but  occasional  examples  that  looked  normal  have  been  found  after  4,000  lbs./in.2 

In  striking  contrast  were  the  results  after  5,000  Ibs.  had  been  applied.  The 
traction  fibers  were  then  reduced  in  length  and  in  diameter  so  that  they  appeared 
as  delicate  structures  (Fig.  30).  Small  numbers  of  faint  and  very  thin  continuous 
fibers  were  usually  visible,  although  not  always.  Ordinarily  6,000  Ibs.  pressure 
obliterated  the  spindle  completely,  but  in  a  small  fraction  of  the  cells  a  fine  residual 
fiber  structure  remained  visible.  Figure  31  is  a  photograph  of  the  heaviest  and 
most  extensive  fibers  which  have  been  observed  in  material  fixed  after  an  exposure 
to  this  pressure.  It  must  be  emphasized  that  this  is  an  entirely  atypical  cell.  No 
sign  of  continuous  fibers  has  been  seen  after  exposures  to  8,000  Ibs.,  and  it  was  the 
very  rare  cell  which  showed  indications  of  traction  fibers.  When  visible,  as  in 
Figure  33  (arrows),  they  were  thin  and  short.  No  oriented  fiber  structure  of  any 
sort  was  ever  observed  after  exposures  to  10,000  or  15,000  lbs./in.2 

In  summary,  it  can  be  said  that  the  first  division  spindle  looked  essentially  nor- 
mal after  treatments  with  4,000  lbs./in.2  pressure,  but  was  profoundly  affected  by 
5,000  Ibs.  This  demarkation  was  really  very  sharp  ! 

Effects  upon  the  second  division  spindle 

The  spindle  of  the  second  meiotic  division  was  considerably  more  resistant  to 
hydrostatic  pressure  than  that  of  the  first  division.  The  spindles  appeared  nearly 
normal  after  4,000  lbs./in.2  pressure,  and  after  6,000  Ibs.  the  spindles  of  some  cells  did 
not  seem  to  be  greatly  affected.  After  6,000  Ibs.  pressure  there  was  a  considerable 
individual  variability  in  different  cells,  even  within  the  same  anther  lobe.  The  best 
spindles  were  somewhat  fainter  than  normal,  and  the  fibers  seemed  generally  thin- 
ner, but  they  sometimes  extended  from  one  pole  to  the  other.  After  8,000  Ibs.  pres- 
sure there  were  occasionally  evidences  of  traction  and  continuous  fibers,  although 
they  were  always  thin  and  faint  if  present.  No  fiber  structure  was  ever  visible 
after  pressures  of  10,000  Ibs.  or  more. 

It  thus  appears  that  the  second  division  spindle  withstood  nearly  2,000  lbs./in.2 
more  pressure  than  the  spindle  of  the  first  division.  It  will  appear  later  that  the 
pressure  required  to  block  anaphase  movement  was  similarly  proportional. 


148  DANIEL  C.  PEASE 

It  may  also  be  noted  here  that  there  was  a  little  evidence  that  the  spindles  of  the 
somatic  cells  in  the  connective  were  even  more  resistant  to  pressure,  and  were  not 
entirely  destroyed  unless  pressures  in  excess  of  8,000  Ibs.  were  applied. 

i 

Effects  upon  the  chromosomes — fusion 

Increasing  hydrostatic  pressures  made  the  chromosomes  progressively  more 
"sticky"  and  "soft."  Chromosomes  tended  to  aggregate  in  fused  masses.  In  Fig- 
ure 27  a  metaphase  plate  is  shown,  fixed  just  after  the  release  of  2,000  Ibs.  pressure. 
It  will  be  noted  that  there  are  stained  "bridges"  connecting  all  of  the  chromosomes. 
At  this  low  pressure,  the  bridges  were,  on  the  average,  only  slightly  heavier  than 
comparable  bridges  which  could  be  found  in  controls  of  the  proper  stage.  However, 
they  persisted  much  longer  than  normally,  well  into  the  anaphase  stages. 

When  pressures  of  3,000  Ibs.  or  more  were  applied,  the  inter-chromosomal 
bridges  tended  to  become  much  thicker,  and  entirely  out  of  the  range  of  normal 
variation.  Figure  32  shows  such  connections  in  a  cell  fixed  just  after  the  release  of 
6,000  Ibs.  pressure.  With  progressively  higher  pressures,  there  was  an  increasing 
tendency  for  the  fusion  of  chromosomes  into  a  single  mass.  This  can  be  seen  in 
Figures  33  and  34.  The  extreme  condition  was  reached  at  15,000  Ibs. /in.2  when 
it  was  nearly  always  quite  impossible  to  recognize  individual  chromosomes.  This 
is  well  shown  in  Figure  36. 

It  must  be  emphasized  that  the  preceding  description  and  the  photographs  are 
typical  of  cells  to  which  the  pressure  was  applied  in  late  metaphase  stages.  When 
the  pressure  was  applied  to  early  metaphases,  the  chromosomes  showed  a  much 
greater  degree  of  fusion  for  corresponding  pressures.  Of  considerable  importance 
must  have  been  the  proximity  of  chromosomes,  and  probably  also  the  initial  pres- 
ence of  thin  connections.  The  existence  of  some  movement  in  the  low  pressure 
range  may  have  aided  the  process. 

Not  only  were  metaphase  chromosomes  fused  together  by  treatment  with  hydro- 
static pressures,  but  a  comparable  effect  was  observed  with  late  diakinesis  chro- 
mosomes before  the  nuclear  membrane  broke  down.  Here  the  chromosomes  are 
apparently  normally  kept  separate  from  one  another  by  gel  structure  within  the  nu- 
cleus, for  nucleoplasm  strands  showed  clearly  enough  in  fixed  preparations.  These 
strands  continued  to  be  visible  until  pressures  of  6,000  or  8,000  Ibs./in.2  were  ap- 
plied. As  long  as  they  were  present  the  chromosomes  kept  apart  and  did  not  fuse. 
After  the  higher  pressures  the  strands  were  no  longer  visible,  and  the  chromosomes 
were  all  in  a  single  clump  together.  But,  as  with  the  metaphase  chromosomes,  the 
individual  chromosomes  did  not  lose  their  visible  identity  until  pressures  of  15.000 
Ibs.  were  applied. 

At  metaphase,  the  chromosomes  were  not  only  found  fused  laterally  in  the  plane 
of  the  equatorial  plate,  but  the  homologous  chromosomes  were  also  fused  together 
so  that  their  separation  was  greatly  complicated.  This  was  very  obvious  when  first 
diyision  anaphases  fixed  just  after  the  release  of  3,000  or  4,000  Ibs.  pressure  were 
studied.  Practically  every  cell  showed  evidences  of  fusion  with  bridges  that  were 
often  long  and  massive  (cf..  Figs.  1-12).  Such  bridges  always  stained  just  as  the 
chromosome  proper  with  hematoxylin  (Fig.  39),  and  the  larger  ones,  at  least,  were 
stained  by  the  Feulgen  reaction.  These  bridges  were  frequently  between  homolo- 
gous chromosomes,  but  also  commonly  involved  lateral  fusion  with  non-homologous 
chromosomes. 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II  149 

Even  more  massive  bridges  were  found  in  the  second  meiotic  division  material 
subjected  to  the  higher  pressures  which  still  allowed  a  good  spindle  to  exist.  Then, 
after  6,000  lbs./in.2  pressure,  most  or  all  of  the  chromosomes  were  frequently  so 
fused  together  that  they  nearly  lost  their  visible  identity.  However,  the  mass  of 
chromosomes  often  would  be  strung  out  from  one  end  of  the  cell  to  the  other  (Fig. 
15). 

It  should  be  noted  that  the  chromosomes  of  somatic  cells  showed  the  same  type 
of  fusion.  These  have  occasionally  been  seen  in  the  tissue  of  the  connective,  and 
Figure  41  shows  one  bridge  out  of  a  total  of  three  present  in  such  a  cell  fixed  just 
after  the  release  of  4,000  Ibs.  pressure. 

Effects  upon  the  chromosomes — rounding 

It  should  be  emphasized  that  all  of  the  fusion  bridges  between  chromosomes  had 
rounded  outlines.  This  shows  well  in  Figures  27  and  32,  and  suggests  a  consider- 
able plasticity. 

In  addition,  the  chromosomes  as  a  whole  tended  to  round  up  under  the  higher 
pressures.  This  was  most  obvious  in  the  second  division  chromatids  which  were 
V-shaped  with  relatively  long  and  thin  arms.  After  3,000  Ibs.  pressure  there  was 
very  little  noticeable  change  in  shape  even  though  there  might  be  some  fusion  (Fig. 
13).  However,  after  4,000  Ibs.  there  was  a  striking  alteration.  The  chromatids 
were  then  decidedly  thickened  and  shortened  (Fig.  14).  This  tendency  became 
more  pronounced  with  increased  pressures  (Fig.  15,  6,000  Ibs.). 

The  short  and  thick  chromosomes  of  the  first  meiotic  division  were  not  as  suited 
for  study,  but  the  same  tendency  was  obviously  present.  Particularly  after  10,000 
Ibs.,  when  the  identity  of  individual  chromosomes  could  still  be  seen,  they  were  de- 
cidedly shortened  and  rounded  except  at  the  kinetochore  region  (Fig.  34). 

Effects  upon  the  chromosomes — the  spindle  attachment  region 

The  first  meiotic  division  material  gave  the  impression  that  1,000-3,000  lbs./in.2 
pressure  allowed  a  greater  extension  of  the  attachment  region  of  the  chromosomes 
than  was  normal  (compare  Fig.  26  with  25).  More  particularly,  this  region  of 
some  chromosomes  was  extended  far  beyond  what  could  be  found  in  the  controls. 
The  attachment  region  gave  the  impression  of  being  unduly  short  in  the  material 
exposed  to  4,000  Ibs.  pressure.  An  attempt  to  measure  statistical  samples  was  de- 
cided upon. 

In  Table  I  the  mean  extensions  of  the  attachment  regions  of  first  division  chro- 
mosomes are  given  for  pressures  up  to  4,000  lbs./in.2  There  were,  of  course,  real 
difficulties  in  measuring  such  small  distances,  but  errors  should  have  cancelled  out 
in  the  averages.  While  no  great  reliance  should  be  placed  on  the  absolute  values, 
they  certainly  indicate  the  general  trend. 

The  measurements  were  made  with  a  filar  micrometer.  In  each  group,  50  meas- 
urements were  made  at  random,  excepting  that  only  cells  in  anaphase  were  selected, 
and  individual  chromosomes  that  had  not  yet  separated  and  left  the  metaphase  plate 
were  measured.  The  micrometer  hair  was  moved  up  to  a  chromosome  until  it  just 
touched  the  distal  tip  of  the  kinetochore  (indicated  by  the  arrows  in  Figs.  25  and 
26),  and  a  reading  made.  Then  the  hair  was  swung  across  the  field,  and  moved 
back  in  the  other  direction  until  the  hair  just  touched  the  base  of  the  attachment 


150 


DANIEL  C.  PEASE 


stalk  which  was  ordinarily  rather  well  defined  from  the  body  of  the  chromosome  by 
its  relative  translucency.  Then  a  second  reading  was  made.  The  difference  meas- 
ured the  length  of  the  stalk  plus  the  width  of  the  hair  in  the  micrometer.  The  hair 
width  was  measured  in  the  same  way  in  relation  to  a  fixed  point,  and  this  value  was 
subtracted  from  all  of  the  measurements.  The  figures  were  then  converted  to  micra. 
The  control  measurements  actually  used  for  comparison  were  combined  from  data 
upon  the  control  anther  lobes  of  the  1,000  and  3,000  Ib.  experimental  material,  and 
a  control  anther  which  was  left  mounted  in  the  bomb  for  one  hour  before  fixation, 
but  without  pressure. 

It  is  to  be- concluded  that  the  mean  length  of  the  attachment  stalk  was  definitely 
increased  by  pressures  from  1,000  to  3,000  lbs./in.2,  and  it  has  also  been  found  that 
there  is  no  overlap  in  the  extreme  extensions  between  control  cells  and  experimental 
cells  exposed  to  this  pressure  range.  With  4,000  Ibs.  pressure  the  mean  extension 
was  significantly  less  than  in  the  controls,  and  the  greatest  extensions  found  after 
this  treatment  did  not  even  approach  the  maxima  found  in  the  controls. 


TABLE  I 


Pounds  pressure 

Mean  extension 
in  micra 

Percentage  increase 
in  length 

Percentage  overlap  with 
control  mean 

control 

0.85 

1,000 

1.4 

59.4 

2 

2,000 

1.2 

38.6 

6 

3,000 

1.2 

42.9 

4 

4,000 

0.68 

-19.9 

22 

The  distance  between  the  tip  of  the  kinetochore  and  the  base  of  its  stalk  is  given  in  the 
second  column.  In  the  experimental  series,  50  measurements  were  made  at  random,  excepting 
only  that  early  anaphase  cells  were  selected.  The  control  average,  however,  is  a  combined  average 
of  three  sets  of  measurements  upon  different  material.  The  mean  percentage  increases  in  length 
are  based  upon  figures  carried  to  the  third  decimal  place.  The  last  column  gives  the  percentage 
of  measurements  which  overlapped  the  mean  of  the  control. 

Effects  upon  the  chromosomes — chromoneinata 

We  have  already  seen  that  late  prophase  and  metaphase  chromosomes  fused  to- 
gether and  rounded  up  under  the  influence  of  hydrostatic  pressure.  This,  however, 
only  applied  to  condensed  chromosomes.  Uncondensed  early  prophase  chromo- 
somes did  not  seem  to  be  affected  by  even  the  highest  pressures  employed.  This 
agrees  with  the  findings  of  Pease  and  Regnery  (1941)  who  were  unable  to  detect 
any  effect  of  15,000  lbs./in.2  pressure  upon  Drosophila  salivary  chromosomes  which 
are  similarly  uncondensed.  It  must  be  admitted  that  no  detailed  study  has  been 
made  of  the  early  prophase  chromosomes.  While  there  was  certainly  no  general 
clumping,  it  is  possible  that  very  local  fusions  could  have  been  overlooked,  but  there 
was  no  indication  of  shortening  or  thickening. 

An  "accidental  experiment"  gave  further  information,  and  additional  reason  for 
believing  that  the  chromonemata  were  not  affected  by  hydrostatic  pressure.  An 
anther  lobe  which  had  been  pricked  was  exposed  to  15,000  lbs./in.2  pressure  for  one 
hour  and  was  then  rapidly  fixed  in  the  usual  fashion.  The  surrounding  sugar  solu- 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II 


151 


tion  had  entered  the  anther,  and  apparently  was  somewhat  hypertonic.  All  of  the 
cells  were  slightly  plasmolized  and  had  more  or  less  swollen  chromosomes.  In  one 
small  section  of  the  anther,  conditions  were  such  that  the  spiral  structure  was  visible. 
Figures  37a  and  b  are  photographs  of  one  of  these  early  anaphase  cells,  and  it  is 
obvious  that  the  spiral  structure  was  unaffected.  Oddly  enough  there  was  no  tend- 
ency for  the  chromosomes  to  fuse  under  these  circumstances. 


First  division  cells.  Figures  1-10  are  of  sections  from  material  which  was  fixed  just  after 
the  release  of  4,000  lbs./in.2  pressure.  Figures  11  and  12  are  of  sections  fixed  just  after  the  re- 
lease of  3,000  Ihs.  pressure.  The  broken  lines  represent  traction  fibers  except  in  Figure  7  where 
they  represent  the  pathways  of  "continuous  fibers."  All  of  the  chromosomes  visible  were  not 
necessarily  included. 

Abnormalities  of  chromosome  movement  under  pressure 

Because  of  the  fusion  of  metaphase  chromosomes,  even  by  relatively  low  pres- 
sures, their  ultimate  distribution  to  the  two  spindle  poles  was  usually  very  abnormal 
whenever  anaphase  movement  took  place  during  the  pressure  treatment.  The  par- 
ticular pattern  which  resulted  apparently  depended  upon  the  balance  between  ana- 


152 


DANIEL  C.  PEASE 


phase  forces  and  the  local  resistances  of  whatever  fused  bridges  happened  to  be  pres- 
ent. Greater  or  lesser  fusions  might  occur  between  homologous  chromosomes  and, 
laterally,  between  non-homologous  chromosomes.  Almost  any  conceivable  vari- 
ation in  the  resulting  pattern  could  be  found  in  all  degrees.  Some  of  the  more 
interesting  variations  which  have  been  seen  are  included  in  Figures  1-15,  which 
are  also  perfectly  typical  of  material  exposed  to  3,000  or  4,000  Ibs.  pressure. 

Homologous  chromosomes  might  be  so  extensively  fused  that  separation  could 
not  occur.     Such  pairs  of  chromosomes,  fused  as  in  Figure  2  in  the  metaphase  plate 


Second  division  cells.  Figure  13  is  from  material  fixed  just  after  the  release  of  3,000 
lbs./in.2  pressure;  Figure  14,  after  4,000  Ibs.  pressure;  and  Figure  15,  after  6,000  Ibs.  pressure. 
Figure  16  is  from  recovery  material,  fixed  30  minutes  after  the  release  of  10,000  Ibs.  pressure. 
The  broken  lines  indicate  traction  fibers  except  in  the  upper  cell  of  Figure  15  in  which  they 
indicate  the  pathways  of  the  "continuous  fibers."  Not  all  visible  chromatids  were  necessarily 
included. 


Figures  17-24  are  all  from  first  division  recovery  material  which  was  fixed  30  minutes  after 
the  release  of  10,000  Ibs. /in.2  pressure.  The  broken  lines  indicate  traction  fibers.  Not  all  visible 
chromosomes  were  included  except  in  the  last  three  figures. 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II 


153 


27 


28 


Figure  25  is  a  first  division  early  anaphase  control  exposed  in  the  bomb  for  an  hour  (but 
without  pressure)  before  fixing.  Figure  26  is  of  a  cell  fixed  just  after  the  release  of  2,000 
lbs./in.2  pressure.  Figure  27  is  a  metaphase  plate  of  the  same  material.  Figure  28  is  of  a  cell 
fixed  just  after  the  release  of  4,000  Ibs.  pressure,  and  note  the  anaphase  separation  of  the  homolo- 
gous chromosomes  a'  and  a".  The  small  arrows  indicate  the  distal  ends  of  the  kinetochores. 
The  magnification  of  these  and  the  following  photographs  is  approximately  X  3,000. 


154  DANIEL  C.  PEASE 

region,  would  presumably  have  remained  there,  and  eventually  formed  micronuclei 
(Figs.  9  and  10). 

Even  though  there  was  no  lateral  fusion  with  other  chromosomes,  there  might 
be  slight  differences  in  the  forces  directed  towards  the  two  poles,  or  possibly  in  the 
strength  of  the  traction  fibers  going  to  opposite  poles.  An  extensively  fused  pair 
of  chromosomes  might  then  go  as  a  unit  to  one  pole  (Fig.  5).  Then  there  would 
always  be  an  abnormally  long,  but  otherwise  normal  looking  traction  fiber  (with 
full  thickness)  going  most  of  the  way  across  the  cell  to  the  other  pole. 

Figures  4  and  5  show  very  extensive  lateral  fusion  between  non-homologous 
chromosomes.  -Such  anaphase  cells  would  probably  have  given  rise  to  extensive 
bridges  in  telophase,  and  between  daughter  nuclei,  such  as  are  shown  in  Figures  8, 
10,  and  12. 

In  Figure  6  the  lower  member  of  a  pair  of  homologous  chromosomes,  indicated 
by  an  arow,  was  laterally  fused  with  a  non-homologous  chromosome  going  to  the 
upper  pole.  Seemingly  it  was  being  carried  to  that  pole  in  spite  of  its  traction  fiber 
to  the  other  pole. 

We  have  already  spoken  of  the  massive  bridges  which  characterized  the  second 
meiotic  division  material  exposed  to  6,000  Ibs.  pressure,  and  which  often  involved 
all  of  the  chromatids  (Fig.  15).  There  was  less  fusion  with  lower  pressures,  and 
the  abnormalities  more  nearly  resembled  what  has  just  been  described  for  the  first 
division. 

The  critical  pressure  blocking  anapliasc  movement 

The  best  evidence  for  chromosome  movement  under  pressure  is  certainly  the 
presence  of  extensive  bridging.  The  author  sees  no  rational  way  of  accounting 
for  the  bridges  other  than  to  suppose  that  anaphase  movement  occurred  after  the 
chromosomes  established  fusions  in  the  metaphase  plate  and  then  pulled  out  the 
bridging  connections. 

With  this  as  a  criterion  of  movement,  it  is  possible  to  state  that  anaphase  move- 
ment continued  at  4,000  Ibs. /in.2  hydrostatic  pressure  in  the  first  meiotic  division, 
but  was  blocked  by  5.000  Ibs.  pressure.  No  extended  bridge  has  been  seen  in  any 
cell  of  this  division  exposed  to  5,000  or  more  pounds  pressure.  Nor  were  there 
ever  signs  of  asynchrony,  or  of  directionally  atypical  movements. 

It  must  also  be  emphasized  that  abnormal  division  resulting  from  fusion  charac- 
terized practically  ez>cry  anaphase  cell  exposed  to  4,000  Ibs.  pressure.  It  was  also 
extremely  common  after  3,000  Ib.  treatments.  Similar  abnormalities  appeared  on 
a  lesser  scale  after  1,000  or  2,000  Ibs.,  but  then  the  separation  was  more  frequently 
fairly  normal,  and  characterized  only  by  loss  of  division  synchrony. 

In  the  second  meiotic  division  very  abnormal  anaphase  movement  involving 
massive  fusions  took  place  in  some  cells  exposed  to  6,000  Ibs. /in.2  pressure  (Fig. 
15),  but  none  wras  possible  at  8,000  Ibs. 

Bridging  has  been  found  even  after  8,000  Ibs.  pressure  in  the  somatic  cells  of 
the  connective.  Figure  42  is  from  a  somatic  cell  forming  daughter  nuclei  at  this 
pressure,  and  two  out  of  a  total  of  five  bridges  are  visible  in  the  plane  of  the 
photograph. 

In  the  meiotic  divisions,  at  least,  the  presence  of  a  good  visible  spindle  was  corre- 
lated with  anaphase  movement.  When  the  spindle  was  obviously  considerably  af- 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II 


155 


. 


30. 


31 


32. 


Figure  29  is  a  late  anaphase  cell  from  the  same  material  as  Figure  28  (exposed  to  4,000 
lbs./in.2  pressure).  Figure  30  is  a  cell  fixed  just  after  the  release  of  5,000  Ibs.  pressure.  Fig- 
ures 31  and  32  are  from  material  fixed  just  after  the  release  of  6,000  Ibs.  pressure. 


156  DANIEL  C.  PEASE 

fected  there  were  no  longer  evidences  of  anaphase  movement.  This  was  also  prob- 
ably true  of  the  somatic  cells,  but  they  have  not  been  carefully  studied.  It  is  clear 
that  movement  is  most  sensitive  to  hydrostatic  pressure  during  the  first  meiotic  divi- 
sion, withstands  nearly  2,000  Ibs.  more  pressure  in  the  second  division,  and  seem- 
ingly about  2,000  Ibs.  more  in  the  somatic  cells.  This,  in  turn,  appears  due  to  dif- 
ferent characteristics  of  the  spindle  gels,  rather  than  being  due  to  differential  pres- 
sure effects  upon  the  chromosomes.  For  in  the  first  and  second  meiotic  divisions, 
and  probably  also  in  the  somatic  divisions,  the  chromosomes  seemed  affected  equally 
by  equal  pressures. 

Spindle  recovery  after  pressure  release 

At  the  time  of  making  these  experiments  the  importance  of  the  recovery  stages 
was  largely  unsuspected,  and  relatively  little  material  was  gathered.  But  after  one 
hour  exposures  to  10,000  and  15,000  Ibs. /in.-  pressures,  some  experimental  material 
was  removed  from  the  bomb  and  given  a  30  minute  recovery  period  before  fixing. 
Many  of  these  cells  showed  excellent  spindles  with  massive  traction  fibers  (Fig.  38). 

Of  particular  interest  is  the  fact  that  the  traction  fibers  of  these  recovery  spindles 
were  de  novo  formations.  Conclusive  evidence  of  this  was  afforded  by  paired  ho- 
mologous chromosomes  (still  fused  as  a  result  of  the  pressure  treatment)  which 
formed  traction  fibers  from  both  kinetochores  that  went  to  the  same  pole.  Figure 
39  is  a  photograph  of  such  a  condition.  Figure  40a  is  a  drawing  of  another  ex- 
ample. Figure  40fr  seems  further  complicated  for  apparently  one  traction  fiber  had 
to  curve  around  a  blocking  chromosome  before  its  direction  to  the  "wrong"  pole 
could  become  definitive.  In  Figure  40r  each  traction  fiber  can  probably  be  con- 
sidered as  having  gone  to  the  "wrong"  pole  so  that  the  original  polarity  of  each 
chromosome  was  entirely  reversed. 

Figures  such  as  those  described  in  the  last  paragraph  were  not  rare,  although 
out  of  the  ordinary.  They  were  never  seen  in  the  controls,  nor  is  the  author  aware 
of  similar  accounts  in  the  literature. 

Most  commonly  the  spindle  appeared  to  re-form  nearly  along  its  original  axis 
if  it  is  assumed  that  the  metaphase  plate  was  not  displaced,  and  remained  as  an 
index  of  that  polarity.  The  pattern  thus  usually  seemed  very  nearly  normal. 
However,  the  long  axis  of  the  new  spindle  was  sometimes  very  oblique  to  the  plate, 
and  presumably  to  the  original  spindle  axis.  In  extreme  cases  a  90°  shift  was 
indicated. 

Also,  not  infrequently  multipolar  spindles  were  found  which  were  very  rare  in 
the  control  material.  Three-pole  spindles  such  as  Figure  24  were  fairly  common, 
and  a  few  four-pole  spindles  have  been  seen.  All  possible  variants  were  seen  with 
equal  or  very  unequal  poles,  spaced  equidistant  from  one  another,  or  barely 
separated. 

These  several  lines  of  evidence  all  imply  that  the  spindle  was  re-formed  de  novo, 
and  was  not  rebuilt  upon  residual  structure  which  had  survived  the  pressure  treat- 
ment and  persisted  to  give  a  framework.  New  patterns  appeared,  and  whatever 
molecules  were  involved,  they  were  at  least  rearranged. 

The  development  of  the  recovery  spindle 

One  can  select  a  series  of  cells  which  apparently  show  the  different  steps  of 
spindle  re-formation  after  the  release  of  pressure.  In  some  cells  fiber  structure  con- 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II 


157 


J 


- 


35. 


36. 


Figure  33  is  of  a  cell  fixed  just  after  the  release  of  8,000  lbs./in.:  pressure  (the  arrows 
indicate  very  faintly  visible  traction  fibers),  and  Figure  34  after  10,000  Ibs.  pressure.  Figure  35 
is  of  a  cell  fixed  just  after  the  release  of  15,000  Ibs.  pressure,  and  the  orientation  is  thought  to 
be  in  the  plane  of  the  original  spindle  axis.  Figure  36  is  from  the  same  material,  but  sectioned 
in  the  plane  of  the  metaphase  plate. 


158  DANIEL  C.  PEASE 

sisted  of  thin  fibrils  tangled  around  the  clumped  chromosomes  of  the  equatorial 
plate,  and  without  any  polar  orientation.  The  fiber  direction  was  roughly  circum- 
ferential to  the  enclosed  mass  of  chromosomes  (as  in  a  cocoon.  Fig.  44).  This 
could  be  regarded  as  the  first  recovery  stage. 

Many  cells  showed  polarized  fibers  as  in  Figure  45a.  The  section  of  Figure  45 
is  oblique  to  the  spindle  axis.  The  focus  of  Figure  45a  is  tangent  to  the  slant  height 
of  the  cone  which  makes  up  one-half  of  the  entire  spindle  (the  "surface"  of  the 
spindle,  so  to  say).  The  visible  fibers  are  the  continuous  fibers  of  the  new  spindle. 
Figure  45b  is  a  lower  focus  of  the  same  cell.  It  should  be  observed  that  there  are 
no  continuous  fibers  in  the  center  of  the  cone.  Instead,  there  are  only  slight  indi- 
cations of  traction  fibers.  The  continuous  fibers  were  thus  largely  peripheral,  but 
the  extensive  lateral  fusion  of  the  chromosomes  to  make  a  practically  solid  meta- 
phase  plate  probably  had  much  to  do  with  this  morphological  pattern  which  was 
typical  of  recovery  material. 

Traction  fibers  were  not  seen  in  cells  without  polarized  continuous  fibers.  But 
when  the  latter  had  formed,  traction  fibers  could  usually  be  found.  In  some  cells 
they  would  be  thin  and  short,  in  others  longer  and  more  massive.  Thus  the  trac- 
tion fibers  appeared  to  "grow"  outward  directly  away  from  the  kinetochore  region, 
and  full  thickness  was  not  achieved  until  they  practically  reached  the  poles. 

It  was  possible  to  find  many  minor  irregularities  in  the  developmental  pattern 
of  traction  fibers.  These  resulted  whenever  the  kinetochore  pointed  in  some  other 
direction  than  directly  towards  a  pole.  A  graded  series  could  be  found,  the  ex- 
treme examples  being  when  kinetochores  pointed  more  or  less  to  "wrong"  poles. 
Invariably  the  base  of  the  traction  fiber  extended  directly  away  from  the  kinetochore, 
and  it  did  not  bend  towards  a  pole  until  it  became  associated  with  continuous  fibers. 
The  bend  would  then  be  towards  the  pole  less  than  90°  away  from  the  initial  growth 
direction  even  if  this  happened  to  be  the  "wrong"  pole.  It  thus  looked  as  though 
the  growth  direction  was  unimpeded  until  the  traction  fiber  became  associated  with 
continuous  fibers,  and  then  the  further  extension  of  the  traction  fiber  followed  the 
path  of  least" resistance  in  the  pattern  expressed  by  the  continuous  fibers.  Thus  the 
traction  fiber'  even  developed  around  obstructions  as  in  Figure  40b. 

The  fusion  of  traction  fibers 

A  very  rare  situation  casts  further  light  on  the  formation  of  traction  fibers  if 
the  interpretation  is  correct.  It  was  possible  to  find  non-homologous  chromosomes 
in  the  recovery  material  which  appeared  to  be  bridged  across  the  kinetochore  re- 
gions. A  photograph  of  such  a  bridge  is  shown  in  Figure  43.  These  bridges  dif- 
fered from  all  the  other  ordinary  bridges  which  have  been  seen  in  that  they  were 
achromatic.  Although  they  were  short,  they  had  exactly  the  appearance  in  the 
fixed  and  stained  preparations  that  traction  fibers  had.  They  certainly  gave  the 
impression  that  they  represented  fused  traction  fibers,  traction  fibers  which  started 
to  develop  from  each  separate  kinetochore  in  opposite  directions,  and  which  grew 
terminally  into  each  other  to  fuse  end  to  end. 

The  author  hesitates  to  emphasize  these  structures.  The  material  has  been 
thoroughly  searched  and  only  two  good  examples  have  been  seen,  plus  another  which 
was  more  questionable  because  overlying  material  partially  obscured  it.  There 
may  be  good  reason  for  their  rarity,  for  it  is  obviously  an  exceptional  situation  to 
have  two  kinetochores  pointed  directly  towards  each  other.  If  we  accept  their 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.    II 


159 


37 


a. 


b.      38. 


39. 


A    40. 


Figure  37  is  from  a  slightly  plasmolized  cell  fixed  just  after  the  release  of  15,000  lbs./in.2 
pressure  (a  and  b  are  different  focal  levels).  Figures  38-40  are  from  recovery  material  fixed 
30  minutes  after  the  release  of  10,000  Ibs.  pressure.  In  Figure  38  note  the  bridge,  br.  In  Fig- 
ures 39  and  40  de  novo  recovery  traction  fibers  of  fused  homologous  chromosomes  go  to  the 
"wrong"  pole.  The  direction  of  a  pole  is  indicated  by  arrows  in  Figure  40. 


160 


DANIEL  C.  PEASE 


42. 


ijte 


a. 


. 


44. 


b. 


Figure  41  is  of  a  somatic  anaphase  cell  fixed  just  after  the  release  of  4,000  Ibs. /in.2  pressure. 
Figure  42  is  of  a  somatic  cell  forming  daughter  nuclei,  fixed  just  after  the  release  of  8,000  Ibs. 
pressure.  Figure  43  is  from  recovery  material  fixed  30  minutes  after  the  release  of  10,000  Ibs. 
pressure,  and  shows  achromatic  bridging  between  non-homologous  chromosomes  (fused  trac- 
tion fibers?).  Figure  44  shows  an  early  stage  of  spindle  recovery  in  material  fixed  30  minutes 
after  the  release  of  10,000  Ibs.  pressure.  Figure  45  is  from  the  same  material,  but  spindle  re- 
covery is  more  advanced  (a  and  />  are  different  focal  levels  of  the  same  cell). 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.    II  161 

reality  and  the  above  interpretation,  however,  the  implications  are  of  considerable 
interest,  for  it  means  that  developing  fibers  can  mutually  terminalize  each  other. 
Yet  there  is  no  effect  as  far  as  lateral  growth  is  concerned,  and  the  fibers  thicken 
as  normally.  There  is  simply  no  growing  end  left.  We  can  say  that  fibers  extend 
by  terminal  additions  rather  than  from  the  kinetochore,  or  by  elongation  from 
within  their  length. 

Having  gone  this  far,  we  can  make  another  deduction  as  to  the  role  of  the 
kinetochore  in  traction  fiber  formation.  We  can  regard  it  as  an  "organizing  center" 
which  initiates  linear  extension  and  controls  fiber  diameter.  The  linear  gowth  is 
self  perpetuating  once  started  until  the  fiber  reaches  a  pole,  or  is  terminalized  as 
above.  The  fiber  thickens  by  further  organization  at  the  kinetochore,  and  additional 
linear  growth  parallel  to  the  initially  thin  fiber,  thus  adding  enclosing  layers.  The 
final  fiber  has  a  thickness  equal  to  the  diameter  of  the  organizing  center.  The  au- 
thor reiterates  that  this  hypothesis  has  a  slender  experimental  basis,  and  depends 
upon  a  correct  interpretation  of  three  figures. 

Chromosome  movement  in  recovery  material 

There  were  obvious  indications  of  chromosome  movement  in  recovery  material 
after  the  spindles  re-formed.  The  movement  was  abnormal  because  of  strong  and 
persistent  fusion  bridges,,  and  in  many  'ways  resembled  the  anaphase  movement 
which  occurred  under  low  pressures  (3,000  and  4,000  Ibs.). 

Frequently  fused  pairs  of  homologous  chromosomes  were  found  going  to,  or 
after  they  had  reached,  a  single  pole  as  in  Figures  38  and  17.  In  such  cases  one 
traction  fiber  extended  all  the  way  across  the  cell  to  the  other  pole  but  seemed  to 
be  of  normal  thickness.  This  type  of  movement  often  seemed  to  be  aided  by  lateral 
fusion  with  non-homologous  chromosomes  as  in  Figures  18  and  21.  Less  fre- 
quently the  fusion  between  homologous  chromosomes  was  relatively  slight,  and  there 
would  be  a  partial  separation  with  the  formation  of  more  or  less  long  and  thin  bridges 
(Figs.  19,  20,  and  38,  /»-.).  Quite  frequently  very  massive  bridges  were  formed 
involving  most  if  not  all  of  the  chromosomes  which  would  be  fused  together  (Figs. 
22  and  23).  There  were  no  important  differences  between  first  and  second  meiotic 
division  cells  (note  Fig.  16). 

None  of  the  material  was  allowed  a  sufficient  recovery  period  so  that  daughter 
nuclei  formed  in  cells  which  began  their  anaphase  movement  after  the  application 
of  pressure.  It  can  be  presumed,  however,  that  many  of  the  cells  would  form  only 
a  single  nucleus  because  of  an  inability  on  the  part  of  the  chromosomes  to  separate. 
Other  cells  would  be  expected  to  form  bridged  nuclei,  and  probably  multiple 
micronuclei.1 

Chromosome  structure  in  recovery  material 

The  persistence  of  chromosome  fusion  in  the  recovery  material  would  seem  to 
suggest  just  one  possibility — that  the  initial  fusion  under  high  pressure  must  have 
been  due  to  at  least  a  partial  liquefaction  of  some  chromosomal  element,  and  that 
the  fusion  bridges  then  gelled  when  the  pressure  was  released.  In  the  recovery 
material  the  chromosomes  were  thus  stuck  together  by  very  viscous  bridges.'  After 
examining  a  great  deal  of  material,  the  author  is  of  the  opinion  that  it  is  very  doubt- 

1  Pease  (1941)  definitely  found  this  to  be  the  case  in  Urechis  eggs. 


162  DANIEL  C.  PEASE 

ful  that  fused  chromosomes  were  ever  able  to  separate  completely  before  the  forma- 
tion of  daughter  nuclei.  Most  commonly  there  were  few  signs  of  any  separation, 
but  even  in  extreme  cases,  thin  and  very  long  bridges  persisted  as  in  Figures  19 
and  20.  The  moderately  thick  bridges,  at  least,  stained  with  Feulgen. 

There  is  another,  and  much  more  puzzling,  aspect  of  chromosome  structure 
which  is  brought  to  light  by  a  study  of  the  recovery  material.  Even  after  the  re- 
lease of  15,000  lbs./in.2  pressure  (which  resulted  in  the  very  complete  fusion  of  the 
chromosomes  as  in  Figure  36)  the  chromosomes  regained  their  visible  identity  and 
their  approximately  normal  shape.  This  tendency  can  be  seen  (in  10,000  Ibs.  mate- 
rial) by  comparing  Figure  38  with  Figure  34,  but  it  is  best  seen  by  comparing  the 
long  chromatids  of  the  second  meiotic  division  (compare  Fig.  16  with  Figs.  14  and 
15).  In  regaining  the  normal  shape,  the  fusion  areas  must  necessarily  have  been 
reduced  in  cross-section,  and  it  is  likely  that  some  fusion  bridges  were  lost  entirely 
during  this  change.  The  effects  of  this  change  were  best  demonstrated  by  the  sepa- 
ration of  the  second  division  chromatids  in  material  recovering  from  10,000  Ibs. 
pressure.  Extensive  separation  sometimes  occurred,  thus  differing  in  degree  from 
the  first  division.  Figure  16  gives  an  indication  of  typical  difficulties  which  were 
essentially  the  same  as  in  the  first  division. 

Absolute  pressure  and  recovery  rate 

In  Urechis  egg  material  Pease  (1941)  found  that  the  rate  of  recovery  was 
roughly  proportional  to  the  absolute  pressure  which  had  been  applied.  In  the 
Tradescantia  PMC  material  we  can  only  compare  the  effects  of  10,000  and  15,000 
lbs./in.2  pressures.  Comparison  is  subjective,  but  there  was  not  the  slightest  doubt 
but  that  the  cells  subjected  to  10,000  Ibs.  pressure  showed  a  much  greater  amount 
of  recovery  of  the  spindle  elements  in  30  minutes  than  the  cells  exposed  to  15,000 
Ibs.  showed  in  the  same  length  of  time.  Fully  developed  new  spindles  were  only 
rarely  found  in  the  15,000  Ib.  material,  but  were  common  in  the  10,000  Ib.  mate- 
rial. In  both,  however,  the  chromosomes  had  regained  their  visible  identity  and 
approximately  normal  shapes. 

CONCLUSIONS 

A  single  hypothesis  readily  accounts  for  most  of  the  manifold  effects  of  hydro- 
static pressure  upon  spindle,  chromosomes,  and  anaphase  movement.  This  sup- 
poses that  increasing  hydrostatic  pressures  progressively  reduce  gel  rigidity,  with 
liquefaction  as  the  end  result.  Conversely,  after  the  release  of  pressure,  conditions 
return  to  a  state  such  that  gel  structures  can  be  re-formed  once  more.  There  is, 
of  course,  an  excellent  experimental  background  for  this  thesis,  particularly  in  so 
far  as  it  applies  to  cytoplasmic  systems.  This  has  been  indicated  in  the  introduction, 
and  has  been  outlined  at  greater  length  in  the  first  paper  of  this  series  (Pease,  1941 ). 

It  is,  however,  unfortunate  that  this  work  depends  upon  an  interpretation  of 
fixed  material.  However,  we  have  every  reason  for  believing  that  the  presence  of 
good  fiber  structures  in  such  material  is  a  good  index  of  oriented  gel  structure  in 
life.  It  is  only  on  that  assumption  that  a  comprehensive  pattern  appears,  consistent 
throughout  its  details.  It  is  true  that  whenever  we  have  contributory  evidence  of 
liquefaction  (such  as  a  block  of  anaphase  movement),  we  do  not  find  fiber  struc- 
tures in  the  cytological  material.  Apparently  extensive  fiber  structures  are  only 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II  163 

precipitated  by  fixation  agents  when  molecules  are  at  least  organized  into  an  oriented 
pattern  and  probably  also  concentrated  in  a  gel. 

Spindle  structure  and  formation 

In  view  of  the  above  considerations,  it  is  not  surprising  to  find  that  the  spindle 
no  longer  appears  in  cytological  preparations  after  a  critical  pressure  has  been  ap- 
plied before  fixation.  This  is  to  be  interpreted  as  indicating  a  liquefaction  of  pre- 
existing gel  structures,  with  a  consequent  loss  of  molecular  organization. 

It  has  been  demonstrated  that  the  pattern  of  the  recovery  spindle  can  be  very 
different  from  that  of  the  original  spindle.  High  hydrostatic  pressure  seems  able 
to  break  down  the  oriented  structure  of  the  original  spindle  so  completely  that  it 
re-forms  de  novo,  and  sometimes  with  a  new  polarity.  In  the  re-formation  of  the 
spindle  much  the  same  protoplasmic  material  may  well  be  used,  but  the  unit  mole- 
cules or  micells  are  rearranged  in  a  different  manner,  just  as  a  pile  of  second-hand 
bricks  might  be  rearranged  to  build  a  new  house.  This  conclusion  can  probably  be 
accepted  as  a  generalization  for  it  agrees  with  the  findings  in  Urechis  eggs  which, 
in  their  formation  of  "half  spindles,"  were  even  more  striking  (Pease,  1941),  with 
certain  other  observations  on  cytoplasmic  systems  (cf.,  Pease,  1940),  and  with  gen- 
eral theory. 

It  is  not  clear  just  what  does  orient  the  new  spindle  axis  in  Tradcscantia  PMC. 
Cytasters  accomplished  this  end  in  Urechis  eggs,  and  obviously  played  the  impor- 
tant role.  These  were  never  observed  in  the  PMC  material.  Instead,  we  find  a 
strong  tendency  for  the  new  axis  to  coincide  more  or  less  with  the  original.  The 
recovery  spindle  encountered  one  unusual  difficulty  in  its  organization  in  that  the 
chromosomes  were  no  longer  completely  separate  entities.  After  the  higher  pres- 
sures there  was  usually  a  continuous  plate  of  fused  chromosomes  in  the  equatorial 
region.  Continuous  fibers  did  not,  indeed  could  not,  penetrate  this  obstruction. 
However,  note  that  homologues  were  not  even  found  as  half  spindle  components. 
Continuous  fibers  were  only  found  sweeping  around  the  blocking  mass  leaving  the 
core  of  the  spindle  devoid  of  visible  oriented  structure  except  for  traction  fibers. 
Apparently,  therefore,  the  continuous  fibers  are  entirely  a  product  of  the  cytoplasm, 
and  are  not  directly  related  to  the  chromosomes.  The  latter,  in  fact,  are  obstacles 
to  be  by-passed.  This  does  not,  however,  preclude  the  possibility  of  a  generalized 
interaction  between  chromosomes  and  cytoplasm  in  that  the  former  may  "activate" 
the  latter  to  form  gel  structures.  Such  an  "activation"  was  quite  definitely  shown 
by  Urechis  eggs  recovering  from  the  effects  of  hydrostatic  pressure  (Pease,  1941). 
A  more  accurate  interpretation  might  be  not  to  stress  the  continuous  fibers  as  such, 
but  to  consider  them  simply  as  an  index  of  a  more  fundamental  structural  organiza- 
tion of  molecules.  They  thus  may  signify  nothing  more  than  the  basic  pattern  of 
an  extensive  gel  framework. 

On  the  other  hand,  the  kinetochore  apparently  quite  specifically  "organizes"  the 
protoplasm  to  form  the  attached  traction  fiber.  This  process  is  partially  separable 
from  the  development  of  continuous  fibers.  We  have  good  reason  for  believing 
that  developing  traction  fibers  simply  follow  the  path  of  least  resistance  in  the  struc- 
tural pattern  of  the  bulk  of  the  spindle,  which,  in  turn,  is  expressed  by  the  distribu- 
tion of  the  continuous  fibers.  Thus  the  structural  pattern  of  the  body  of  the  spindle 
limits  the  course  taken  by  the  traction  fibers  as  they  develop  outwards  away  from 
the  kinetochores.  It  seems  likely  that  this  is  a  progressive  wave  of  molecular  or- 


164  DANIEL  C.  PEASE 

ganization.  This  view  is  quite  similar  to  that  of  Schrader  (1932),  although  based 
upon  different  evidence.  However,  it  is  fundamentally  distinct  from  that  of  Belar 
(1929)  who  supposed  a  very  different  relationship  between  traction  and  continuous 
fiber.  Further  tentative  conclusions  on  the  growth  of  traction  fibers  have  already 
been  given  in  describing  the  experimental  results. 

The  extension  of  the  attachment  region  in  chromosomes  subjected  to  relatively 
low  pressures  indicates  a  real  pull  by  or  through  the  traction  fibers.  It  is  almost 
impossible  to  imagine  that  it  could  be  due  to  "repulsive  forces"  between  the  kineto- 
chores  for,  if  that  was  so,  the  extension  should  continue  to  increase  with  progres- 
sively higher,  pressures  which  further  soften  the  chromosomes.  Instead,  we  find  the 
extension  to  be  subnormal  while  we  still  have  evidence  of  traction  fibers  and  ana- 
phase  movement  (at  4,000  Ibs.  in  the  first  meiotic  division).  Our  conclusion,  then, 
is  that  the  traction  fiber  is  a  reasonably  stiff  gel.  No  doubt  it  progressively  loses 
rigidity  with  increasing  pressure,  but  it  has  a  margin  of  strength,  and  there  is  no 
important  weakness  until  a  pressure  threshold  is  passed.  The  extension  of  the 
attachment  stalk  is  therefore  thought  due  to  a  pressure  effect  upon  the  chromosome 
itself  so  that  it  is  softened,  and  can  be  unduly  pulled  out.  The  subnormal  exten- 
sion at  4,000  Ibs.  indicates  a  significant  weakness  of  either  the  traction  fiber  or 
available  force.  It  is  interesting  for  comparison  that  the  centrifuging  experiments 
of  Shimamura  (1940)  with  comparable  material  (Liliwn  PMC)  also  lead  to  the 
conclusion  that  the  traction  fiber  is  a  fairly  stiff  gelled  structure.  The  latter's 
work  seems  to  the  author  to  be  quite  conclusive. 

Chromosome  structure 

It  seems  obvious  that  some  portion  of  the  condensed  chromosome  tends  to  be 
softened,  and  finally  liquefied,  by  hydrostatic  pressures.  Since  there  was  no  ap- 
parent effect  upon  uncondensed  chromosomes,  or  upon  the  spirally  coiled  chro- 
monemata,  the  portion  affected  would  seem  to  be  the  "matrix"  (no  morphologically 
separable  "sheath"  is  visible,  and  presumably  more  than  a  sheath  would  be  involved 
when  the  attachment  region  is  extended).2 

A  critical  analysis  of  the  data,  however,  discloses  some  relationships  that  cannot 
yet  be  interpreted  with  any  assurance  of  certainty.  The  normal  presence  of  an  at- 
tachment stalk,  and  its  further  extension  under  relatively  low  pressures,  suggests 
that  the  rigidity  of  the  matrix  is  normally  low,  but  is  further  reduced  by  pressure. 
One  might  suppose  it  to  be  viscous  rather  than  a  stiff  gel.  While  the  spindle  gels 
are  liquefied  by  moderate  pressures,  the  matrix  is  not  entirely  liquefied  until  pres- 
sures of  about  15,000  Ibs. /in.2  are  applied  when  the  chromosomes  so  fuse  that  they 
lose  their  visible  identity.  Thus  a  structural  viscosity  appears  to  persist  and  with- 
stand very  considerable  pressures. 

It  is  a  fair  assumption  that  the  spindle  gels  obey  Marsland's  (1939)  law,  so  that 
their  rigidity  is  reduced  24  per  cent  by  each  pressure  increment  of  1,000  lbs./in.- 
Liquefaction  then  occurs  at  a  critical  pressure,  when  gel  linkages  tend  to  break  more 

2  In  the  first  paper  of  this  series  (Pease,  1941)  chromosome  aggregation  was  described  in 
Urechis  eggs  subjected  to  hydrostatic  pressure.  The  cytological  appearance  suggested  that  a 
"sheath"  was  involved  in  this  fusion  rather  than  the  matrix.  The  Urechis  chromosomes  were 
so  small,  though;  that  the  details  were  not  visible.  In  view  of  the  present  work  it  seems  more 
likely  that  the  matrix  as  a  whole  was  involved. 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II  165 

rapidly  than  they  can  be  formed.  Whereas  we  can  probably  apply  Mainland's  law 
to  the  spindle  gels,  it  does  not  seem  applicable  to  the  chromosome  matrix,  unless  we 
assume  that  the  matrix  material  has  a  much  lower  pressure/rigidity  constant  than 
cytoplasmic  or  spindle  gels,  i.e.,  much  less  than  24  per  cent  per  1,000  lbs./in.2 
That  other  different  gels  in  vitro  do,  in  fact,  have  different  constants  has  been  dem- 
onstrated by  Marsland  and  Brown  (1942). 

There  is  yet  another  aspect  of  chromosome  structure  to  be  considered.  Why  is 
it  that  with  increasing  pressures  we  find  chromosomes  rounding  up  and  tending  to 
fuse  into  a  single  mass?  This  looks  like  an  interfacial  phenomenon  to  be  exp' lined 
on  the  basis  of  surface  tension  laws.  We  do  not  observe  this  with  uncondensed 
chromosomes.  The  author  does  not  see  how  these  and  related  observations  can 
be  explained  except  by  the  assumption  that  a  true  interface  does  exist  between  con- 
densed chromosome  and  surrounding  protoplasm  (cf.,  Hirschler,  1942).  Many 
workers  do  not  believe  that  there  is  an  osmotically  active  membrane  separating 
chromosome  from  protoplasm,  although  this  could  explain  many  of  the  observa- 
tions of  chromosome  swelling.  However,  a  real  interfacial  boundary  would  not 
necessarily  imply  an  osmotically  active  system. 

In  any  case,  it  can  be  presumed  safely  that  any  intracellular  interface  would  exert 
only  a  very  low  tension,  certainly  not  more  than  a  fraction  of  a  dyne,  or  the  very 
few  dynes,  that  have  invariably  been  recorded  for  water/cell  interfaces,  or  intra- 
cellular oil/protoplasm  interfaces  (cf.,  Harvey  and  Shapiro,  1934  and  Harvey  and 
Schoepfle,  1939).  The  presence  and  properties  of  dissolved  proteins  would  always 
prevent  high  values.  Thus  any  interfacial  tension  at  the  surface  of  a  chromosome 
would  be  so  low  that  complete  rounding  of  the  aspherical  shape  would  occur  only 
when  both  chromosome  and  surrounding  protoplasm  were  essentially  fluid,  and 
practically  without  structural  viscosity.  It  is  only  at  a  pressure  of  about  15,000 
Ibs./in.2  that  the  observed  effect  indicates  these  conditions  as  being  nearly  fulfilled. 

The  spindle  in  chromosome  •movement 

It  has  already  been  pointed  out  that  there  is  a  direct  and  definite  correlation  be- 
tween anaphase  movement  and  the  presence  of  a  good  visible  spindle.  Hence,  our 
outstanding  conclusion  is  that  the  presence  of  gel  structure  in  a  spindle  is  essential 
for  anaphase  movement.  When  the  gel  rigidity  is  sufficiently  reduced,  the  move- 
ment necessarily  ceases.  Other  types  of  experimentation  have  less  directly  led  to 
the  same  conclusion  (cf.,  particularly  the  work  of  von  Mollendorff,  1938  and  1939, 
on  the  specific  effects  of  chemical  agents).  On  the  other  hand,  hypotheses  involving 
attractive  or  repulsive  forces  are  well  nigh  incompatible  with  the  results.  It  is 
hard  to  imagine  hydrostatic  pressure  affecting  such  forces,  particularly  in  the  low 
pressure  range.  Under  pressure,  with  conditions  of  reduced  viscosity,  the  chro- 
mosomes should  move  apart  all  the  more  rapidly  and  easily  if  such  forces  were  in- 
volved. Furthermore,  since  Marsland's  law  relating  pressure  and  viscosity  ex- 
presses a  logarithmic  relationship,  the  effect  should  be  most  noticeable  in  the  low 
pressure  range.  Obviously  this  is  in  direct  disagreement  with  the  present  findings. 

But  what  is  the  role  of  gel  structure  in  anaphase  movement?  Certainly  there 
are  at  least  two  separable  structures  to  be  considered — the  traction  fibers  and  the 
spindle  mass. 

Considering  the  traction  fibers  first,  Cornman  (1944)  in  a  thought-provoking 
review  comes  to  the  conclusion  that  they  are  contractile  structures  and  supply  the 


166  DANIEL  C.  PEASE 

force  for  movement.  However.  Cornman  ignores  one  major  difficulty  in  his  other- 
wise excellent  analysis.  No  one  has  yet  been  able  to  demonstrate  that  traction 
fibers  thicken  as  they  shorten,  although  this  would  be  expected  if  we  were  dealing 
with  contractile  bodies.  The  author  has  certainly  seen  no  evidence  of  this  in  his 
own  preparations,  nor  has  he  been  able  to  observe  the  converse  of  any  visible  thin- 
ning when  a  traction  fiber  was  extended  all  the  way  across  the  cell  from  one  pole 
to  the  other.  We,  therefore,  seem  to  require  a  different  explanation. 

It  is  the  author's  thought  that  Schrader  (1932)  was  correct  in  regarding  trac- 
tion fibers  as  being  no  more  than  passively  semi-elastic  structures. .  This  has  been 
given  excellent  experimental  foundation  by  Ris  (1943)  who  has  been  able  to  meas- 
ure directly  anaphase  movement  in  living  cells  (insect  spermatogonia  and  spermato- 
cytes).  In  some  cases  he  has  demonstrated  that  anaphase  movement  is  very  defi- 
nitely a  two  step  process.  The  first,  relatively  rapid  movement  can  be  explained 
as  due  to  the  release  of  elastic  tension  so  that  the  traction  fibers  do  actually  shorten. 
The  remaining  movement  is  then  due  to  the  spindle  mass,  with  the  traction  fibers 
serving  simply  as  passive  connections  to  the  chromosomes.  Lewis '(1939)  pro- 
duced an  accelerated  motion  picture  of  dividing  fibroblasts  in  vitro  which  beauti- 
fully showed  the  same  phenomenon,  although  he  has  not  commented  upon  it. 

A  general  hypothesis  of  anaphase  movement  can  be  advanced  on  the  assumption 
that  the  traction  fiber  is  anchored  at  one  end  to  the  chromosome,  and  along  some  of 
its  length  to  the  larger  gelled  mass  of  the  spindle  which,  in  turn,  is  in  motion.  Thus 
it  is  simply  a  more  or  less  elastic  connection  from  the  spindle  body  to  the  chromo- 
some— a  rope,  so  to  say,  between  the  machine  and  the  load.  This  interpretation 
forces  our  attention  to  the  body  of  the  spindle. 

The  analysis  of  anaphase  movement  by  Belar  (1929)  does  much  to  delimit  the 
problem,  even  though  we  cannot  accept  his  general  hypothesis.  He  demonstrated 
that  it  was  impossible  to  account  for  the  total  movement  on  the  basis  of  simple 
swelling  or  elongation  of  the  main  spindle  mass  (or,  more  specifically,  the  Stemm- 
korper).  There  is,  however,  an  obvious  way  to  avoid  the  difficulties  outlined  by 
Belar  (other  than  his  own  solution),  and  still  be  consistent  with  his  findings  and 
other  knowledge. 

It  is  proposed  that  motion  and  force  may  be  imparted  to  the  spindle  mass  by 
means  of  two  phase  transformations.  The  postulate  supposes  that  gel  material  is 
added  either  in  the  interzonal  region  3  or  along  the  greater  part  of  the  spindle, 
while  a  proportional  solation  occurs  at  the  poles.  Thus  a  material  circulation  is 
established,  but  a  circulation  by  means  of  sol-gel-sol  transformations  rather  than 
within  a  single  phase.  Actually  a  somewhat  comparable  idea  has  been  proposed 
by  Wassermann  (1929  and  1939).  Such  an  idea  would  be  regarded  by  many  as 
entirely  too  speculative,  and  not  subject  to  either  proof  or  disproof.  The  author, 
however,  wishes  to  point  out  some  comparable  effects  which  are  not  likely  to  be 
known  to  most  cytologists. 

Dan  ct  al.  (1938  and  1940)  discovered  a  remarkable  phenomenon  in  dividing 
sea  urchin  eggs.  After  the  furrow  completes  its  intrusion,  an  entirely  new  region 
of  gelled  cortex  is  added  in  the  center  of  the  furrow  region  as  the  original  cortical 

3  Note  that  Schmidt  (1939)  did  not  find  birefringence  with  polarized  light  in  the  mid-region 
of  sea  urchin  egg  spindles,  and  that  Shimamura  (1940)  found  this  to  be  the  "weak"  region  in 
centrifuging  experiments  upon  Lilium  PMC. 


ANAPHASE  MOVEMENT  UNDER  PRESSURE.     II  167 

material  backs  out.  Pease  (1943)  calculated  that  this  de  novo  cortex  came  to  cover 
about  11  per  cent  of  the  cell  surface.  This  gel  growth  is  obviously  analogous  to  a 
system  that  could  very  well  work  within  a  spindle. 

Since  the  advent  of  hydrostatic  pressure  techniques,  it  has  also  become  clear 
that  all  sorts  of  other  cell  processes  involving  movement  are  dependent  upon  gel 
structure.  Thus  amoeboid  movement,  cyclosis,  streaming,  cytoplasmic  division, 
the  movement  of  pigment  granules,  and  the  pole  cell  nuclei  of  Drosophila  eggs,  and 
even  sperm  penetration  both  through  the  egg  surface  and  also  to  their  final  central 
position  all  cease  (reversibly)  when  the  gel  is  liquefied.  All  of  these  movements 
depend  upon  the  rather  unexpected,  and  admittedly  little  understood,  properties  of 
protoplasmic  gels.  Obviously  the  gel  rearranges  itself,  and  is  itself  in  motion  (cf., 
the  review  of  Marsland,  1942).  No  doubt  gel-sol  transformations  are  usually  if 
not  always  involved  along  with  the  rearrangement.  Thus  we  do  find  empirically 
a  common  denominator  for  all  movements  other  than  such  specialized  activities  as 
muscle  contraction  and  ciliary  motion.  The  author  believes  that  a  general  theory 
of  anaphase  movement  is  in  sight,  and  that  it  will  come  from  a  better  physico- 
chemical  understanding  of  protoplasmic  gel-sol  systems. 

SUMMARY 

Hydrostatic  pressures  have  been  applied  to  Tradescantia  pollen  mother  cells  as 
a  technique  for  studying  the  structure  of  division  spindles  and  chromosomes  and 
the  mechanics  of  anaphase  movement.  The  procedure  has  given  pertinent  informa- 
tion by  virtue  of  the  fact  that  increasing  pressures  progressively  reduce  gel  rigidity. 
Sufficiently  high  pressure  results  in  liquefaction.  Yet  the  effects  are  reversible. 

The  spindle  of  the  first  meiotic  division  was  but  slightly  affected  by  4,000 
lbs./in.2  pressure,  yet  was  mostly  liquefied  by  5,000  Ibs.  The  spindle  of  the  second 
meiotic  division  withstood  about  2,000  Ibs.  more  pressure.  The  somatic  cells  were 
even  more  resistant. 

Condensed  chromosomes  were  significantly  softened  by  even  1,000  lbs./in.2 
pressure  as  indicated  by  an  undue  elongation  of  the  kinetochore  stalk.  Fusion 
bridges  became  particularly  obvious  when  3,000  Ibs.  was  applied.  Significant  short- 
ening and  rounding  occurred  at  4,000  Ibs.  Total  fusion  and  rounding,  indicating 
complete  liquefaction  of  the  matrix,  did  not  occur  until  pressures  of  15,000  lbs./in.2 
were  applied.  The  fusion  and  rounding  appeared  to  be  a  surface  tension  effect,  and 
suggested  the  existence  of  a  true  interfacial  membrane  between  condensed  chromo- 
some and  cytoplasm.  Not  even  these  highest  pressures,  however,  affected  the  un- 
condensed  prophase  chromosomes  so  that  the  effect  of  pressure  was  thought  to  be 
only  upon  the  matrix  material. 

Chromosome  movement  was  limited  to  those  pressures  which  did  not  liquefy 
the  spindle.  The  presence  of  fusion  bridges,  however,  resulted  in  very  abnormal 
movement. 

After  the  release  of  high  pressures,  spindles  re-formed.  That  these  were  de 
novo  structures  was  indicated  by  their  sometimes  abnormal  orientation,  by  the  fre- 
quency of  multipolar  spindles,  and  by  abnormalities  in  the  course  of  traction  fibers. 
Thus,  the  traction  fibers  of  two  homologous  chromosomes  might  go  to  a  single 
pole.  Abnormalities  made  it  seem  likely  that  the  growth  of  traction  fibers  was  in 
a  large  measure  independent  of  the  growth  of  the  body  of  the  spindle.  The  direc- 


168  DANIEL  C.  PEASE 

tion  of  growth  of  the  traction  fiber  was  not  specifically  oriented  until  it  reached  the 
oriented  bulk  of  the  spindle. 

Chromosome  movement  in  recovery  material  was  abnormal  in  that  the  fusion 
bridges  persisted.  Thus  the  chromosome  matrix  which  had  been  liquefied,  had 
become  highly  viscous  once  more.  Under  such  circumstances,  homologous  chromo- 
somes frequently  went  to  a  single  pole,  and  the  traction  fiber  to  the  other  pole  ex- 
tended all  the  way  across  the  cell.  However,  such  traction  fibers  were  not  thinner 
than  normal. 

The  outstanding  conclusion  is  that  a  gel  structure  in  the  spindle  is  essential  for 
anaphase  movement.  The  traction  fiber  apparently  serves  as  nothing  more  than 
a  semi-elastic  connection  between  the  chromosome  and  the  main  mass  of  the  spindle 
which,  in  turn,  is  in  motion.  It  is  suggested  that  motion  and  force  is  imparted  by 
means  of  sol-gel-sol  transformations,  with  gel  being  added  to  the  central  bulk  of  the 
spindle  while  a  proportional  solation  goes  on  at  the  poles. 

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THE  COMPARATIVE  DISTRIBUTION  OF  TWO  CHROMA- 

TOPHOROTROPIC  HORMONES   (CDH  AND  CBLH) 

IN  CRUSTACEAN  NERVOUS  SYSTEMS 

FRANK  A.  BROWN,  JR.,  AND  LORRAINE  M.  SAIGH 

Department  of  Zoology,  N  or  thivc  stern  University,  Evanston,  111.,  and 
Marine  Biological  Laboratory,  Woods  Hole,  Mass. 

INTRODUCTION 

It  was  demonstrated  by  Brown  (1933)  that  sea-water  extracts  of  the  crustacean 
central  nervous  organs  contained  material  having  a  definite  and  characteristic  effect 
upon  certain  chromatophores  of  the  body.  The  nervous  organs  were  the  only  tis- 
sues of  the  body  other  than  the  eyestalks,  with  their  included  sinus  glands,  that 
yielded  such  a  chromatophorotropically  active  substance,  thus  suggesting  that  the 
former  possibly  contained  a  source  or  sources  of  normal,  color-changing  hormonal 
material.  In  the  shrimp,  Palaeinonetes,  injection  of  extracts  of  the  nervous  system 
were  shown  to  bring  about  a  rapid  blanching  of  dark-colored  specimens  through 
concentration  of  the  red  and  yellow  pigments  within  the  chromatophores,  an  action 
similar  to  that  which  could  be  induced  by  extracts  of  the  sinus  gland  of  the  eyestalk. 

Similar  activity  of  the  nervous  system  was  described  by  Hosoi  (1934)  for 
Penacus  japonicus  and  by  Hanstrom  (1937)  for  Penacus  brasilicnsis.  Knowles 
(1939)  found  that  extracts  of  the  central  nervous  system  of  Lcander  adspersus 
caused  concentration  of  the  white  pigment  within  that  species.  Concentration  of 
white  pigment  by  extracts  of  central  nervous  system  was  also  reported  for  Cambarus 
by  Brown  and  Meglitsch  (1940)  who  worked  with  the  chromatophores  in  isolated 
pieces  of  integument.  Sinus  gland  extracts  had  an  antagonistic  action  upon  this 
pigment,  thus  proving  that  the  sinus  glands  and  nervous  system  did  not  yield  exclu- 
sively identical  chromatophorotropic  substances. 

Evidence  that  the  central  nervous  organs  contained  sources  of  hormones  nor- 
mally involved  in  the  adaptive  color-changes  of  Palacmonetcs  was  presented  by 
Brown  (1935)  who  found  that  any  vigorous  stimulation  of  the  cut  ends  of  the  optic 
nerves  in  darkened  eyestalkless  specimens  would  induce  a  blanching  characteristic 
of  that  following  injection  of  extracts  of  central  nervous  organs.  Roller  (1930) 
had  also  observed  comparable  responses  of  eyestalkless  Crago  but  did  not  at  that 
time  consider  the  central  nervous  organs  to  be  a  source  of  the  active  material. 

More  convincing  evidence  for  the  production  of  a  normal  chromatophorotropic 
hormone  in  the  crustacean  nervous  system  was  presented  by  Brown  and  Ederstrom 
(1940).  Their  observations  concerned  the  reactions  of  the  particularly  sensitive 
melanophores  in  the  telson  and  uropods  of  the  shrimp,  Crago.  Amputation  of  the 
eyestalks  of  a  white-adapted  animal  brought  about,  within  3-6  minutes,  a  complete 
dispersion  of  black  pigment  in  the  melanophores  giving  the  animal  a  "black-tailed" 
appearance.  The  condition  persisted  for  about  an  hour  whereupon  the  pigment  re- 
turned to  its  former  concentrated  state,  the  latter  condition  typically  lasting  for 
several  days.  Brown  and  Ederstrom  found  that  the  black  pigment  could  be  caused 

170 


HORMONES  IN  CRUSTACEAN   NERVOUS  SYSTEMS  171 

to  disperse  again  by  stimulation  of  the  eyestubs  or  by  the  injection  of  extracts  of 
the  circumoesophageal  connectives.  Upon  more  extensive  experimentation  they 
concluded  that  the  mid-region  of  the  connectives,  including  the  connective  ganglia, 
contained  the  origin  of  the  Crago  tail-darkening  hormone  (CDH)  involved  here. 
The  results  of  these  investigators  were  confirmed  and  extended  when  Brown  and 
Wulff  (1941)  gave  evidence  for  a  second  chromatophorotropic  principle  within  the 
central  nervous  system,  namely  a  Crago  body-lightening  hormone  (CBLH)  by  de- 
scribing that  strong  stimulation  of  the  eyestubs  simultaneously  darkened  the  telson 
and  uropods  and  lightened  the  remainder  of  the  body,  an  action  duplicated  by  in- 
jection of  extracts  of  the  central  nervous  system  as  a  whole.  It  was  shown  that 
these  two  actions  were  due  to  two  separable  principles  in  that  injection  of  ethyl  - 
alcohol  extracts  of  the  nervous  system  gave  only  body-lightening  action,  the  tail- 
darkening  principle  remaining  in  the  alcohol-insoluble  residue,  and,  that  mild  stimu- 
lation of  the  eyestubs  of  eyestalkless  animals  produced  both  tail-darkening  and 
body-darkening.  Brown  and  Wulff  speculated  that  CDH  was.  in  the  absence  of 
CBLH,  a  general  body-darkening  principle.  This  hypothesis  was  more  specifically 
set  forth  and  given  experimental  support  by  Brown  (1946)  who  clearly  demon- 
strated the  source  of  this  darkening  principle  to  lie,  not  in  the  circumoesophageal 
connectives  proper,  but  in  the  minute  tritocerebral  commissure  interconnecting  the 
connectives  immediately  posterior  to  the  oesophagus.  Injection  of  sea-water  ex- 
tract of  this  commissure  in  various  experiments  produced  in  every  case  tail- 
darkening  but  various  degrees  of  either  body-lightening  or  body-darkening.  The 
variable  effects  upon  the  body  seemed  reasonably  explained  in  terms  of  varying 
concentrations  of  an  antagonistic  body-lightening  principle. 

In  the  following  experiments  a  survey  was  made  of  the  effects  of  sea- water  ex- 
tracts of  the  central  nervous  systems  of  thirteen  species  of  higher  crustaceans  repre- 
senting the  Isopoda,  Natantia,  Ashicura,  Anomura,  and  Brachyura  upon  Crago 
color-change.  The  distribution  of  both  the  Crago  tail-darkening  hormone,  CDH, 
and  the  Crago  body-lightening  hormone,  CBLH,  was  considered.  We  have  con- 
cerned ourselves  primarily  with  the  presence  or  absence  of  each  substance  within 
the  centra]  nervous  systems  and,  when  the  hormones  are  present  in  a  particular 
species,  with  a  survey  of  the  relative  concentrations  of  the  principles  within  the 
parts  containing  the  hormone  in  question. 

EXPERIMENTS  AND  RESULTS 

The  experiments  to  determine  the  distribution  of  CDH  and  CBLH  were  con- 
ducted in  the  following  manner.  Animals  for  use  in  assaying  the  concentration 
of  active  principles  in  extracts  of  nervous  tissue  were  first  prepared.  The  eyestalks 
of  a  number  of  Crago  septevnspinosus,  ranging  from  3-6  cm.  in  length,  were  ampu- 
tated by  means  of  a  sharp  scalpel  and  the  eyestubs  cauterized  with  an  electric  cautery 
needle.  No  animals  were  used  for  assay  purposes  until  at  least  twelve  hours  fol- 
lowing this  operation,  at  which  time  they  could  best  be  described  as  possessing 
mottled  black  and  white  bodies  and  light  telson  and  uropods  (see  Fig.  \A,  control). 

A  relatively  simple  but  effective  method  was  used  in  the  preparation  of  central- 
nervous-system  extracts.  The  donor  of  the  nervous  tissue  first  had  eyestalks  re- 
moved and  stubs  cauterized  in  the  same  manner  as  described  above  for  Crago.  The 
dorsal  portion  of  the  exoskeleton  was  then  cut  away.  After  removing  surrounding 


172 


FRANK  A.  BROWN,  JR.,  AND  LORRAINE  M.  SAIGH 


viscera  and  muscles  the  nervous  organs  were  removed  under  a  dissecting  micro- 
scope by  carefully  severing  the  nerves  about  the  brain,  thoracic  and  abdominal  cords 
and  gently  lifting  the  entire  system  out  of  the  animal.  Particular  caution  was  ob- 
served in  the  removal  of  the  circumoesophageal  connectives  so  as  to  prevent  any 
damage  to  the  tritocerebral  commissure.  The  nervous  system  was  then  placed  in 
a  watchglass  containing  a  small  amount  of  sea-water  and  divided  by  means  of  a 
sharp  scalpel  into  the  desired  portions  which  usually  comprised  brain,  connectives, 
thoracic  cord,  and  abdominal  cord. 


A 


B 

FIGURE  1.  ^.Darkening  of  eyestalkless  Crago  following  injection  of  a  sea-water  extract 
of  the  abdominal  nerve  cord  of  Homarns  (cone.  =  1  cord/0.5  ml.  sea-water).  The  two  speci- 
mens on  the  left  are  two  uninjected  ones  used  for  a  control.  The  injections  for  the  animals  on 
the  right  were  made  15  min.  before  the  photographs  were  made.  B.  Lightening  of  eyestalkless 
Crago  following  injection  of  a  sea-water  extract  of  the  circumoesophageal  connectives  of  fY<; 
(cone.  =  3  pr.  conn,  to  0.2  ml.  sea-water).  The  two  specimens  on  the  right  were  injected  8 
minutes  before  the  photographs  were  made. 


HORMONES  IN  CRUSTACEAN  NERVOUS  SYSTEMS 


173 


Following  this  procedure  the  organs  were  transferred  to  individual  glass  mortars 
where  excess  sea-water  was  removed  and  the  tissues  allowed  to  dry  partially.  The 
tissue  was  then  triturated  with  a  measured  amount  of  sea-water  varying  in  quantity 
with  the  different  species  from  0.1-0.5  cc.  per  portion  depending  upon  the  size  of 
the  nervous  system  as  a  whole.  In  some  cases,  such  as  that  of  Idothca,  it  was 
necessary  to  use  the  parts  of  several  nervous  systems  in  the  preparation  of  each 
extract  in  order  to  obtain  adequate  concentration  and  amount  for  assay.  All  ex- 
tracts were  centrifuged  for  three  minutes  at  approximately  3,500  R.P.M.  and  the 
supernatant  liquid  of  each  injected  into  the  dorsal  musculature  of  the  abdomen  of 
at  least  two  test-animals  prepared  as  described  above.  The  amount  of  extract  in- 
jected into  each  varied  with  the  size  of  the  test-animal,  but  was  normally  between 

TABLE  I 

Responses  of  eyestalkless  Crago  to  injection  of  extracts  of  various  portions  of  the  central  nervous  system 
of  other  crustaceans.     No.  of  cases  signifies  the  number  of  donors 

Body-lightening  ©  or 

Tail-darkening  darkening  © 

Time  (min.)  Time  (min.) 


Species 

Organ 

No. 
cases 

0 

5 

10 

15 

30 

45 

60 

0 

5 

10 

15 

30 

45 

60 

Homarus 

Brain 
Connectives 
Thoracic  cord 
Abdominal  cord 

7 
8 
8 
2 

0.0 
0.0 
0.0 
0.0 

3.3 
1.9 
1.6 
1.0 

3.6 
2.3 
2.1 
1.5 

3.9 
2.3 
2.2 
2.5 

3.7 
2.3 
2.8 
3.0 

3.7 
2.1 
2.7 
2.5 

1.6 
1.4 

2.5 
1.5 

0.0 
0.0 
0.0 
0.0 

0.0 
-1.4 
-0.8 
+3.0 

+0.7 
-1.0 
-0.3 
+3.5 

+2.0 
-    0.0 
+0.4 
+4.0 

+2.6 
+0.6 
+2.8 
+4.0 

+  1.9 
+0.4 
+2.8 
+4.0 

+0.5 
0.0 

+4.0 

Cambarus 

Brain 
Connectives 
Thoracic  cord 
Abdominal  cord 

10 
10 
10 
10 

0.0 
0.0 
0.0 
0.0 

3.4 
2.5 
2.7 
2.6 

3.4 
2.9 
3.1 
2.8 

3.4 
3.2 
3.4 
2.8 

2.6 
2.2 
2.9 
2.5 

1.3 
0.9 
2.1 
1.4 

0.4 
0.3 
1.3 
1.0 

0.0 
0.0 
0.0 
0.0 

+0.8 
-1.7 
-1.9 
+0.8 

+  1.2 
-0.9 
-1.7 

+  1.2 

+  1.7 
-0.6 
-0.9 

+  1.4 

+0.9 
-0.1 
+0.1 
+  1.1 

+0.4 
+0.1 
+0.3 
+0.6 

+0.2 
+0.3 
0.0 
+0.2 

Upogebia 

Brain 
Connectives 
Thoracic  cord 
Abdominal  cord 

7 
6 

7 
7 

().() 

0.0 
0.0 
0.0 

1.3 
0.2 
1.5 
1.0 

2.3 
0.6 
2.1 
1.4 

2.3 
0.0 
2.7 
1.4 

2.2 
0.0 
2.5 
1.2 

0.8 
0.0 
2.0 
0.9 

0.0 
0.0 
1.4 
0.4 

0.0 
0.0 
0.0 
0.0 

-2.0 
-2.3 
-0.2 
-0.6 

-1.7 
-2.6 
+0.7 
-0.6 

-1.5 
-2.0 
+  1.2 
-0.3 

-0.4 
-1.7 
+0.8 
0.0 

-0.2 
-0.4 
+0.5 
0.0 

0.0 
-0.2 
+0.2 
0.0 

Pagurus 

Brain 
Connectives 
Thoracic  cord 

8 
8 
8 

0.0 
0.0 
0.0 

0.4 
0.0 
1.9 

0.5 
0.0 
2.9 

0.4 
0.0 
3.1 

0.2 
0.0 

2.4 

0.2 
0.0 
1.4 

0.0 
0.0 
0.2 

0.0 
0.0 
0.0 

-  .6 
-  .8 
-  .3 

-1.6 
-1.6 
-1.3 

-1.1 
-1.0 
-1.3 

-0.5 
0.0 
-0.5 

-0.1 
+0.1 
+0.2 

0.0 
+0.1 
0.0 

Emerita 

Brain 
Connectives 
Thoracic  cord 

8 
8 
8 

0.0 
0.0 
0.0 

0.0 
0.0 
1.0 

0.0 
0.0 
1.6 

0.0 
0.0 
1.6 

0.0 
0.0 
1.0 

0.0 
0.0 
0.5 

0.0 
0.0 
0.1 

0.0 
0.0 
0.0 

-  .5 
-  .4 
-0.9 

-0.6 
-0.6 
-0.6 

-0.5 
-0.2 
-0.1 

-0.2 
0.0 
-0.1 

0.0 
0.0 
0.0 

0.0 
0.0 
0.0 

Libinia 

Brain 
Connectives 
Thoracic  cord 

7 
7 
7 

0.0 
0.0 
0.0 

0.0 

0.0 
0.0 

0.0 
0.0 
0.0 

0.0 
0.0 
0.0 

0.0 
0.0 
0.0 

0.0 
0.0 
0.0 

0.0 
0.0 
0.0 

0.0 
0.0 
0.0 

-2.6 
-1.7 

-1.7 

-2.8 
-1.8 
-1.7 

-2.0 
-1.7 
-1.8 

-1.0 
-0.4 
-0.9 

-0.3 
-0.1 
-0.4 

0.0 
0.0 
0.0 

0.025  and  0.04  cc.     Sea-water  injected  or  uninjected  controls  were  observed  simul- 
taneously with  all  test-animals. 

Observations  of  the  color  changes  in  both  body  and  tail  were  taken  at  five- 
minute  intervals  up  to  fifteen  minutes  and  at  fifteen-minute  intervals  thereafter. 
The  degree  of  darkness  of  the  tail  or  body  was  described  within  the  range,  +  1  to 
+  4,  the  number  +  4  representing  the  maximum  extent  of  darkening  and  the  num- 
ber +  1,  the  minimum  observable  one.  In  a  similar  manner  body-lightening  was 
indicated  by  the  range,  -  -  1  to  --  4,  with  --  4  denoting  the  greatest  extent  of  body- 
lightening.  Final  results  for  a  number  of  experiments  were  averaged  and  are  pre- 
sented in  tabular -form  in  Table  I.  These  results  have  been  further  analyzed  so  as 
to  present  the  distribution  of  CDH  and  CBLH  within  the  central  nervous  system 


174 


FRANK  A.  BROWN,  JR.,  AND  LORRAINE  M.  SAIGH 


of  each  of  the  species  considered  (see  Tables  II  and  III).  In  these  tables,  the 
relative  distribution  of  activity  of  the  hormones  is  calculated  for  the  various  portions 
of  the  nervous  system  for  each  species. 

This  was  done  as  follows.  The  average  values  of  the  chromatophores  at  5,  10, 
15,  and  30  minutes  following  extract-injection  were  of  themselves  averaged.  Then 
for  Table  II  the  portion  of  the  nervous  system  producing  maximum  darkening  was 

TABLE  II 

The  quantitative  distribution  of  CDH  activity  within  the  central  nervous  systems  of  a  number  of 
crustaceans.  The  region  of  maximum  activity  is  arbitrarily  given  the  value  1.00.  It  is  important  to 
note  that  each  portion  of  the  nervous  system,  regardless  of  size,  is  extracted  in  an  equal  volume  of  sea- 
water,  and  the  relative  concentrations  of  the  principles  investigated  are  expressed  solely  in  terms  of  their 
activities.  This  note  applies  equally  to  Table  III. 


Classification 

Species  or  genus 

Brain 

Connec- 
tives 

Thoracic 
cord 

Abdominal 
cord 

Isopoda 

Decapoda 
Natantia 

Reptantia 
Astacura 

Anomura 

Brachyura 
Oxyrhyncha 

Brachyrhyncha 

Idothea  baltica 

Crago  scptemspinosus 
Palaemonetes  vulgaris 

Homarus  americanus 
Cambarus  virilis 
Upogebia  affinis 
Pagurus  sp. 
Emerita  talpoidea 

Libinia  sp. 
Cancer  irroratus 
Carcinides  maenas 
Ovalipes  ocellatus 
Uca  pugilator 

1.00 

1.00 

1.00 

1.00 

some 
0.06 

1.00 

0.22 

0.21 

0.85 

1.00 

0.97 

0.98 

1.00 

0.61 

0.61 

0.53 

1.00 

0.84 

0.94 

0.84 

1.00 

0.10 

0.80 

0.65 

0.17 

0 

1.00 

— 

0 

0 

1.00 

— 

0 

0 

0 

— 

0 

0 

0 

— 

0 

0 

0 

— 

0 

0 

0 

— 

0 

0 

0 

— 

arbitrarily  given  the  value  1.00,  the  activity  of  the  other  parts  being  expressed  in 
terms  of  simple  proportions  of  this.  For  Table  III  the  part  showing  maximum 
lightening  was  given  the  value  --  1.00  with  the  activity  of  other  parts  similarly  ex- 
pressed proportionately.  The  positive  values  in  the  latter  table  obviously  indicate 
darkening  rather  than  lightening. 

Within  the  single  species  of  Isopoda  investigated,  Idothea  baltica,  there  appears 
to  be  roughly  a  uniform  distribution  of  CDH  throughout  the  central  nervous  sys- 
tem, all  organs  darkening  the  telson  and  uropods  of  Crago  to  approximately  the 


HORMONES  IN  CRUSTACEAN  NERVOUS  SYSTEMS 


175 


same  degree.  Great  variations  in  distribution  of  the  hormones  occur  among  the 
decapods.  The  Natantian,  Crago  apparently  possesses  significant  CDH  activity  only 
in  the  regions  of  the  circumoesophageal  connectives.  CDH  is  differentially  distrib- 
uted throughout  the  central  nervous  system  of  the  anomurans  with  highest  quantity 
usually  in  the  posterior  region  of  the  thoracic  cord,  is  relatively  uniformly  distributed 
within  the  central  nervous  system  of  the  astacurans  and  Palaemonetes,  and  is  en- 
tirely absent  within  that  of  the  brachyurans. 

The  quantitative  distribution  of  CBLH  was  considered  here  solely  within  the 
reptantian  nervous  system,  although  it  is  known  to  be  present  throughout  the  cen- 
tral nervous  system  of  the  natantians  (Brown  and  Wulff,  1941).  Both  the  anomu- 
rans and  brachyurans  show  wide  distribution  of  this  principle  throughout  brain. 


TABLE  III 

The  quantitative  distribution  of  CBLH  activity  within  the  central  nervous  systems  of  a  number 
of  crustaceans.  The  region  of  maximum  body -lightening  is  arbitrarily  assigned  the  value  —  1.00. 
The  +  values  indicate  body-darkening. 


Classification 

Species  or  genus 

Brain 

Connec- 
tives 

Thoracic 
cord 

Abdominal 
cord 

Isopoda 

Idothea  baltica 

pres. 

pres. 

pres. 

pres. 

Decapoda 

Natantia 

Crago  septemspinosus 

pres. 

pres. 

pres. 

pres. 

Palaemonetes  vulgaris 

pres. 

pres. 

pres. 

pres. 

Reptantia 

Astacura 

Homarus  americanus 

+  2.40 

-1.00 

+  2.20 

+  7.20 

Cambarus  virilis 

+  1.09 

-0.73 

-1.00 

+  1.00 

Anomura 

Upogebia  affinis 

-0.64 

-1.00 

+0.27 

-0.18 

Pagurus  sp. 

-0.92 

-0.85 

-1.00 

Emerita  talpoidea 

-1.00 

-0.86 

-0.57 

Brachyura 

Oxyrhyncha 

Libinia  sp. 

-1.00 

-0.67 

-0.71 

Brachyrhyncha 

Cancer  irroratus 

pres. 

pres. 

pres. 

Carcinides  maenas 

pres. 

pres. 

pres. 

Ovalipes  ocellatus 

pres. 

pres. 

pres. 

Uca  pugilator 

pres. 

pres. 

pres. 

connectives,  and  thoracic  cord.  However,  a  striking  feature  is  noted  in  the  asta- 
curans and  the  natantian,  Palaemonetes,  in  which  a  darkening  (see  Fig.  I  A),  as  well 
as  a  lightening,  of  the  body  occurs. 

The  two  species  of  astacurans  with  which  we  have  concerned  ourselves  more  or 
less  parallel  one  another  with  respect  to  the  distribution  of  CDH.  In  Homarus  and 
Cambarus  the  region  of  greatest  quantity  of  this  principle  is  the  brain,  and  is  fol- 
lowed by  an  apparent  gradual  diminution  of  the  substance  from  anterior  to  posterior 
within  the  nervous  system.  The  problem  of  CBLH  distribution  seems  somewhat 
more  complex  since,  as  has  been  previously  mentioned,  certain  of  these  nervous- 
system  extracts  appear  to  produce  body-darkening  preceded  by  a  body-lightening. 
The  abdominal-cord  extract  is  particularly  active  in  body-darkening  and  only  the 


176 


FRANK  A.  BROWN,  JR.,  AND  LORRAINE  M.  SAIGH 


connectives  and  thoracic  cords  of  Homarus  and  Cambarus  show  any  body-lightening 
activity  at  all.  In  these  cases  where  body-lightening  is  indicated,  the  lightening 
persists  for  only  a  short  time  and  is  followed  by  a  definite  darkening.  These  ob- 
servations suggest  that  the  body-darkening  activity  observable  for  extracts  of  the 
astacuran  central  nervous  system  is  explainable  in  terms  of  CDH.  It  is  significant 
that  in  no  case  is  body-darkening  ever  obtained  from  a  portion  of  the  nervous 
system  lacking  tail-darkening  activity.  However,  since  there  is  no  essential  direct 
correlation  between  the  degree  of  tail-darkening  and  the  degree  of  body-darkening 
even  within  a  single  species,  the  observed  results  must  be  the  consequences  of  vary- 
ing proportions  of  the  two  principles  within  the  extracts,  with  the  degree  of  influ- 
ence of  either  one  being  a  function  of  its  relative  concentration  at  any  given  instant. 
There  are  significant  differences  in  the  distribution  of  CDH  within  the  group 
of  anomurans.  Pagurus  and  Emcrita  exhibit  similar  tail-darkening  activities  and 
these  are  shown  chiefly  by  thoracic  cord  extracts.  On  the  other  hand,  extracts  of 


TABLE  IV 

The  responses  of  eyestalkless  Crago  to  injections  of  extracts  of  parts  of  the  thoracic  cord  of  some 
anomurans,  showing  the  differing  distributions  of  CBLH  and  CDH  activity.  No.  of  cases  signifies 
number  of  donors. 

Body-lightening  0  or 

Tail-darkening  darkening  © 

Time  (min.)  Time  (min.) 


Species 

Part  of  thor. 

No. 

0 

5 

10 

15 

30 

45 

60 

0 

5 

10 

15 

30 

45 

60 

cases 

Pagurus  pollicaris 

Anterior  J£ 

8 

0.0 

0.3 

0.3 

0.4 

0.3 

0.0 

0.0 

0.0 

-2.3 

-2.2 

-2.0 

-0.6 

-0.3 

0.0 

Second  J4 

8 

0.0 

0.6 

0.7 

0.7 

0.2 

0.0 

0.0 

0.0 

-0.7 

-0.6 

-0.3 

-0.3 

0.0 

0.0 

Third  y± 

8 

0.0 

1.5 

1.6 

1.6 

0.5 

0.5 

0.4 

0.0 

-0.4 

-0.4 

-0.3 

0.0 

0.0 

0.0 

Posterior  J£ 

8 

0.0 

2.6 

2.6 

2.4 

0.9 

0.5 

0.0 

0.0 

-0.5 

-0.5 

-0.3 

-0.1 

0.0 

0.0 

Pagurus 

Anterior  J£ 

6 

0.0 

0.5 

0.5 

0.5 

0.0 

0.0 

0.0 

0.0 

-1.7 

-1.6 

-1.2 

-0.4 

-0.2 

0.0 

longicarpus 

Second  J4 

6 

0.0 

0.2 

0.2 

0.2 

0.0 

0.0 

0.0 

0.0 

-1.0 

-1.0 

-0.3 

0.0 

0.0 

0.0 

Third  M 

6 

0.0 

1.3 

1.4 

1.5 

0.8 

0.5 

0.2 

0.0 

-1.2 

-0.8 

-0.2 

0.0 

+0.2 

+0.2 

Posterior  J£ 

6 

0.0 

2.0 

2.0 

2.0 

1.2 

0.5 

0.0 

0.0 

-0.5 

-0.4 

-0.2 

-0.2 

0.0 

0.0 

Emerila  talpoidea 

Anterior  J^ 

4 

0.0 

0.8 

0.8 

0.8 

0.3 

0.0 

0.0 

0.0 

-2.3 

-2.3 

-1.8 

-0.5 

0.0 

0.0 

Second  }-£ 

4 

0.0 

0.0 

0.0 

0.0 

0.0 

().() 

0.0 

().() 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

Third  H 

4 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

-0.3 

-0.3 

0.0 

0.0 

0.0 

0.0 

Fourth  Ji 

4 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

-0.5 

-0.5 

0.0 

0.0 

0.0 

0.0 

Posterior  J£ 

4 

0.0 

0.3 

1.8 

1.8 

0.3 

0.1 

0.0 

0.0 

-0.3 

+0.8 

+0.8 

0.0 

0.0 

0.0 

brain,  thoracic  and  abdominal  cords  of  Upogebia  all  contain  notable  amounts  of 
CDH.  Another  similarity  between  extracts  from  Pagurus  and  Emerita  is  seen  in 
the  distribution  of  CBLH.  CBLH  is  found  in  considerable  amounts  in  brain,  con- 
nectives, and  thoracic  cord  of  both  genera.  However,  the  thoracic  cord  extracts 
of  Upogebia  show  almost  a  complete  absence  of  CBLH  activity  while  the  extracts 
of  the  remaining  parts  of  the  central  nervous  system  produce  a  definite  body- 
lightening,  the  connectives  being  most  active  in  this  respect. 

The  absence  of  CDH  within  the  brachyurans  investigated  as  well  as  the  restric- 
tion of  this  principle  to  the  connective  region  of  the  natantians  studied  confirms  the 
results  of  Brown  and  Ederstrom  (1940).  Experimental  data  show  that  moderate 
amounts  of  CBLH  are  found  in  brain,  thoracic  cord,  and  connectives.  Although 
results  for  CBLH  distribution  for  brachyurans  are  shown  only  for  Libinia,  it  has 
been  found  that  they  are  qualitatively  the  same  for  Uca,  Cancer,  Carcinides,  and 


HORMONES  IN  CRUSTACEAN  NERVOUS  SYSTEMS  177 

Oval  i  pcs.  The  striking  body -lightening  effect  of  a  strong  extract  of  Uca  connec- 
tives and  commissures  is  illustrated  in  Figure  IB. 

An  attempt  was  made  to  analyze  further  the  localization  of  CDH  and  CBLH 
within  the  thoracic  cords  of  Emcrita  and  two  species  of  Pagnnis:  pollicaris  and 
longicarpns  (Table  IV).  The  procedure  consisted  of  dividing  the  thoracic  cords 
into  a  number  of  approximately  equal  portions,  four  in  the  case  of  Pagunis  and 
five  in  that  of  Emerita.  It  was  observed  that  the  concentration  of  CDH  within 
the  thoracic  cord  of  both  P.  pollicaris  and  P.  longicarpus  is  greatest  in  the  posterior 
fourth  of  the  cord  and  decreases  gradually  along  the  cord  as  one  proceeds  anteriorly. 
In  Emcrita  the  highest  region  of  CDH  concentration  is  also  the  posterior  portion 
of  the  thoracic  cord.  However,  there  is  a  lack  of  CDH  in  any  of  the  central  por- 
tions of  the  thoracic  cord  in  Emcrita.  It  would  seem  then  that  the  distribution  of 
CDH  in  the  thoracic  cord  of  Emcrita  is  more  restricted  than  in  Pagunis. 

The  distribution  of  CBLH  in  the  thoracic  cord  of  P.  pollicaris  and  P.  longicarpus 
is  similar.  The  most  intense  body-lightening  effect  is  brought  about  by  extracts 
of  the  anterior  fourth  of  the  cord  while  less  intense  reactions  are  produced  by  ex- 
tracts of  the  remaining  portions.  Experiments  with  extracts  of  Emcrita  thoracic 
cord  indicate  a  higher  concentration  of  CBLH  in  the  anterior  portion  of  the  cord, 
and  apparent  absence  of  CBLH  in  the  second  portion  and  only  slight  amounts  of 
the  principle  in  the  third,  fourth  and  fifth  divisions  of  the  cord.  In  summarizing 
the  distribution  of  CDH  and  CBLH  within  the  thoracic  cords  of  Pagnnis  and 
Emcrita  we  can  say  that  CDH  is  relatively  more  concentrated  posteriorly  in  the 
thoracic  cord  while  CBLH  appears  more  concentrated  anteriorly. 

DISCUSSION  OF  RESULTS 

The  effect  of  the  extracts  of  the  central  nervous  system  upon  the  dark  pigments 
of  the  telson  and  uropods  of  Crago  possesses  a  characteristic  pattern  in  each  of  the 
major  groups  of  the  order  Decapoda.  In  the  Xatantian,  Crago,  we  have  observed 
the  restriction  of  CDH  activity  to  the  circumoesophageal  connectives,  whereas  the 
Astacnra  and  Palacmonctcs  exhibit  a  more  generalized  occurrence  of  the  hormone 
within  the  organs  of  the  central  nervous  system.  However,  as  one  proceeds  to  the 
Anoinnra.  these  contain  changes  from  the  widespread  condition  in  the  astacurans  to 
a  more  specialized  one  as  evidenced  by  the  restriction  of  CDH  in  the  thoracic  cord  of 
two  of  the  three  genera  examined.  Finallv  there  is  an  entire  lack  of  CDH  among  the 

O  J 

brachyurans. 

Experimental  data  concerning  the  distribution  of  CBLH  in  the  reptantians  pre- 
sent an  interesting  problem.  Although  both  the  anomurans  and  brachyurans  pos- 
sess the  body-lightening  hormone  in  varying  amounts  throughout  the  entire  central 
nervous  system,  the  astacurans  appear  to  limit  the  hormone  to  connectives  and 
thoracic  cord.  The  simplest  explanation  for  the  body-darkening  activity  of  the 
astacuran  central-nervous-system  extracts  involves  action  of  the  tail-darkening  prin- 
ciple. It  is  thought  that  CDH  produces  body-darkening  after  CBLH  has  been  ex- 
hausted or  in  the  absence  of  CBLH.  This  is  indicated  in  Figure  2  in  which  selected 
portions  of  the  central  nervous  system  of  Libinia,  Cainbanis,  and  Homarus  are 
shown  to  produce  a  graded  series  of  differential  effects  upon  the  coloration  of  the 
body  of  eyestalkless  Crago.  These  range  all  the  \vay  from  maximum  body-lightening 
and  no  trace  of  darkening  (Libinia  brain)  through  initial  lightening  followed  by 


178 


FRANK  A.  BROWN,  JR.,  AND  LORRA1NK  M.  SAIGH 


darkening,  to  immediate  and  extensive  body-darkening  (Hoinanis  abdominal  cord). 
These  results  are  believed  to  be  explained  in  terms  of  different  relative  amounts  of 
CDH  and  CBLH.  The  former  is  known  to  be  absent  in  the  case  of  Libinia,  and 
it  is  assumed  that  the  latter  is  absent  or  nearly  so  in  the  case  of  Honianis  abdominal 
cord.  In  the  case  of  the  extracts  of  Honuinis  thoracic  cord  and  connectives  and 
those  of  Cainbarus  thoracic  cord,  CBLH  is  present  in  small  amounts  and  lightens 
the  body  for  a  short  time,  thereby  delaying  the  darkening  influence  of  CDH  on  the 
body. 

A  comparison  of  tail-darkening  and  body-darkening  within  Craf/o  injected  with 
nervous  system  extracts  from  numerous  sources  suggests  a  rough  positive  correla- 
tion between  the  two  (Fig.  3).  Generally  speaking,  we  may  infer  from  these  data 
that  the  tendency  towards  body-darkening  is  greater  in  those  animals  showing  a 
high  degree  of  tail-darkening.  This  gives  further  support  for  an  active  role  of 
CDH  in  body-darkening. 

Unlike  the  Dccapoda  the  Isof>oda  apparently  exhibit  a  uniform  distribution  of 
CDH  within  the  central  nervous  system.  However,  since  only  a  single  species  was 
considered,  further  experimentation  is  deemed  necessary  before  any  decisive  state- 
ment is  made  concerning  CDH  distribution  within  this  group. 


10 


20 


TIME 


30 

IN  MINUTES 


FIGURE  2.  The  influences  of  extracts  of  selected  portions  of  the  central  nervous  system  of 
some  crustaceans  upon  the  body  coloration  of  eyestalkless  Crago. 

From  most  positive  to  most  negative  at  the  end  of  10  minutes  are  shown,  respectively,  Huniants 
abdominal  cord,  Homarus  brain,  Hoiuanis  thoracic  cord,  Huniants  circumoesophageal  connectives, 
Cainbarus  thoracic  cord,  and  J.ibinia  brain.  Concentration  in  each  experiment  was:  organs  of 
one  specimen/0.5  ml.  sea-water. 


HORMONES  IN  CRUSTACEAN  NERVOUS  SYSTEMS 


179 


20 


15 


O 


10 


o- 


o  o 


0°° 


o 


o 
o 


00 


o  o   o  oo 


-IO  -5  O  +5 

BODY  LIGHTENING  (-)  OR  DARKENING 


•HO 


FIGURE  3.  The  general  relationship  between  the  degree  of  darkening  or  lightening  of  the 
body  proper  of  eyestalkless  Crago  and  the  degree  of  darkening  of  the  telson  and  uropods. 
Darkening  of  the  tail  is  expressed  as  the  algebraic  sum  of  the  intensities  of  the  reactions  at  5, 
10,  15,  30,  45,  and  60  min.  following  extract  injection,  thereby  including  a  measure  of  both  in- 
tensity and  duration  of  the  effect.  Body-lightening,  being  more  rapidly  transitory,  is  expressed 
as  the  algebraic  sum  of  the  values  at  5,  10,  15,  and  30  min. 


SUMMARY 

1.  A  survey  was  made  of  the  effects  upon  Crago  color-change  of  sea-water  ex- 
tracts of  various  parts  of  the  central  nervous  system  of 'thirteen  species  of  higher 
crustaceans.     The  crustaceans  represented  the  groups  Isopoda,  Natantia,  Astacura, 
Anomura,  and  Brachyura. 

2.  Extracts  of  various  portions  of  the  nervous  system  among  the  various  groups 
showed  wide  differences  in  their  total  chromatophorotropic  activities,  producing 
various    degrees    of   telson    and    uropod    darkening    and    of    body-lightening    and 
darkening. 

3.  An  analysis  of  the  results  gave  support  to  the  hypothesis  that  most  crustacean 
nervous  systems  possess  at  least  two  principles,  a)  a  Crago  body-lightening  prin- 
ciple, CBLH,  lightening  all  portions  of  the  body  except  telson  and  uropods,  and  b} 
a  CVa<70-darkening  hormone,  CDH,  darkening  the  telson  and  uropods,  and,  in  the 
absence  of  CBLH,  the  body  as  well. 


180  FRANK  A.  BROWN,  JR.,  AND  LORRAINE  M.  SAIGtt 

4.  CBLH  is  more  or  less  uniformly  distributed  throughout  the  nervous  systems 
of  all  the  species  examined  except  the  astacurans  in  which  it  is  demonstrated  only 
for  the  circumoesophageal  connectives  and  thoracic  cord. 

5.  CDH  is  restricted  to  the  circumoesophageal  connective  region  of  the  Natantia, 
is  differentially  distributed  throughout  the  nervous  systems  of  anomurans,  with 
highest  concentration  in  the  posterior  region  of  the  thoracic  cord,  and  is  distributed 
throughout  the  nervous  systems  of  the  other  species  except  the  brachyurans  in 
wrhich  it  is  absent. 

LITERATURE  CITED 

BROWN,  F.  A.,  JR.,  1933.  The  controlling  mechanism  of  chromatophores  in  Palaemonetes. 
Proc.  Nat.  Acad.  Sci,  Washington,  19:  327-329. 

BROWN,  F.  A.,  JR.,  1935.  Control  of  pigment  migration  within  the  chromatophores  of  Palaemo- 
netes vulgaris.  Jour.  Exp.  Zool.,  71 :  1-15. 

BROWN,  F.  A.,  JR.,  1946.  The  source  and  activity  of  Crago-darkening  hormone  (CDH). 
Physiol.  Zool.,  19:  215-223. 

BROWN,  F.  A.,  JR.,  AND  H.  E.  EDERSTROM,  1940.  Dual  control  of  certain  black  chromatophores 
of  Crago.  Jour.  Exp.  Zool.,  85 :  53-69. 

BROWN,  F.  A.,  JR.,  AND  A.  MEGLITSCH,  1940.  Comparison  of  the  chromatophorotropic  activity 
of  insect  corpora  cardiaca  with  that  of  crustacean  sinus  glands.  Biol.  Bull.,  79 :  409- 
418. 

BROWN,  F.  A.,  JR.,  AND  V.  J.  WULFF,  1941.  Chromatophore  types  in  Crago  and  their  endo- 
crine control.  Jour.  Cell.  Comp.  Physiol.,  18 :  339-353. 

HANSTROM,  B.,  1937.  Die  Sinusdruse  und  der  hormonal  bedingte  Farbwechsel  der  Crustaceen. 
Kungl.  Svenska  Vetenskap.  Handl,  16:  Nr.  3,  1-99. 

Hosoi,  T.,  1934.  Chromatophore  activating  substance  in  the  shrimps.  Jour.  Fac.  Sci.  Imp. 
Univ.  Tokyo,  3  :  265-270. 

KNOWLES,  F.  G.  W.,  1939.  The  control  of  white-reflecting  chromatophores  in  Crustacea.  Pub- 
bli.  Stas.  Napoli,  17:  174-182. 

ROLLER,  G.,  1930.  Weitere  Untersuchungen  iiber  FaYw'echsel  und  Farbwechsel-hormonen. 
Biol.  Centralbl,  50:  759-768. 


PHYSIOLOGICAL  OBSERVATIONS  ON  WATER  LOSS  AND 
OXYGEN  CONSUMPTION  IN  PERIPATUS  x 

PETER  R.  MORRISON 

Biological  Laboratories,  Harvard  University,  Cambridge,  Massachusetts 

The  small  group  of  species  which  comprises  the  Onychophora  have  long  been 
of  interest  because  of  their  unique  combination  of  arthropod  and  annelid  characters 
which  places  them  in  a  phylogenetic  position  intermediate  to  those  two  extensive 
groups  (Snodgrass,  1938).  They  are  further  of  interest  because  of  their  close 
homogeneity  despite  a  sporadic  distribution  that  encompasses  a  large  portion  of  the 
world  and  points  to  an  ancient  separation  of  some  of  the  genera  (Clark,  1915  ;  Brues, 
1923).  This  homogeneity  appears  to  be  physiological  and  ecological  as  well  as 
morphological,2  since  Peripatus  is  restricted  everywhere  to  a  moist  but  terres'rial 
environment.  Further,  their  sporadic  and  fluctuating  local  distribution  suggests 
that  environmental  variation,  presumably  in  moisture,  is  actively  limiting  their  oc- 
currence even  in  the  regions  where  they  are  found. 

Physiological  observations  on  members  of  this  group,  then,  are  of  interest,  and 
it  is  of  particular  interest  to  examine  the  process  of  water  loss  and  to  contrast  Perip- 
atus in  this  respect  to  comparable  annelids  and  arthropods.  Manton  and  Ramsay 
(1937)  have  reported  a  value  for  water  loss  in  Peripatus  (Pcripatopsis)  at  30° 
and  with  wind  velocity  of  7.0  m./sec.  (16  m.p.h.).  These  conditions,  however, 
seem  rather  severe  for  a  species  which  is  uncomfortable  at  temperatures  above  20° 
(Manton,  1938)  and  which,  living  in  crevices,  must  have  little  exposure  to  wind. 
The  experiments  reported  here  were  made  under  conditions  which  more  nearly  ap- 
proximate those  encountered  by  the  animal  in  nature. 

In  this  connection  reports  in  the  literature  suggest  that  there  may  have  been 
some  temperature  adaptation  in  the  Onychophora.  Thus  the  two  species  studied 
here,  both  from  Panama  (lat.  9°  N.),  stayed  in  good  condition  at  a  temperature  of 
25°  =t.  In  contrast  as  already  noted,  Peripatus  from  near  Cape  Town  (lat.  34°  S.) 
became  uneasy  at  temperatures  above  20°  although  low  temperatures,  even  down  to 
freezing,  did  not  bother  them.  They  survived  very  well  in  England  (Manton, 
1938;  Sedgwick,  1885)  as  have  specimens  from  New  Zealand  (lat.  40°  S.).  The 
latter  were  only  successfully  transported  through  the  intervening  tropical  regions 
with  the  aid  of  refrigeration  (Sedgwick,  1887).  Peripatus  from  New  Zealand 
(Hutton,  1876)  and  from  Australia  (Steel,  1896)  are  reported  to  become  torpid 
during  the  winter  but  with  no  subsequent  ill  effects.  On  the  other  hand  Sclater 
(1887)  reported  that  his  specimens  from  British  Guiana  (lat.  7°  N.)  successfully 

1  These  observations  are  by  no  means  complete,  but  because  the  literature  contains  little  data  on 
living  Onychophora,  particularly  on  New  World  species,  and  because  there  was  no  immediate 
prospect  of  obtaining  a  further  supply  of  these  unusual  animals,  it  seemed  advisable  to  present 
them  at  this  time. 

2  It  should  be  noted,  however,  that  for  such  a  small  group  of  Onychophora  show  remarkable 
diversity  in  their  embryological  development  and  their  mode  of  reproduction. 

181 


182  PETER  R.  MORRISON 

survived  the  trip  to  England,  "but  unfortunately  were  much  affected  by  the  cold, 
and  were  therefore  killed." 

MATERIAL 

These  Peripatus  were  secured  on  Barro  Colorado  Island,  Canal  Zone,  through 
the  great  kindness  of  Mr.  James  Zetek.  Two  species  (note  Clark  and  Zetek,  1946) 
were  obtained,  the  larger  of  which  (Epipcripatus  brasiliensis  varians)  had  a  con- 
tracted length  of  50  mm.  and  was  uniformly  colored  a  rich  red-brown.  The  smaller 
species  (Oroperipatus  corradi)  had  a  contracted  length  of  25  mm.  and  was  a  choco- 
late color  with  lighter  underside  and  with  darker  legs  and  a  dark,  median,  dorsal 
stripe  0.3  mm.  in  width.  The  animals  were  taken  in  -early  September  and  these 
observations  were  made  in  Cambridge  about  a  month  later.  During  the  interim 
they  were  kept  in  moist  forest  debris  but  were  not  given  suitable  food  other  than  the 
supply  of  termites  initially  in  the  debris.  The  animals  survived  the  trip  well  and 
apparently  stayed  in  good  health  until  just  before  death  which  presumably  occurred 
through  starvation. 

The  general  behavior  of  these  individuals  corresponded  to  that  described  for 
other  species  (Manton,  1938;  Holliday,  1942;  Andrews,  1933;  Steel,  1896;  Sedg- 
wick,  1885;  etc.).  They  were  retiring  and  preferred  to  remain  inactive  in  some 
dark  crevice.  They  are  sensitive  to  light  but  react  even  more  sharply  to  dryness 
which  stimulates  them  to  constant  activity.  The  smaller  species  were  definitely 
more  sensitive  in  this  respect  and  could  not  be  held  still  even  for  a  moment. 

An  occurrence  involving  an  individual  of  the  larger  species  may  be  of  particular 
interest.  On  the  occasion  of  mechanical  injury  to  one  of  its  antennae  that  member 
was  placed  in  the  mouth  and  the  injured  portion,  about  half  the  length,  was  removed. 
The  stump  healed  and  the  individual  did  not  appear  to  be  inconvenienced  by  the 
loss.  Parturition  as  observed  in  these  specimens  has  been  described  elsewhere 
(see  Morrison,  1946). 

The  rate  of  oxygen  consumption  and  water  loss  in  Peripatus  was  compared 
to  several  arthropods  and  annelids  of  fairly  similar  size,  habitat  and  body  form : 
centipeds  (Lithobius)  ;  millipeds  (Julits}  ;  sow  bugs  (Onlscus)  ;  and  earthworms. 
These  were  all  collected  locally  with  the  exception  of  one  small  tropical  earthworm 
found  among  the  debris. 

OBSERVATIONS 

Sensory  responses 

With  the  exception  of  the  antennae  the  animals  showed  equal  tactile  sensitivity 
all  over  the  body,  on  the  dorsal  and  ventral  surfaces  and  on  the  legs.  A  very  light 
stimulation  could  be  applied  with  no  response,  a  light  one  produced  a  local  with- 
drawal of  a  leg  or  small  section  of  the  body,  while  a  strong  stimulus  led  to  a  general 
withdrawal.  Holliday  (1942)  noted  that  fairly  large  wood  lice  and  centipeds  could 
crawl  over  the  body  of  a  Peripatus  without  evoking  any  response.  The  antennae 
are  much  more  sensitive  and  the  lightest  touch  here  results  in  the  retraction  of  one 
or  both.  With  stronger  or  repeated  stimulation  the  animal  will  completely  contract 
and  change  its  direction  of  progression ;  further  irritation  provoked  the  well  known 
ejection  from  the  slime  glands.  These  responses  are  in  accord  with  the  histological 
findings  of  Manton  (1937)  that  while  a  single  well  ensconced  sense  capsule  was 


OBSERVATIONS  ON  PERIPATUS  183 

found  in  each  primary  body  papilla,  each  antennal  papilla  bore  at  least  three  much 
more  exposed  capsules  with  much  heavier  innervation. 

The  animals  usually  walked  forwards  but  when  startled  would  often  reverse 
their  direction,  apparently  walking  backwards  with  equal  ease.  Occasionally  they 
would  half  turn  backwards  and  then  move  in  the  form  of  a  "U"  with  the  legs  of 
the  anterior  half  walking  forward  and  those  of  the  posterior  half  walking  backwards. 
This  mode  of  progression  must  impose  an  interesting  problem  in  coordination. 

The  response  of  the  animals  to  a  single  point  source  of  light  (a  two-cell  flashlight 
with  reflector  and  glass  removed,  at  a  distance  of  0.5  to  1  m.)  was  recorded  by  trac- 
ing their  path  on  a  large  underlying  sheet  of  paper.  A  number  of  records  were 
made  both  with  the  light  fixed  and  with  it  moved  through  90  or  180°  halfway 
through  the  record.  Examination  of  the  records  showed  no  oriented  negative 
phototropism ;  indeed,  the  animals  actually  travelled  towards  the  light  more  often 
than  away  from  it.  Thus  these  animals  would  appear  to  be  unable  to  localize  light 
but  only  to  be  aware  of  it.  This  corresponds  to  the  observations  of  Manton  (1938) 
that  the  movement  of  objects  near  Peripatus  elicited  a  response  only  when  accom- 
panied by  air  movement.  These  experiments  were  not  carried  out  in  a  saturated 
environment,  however,  and  it  is  possible  that  with  the  very  strong  stimulus  of  dry- 
ness  removed,  some  phototropic  pattern  might  be  observed. 

Water  balance 

In  measuring  water  loss  the  animals  were  placed  in  large  (D  =  5  cm.)  flat, 
weighing  bottles  containing  a  layer  of  calcium  chloride  covered  by  a  floor  of  brass 
gauze.  Measurements  were  made  at  24°  which  is  within  the  range  normally  en- 
countered by  these  species  (Kenoyer,  1929),  and  for  periods  of  30  minutes.  No 
circulation  was  supplied,  the  movement  of  the  animals  themselves  providing  for 
convection.  The  Peripatus  were  particularly  uneasy  in  this  very  dry  atmosphere 
and  kept  in  constant  and  vigorous  motion. 

The  values  obtained  for  the  two  species  of  Peripatus  and  for  several  other  ani- 
mals are  summarized  in  Table  I.  Water  loss  has  been  computed  on  the  basis  of 
both  body  weight,  and  the  two-thirds  power  of  the  body  weight.3  The  latter  is 
perhaps  a  more  reasonable  basis  for  comparing  animals  of  different  size.  The  two 
values  for  Peripatus  agree  well  and  lie  between  those  found  for  the  annelids  and 
arthropods.  They  indicate  that  Peripatus  has  a  twofold  advantage  over  the  earth- 
worm 4  in  the  conservation  of  water ;  and  that  it  is  at  a  twofold  disadvantage  as 
compared  to  the  centiped,  the  most  xerosensitive  arthropod  studied.  Other  arthro- 
pods showed  values  ranging  down  to  one-twentieth  that  observed  in  Peripatus. 
These  data  are  presented  graphically  in  Figure  1. 

Manton  and  Ramsay  (1937)  reported  on  water  loss  in  Peripatus  under  the 
much  more  rigorous  conditions  of  30°  with  a  7  m./sec.  (16  m.p.h.)  wind  and  a  rela- 

3  This   quantity   is   proportional   to   the   surface   area    in   animals    of    similar   body   form.     In 
Peripatus  and  the  arthropods  where  the  actual  body  surface  is  increased  by  appendages  and 
papillae,  loss  of  water  very  probably  takes  place  largely  through  the  trachae   (note  Mellanby, 
1935).     Water  loss  will  therefore  be  related  to  respiration  which  is  also  roughly  proportional 
to  the  two-thirds  power  of  the  body  weight  in  animals  of  different  size   (Krogh,  1916). 

4  This  will  be  a   minimum   figure  since  the  body  weight  of   the   earthworm   includes  a   con- 
siderable amount  of  dirt  in  the  gut.     These  earthworms  were  kept  in  clean  wet  containers  for 
l1/^  days  before  use,  during  which  time  they  evacuated  up  to  15  per  cent  of  their  weight,  but 
more  undoubtedly  remained. 


184 


PETER  R.  MORRISON 


tive  humidity  of  27.5  per  cent.  They  found  a  value  of  13.0  mg./g.  min.  or  2  to  3 
times  our  value.  A  similarly  measured  value  for  an  earthworm  was  about  half  as 
large  on  a  weight  basis  or  of  equal  magnitude  on  the  basis  of  surface  area.  How- 
ever, the  advisability  of  making  measurements  under  physiological  conditions  should 
be  stressed  since  under  abnormal  circumstances  quite  different  relations  may  hold. 
Thus,  for  example,  Ramsay  (1935)  showed  that  in  the  cockroach  water  was  lost 


O 


UJ 
Q_ 

i 


25 


20 


*       15 


c/) 
O 

.J 

CL 
UJ 


10 


0.2 


0.4 


0.6 


0.8 


BODY    WEIGHT     IN     GRAMS 

FIGURE  1.  Water  loss  in  Peripatus  and  other  animals  at  24°  over  calcium  chloride  as  a 
function  of  the  body  weight.  Open  circles,  earthworms ;  crossed  circles,  Peripatus ;  half-closed 
circles,  centipeds ;  lined  circles,  sow  bugs ;  closed  circles,  millipeds.  The  curves  represent 
}" -=  K  (X)2/3,  where  the  values  for  K  are  the  average  values  given  in  Table  I. 

much  more  rapidly  at  temperatures  above  30°  with  an  apparent  breakdown  of  the 
hydrophobic  character  of  the  body  surface. 

In  considering  this  function  it  is  of  interest  to  note  that  Clark  (1915)  concluded 
on  the  basis  of  distributional  and  taxonomic  considerations  that  the  Onychophora 
had  originally  evolved  in  a  cooler  rather  than  a  warmer  environment.  Thus,  the 
more  primitive  groups  are  found  on  mountains  or  in  the  "temperate"  regions  while 


OBSERVATIONS  ON  PERIPATUS 


185 


the  more  recent  forms  are  tropical.  This  is,  of  course,  entirely  in  accord  with  the 
physiological  considerations  since  the  xerotic  stress  would  be  reduced  at  a  lower 
temperature  and  such  an  environment  would  be  more  favorable  for  evolution  from 
an  aquatic  to  a  terrestrial  mode  of  life. 

TABLE  I 

Water  loss  in  Peripatus  and  other  animals  at  24°  over  calcium  chloride 


Water  loss 

.    .      . 

Number  and  weight 

Duration  of  experi- 

in mg. 

ment  in  min. 

mg./g.min. 

mg./g.2/3min. 

Earthworm 

884 

15 

7.4 

7.1 

703 

15 

12.0 

10.4 

360 

15 

13.4 

9.6 

208 

10 

17.3 

;  10.4 

105 

8 

22.6 

10.7 

Peripatus 

Epiperipatus 

788 

30 

5.2 

4.7 

Oroperipatiis 

423 

30 

6.6 

5.0 

Centiped 

150 

60 

5.6 

3.0 

135 

20 

5.9 

3.1 

4X95 

60 

4.1 

1.9 

4X63 

30 

7.2 

2.8 

Sow  bug 

158 

25 

3.0 

1.6 

97 

40 

2.5 

1.2 

6X49 

60 

3.0 

1.1 

48 

20 

4.5 

1.6 

Milliped 

3X76 

120 

0.56 

0.24 

3X98 

600 

0.44 

0.21 

A  verages 

Earthworm 

15  Experiments 

9.9 

Peripatus 

2  Experiments 

4.9 

Centiped 

4  Experiments 

2.5 

Sow  bug 

5  Experiments 

1.3 

Milliped 

2  Experiments 

0.22 

Respiration 

The  oxygen  consumption  of  the  larger  species  of  Peripatus  and  of  various  other 
animals  was  measured  in  a  Warburg  apparatus.5  Carbon  dioxide  was  absorbed  in 
sodium  hydroxide  in  a  small  cup  fused  to  the  bottom  of  the  chamber.  The  animals 
were  placed  directly  in  the  chamber  and  were  kept  from  the  lye  by  a  small  screen 
shield.  Measurements  were  made  at  25.0°  C.  over  a  period  of  60  minutes. 

The  results  on  Peripatus  are  shown  in  Figure  2.  After  a  restless  initial  period 
(10  minutes)  it  settled  down  to  a  very  uniform  rate  of  oxygen  consumption.  The 
centipeds,  also  shown  in  Figure  2,  were  less  regular.  The  results  for  the  various 

5  I  am  indebted  to  Dr.  William  Carroll  for  the  use  of  his  calibrated  Warburg  assembly. 


186 


PETER  R.  MORRISON 


animals  are  summarized  in  Table  II.  The  exact  significance  of  the  "resting"  or 
"basal"  oxygen  consumption  is  not  known  but  some  correlation  between  it  and  the 
"intensity"  of  the  organism  has  been  observed.  Compared  on  a  weight  basis  Perip- 
atus  consumes  oxygen  at  the  same  rate  as  the  earthworm  and  at  about  half  the  rate 
of  the  arthropods.  It  has  been  observed,  however,  that  within  a  given  group,  the 
metabolism  per  unit  of  weight  varies  with  the  size  of  the  animal  (note  Edwards, 
1946,  for  example),  and  that  the  metabolism  is  more  nearly  proportional  to  some 


16 


ID 
O 


Q. 

2 
ID 
(/) 
Z 
O 

o 


LL) 
O 

X 

o 


8 


20 


40 


60 


TIME      IN      MINUTES 


FIGURE  2.  Oxygen  consumption  in  Peripatus  and  the  centiped  as  a  function  of  time.  Open 
circles,  Peripatus  (Epiperipatus),  0.68  g. ;  closed  circles,  3  centipeds,  total  weight  0.33  g. ; 
temperature,  25°. 

lower  power  of  the  weight.  As  a  first  approximation  this  may  be  taken  as  the 
two-thirds  power  (Krogh,  1916).  When  the  oxygen  consumption  is  compared  on 
this  basis,  Peripatus  agrees  more  closely  with  the  arthropods  and  has  a  higher  value 
than  the  earthworm. 

The  hydrophobic  character  of  the  body  surface  has  been  noted  by  many  ob- 
servers. It  is  particularly  evident  when  the  animal  is  submerged  since  the  body 
papillae  hold  the  water  away  from  the  body  surface  and  leave  the  animal  entirely 


OBSERVATIONS  ON  PERIPATUS 


187 


surrounded  by  a  sheath  of  air.  It  would  seem  entirely  possible  that  this  air  sheath 
may  function  as  a  respiratory  surface  under  water.  Such  a  mechanism  has  been 
demonstrated  in  certain  aquatic  insects  which  carry  down  an  air  supply  by  means 
of  hydrophobic  hairs  and  which,  by  this  means,  greatly  extend  their  periods  under 
water  (Krogh,  1941 ;  Wigglesworth,  1931).  Since  Peripatus  must  be  often  covered 
by  water  in  rainstorms,  particularly  as  its  lack  of  resistance  to  dessication  forces  it 
to  frequent  wet  places,  this  mechanism  could  be  of  real  utility  and  have  a  consider- 
able survival  value.  This  would  provide  a  functional  explanation  for  the  papilla- 
covered  body  surface  which  is  characteristic  and  unique  in  the  Onychophora. 

TABLE  II 

Oxygen  consumption  in  Peripatus  and  other  animals  at  25°  C. 


Oxygen  consumption 

Animal 

Weight  in  mg. 

cc./g.hr. 

cc./g.2«hr. 

Earthworm 

96 

0.22 

0.10  ' 

Peripatus 

(Epiperipatus) 

680 

0.23 

0.20 

Millipeds 

3X111 

0.46 

0.22 

Centipeds 

2X69 

0.56 

0.22 

Pill  bugs 

5X61 

0.35  2 

0.14 

1  Lesser  (1908)  reported  values  of  0.4  cc.  per  g.2/3hr.  at  19°  at  which  temperature  the  oxygen 
consumption  should  be  about  half  that  measured  at  25°  (Vernon,  1897). 

2  Edwards  (1946)  reports  a  similar  value  but  at  a  temperature  of  17°. 

SUMMARY 

The  Onychophora  represent  a  morphological  transition  between  the  annelids  and 
the  arthropods.  They  also  represent  a  physiological  transition  between  the  aquatic 
and  the  terrestrial  environment.  In  the  latter  transition  the  most  important  adapta- 
tions are  those  involving  the  functions  of  water  conservation  and  respiration. 

The  ability  of  Peripatus  to  conserve  water  has  been  compared  to  that  of  com- 
parable annelids  and  arthropods.  Peripatus  is  shown  to  be  intermediate  to  those 
two  groups  in  this  function,  losing  twice  as  much  water  as  the  centiped,  but  only 
one-half  as  much  as  the  earthworm.  This  corresponds  to  its  taxonomic  and  ecologi- 
cal positions. 

The  "resting"  rate  of  oxygen  consumption  has  also  been  compared  to  other  ani- 
mals. The  rate  in  Peripatus  is  comparable  to  that  in  the  arthropods  and  larger  than 
that  in  the  earthworm. 

It  is  suggested  that  the  unique  papilla-covered  body  surface  may  represent  an 
adaptation  for  underwater  respiration  to  meet  the  environmental  restriction  imposed 
by  the  inadequate  regulation  of  water  loss. 

LITERATURE  CITED 

ANDREWS,  E.  A.,  1933.     Peripatus  in  Jamaica.     Quart.  Rev.  Biol.,  8 :  155. 

BRUES,  C.  T.,  1923.     The  geographical  distribution  of  the  Onychophora.     Amcr.  Nat.,  57:  210. 
CLARK,  A.  H.,  1915.     The  present  distribution  of  the  Onychophora,  a  group  of  terrestrial  in- 
vertebrates.    Smithsonian  Misc.  Coll.,  65 :  1-25. 


188  PETER  R.  MORRISON 

CLARK,  A.  H.,  AND  J.  ZETEK,  1946.     The  Onychophores  of  Panama  and  the  Canal  Zone.     Proc. 
U.  S.  Nat.  Mus.,  96 :  205. 

EDWARDS,  G.  A.,  1946.     The  influence  of  the  temperature  upon  the  oxygen  consumption  of  sev- 
eral arthropods.    Jour.  Cell.  Comp.  Physiol.,  27 :  53. 

HOLLIDAY,  R.  A.,  1942.     Some  observations  on  Natal  Onychophora.    Ann.  Natal.  Mus.,  10:  237. 

HUTTON,  F.  W.,  1876.     On  Peripatus  Novae-Zealandiae.    Ann.  Mag.  Nat.  Hist.   (4)    18:  361. 

KENOYER,  L.  A.,  1929.     General  and  successional  ecology  of  the  lower  tropical  rain  forest  at 
Barro  Colorado  Island,  Panama.     Ecology,  10:  210. 

KROGH,  A.,  1916.     The  respiratory  exchange  of  animals  and  man.     London,  Longmans,  Green 
and  Co.,  173  pp. 

KROGH,  A.,  1941.     The  comparative  physiology  of  respiratory  mechanisms.     Philadelphia,  The 
University  of  Pennsylvania  Press,   172  pp. 

LESSER,  E.  J.,  1908.     Chemische  Prozesse  bei  Regenwiirmern.     Zeit,  f.  Biol.,  50:  421. 

MANTON,  S.  M.,  1937.     Studies  on  the  Onychophora.     II.  The  feeding,  digestion,  excretion  and 
food  storage  of  Peripatopsis.     Proc.  Roy.  Soc.  London,  B,  227  :  411. 

MANTON,   S.   M.,   1938.     Studies   on  the   Onychophora.     VI.   The   life  history  of   Peripatopsis. 
Ann.  Mag.  Nat.  Hist.  (11),  1 :  515. 

MANTON,  S.  M.,  AND  J.  A.  RAMSAY,  1937.     Studies  on  the  Onychophora.     III.  The  control  of 
water  loss  in  Peripatopsis.     Jour.  Exp.  Bio!.,  14:  470. 

MELLANBY,  K.,  1935.     The  evaporation  of  water  from  insects.    Biol.  Rev.,  10:  317. 

MORRISON,  P.  R.,  1946.     Parturition  in  Peripatus.     Psyche    (In  press). 

RAMSAY,  J.  A.,  1935.     The  evaporation  of  water  from  the  cockroach.    /.  Exp.  Biol.,  12 :  373. 

SCLATER,  W.  L.,  1887.     Notes  on  the  Peripatus  of  British  Guiana.    Proc.  Zool.  Soc.  London, 
130. 

SEDGWICK,  A.,  1885.     A  monograph  on  the  development  of  Peripatus  capensis.     Quart.  Jour. 
Micr.  Sci.,  25  :  449. 

SEDGWICK,  A.,  1887.     A  monograph  of  the  genus  Peripatus.     Quart.  Jour.  Micr.  Sci.,  28:  431. 

SNODGRASS,  R.  E.,  1938.     Evolution  of  the  Annelida,  Onychophora,  and  Arthropoda.    Smith- 
sonian Misc.  Coll.,  97  :  6. 

STEEL,  T.,  1896.     Observations  on  Peripatus.     Proc.  Linn  can  Soc.  New  South  Wales,  21 :  94. 

VERNON,  H.  M.,   1897.     The  relation  of  the  respiratory  exchange  of  cold  blooded  animals  to 
temperature.     Jour.  Physiol.,  21  :  443. 

WIGGLESWORTH,  V.  B.,  1931.     The  respiration  of  insects.    Biol.  Rev.,  6:   181. 

J 


STUDIES  ON  CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE 

CHATTON  AND  LWOFF  (ORDER  HOLOTRICHA, 

SUBORDER  THIGMOTRICHA) 

III.    ANCISTROCOMA  PELSENEERI  CHATTON  AND  LWOFF, 

ANCISTROCOMA  DISSIMILIS  SP.  NOV.,  AND 

HYPOCOMAGALMA  PHOLADIDIS  SP.  NOV. 

EUGENE  N.  KOZLOFF 
Lewis  and  Clark  College,  Portland,  Oregon 

INTRODUCTION 

Chatton  and  Lwoff  described  in  1926  two  ciliates  for  which  they  created  the 
genus  Ancistrocorna:  A.  pelseneeri,  from  the  gills  and  palps  of  Macoma  balthica 
(L.)  ;  and  A.  pholadis,  from  Barnea  (Pholas)  Candida  (L.).  Their  descriptions  of 
these  two  species  are  of  a  preliminary  nature  and  are  not  accompanied  by  illustra- 
tions. More  detailed  descriptions  of  A.  pelseneeri,  together  with  illustrations,  are 
given  in  two  papers  of  Raabe  (1934,  1938). 

Kofoid  and  Bush  (1936)  described  as  Parachaenia  myae  a  ciliate  from  the  peri- 
cardial  cavity  and  excurrent  siphon  of  My  a  arenaria  L.  which  Kirby  (1941)  noted 
was  in  several  respects  apparently  identical  with  A.  pelseneeri.  Kudo  (1946), 
however,  listed  Parachaenia  myae  as  a  valid  species  in  the  suborder  Gymnostomata. 
Kofoid  and  Bush  stated  that  they  did  not  find  P.  myae  in  any  other  molluscs  which 
were  present  in  the  same  localities  as  the  host  species.  I  have  studied  the  ciliate 
associated  with  Mya  arenaria  in  San  Francisco  Bay  and  have  compared  it  with 
similar  forms  from  Cryptomya  calif ornica  (Conrad),  Macoma  inconspicua  Broderip 
and  Sowerby,1  Macoma  nasuta  (Conrad),  and  Macoma  ims  (Hanley)  from  San 
Francisco  Bay,  and  from  Macoma  sect  a  (Conrad)  from  Tomales  Bay,  California. 
I  have  concluded  that  the  ciliate  described  by  Kofoid  and  Bush  as  Parachaenia  myae 
is  not  specific  in  Mya  arenaria  and  that  P.  myae  is  identical  with  Ancistrbcoma 
pelseneeri  Chatton  and  Lwoff. 

On  the  gills  and  palps  of  the  rock-boring  piddock  Pholadidea  penita  (Conrad) 
there  occurs  a  species  of  Ancistrocoma  which  is  clearly  distinct  from  A.  pelseneeri 
and  which  I  will  describe  in  this  paper  as  Ancistrocoma  dissimilis  sp.  nov.  Another 
ciliate  I  have  studied  from  P.  penita  is  referable  to  the  genus  Hypocomagalma,  cre- 
ated by  Jarocki  and  Raabe  (1932)  for  H.  dreissenae,  from  the  fresh  water  mussel 
Drcissena  polyrnorpha  (Pall.).  It  will  be  described  herein  as  Hypocomagalma 
pholadidis  sp.  nov. 

1  By  some  malacologists  the  small  species  of  Macoma  referred  to  in  this  paper  as  M.  incon- 
spicua is  considered  to  be  conspecific  with  M.  balthica;  by  others  it  is  considered  to  be  a  sub- 
species of  M.  balthica.  No  conclusive  evidence  has  been  presented  in  the  literature  in  recent 
years  either  to  support  or  refute  these  contentions. 

189 


190 


EUGENE  N.  KOZLOFF 


ANCISTROCOMA  PELSENEERI  CHATTON  AND  LWOFF 
(Figure  1 ;  Plate  I,  Figs.  1,  2) 

The  body  is  elongated  and  somewhat  flattened  dorso-ventrally.2  As  seen  in 
lateral  view,  the  ciliate  is  banana-shaped,  the  ventral  surface  being  incurved.  The 
anterior  end  is  more  or  less  attenuated.  The  body  is  usually  wjdest  and  thickest 
in  its  posterior  third.  Forty  living  individuals  taken  at  random  from  Mya  arenaria 
ranged  in  length  from  50^  to  83  p.,  in  width  from  14 /A  to  20  /*,  and  in  thickness 
from  11 /A  to  16^,,  averaging  about  62  ,11  by  16 /x  by  12.5  //.  Twenty  individuals 
from  Macoma  inconspiaia  ranged  in  length  from  52  /u.  to  78  /*,  in  width  from  14  ju, 
to  19  p.,  and  in  thickness  from  11  p.  to  15  /JL. 


FIGURE  1.    Ancistrocoma  pelsenceri  Chatton  and  Lwoff.     Distribution  of  ciliary  rows,  somewhat 
diagrammatic.3     A,  dorsal  aspect ;   B,  ventral  aspect. 

The  anterior  end  is  provided  with  a  contractile  suctorial  tentacle  which  enables 
the  ciliate  to  attach  itself  to  the  epithelial  cells  of  the  gills  and  palps  of  the  host  and 
to  suck  out  their  contents.  The  internal  tubular  canal  continuous  with  the  tentacle 
is  directed  at  first  dorsally  and  then  ventrally  and  obliquely  toward  the  right  side 
of  the  body.  It  can  usually  be  traced  in  fixed  individuals  stained  with  iron  hema- 
toxylin  for  about  two-thirds  the  length  of  the  body.  Kofoid  and  Bush  suggested 
only  in  the  title  of  their  paper  that  the  form  which  they  named  Parachaenia  myae 
was  parasitic  in  Mya  arenaria,  but  did  not  describe  attachment  of  the  ciliate  to  the 
epithelium.  They  found  the  ciliate  in  the  pericardial  cavity  and  excurrent  siphon 

2  Kofoid  and  Bush  stated  in  their  description  of  "Parachaenia  myae"  that  the  body  of  this 
ciliate  is  bilaterally  compressed,  the  transverse  diameter  being  about  two-thirds  the  dorso-ventral 
diameter.     Obviously  their  orientation  of  the  form  in  question  is  not  in  agreement  with  the 
orientation  assigned  to  it  by  Chatton  and  Lwoff,  Raabe,  and  myself. 

3  All  text-figures  illustrating  this  paper  are  based  on  camera  lucida  drawings  of  individuals 
fixed  in  Schaudinn's  fluid  and  impregnated  with  activated  silver  albumose   (protargol). 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.    Ill  191 

oi  the  clams  and  apparently  believed  it  to  be  unattached  and  to  feed  as  a  gymno- 
stome,  by  producing  a  current  in  the  medium  by  means  of  vigorous  ciliary  activity 
which  carries  food  particles  to  the  mouth.  They  stated  that  they  observed  a  few 
instances  of  food  taking,  in  which  "debris  containing  bacteria  enters  the  mouth  and 
moves  along  the  cytopharynx,  forming  little  globules  which  continue  back  and  aggre- 
gate in  the  large  food  vacuoles  which  distend  the  posterior  part  of  the  body."  They 
stated  further  that  "stained  specimens  show  some  vacuoles  containing  broken-up 
nuclear  material  similar  to  that  of  the  epithelial  cells  which  are  removed  when  the 
fluid  is  taken  from  the  clam."  I  have  not  observed  any  instances  of  ingestion  of 
food  such  as  that  described  by  Kofoid  and  Bush,  and  although  I  admit  it  is  perhaps 
possible  for  the  ciliates  to  ingest  food  in  this  manner,  I  believe  that  they  are  pri- 
marily branchial  parasites  which  feed  by  means  of  the  suctorial  tentacle. 

The  cilia  of  A.  pelseneeri  are  disposed  on  the  ventral,  lateral,  and  dorso-lateral 
surfaces  of  the  body  in  longitudinal  rows  originating  at  the  anterior  end.  In  all 
individuals  which  I  examined  carefully  the  number  of  ciliary  rows  was  fourteen, 
but  Raabe  stated  that  in  some  specimens  there  are  but  thirteen  rows.  According  to 
Raabe  the  ciliary  system  is  composed  of  three  separate  complexes,  the  first  consist- 
ing of  eight  or  nine  rows  spiralling  from  the  left  side  of  the  body  toward  the  right 
and  terminating  progressively  more  posteriorly  on  the  ventral  surface,  the  second 
consisting  of  two  approximately  meridional  rows  situated  on  the  central  part  of  the 
ventral  surface,  and  the  third  consisting  of  three  rows  spiralling  from  the  right  side 
of  the  body  toward  the  left  and  terminating  on  the  ventral  surface.  After  studying 
a  large  number  of  the  ciliates  from  Mya  arenaria  and  Macoma  inconspicua  I  cannot 
agree  with  Raabe  on  this  matter.  The  ciliary  rows  appear  collectively  to  form  a 
single  complex.  There  are  usually  five  approximately  equal  rowrs  about  two-thirds 
the  length  of  the  body  occupying  the  central  portion  of  the  ventral  surface ;  these  are 
bounded  on  the  right  by  three  progressively  longer  and  more  widely-spaced  rows 
and  on  the  left  by  six  progressively  longer  and  more  widely-spaced  rows.  In  some 
specimens  the  number  of  longer  rows  on  the  left  side  is  greater  than  six,  in  which 
case  the  number  of  approximately  equal  and  more  or  less  meridional  rows  is  pro- 
portionately decreased.  Some  of  the  outer  rows  on  either  side  of  the  body,  which 
originate  on  the  lateral  margins  or  on  the  dorsal  surface,  curve  ventrally  as  they  ex- 
tend posteriorly,  but  the  last  two  rows  on  the  left  side  and  the  last  row  on  the  right 
side  are  typically  dorso-lateral  in  position  over  their  entire  length.  The  outermost 
row  on  either  side  extends  almost  to  the  posterior  tip  of  the  body.  Kofoid  and 
Bush  stated  that  the  ciliary  rows  of  "Parachacnia  myac"  may  unite  with  one  an- 
other, but  I  have  never  observed  this  to  be  the  case,  although  in  some  seriously 
shrunken  fixed  individuals  a  few  of  the  rows  converge  in  such  a  way  that  they  ap- 
pear to  be  united. 

In  one  of  the  illustrations  accompanying  the  first  of  Raabe's  papers  in  which 
there  is  a  detailed  discussion  of  A.  pelseneeri  (1934)  the,  outermost  ciliary  row  on 
the  right  side  of  the  body  is  shown  to  originate  as  far  anteriorly  as  the  more  cen- 
tral rows,  while  the  outer  three  or  four  rows  on  the  left  side  are  shown  to  originate 
progressively  more  posteriorly.  According  to  my  own  observations,  however,  the 
outermost  row  on  the  right  side  originates  at  about  the  same  level  as  the  last  row 
on  the  left  side.  In  all  suitably  impregnated  individuals  which  I  have  studied  the 
eighth  row  from  the  right  side  originates  a  little  posterior  to  the  level  of  origin  of 
the  adjacent  ventral  rows. 


192  EUGENE  N.  KOZLOFF 

The  cilia  of  A.  pelseneeri  are  8  ^  to  10  p.  in  length.  Those  at  the  anterior  end 
of  the  body  are  usually  the  more  active  and  may  be  employed  for  thigmotactic  at- 
tachment. Kofoid  and  Bush  stated  that  the  cilia  of  the  "dorso-bilateral  region"  of 
"Parachaenia  myae"  are  about  20  /x  long  near  the  anterior  end,  becoming  somewhat 
shorter  posteriorly ;  the  cilia  of  the  ventral  surface,  on  the  other  hand,  were  said 
by  them  to  be  about  one-half  the  length  of  those  of  the  dorso-bilateral  area.  I  have 
noted,  however,  no  significant  disparity  between  the  lengths  of  the  cilia  of  various 
parts  of  the  ciliary  system.  When  dissociated  from  the  host  the  ciliate  swims  ener- 
getically, rotating  on  its  longitudinal  axis  or  swaying  from  side  to  side. 

In  the  original  description  of  A.  pelseneeri  given  by  Chatton  and  Lwoff  refer- 
ence is  made  to  a  "frange  peristomienne"  which  they  supposed  corresponded  to  the 
peristomal  fringe  of  cilia  in  species  of  Ancistrmna.  In  his  paper  of  1934,  Raabe 
described  a  short  (approximately  13  /x  long)  row  of  basal  granules  lying  in  a  dorsal 
anterior  depression  just  above  the  anterior  part  of  the  internal  tubular  canal  which 
he  thought  may  represent  the  "frange  peristomienne"  described  by  Chatton  and 
Lwoff.  In  his  paper  of  1938,  however,  Raabe  stated  that  on  certain  of  his  prepara- 
tions of  this  ciliate  he  could  distinguish  a  row  of  basal  granules  such  as  he  described 
in  1934,  but  did  not  refer  to  it  as  the  peristomal  fringe,  and  suggested  that  Chatton 
and  Lwoff  may  have  mistaken  the  stained  outline  of  the  internal  tubular  canal  for 
a  row  of  basal  granules  homologous  with  those  of  the  peristomal  fringe  of  ancis- 
trumid  ciliates.  In  my  study  of  living,  stained,  and  impregnated  individuals  of  the 
ciliate  I  believe  to  be  A.  pelseneeri  I  have  found  no  evidence  whatever  of  a  dorsal 
anterior  depression  or  a  row  of  basal  granules  such  as  that  described  by  Raabe. 

Kofoid  and  Bush  described  internal  fibrillar  structures,  wrhich  they  believed  to 
represent  elements  of  the  neuromotor  system,  extending  for  a  short  distance  poste- 
riorly from  an  annular  commissure  ("cytostomal  ring")  around  the  "cytostome." 
One  of  the  fibrils  was  said  by  them  to  pass  along  the  internal  tubular  canal  ("cyto- 
pharynx")  to  a  slight  thickening  on  the  surface  of  the  canal,  then  "towards  the 
dorsal  surface  where  it  joins  a  relatively  large  granule  which  is  closely  associated 
with  the  mid-dorsal  ciliary  fibril."  They  stated  further  that  "from  points  of  the 
cytostomal  ring  on  the  ventral  side,  two  fibrils  are  given  off  which  soon  unite  and 
continue  as  a  slender  thread  along  the  ventral  surface  of  the  cytopharynx."  I  have 
been  unable  to  detect  any  structures  in  A.  pelseneeri  which  might  be  construed  as 
elements  of  a  neuromotor  system,  but  perhaps  it  is  a  siderophilic  fibril-like  structure 
of  the  type  that  Kofoid  and  Bush  described  that  Raabe  may  have  thought  to  repre- 
sent a  series  of  basal  granules.  The  "cytostomal  ring"  around  the  "cytopharynx" 
was  stated  by  Kofoid  and  Bush  to  be  connected  with  the  longitudinal  ciliary  rows, 

EXPLANATION  OF  PLATE  I 

FIGURE  1.  Ancistrocoma  pelseneeri  Chatton  and  Lwoff  (from  Mya  arenaria).  Ventral 
aspect.  Heidenhain's  "susa"  fixative-iron  hematoxylin.  X  1,680. 

FIGURE  2.  Ancistrocoma  pelseneeri  Chatton  and  Lwoff  (from  Macoma  inconspicua) . 
Lateral  aspect  from  left  side,  from  life. 

FIGURE  3.  Ancistrocoma  dissimilis  sp.  nov.  Ventral  aspect.  Schaudinn's  fixative-iron 
hematoxylin.  X  1,680. 

FIGURE  4.  Hypocomagalma  pholadidis  sp.  nov.  Dorsal  aspect.  Schaudinn's  fixative-iron 
hematoxylin.  X  1,260. 

FIGURE  5.  Hypocomagalma  pholadidis  sp.  nov.  Ventral  aspect.  Schaudinn's  fixative-iron 
hematoxylin.  X  1,260. 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.     Ill 


193 


ii\\ 


- 


ii 

. 

r 


JK; 


PLATE  I 


194  EUGENE  N.  KOZLOFF 

but  I  have  not  observed  this  to  be  the  case  in  A.  pclscnccri.  As  has  been  pointed 
out  above,  some  of  the  rows  do  not  originate  as  close  to  the  base  of  the  suctorial 
tentacle  as  others.  It  is  possible  that  the  structure  referred  to  by  Kofoid  and  Bush 
as  the  "cytostomal  ring"  represents  the  siderophilic  anterior  edge  of  the  contracted 
suctorial  tentacle. 

The  cytoplasm  is  colorless  and  contains  numerous  small  refractile  granules  of  a 
lipoid  substance.  In  the  posterior  part  of  the  body  there  are  in  addition  to  typical 
food  vacuoles  containing  ingested  fragments  of  epithelial  cells  one  or  more  large 
vacuoles  containing  globular  masses  usually  of  a  dense,  homogeneous  character. 
Raabe  referred  to  this  type  of  vacuole  as  "Konkrementenvacuole"  and  suggested 
that  since  he  observed  the  internal  tubular  canal  to  terminate  very  near  the 
"Konkrementenvacuole"  the  material  within  the  vacuole  may  represent  an  accumula- 
tion of  waste  material  which  was  not  digested  and  absorbed  as  the  ingested  food 
material  passed  backward  down  the  canal.  It  is  quite  true  that  these  concrement 
vacuoles  do  not  resemble  the  typical  food  vacuoles  of  most  other  ancistrocomid 
ciliates  which  I  have  studied.  It  would  be  interesting  to  determine  whether  or  not 
digestion  and  absorption  take  place  in  the  internal  tubular  canal,  and  how  the  mate- 
rial in  the  concrement  vacuole,  if  it  represents  undigested  wastes,  is  gotten  rid  of 
by  the  ciliate. 

The  macronucleus  is  usually  sausage-shaped,  rarely  ovoid,  and  typically  is  situ- 
ated dorsally  near  the  middle  of  the  body.  In  some  fixed  specimens  stained  with 
iron  hematoxylin  the  chromatin  appears  to  be  distributed  in  irregular  masses  scat- 
tered through  the  macronuclear  material ;  in  other  iron  hematoxylin  preparations 
and  in  most  specimens  stained  by  the  Feulgen  reaction  the  chromatin  is  aggregated 
into  a  dense  reticulum  enclosing  vacuole-like  clear  spaces.  In  twenty  individuals 
from  Mya  arcnaria  fixed  in  Schaudinn's  fluid  and  stained  by  the  Feulgen  reaction 
the  macronucleus  ranged  in  length  from  11  ^  to  16  p.  and  in  width  from  4  p.  to  7  p. 

The  micronucleus  is  ovoid,  fusiform,  or  sausage-shaped,  and  usually  is  seen  to 
lie  to  the  right  of  the  macronucleus.  In  fixed  and  stained  specimens  the  chromatin 
is  ordinarily  aggregated  into  granules.  In  twenty  individuals  from  Mya  arcnaria 
fixed  in  Schaudinn's  fluid  and  stained  by  the  Feulgen  reaction  the  micronucleus 
ranged  in  size  from  1.2  /x  by  3  ^  to  2.1  /j,  by  3.2  ju. 

Ancistrocoina  pclscnccri  is  very  common  in  M\a  arcnaria  in  all  localities  in  San 
Francisco  Bay  where  I  have  collected  this  mollusc.  I  have  found  it  to  be  present, 
although  usually  in  smaller  numbers,  also  in  Cryptoniya  calljornlca,  Maconia  incon- 
splcua,  M.  nasuta,  and  M.  irus  from  several  localities  in  San  Francisco  Bay,  and  in 
Macoma  sccta  from  Tomales  Bay.  It  is  peculiar  that  this  ciliate  was  not  recorded 
by  Raabe  from  Mya  arenaria  at  the  marine  biological  station  at  Hel.  Raabe  listed 
Sphcnophyra  doslniac  Chatton  and  LwofF.  Hypocomidium  grainun  Raabe,  and  a 
species  of  Ancistruma  which  he  provisionally  referred  to  A.  cyclidioides  (Issel), 
from  M.  arcnaria.  I  have  found  6".  doslniac  in  a  small  percentage  of  M.  arcnaria 
and  in  a  fairly  large  percentage  of  Cryptomya  calijornica  from  San  Francisco  Bay. 
I  have  also  found  in  M.  arcnaria  the  ciliate  thought  by  Raabe  to  be  A.  cyclidioides, 
but  not  Hypocomidium  graimin. 

Ancistrocoina  pclscnccri   Chatton  and  Lzvoff    (=  Parachacnia  myac  Kofoid  and 
Bush}  • 

Diagnosis:  Length  50  /*-83  /JL  (according  to  Kofoid  and  Bush  40/x-100/i),  aver- 
age about  62  p.;  width  14//.-20/*,  average  about  16^;  thickness  11  ^-16  /x,  average 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.     Ill  195 

about  12.5  JJL.  The  ciliary  rows  are  fourteen  (according  to  Raabe  thirteen  or  four- 
teen) in  number  and  are  distributed  on  the  ventral,  lateral,  and  dorso-lateral  sur- 
faces of  the  body.  There  are  usually  five  approximately  equal  rows  about  two- 
thirds  the  length  of  the  body  on  the  ventral  surface,  bounded  on  the  right  by  three 
progressively  longer  and  more  widely-spaced  rows  and  on  the  left  by  six  progres- 
sively longer  and  more  widely-spaced  rowrs.  The  outermost  row  on  either  side 
extends  almost  to  the  posterior  tip  of  the  body.  The  more  central  rows  originate 
close  to  the  base  of  the  suctorial  tentacle,  while  the  several  outer  rows  on  either  side 
originate  progressively  more  posteriorly  on  the  lateral  margins  and  the  dorsal  sur- 
face. Some  of  these  rows  curve  ventrally  as  they  extend  posteriorly,  but  the  two 
outer  rows  on  the  left  side  and  the  outermost  row  on  the  right  side  are  typically 
dorso-lateral  in  position  over  their  entire  length.  The  macronucleus  is  usually 
sausage-shaped.  The  micronucleus  is  ovoid,  fusiform,  or  sausage-shaped.  Para- 
sitic on  the  epithelium  of  the  gills  and  palps  of  Maconia  balthica  (L.)  (Wimereux 
[Chatton  and  Lwroff]  ;  Hel  [Raabe] )  ;  J\Iacoina  inconspicua  Broderip  and  Sowerby, 
Macoma  uasuta  (Conrad),  Maconia  inis  (Hanley),  Cryptomya  calijornica  (Con- 
rad) (San  Francisco  Bay,  California)  ;  Maconia  sccta  (Conrad)  (Tomales  Bay, 
California)  ;  My  a  arcnaria  L.  (Tomales  Bay  [Kofoid  and  Bush]  ;  San  Francisco 
Bay). 

ANCISTROCOMA  DISSIMILIS  SP.  NOV. 
(Figure  2;  Plate  I,  Fig.  3) 

The  body  is  elongated,  attenuated  anteriorly,  and  somewhat  flattened  dorso- 
ventrally.  The  ciliary  system,  to  be  described  presently,  is  disposed  for  the  most 
part  on  the  incurved  and  slightly  concave  ventral  surface.  The  body  is  widest  and 
thickest  in  its  posterior  third  and  rounded  posteriorly.  Twenty  living  individuals 
taken  at  random  ranged  in  length  from  33 /x  to  51  /x,  in  width  from  10  ^  to  14.5 /x, 
and  in  thickness  from  8  /x  to  12  /x,  averaging  about  44  ^  by  13  ^  by  10  /x. 

The  anterior  end  is  provided  with  a  contractile  suctorial  tentacle  continuous 
with  an  internal  tubular  canal.  The  canal  is  directed  at  first  dorsally  and  then 
ventrally  and  obliquely  toward  the  right  side  of  the  body.  In  fixed  specimens 
stained  with  iron  hematoxylin  it  can  usually  be  traced  posteriorly  for  about  one-half 
the  length  of  the  body. 

The  cilia  of  A.  disshnilis  are  7  it  to  8  /x  in  length  and  are  disposed  in  longitudinal 
rows  originating  at  the  anterior  end.  The  typical  number  of  ciliary  rows  is  eleven, 
but  specimens  with  twelve  rows  are  not  uncommon,  and  I  have  seen  some  with  four- 
teen rows.  There  are  usually  five  approximately  equal  rows  about  three-fifths  the 
length  of  the  body  occupying  the  central  portion  of  the  ventral  surface ;  these  are 
bounded  on  either  side  by  three  progressively  longer  rows,  the  outermost  rows  being 
three-fourths  to  four-fifths  the  length  of  the  body.  In  specimens  having  twelve 
ciliary  rows  there  are  four  longer  ro\vs  on  the  left  side  instead  of  three ;  in  speci- 
mens having  fourteen  rows  there  are  four  longer  rows  on  the  right  side  and  five 
longer  rows  on  the  left.  In  some  cases,  particularly  if  the  number  of  ciliary  rows 
exceeds  eleven,  the  five  central  rows  are  of  unequal  length,  becoming  progressively 
longer  from  right  to  left.  One  or  two  of  the  outer  rows  on  either  side  originate 
on  the  lateral  margin  or  the  dorsal  surface,  usually  a  short  distance  posterior  to  the 
level  of  origin  of  the  other  rows.  These  rows  curve  ventrally  and  inward  as  they 
extend  posteriorly,  so  that  at  least  their  distal  portions  are  visible  in  ventral  view. 


196 


EUGENE  N.  KOZLOFF 


The  cytoplasm  is  colorless  and  contains  numerous  small  refractile  granules  of  a 
lipoid  substance  in  addition  to  food  inclusions.  One  or  more  larger  food  vacuoles 
are  usually  present  in  the  posterior  part  of  the  body.  The  contractile  vacuole  lies 
near  the  middle  of  the  body  and  opens  to  the  exterior  on  the  ventral  surface. 

The  macronucleus  is  ovoid  and  situated  dorsally  near  the  middle  of  the  body. 
In  fixed  and  stained  preparations  the  outline  of  the  macronucleus  is  nearly  always 
very  irregular  and  the  chromatin  appears  to  be  aggregated  into  a  dense  reticulum 
enclosing  vacuole-like  clear  spaces  of  varying  size.  In  twenty  individuals  fixed  in 
Schaudinn's  fluid  and  stained  with  iron  hematoxylin  the  macronucleus  ranged  in 
length  from  6.8  ^  to  13.7  /n  and  in  width  from  5.4  ^  to  7.2  /A. 

The  micronucleus  is  typically  ovoid,  rarely  spherical,  and  commonly  is  situated 
a  short  distance  anterior  to  or  to  one  side  of  the  macronucleus.  In  fixed  and  stained 


FIGURE  2.    Ancistrocoma  dissimilis  sp.  nov.     Distribution  of  ciliary   rows,   somewhat 
diagrammatic.    A,  dorsal  aspect ;  B,  ventral  aspect. 

preparations  the  chromatin  appears  to  be  dispersed  in  granules  of  varying  size.  In 
twenty  individuals  fixed  in  Schaudinn's  fluid  and  stained  with  iron  hematoxylin 
the  micronucleus  ranged  in  size  from  2.2  ^  by  2.4  ,u  to  2.2  ^  by  3.2  p.. 

I  found  Ancistrocoma  dissimilis  to  be  present  on  the  gills  and  palps  of  twenty- 
one  of  thirty-six  specimens  of  Pholadidca  pcnita  which  I  examined  from  localities 
near  Moss  Beach,  California.  It  is  sometimes  found  in  association  with  Hypo- 
comagalma  pholadidis.  In  some  individuals  of  P.  penita  I  have  encountered  a  ciliate 
of  the  genus  Sphenophrya  which  I  hope  to  describe  in  a  later  paper  and  a  species 
of  Boveria  which  may  also  be  new. 

Ancistrocoma  disshnilis  sp.  nov. 

Diagnosis:  Length  33^-51^,  average  about  44^;  width  lO/x-14.5^,  average 
about  13  ju,;  thickness  8  fi-12  /JL,  average  about  10 /x.  The  ciliary  rows  are  eleven  to 
fourteen  (typically  eleven)  in  number  and  are  distributed  for  the  most  part  on  the 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.    Ill  •  197 

ventral  surface  and  lateral  margins  of  the  body.  Most  of  the  rows  originate  on  the 
ventral  surface  close  to  the  base  of  the  suctorial  tentacle,  while  one  or  two  outer 
rows  on  either  side  originate  on  the  lateral  margin  or  the  dorsal  surface  and  curve 
ventrally  and  inward  as  they  extend  posteriorly.  There  are  usually  five  approxi- 
mately equal  rows  about  three-fifths  the  length  of  the  body  bounded  on  the  right 
by  three  progressively  longer  rows  and  on  the  left  by  four  progressively  longer 
rows.  The  outermost  row  on  either  side  is  three-fourths  to  four-fifths  the  length  of 
the  body.  The  macronucleus  is  ovoid.  The  micronucleus  is  typically  ovoid.  Para- 
sitic on  the  gills  and  palps  of  Pholadidea  penita  (Conrad)  (Moss  Beach,  California). 
Syntypes  are  in  the  collection  of  the  author. 

HYPOCOMAGALMA  PHOLADIDIS  SP.  NOV. 
(Figure  3;  Plate  I,  Figs.  4,  5) 

The  body  is  elongated,  strongly  attenuated  anteriorly,  and  markedly  asymmetri- 
cal. The  anterior  end  is  deflected  toward  the  left  and  bent  ventrally.  The  dorso- 
ventral  flattening  characteristic  of  most  ancistrocomid  ciliates  is  not  conspicuous  in 
this  species.  As  viewed  from  the  posterior  end  the  body  appears  in  its  middle  and 
posterior  portions  to  be  almost  as  thick  as  wide.  In  its  anterior  third  the  body  is 
nearly  round  in  cross  section.  Most  fixed  specimens  are  considerably  distorted  and 
compressed  in  such  a  way  that  they  appear  to  be  widest  near  the  middle.  Twenty 
living  individuals  taken  at  random  ranged  in  length  from  63  /*  to  89  ju,  in  width  from 
18  ju  to  25  JJL,  and  in  thickness  from  16  /z  to  21  /*,  averaging  about  76  ^,  by  22  /x  by  19  //.. 

The  anterior  end  is  provided  with  a  contractile  suctorial  tentacle  continuous 
with  an  internal  tubular  canal.  The  canal  can  usually  be  traced  in  fixed  specimens 
stained  with  iron  hematoxylin  down  the  middle  of  the  attenuated  anterior  part  of 
the  body  and  then  obliquely  toward  the  right  side.  I  have  not  succeeded  in  demon- 
strating the  course  of  the  canal  beyond  the  anterior  one-third  of  the  body. 

The  cilia  of  Hypocomagalma  pJwladidis  are  approximately  9/x  to  10  ^  in  length. 
The  ciliary  system  consists  of  twenty-four  or  twenty-five  longitudinal  rows.  The 
body  is  almost  completely  invested  by  cilia  except  for  a  cilia-free  "cap"  at  the  pos- 
terior end.  Two  rows  on  the  right  side  of  the  body  usually  appear  to  be  set  apart 
from  the  others,  but  in  some  specimens  the  spacing  between  these  rows  and  the  adja- 
cent rows  on  either  side  is  not  significantly  wider  than  the  spacing  between  some 
of  the  other  rows.  Perhaps  these  two  rows  are  homologous  with  the  one  or  two 
rows  constituting  the  right  ciliary  complex  of  Crebricoma  carinata  (Raabe),  Insig- 
nicoma  venusta  Kozloff,  and  species  of  Hypocomides.  They  originate  near  the  base 
of  the  suctorial  tentacle  on  the  right  margin  or  the  dorsal  surface  close  to  the  right 
margin  and  curve  ventrally  and  to  the  left  as  they  extend  backward.  The  outer 
row,  as  seen  in  ventral  view,  is  the  longer  and  extends  almost  to  the  posterior  end 
of  the  body.  The  inner  row  terminates  a  short  distance  more  anteriorly  than  the 
outer  row,  but  is  conspicuously  longer  than  the  first  of  the  next  series  of  rows, 
which  usually  is  about  two-thirds  the  length  of  the  body.  The  first  eight  to  ten 
rows  to  the  left  of  the  two  longer  rows  all  originate  at  about  the  same  level  on  the 
ventral  surface  close  to  the  base  of  the  suctorial  tentacle.  The  remaining  rows, 
which  are  disposed  along  the  left  margin  of  the  body  and  on  the  dorsal  surface, 
originate  progressively  more  posteriorly.  The  tenth  or  eleventh  row  of  this  com- 
plex is  usually  the  longest,  although  some  of  the  shorter  rows  on  the  dorsal  surface 


198 


EUGENE  N.  KOZLOFF 


may  terminate  more  posteriorly.  The  last  ciliary  row  on  the  right  side  of  the 
dorsal  surface  is  always  the  shortest  row,  originating  at  a  point  about  one-third  the 
distance  from  the  anterior  end  of  the  body  to  the  posterior  end  and  terminating  at 
a  point  about  three-fourths  or  four-fifths  the  distance  from  the  anterior  end  to  the 
posterior  end. 

The  cytoplasm  is  colorless  and  contains  numerous  small  refractile  granules  of  a 
lipoid  substance  in  addition  to  food  inclusions.  One  or  more  larger  food  vacuoles 
containing  fragments  of  cells  from  the  epithelial  tissues  of  the  gills  or  palps  of  the 
host  are  usually  evident  in  the  posterior  part  of  the  body.  The  contractile  vacuole, 
when  single,  is  located  near  the  middle  of  the  body  and  opens  to  the  exterior  on 
the  ventral  surface.  In  a  larger  percentage  of  the  living  specimens  of  H.  pholadidis 
which  I  examined  there  were  two  or  more  contractile  vacuoles  scattered  through 


FIGURE  3.     Hypocomagaluia  pholadidis  sp.  nov.     Distribution  of  ciliary  rows,  somewhat 
diagrammatic.     A,  dorsal  aspect;  B,  ventral  aspect. 

the  body  which  emptied  their  contents  to  the  exterior  on  the  ventral  surface.  In  a 
large  percentage  of  the  living  specimens  of  H.  pholadidis  which  I  examined  there 
were  two  or  more  contractile  vacuoles  scattered  through  the  body  which  emptied 
their  contents  to  the  exterior  independently  of  one  another.  Jarocki  and  Raabe 
(1932)  reported  that  in  H.  dreissenae  the  contractile  vacuole  was  sometimes  single, 
but  that  in  some  specimens  there  were  several  smaller  ones. 

The  macronucleus  typically  is  sausage-shaped  and  lies  in  the  posterior  third  of 
the  body,  its  longitudinal  axis  placed  obliquely  to  the  longitudinal  axis  of  the  body. 
In  light  iron  hematoxylin  preparations  and  in  specimens  stained  by  the  Feulgen 
reaction  the  chromatin  of  the  macronucleus  appears  to  be  aggregated  into  a  dense 
reticulum  enclosing  vacuole-like  spaces  which  frequently  contain  globular  masses 
of  deeply-staining  material.  In  ten .  individuals  fixed  in  Schaudinn's  fluid  and 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.    Ill  199 

stained  by  the  Feulgen  reaction  the  macronucleus  ranged  in  length  from  12.5  ju,  to 
20  p.  and  in  width  from  5  /*  to  8.9  /*. 

The  micronucleus  is  spherical  and  usually  is  situated  a  short  distance  anterior 
to  or  to  one  side  of  the  macronucleus.  In  most  fixed  and  stained  preparations  the 
chromatin  appears  to  be  homogeneous,  although  in  some  the  chromatin  appears  to 
be  in  part  aggregated  into  granules  or  peripheral  strands.  In  ten  individuals  fixed 
in  Schaudinn's  fluid  and  stained  by  the  Feulgen  reaction  the  diameter  of  the  micro- 
nucleus  ranged  from  2.4  p.  to  3.3  p.. 

I  found  Hypocomagalma  pholadidis  to  be  present  on  the  gills  and  palps  of 
twenty-eight  of  thirty-six  specimens  of  Pholadidea  penita  which  I  examined  from 
localities  near  Moss  Beach,  California.  When  the  ciliate  is  dissociated  from  the 
host  it  swims  erratically,  usually  rotating  on  its  longitudinal  axis  and  tracing  wide 
arcs  with  its  attenuated  anterior  end.  The  cilia  of  the  anterior  half  of  the  body  are 
more  active  than  those  of  the  posterior  half  and  are  sometimes  observed  to  beat 
metachronously.  The  ventral  cilia  near  the  base  of  the  suctorial  tentacle  are  mark- 
edly thigmotactic. 

Hypocomagalma  pholadidis  sp.  nov. 

Diagnosis:  Length  63  /*-S9  /u,  average  about  76  /*;  width  18^-25^,  average 
about  22  p;  thickness  16,u-21  /x,  average  about  19ft.  The  anterior  end  of  the  body 
is  attenuated,  conspicuously  deflected  toward  the  left,  and  bent  ventrally.  The 
ciliary  system  consists  of  twenty-four  or  twenty-five  rows.  Two  long  rows  on  the 
right  side  of  the  body  appear  in  most  specimens  to  be  set  apart  from  the  remaining 
rows ;  these  two  rows  originate  near  the  base  of  the  suctorial  tentacle  and  extend 
almost  to  the  posterior  end  of  the  body.  The  first  eight  to  ten  rows  to  the  left  of 
these  two  longer  rows  originate  at  about  the  same  level  on  the  ventral  surface,  while 
the  remaining  rows,  disposed  along  the  left  lateral  margin  and  the  dorsal  surface, 
originate  progressively  more  posteriorly.  The  contractile  vacuole  may  be  single  or 
represented  by  several  independent  vacuoles  opening  to  the  exterior  on  the  ventral 
surface.  The  macronucleus  is  sausage-shaped.  The  micronucleus  is  spherical. 
Parasitic  on  the  epithelium  of  the  gills  and  palps  of  Pholadidea  penita  (Conrad) 
(Moss  Beach,  California).  Syntypes  are  in  the  collection  of  the  author. 

LITERATURE  CITED 

CHATTON,  E.,  AND  A.  LWOFF,  1926.     Diagnoses  de  cilies  thigmotriches  nouveaux.     Bull.  Soc. 

Zool.  France,  51 :  345. 
JAROCKI,  J.,  AND  Z.  RAABE,  1932.    t)ber  drei  neue  Infusorien-Genera  der  Familie  Hypocomidae 

(Ciliata  Thigmotricha),  Parasiten  in  Susswassernmuscheln.    Bull.  int.  Acad.  Cracovie, 

Cl.  Sci.  math,  not.,  B(II),  1932:  29. 
KIRBY,  H.,  1941.     Relationships  between  certain  Protozoa  and  other  animals.     In  Calkins,  G., 

and  F.  Summers    (editors)  :  Protozoa  in  Biological  Research.     Columbia  University 

Press,  New  York. 
KOFOID,  C.,  AND  M.  BUSH,  1936.     The  life  cycle  of  Parachaenia  myae  gen.  nov.,  sp.  nov.,  a  ciliate 

parasitic  in  Mya  arenaria  Linn,  from  San  Francisco  Bay,  California.    Bull.  Mus.  Hist. 

not.  Belgique,  12,  No.  22. 

KUDO,  R.,  1946.     Protozoology.    3rd  ed.     Charles  C.  Thomas,  Springfield. 
RAABE,  Z.,  1945.    tiber  einige  an  den  Kiemen  von  Mytilus  edulis  L.  und  Macoma  balthica  (L.) 

parasitierende  Ciliaten-Arten.     Ann.  Mus.  sool.  polon.,  10:  289. 
RAABE,  Z.,  1938.     Weitere  Untersuchungen  an  parasitischen  Ciliaten  aus  dem  polnischen  Teil 

der  Ostsee.    II.  Ciliata  Thigmotricha  aus  den  Familien:   Hypocomidae  Biitschli  und 

Sphaenophryidae  Ch.  &  Lw.    Ann.  Mus.  zo.ol.  polon.,  13:  41. 


STUDIES  ON  CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE 

CHATTON  AND  LWOFF  (ORDER  HOLOTRICHA, 

SUBORDER  THIGMOTRICHA) 

IV.    HETEROCINETA  JANICKII  JAROCKI,  HETEROCINETA 

GONIOBASIDIS  SP.  NOV.,  HETEROCINETA  FLUMINI- 

COLAE  SP.  NOV.,  AND  ENERTHECOMA 

PROPERANS  JAROCKI 

EUGENE  N.  KOZLOFF 
Lewis  and  Clark  College,  Portland,  Oregon 

INTRODUCTION 

The  genus  Heterocineta  was  established  by  Mavrodiadi  (1923)  for  a  ciliate 
which  he  named  Heterocincta  anodontae,  and  which  he  had  formerly  believed  to 
represent  a  gregariniform  stage  in  the  development  of  Conchaphthirus  anodontae 
(Ehrenberg).  Unaware  of  the  fact  that  Mavrodiadi  had  abandoned  his  earlier  con- 
ception and  applied  the  name  Heterocincta  anodontae  to  this  ciliate,  Jarocki  and 
Raabe  (1932)  described  the  same  species,  from  Anodonta  cygnca  (L.)  and  Unio 
pictorum  L.,  as  Hypocomatophora  unionidarum.  Jarocki  later  (1934)  pointed  out 
that  Hypocomatophora  unionidarum  was  a  synonym  of  Hcterocineta  anodontae. 

In  his  papers  of  1934  and  1935  Jarocki  described  seven  additional  species  of 
the  genus  Heterocincta  ectoparasitic  on  fresh  water  gastropods :  H.  janickii,  from 
Physa  fontinalis  (L.)  ;  H.  Iwoffi,  from  Viviparus  jasciatus  Miiller ;  H.  chattoni,  from 
Radix  ovata  (Drap.)  ;  H.  krsysiki,  from  Bithynia  tcntaculata  (L.)  ;  H.  maziarskii, 
from  Coretus  corncus  (L.)  ;  H.  turi,  from  Tropidiscus  planorbis  (L.)  and  Spiralina 
vortex  (L.)  ;  and  H.  siedlcckii,  from  Acroloxus  lacustris  (L. ).  In  1945  I  de- 
scribed as  Heterocineta  phoronopsidis  a  ciliate  from  the  tentacles  of  Phoronopsis 
viridis  Hilton.  This  species  is  the  only  representative  of  the  genus  thus  far  de- 
scribed which  is  not  a  parasite  of  fresh  water  molluscs  or  of  the  annelid  commensal 
Chaetogastcr  limnaci  von  Baer  when  the  latter  is  associated  with  infected  snails. 

On  the  fresh  water  prosobranch  snails  Goniobasis  plicijera  silicula  (Gould)  and 
Fluminicola  virens  (Lea)  I  have  found  two  new  species  of  Heterocincta  which  will 
be  described  herein  as  H.  goniobasidis  sp.  nov.  and  H.  fluminicolae  sp.  nov.  I  have 
also  studied  a  species  of  Heterocineta  from  Physa  cooperi  Tryon  which  agrees  with 
the  original  description  of  H.  janickii.  It  seems  advisable,  for  comparative  pur- 
poses, and  in  view  of  the  fact  that  Jarocki's  description  of  H.  janickii  is  not  accom- 
panied by  illustrations,  to  include  an  account  of  the  morphology  of  this  form  in  the 
present  paper. 

The  genus  Encrthccoina  was  proposed  by  Jarocki  (1935)  for  a  single  species, 
E.  propcrans,  parasitic  on  the  gills  of  Viviparus  jasciatus.  Although  the  original 
description  of  this  species  is  quite  adequate,  it  is  not  supplemented  by  illustrations, 
and  the  second  installment  of  Jarocki's  "Studies  on  ciliates  from  fresh-water  mol- 
luscs," in  which  figures  of  E.  properans  and  several  other  ciliates  were  to  be  pub- 

200 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.    IV 


201 


lished,  has  not  come  to  my  attention.  A  ciliate  which  I  have  found  to  infest  Vivi- 
parus  malleatus  (Reeve)  apparently  is  identical  with  E.  properans.  This  ciliate 
will  be  described  and  illustrated  here. 

HETEROCINETA  JANICKII  JAROCKI 
(Figure  1;  Plate  I,  Fig.  1) 

The  body  is  elongated  and  flattened  dorso-ventrally.  The  anterior  end  is  attenu- 
ated, bent  ventrally,  and  deflected  slightly  toward  the  left.  The  anterior  one-half 
of  the  left  margin  is  not  quite  so  rounded  as  the  right  margin  and  typically  is  nearly 
straight  or  weakly  indented.  The  body  is  widest  a  short  distance  behind  the  mid- 
dle and  rounded  posteriorly.  The  ciliary  system,  to  be  described  presently,  is  dis- 
posed on  a  shallow  concavity  occupying  the  anterior  two-thirds  of  the  ventral  sur- 
face ;  the  dorsal  surface  and  that  part  of  the  ventral  surface  posterior  to  the  ciliary 
area  are  convex.  Twenty  living  individuals  from  Pliysa  cooperi  ranged  in  length 
from  25  fj.  to  32  //.,  in  width  from  12 /x  to  15^,,  and  in  thickness  from  10 /x  to  12  /j., 


FIGURE  1. 


^> X- 

Hcterocineta  janickii  Jarocki.     Distribution  of  ciliary  rows,  somewhat  diagram- 
matic.1    Ventral  aspect. 


averaging  about  30^  by  14  ^  by  11  /x.  The  specimens  of  H.  janickii  from  Pliysa 
joiitinalis  which  were  studied  by  Jarocki  ranged  in  length  from  23^  to  32  (*,,  in 
width  from  12^  to  17  /*,  and  in  thickness  from  10^,  to  13^. 

The  anterior  end  of  the  body  is  provided  with  a  short  contractile  suctorial  ten- 
tacle which  enables  the  ciliate  to  attach  itself  to  the  epithelial  cells  of  the  host  and 
to  feed  upon  their  contents.  When  fully  extended  the  tentacle  is  about  3  /*  to  4  ju, 
(according  to  Jarocki  about  4.5  p.)  in  length.  The  internal  tubular  canal  continuous 
with  the  tentacle  is  directed  at  first  dorsally  and  then  ventrally  and  obliquely  toward 
the  right  side,  and  in  specimens  stained  with  iron  hematoxylin  can  usually  be  traced 
for  about  one-half  the  length  of  the  body. 

The  ciliary  system  consists  of  eight  longitudinal  rows  originating  close  to  the 
base  of  the  suctorial  tentacle.  The  first  four  rows  from  the  right  side  are  approxi- 
mately one-half  the  length  of  the  body.  The  remaining  four  rows  become  increas- 
ingly longer  and  terminate  one  behind  the  other  a  little  to  the  left  of  the  midline. 

1  The  text  figures  illustrating  this  paper  are  based  on  camera  lucida  drawings  of  specimens 
impregnated  with  silver  nitrate  by  Klein's  method. 


202  EUGENE  N.  KOZLOFF 

The  longest  row  is  about  two-thirds  the  length  of  the  body.  The  cilia  are  about 
6  p.  to  7  fji  (according  to  Jarocki  about  5  p.  to  7  /x)  in  length.  While  attached  to  the 
skin  of  the  host  the  parasites  are  as  a  rule  almost  immobile,  their  cilia  exhibiting 
only  a  feeble  motion.  When  dissociated  from  the  host  Hetcrocineta  janickii  swims 
sluggishly,  usually  rotating  on  its  longitudinal  axis  and  tracing  wide  arcs  with  its 
attenuated  anterior  end. 

The  cytoplasm  is  colorless  and  contains  numerous  small  refractile  granules  in 
addition  to  food  inclusions.  One  or  more  large  food  vacuoles  are  present  in  the 
posteriorpart  of  the  body  behind  the  macronucleus.  The  contractile  vacuole  is  situ- 
ated near  the  middle  of  the  body  and  opens  to  the  exterior  on  the  ventral  surface. 
I  have  observed  no  permanent  opening  in  the  pellicle. 

The  macronucleus  is  typically  sausage-shaped  and  is  located  near  the  middle  of 
the  body  or  somewhat  posterior  to  the  middle.  As  seen  in  dorsal  or  ventral  view 
the  longitudinal  axis  of  the  macronucleus  is  placed  obliquely  to  the  longitudinal  axis 
of  the  body.  As  seen  in  lateral  view,  the  anterior  end  of  the  macronucleus  is  di- 
rected dorsally,  while  the  posterior  end  is  directed  ventrally.  In  fixed  and  stained 
preparations  the  chrbmatin  appears  to  be  more  or  less  homogeneous.  In  ten  indi- 
viduals fixed  in  Schaudinn's  fluid  and  stained  by  the  Feulgen  reaction  the  macro- 
nucleus  ranged  in  length  from  7  //.  to  1 1  ju,  and  in  width  from  4  /*  to  5  /JL. 

The  micronucleus  is  ovoid  or  spherical  and  is  situated  near  the  dorsal  surface 
anterior  to  or  to  one  side  of  the  macronucleus.  In  most  fixed  and  stained  specimens 
the  chromatin  is  homogeneous,  although  in  some  it  appears  to  be  concentrated  in 
peripheral  granules.  In  ten  individuals  fixed  in  Schaudinn's  fluid  and  stained  with 
iron  hematoxylin  the  size  of  the  micronucleus  ranged  from  1 .4  p.  by  1 .4  ^  to  A  .6  /A 
by  2  p.. 

Hetcrocineta  janickii  was  present  in  very  small  numbers  on  the  tentacles,  mantle, 
and  margins  of  the  foot  of  most  of  the  specimens  of  Physa  coopcri  which  I  col- 
lected in  a  stream  near  Mt.  Eden,  California.  The  degree  of  infestation  increased 
rapidly  on  snails  kept  in  laboratory  aquaria  for  a  period  of  six  weeks. 

Heterocineta  janickii  Jarocki 

Diagnosis:  Length  25  ju-32  p.  (according  to  Jarocki  23/x.-32/x),  average  about 
30  p;  width  12/^-15  p.  (according  to  Jarocki  12/x-17/t),  average  about  14/x;  thick- 
ness 10/x-12/x  (according  to  Jarocki  10/x-13/x),  average  about  11  p..  The  ciliary 
system  consists  of  eight  rows  originating  close  to  the  base  of  the  suctorial  tentacle. 
The  first  four  rows  from  the  right  are  about  one-half  the  length  of  the  body,  while 
the  remaining  four  rows  become  progressively  longer  and  terminate  one  behind  the 
other  a  little  to  the  left  of  the  midline.  The  longest  row  is  about  two-thirds  the 
length  of  the  body.  Parasitic  on  the  epithelium  of  the  tentacles,  mantle,  and  foot 
of  Physa  jontinalis  (L.)  (Warsaw  [Jarocki])'  and  Physa  coopcri  Tryon  (Mt. 
Eden,  California). 

HETEROCINETA  GONIOBASIDIS  SP.  NOV. 
(Figure  2;  Plate  I,  Figs.  2,  3) 

The  body  is  elongated  and  flattened  dorso-ventrally.  The  anterior  end  is  at- 
tenuated, bent  ventrally,  and  deflected  slightly  toward  the  left.  The  anterior  one- 
half  of  the  left  margin  is  not  so  rounded  as  the  right  margin  and  typically  is  nearly 


CILIATES  OF  THE  FAMILY  ANC1STROCOMIDAE.     IV 


203 


straight  or  weakly  indented.  The  body  is  widest  at  the  middle  or  a  short  distance 
anterior  to  the  middle.  The  ciliary  system  is  disposed  on  a  shallow  concavity  oc- 
cupying the  anterior  two-thirds  of  the  ventral  surface ;  the  dorsal  surface  and  that 
part  of  the  ventral  surface  posterior  to  the  ciliary  area  are  convex.  Twenty-five 
living  specimens  taken  at  random  ranged  in  length  from  36  /j.  to  48  /JL,  in  width  from 
15,u,  to  20  fj.,  and  in  thickness  from  11 /A  to  14  /JL,  averaging  about  43 /x  by  18 /A 
by  13  fjL. 

The  anterior  end  is  provided  with  a  contractile  suctorial  tentacle  continuous  with 
an  internal  tubular  canal.  The  nature  of  the  canal  is  very  similar  to  that  of  other 
members  of  the  genus.  It  is  directed  at  first  dorsally  and  then  ventrally  and  ob- 
liquely toward  the  right  side  of  the  body.  It  can  be  traced  in  most  fixed  specimens 
stained  with  iron  hematoxylin  for  about  one-half  to  two-thirds  of  the  length  of  the 
body. 


FIGURE  2. 


Hetcrocineta  goniobasidis  sp.  nov.     Distribution  of  ciliary  rows,  somewhat 
diagrammatic.     Ventral  aspect. 


The  cilia  of  H.  goniobasidis  are  about  9  /A  long.  Those  of  the  anterior  part  of 
the  ciliary  system  are  markedly  thigmotactic.  The  ciliary  system  consists  of  ten 
longitudinal  rows.  The  first  six  rows  are  approximately  the  same  length,  being 
about  one-half  the  length  of  the  body,  although  on  careful  examination  the  first 
row  is  seen  to  originate  some  distance  posterior  to  the  level  of  origin  of  the  other 
five  rows.  The  seventh,  eighth,  ninth,  and  tenth  rows  originate  progressively  more 
posteriorly  and  become  increasingly  longer,  terminating  one  behind  the  other  a  little 
to  the  left  of  the  midline.  The  longest  row  is  two-thirds  to  three-fourths  the  length 
of  the  body.  The  last  one  or  two  rows  usually  originate  on  the  left  margin  and 
curve  ventrally  as  they  extend  backward.  The  cilia  of  the  distal  portions  of  the 
longer  rows  are  nearly  always  practically  motionless  and  directed  posteriorly.  When 
dissociated  from  the  host  the  ciliate  swims  sluggishly  and  erratically,  rotating  on  its 
longitudinal  axis. 

The  cytoplasm  is  colorless  and  contains  numerous  refractile  granules  of  a  lipoid 
substance  in  addition  to  food  inclusions.  There  are  usually  one  or  two  large  food 
vacuoles  in  the  posterior  part  of  the  body  behind  the  macronucleus.  The  contractile 
vacuole  is  central  and  opens  to  the  exterior  on  the  ventral  surface. 


204  EUGENE  N.  KOZLOFF 

The  macronucleus  is  situated  in  the  middle  portion  of  the  body.  It  is  elongated 
and  typically  somewhat  narrower  at  its  anterior  end  than  at  its  posterior  end.  As 
seen  in  dorsal  or  ventral  aspect,  the  longitudinal  axis  of  the  macronucleus  is  placed 
obliquely  to  the  longitudinal  axis  of  the  body.  As  seen  in  lateral  view,  the  anterior 
end  of  the  macronucleus  is  directed  dorsally,  while  the  posterior  end  is  directed 
ventrally.  In  ten  individuals  fixed  in  Schaudinn's  fluid  and  stained  with  iron 
hematoxylin  the  macronucleus  ranged  in  length  from  10^  to  13. 5 /A  and  in  width 
from  4  ^  to  5.5  /x. 

The  spherical  or  ovoid  micronucleus  is  very  difficult  to  distinguish  in  the  living 
ciliates.  It  is  usually  situated  near  the  dorsal  surface  a  short  distance  anterior  to 
the  macronucleus.  In  fixed  and  stained  preparations  the  micronucleus  is  vesicular, 
the  chromatin  being  concentrated  along  the  periphery.  In  ten  individuals  fixed  in 
Schaudinn's  fluid  and  stained  with  iron  hematoxylin  the  micronucleus  ranged  in 
size  from  1 .2  ^  by  1 .5  ;u,  to  1 .5  p,  by  1.7  /i. 

Heterocineta  goniobasidis  was  found  to  be  present  on  the  epithelium  of  the  gills 
and  mantle  of  a  small  percentage  of  the  specimens  of  Goniobasis  plicifcra  silicula 
which  I  collected  in  Crystal  Springs  Creek,  in  Portland,  Oregon.  The  degree  of 
infestation  on  freshly  collected  snails  was  very  low,  but  increased  during  the  four 
weeks  the  specimens  were  kept  in  laboratory  aquaria. 

Heterocineta  goniobasidis  sp.  nov. 

Diagnosis:  Length  36^ — 1-8//.,  average  about  43  yu.;  width  15^-20/x,  average 
about  18/x;  thickness  ll/x-14/x,  average  about  13/x.  The  ciliary  system  h  com- 
posed of  ten  rows.  The  first  six  rows  from  the  right  side  are  about  one-half  the 
length  of  the  body  and,  with  the  exception  of  the  first  row,  originate  close  to  the 
base  of  the  suctorial  tentacle.  The  remaining  rows  originate  progressively  more 
posteriorly  and  become  increasingly  longer,  terminating  one  behind  the  other  a  little 
to  the  left  of  the  midline.  The  longest  row  is  two-thirds  to  three-fourths  the  length 
of  the  body.  Parasitic  on  the  gills  and  mantle  of  Goniobasis  plicifera  silicula 
(Gould)  (Portland,  Oregon).  Syntypes  are  in  the  collection  of  the  author. 

HETEROCINETA  FLUMINICOLAE  SP.  NOV. 
(Figure  3;  Plate  I,  Fig.  4) 

The  body  is  elongated  and  flattened  dorso-ventrally.  The  anterior  end  is  at- 
tenuated, bent  ventrally,  and  deflected  slightly  toward  the  left.  The  anterior  part 

• 

EXPLANATION  OF  PLATE  I 
All  figures  except  Figure  2  have  been  prepared  with  the  aid  of  a  camera  lucida. 

FIGURE  1.  Heterocineta  janickii  Jarocki.  Ventral  aspect.  Schaudinn's  fixative-iron  hema- 
toxylin. X  1,720. 

FIGURE  2.   'Heterocineta  goniobasidis  sp.  nov.     Lateral  aspect  from  left  side,  from  life. 

FIGURE  3.  Heterocineta  goniobasidis  sp.  nov.  Ventral  aspect.  Schaudinn's  fixative-iron 
hematoxylin.  X  1,720. 

FIGURE  4.  Heterocineta  fluniinicolae  sp.  nov.  Ventral  aspect.  Schaudinn's  fixative-iron 
hematoxylin.  X  1,720. 

FIGURE  5.  Encrthccoma  properans  Jarocki.  Macro-  and  micronuclei  from  three  specimens. 
Schaudinn's  fixative-Feulgen  reaction.  X  1,720. 

FIGURE  6.  Enerthecoma  properans  Jarocki.  Ventral  aspect.  Schaudinn's  fixative-iron 
hematoxylin.  X  1,720. 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.    IV 


205 


ff. 


PLATE  I 


206 


EUGENE  N.  KOZLOFF 


of  the  left  margin  is  not  so  rounded  as  the  right  margin  and  typically  is  weakly 
indented.  The  body  is  widest  a  short  distance  behind  the  middle  and  rounded 
posteriorly.  The  ciliary  system  is  disposed  on  a  shallow  concavity  occupying  the 
major  portion  of  the  ventral  surface ;  the  dorsal  surface  and  that  part  of  the  ventral 
surface  posterior  to  the  ciliary  area  are  convex.  Twenty-five  living  individuals 
taken  at  random  ranged  in  length  from  30^  to  36  /x,  in  width  from  13 /x  to  17  /M, 
and  in  thickness  from  10 /A  to  12  //,,  averaging  about  33  /A  by  15/u  by  11  /*. 

The  anterior  end  is  provided  with  a  contractile  suctorial  tentacle  continuous 
with  an 'internal  tubular  canal.  The  canal  is  directed  at  first  ventrally  and  then 
obliquely  toward  the  right  side  of  the  body.  It  can  be  traced  in  most  fixed  speci- 
mens stained  with  iron  hematoxylin  for  about  one-half  the  length  of  the  body. 

The  cilia  of  H.  fluminicolae  are  about  6/x  or  7 p.  long.  Those  of  the  anterior 
part  of  the  ciliary  system  are  strongly  thigmotactic.  The  ciliary  system  consists  of 
ten  longitudinal  rows.  The  first  row  on  the  right  side  of  the  ciliary  complex  origi- 


FIGURE  3.     Hctcrochicla  flnininicolac  sp.  nov.     Distribution  of  ciliary  rows,  somewhat 

diagrammatic.     Ventral  aspect. 

nates  close  to  the  base  of  the  suctorial  tentacle ;  each  of  the  remaining  rows  origi- 
nates progressively  more  posteriorly.  The  first  six  rows  from  the  right  side  are 
approximately  the  same  length,  being  about  two-thirds  the  length  of  the  body.  The 
last  four  rows  become  increasingly  longer  and  incurved  in  such  a  \vay  that  they 
terminate  one  behind  the  other  not  far  to  the  left  of  the  midline.  The  longest  row 
usually  extends  almost  to  the  posterior  end  of  the  body.  The  cilia  of  the  distal 
portions  of  these  longer  rows  are  usually  directed  posteriorly.  When  the  ciliate  is 
dissociated  from  the  host  it  swims  erratically,  rotating  on  its  longitudinal  axis  and 
tracing  wide  arcs  with  its  anterior  end. 

The  cytoplasm  is  colorless  and  contains  numerous  small  refractile  granules  of 
a  lipoid  substance  in  addition  to  food  inclusions.  One  or  more  large  food  vacuoles 
are  usually  present  in  the  posterior  part  of  the  body  behind  the  macronucleus.  The 
contractile  vacuole  is  central  and  opens  to  the  exterior  on  the  ventral  surface.  I 
have  not  observed  a  permanent  opening  in  the  pellicle. 

The  sausage-shaped  macronucleus  is  situated  dorsally  a  short  distance  behind 
the  middle  of  the  body  with  its  longitudinal  axis  placed  obliquely  to  the  longitudinal 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.     IV  207 

axis  of  the  body.  In  fixed  and  stained  preparations  the  chromatin  appears  to  be 
more  or  less  homogeneous.  In  ten  individuals  fixed  in  Schaudinn's  fluid  and 
stained  with  iron  hematoxylin  the  macronucleus  ranged  in  length  from  7.4  p,  to  10  p. 
and  in  width  from  3.9  p,  to  4.4  /JL. 

The  micronucleus  is  round,  fusiform,  or  ovoid,  and  .is  usually  placed  dorsally 
near  the  middle  of  the  body  anterior  to  or  to  one  side  of  the  macronucleus.  In 
fixed  and  stained  specimens  the  chromatin  is  seen  to  be  concentrated  primarily  along 
the  periphery.  In  ten  individuals  fixed  in  Schaudinn's  fluid  and  stained  with  iron 
hematoxylin  the  micronucleus  ranged  in  size  from  1.5^  by  1.2  /JL  to  1.7  p,  by  1.5  /A. 

Heterocineta  fluminicolae  was  present  in  small  numbers  on  the  epithelium  of  the 
gills  and  the  edge  of  the  mantle  of  nearly  all  specimens  of  Fluminicola  virens  which 
I  collected  in  Crystal  Springs  Creek  in  Portland,  Oregon. 

Heterocineta  fluminicolae  sp.  nov. 

Diagnosis  :  Length  30  /A-36  /JL,  average  about  33  p. ;  width  13  p.-\7  p.,  average  about 
15  p.;  thickness  lO/t-12//,,  average  about  11  p.  The  ciliary  system  is  composed  of 
ten  rows  originating  progressively  more  posteriorly  from  the  right  side  to  the  left. 
The  first  six  rows  from  the  right  side  are  about  two-thirds  the  length  of  the  body. 
The  remaining  four  rows  become  increasingly  longer  and  terminate  one  behind  the 
other  a  little  to  the  left  of  the  midline.  The  longest  row  extends  almost  to  the  pos- 
terior end  of  the  body.  Parasitic  on  the  gills  and  mantle  of  Fluminicola  virens 
(Lea)  (Portland,  Oregon).  Syntypes  are  in  the  collection  of  the  author. 

ENERTHECOMA  PROPERANS  JAROCKI 
(Figure  4;  Plate  I,  Figs.  5,  6) 

The  body  is  elongated,  nearly  symmetrical  as  seen  in  dorsal  or  ventral  view,  at- 
tenuated anteriorly,  and  flattened  dorso-ventrally.  The  anterior  end  is  bent  ven- 
trally  and  deflected  inconspicuously  toward  the  left.  The  ciliary  system  is  disposed 
on  a  narrow,  relatively  flat  area  occupying  the  anterior  two-thirds  of  the  ventral 
surface ;  the  dorsal  surface  and  that  part  of  the  ventral  surface  posterior  to  the 
ciliary  area  are  convex.  The  body  is  widest  at  a  point  about  two-thirds  the  dis- 
tance from  the  anterior  end  to  the  posterior  end.  Twenty-five  living  individuals 
taken  at  random  from  Viviparus  malleatus  ranged  in  length  from  32  p,  to  56  p,  in 
width  from  13  ^  to  21  p.,  aiid  in  thickness  from  10  p.  to  13 /x,  averaging  about  44  p. 
by  18  p.  by  11.5^.  Specimens  from  Viviparus  fasciatus  which  were  measured  by 
Jarocki  ranged  in  length  from  33  p.  to  60  p.,  in  \vidth  from  15  p.  to  22  p,  and  in  thick- 
ness from  10  p.  to  13  p.. 

The  contractile  suctorial  tentacle  is  continuous  with  an  internal  tubular  canal 
which  is  directed  at  first  dorsally  and  then  ventrally  and  obliquely  toward  the  right 
side  of  the  body.  In  specimens  stained  with  iron  hematoxylin  the  canal  can  usually 
be  traced  for  about  two-thirds  or  three-fourths  the  length  of  the  body. 

The  ciliary  system  is  composed  of  eight  approximately  equal  rows  about  two- 
thirds  the  length  of  the  body.  These  rows  originate  close  to  the  base  of  the  suc- 
torial tentacle.  The  first  five  rows  from  the  right  side  are  usually  a  little  more 
widely  spaced  than  the  last  three  rows.  This  was  noted  also  by  Jarocki.  who  stated 
that  the  ciliary  system  was  separated  into  two  complexes  by  an  "inconsiderable 
eminence  stretching  from  the  base  of  the  tentacle  to  the  end  of  the  system,"  which 


208 


EUGENE  N.  KOZLOFF 


segregated  the  five  rows  on  the  right  from  the  three  rows  on  the  left.  This  eminence 
was  evident  on  many  of  the  living  specimens  which  I  examined  but  is  never  con- 
spicuous. The  cilia  of  E.  proper ans  are  about  9  ^  in  length  and  exhibit  a  feeble 
undulatory  motion  while  the  parasites  are  attached  to  the  epithelium  of  the  gills  of 
the  host.  When  dissociated  from  the  host  the  ciliates  swim  slow  and  erratically, 
usually  rotating  on  their  longitudinal  axes. 

The  cytoplasm  is  colorless  and  contains  numerous  small  refractile  granules  ot 
a  lipoid  substance  in  addition  to  food  inclusions.  One  or  more  larger  food  vacuoles 
are  usually  present  in  the  posterior  part  of  the  body.  The  contractile  vacuole  is 
situated  a  short  distance  behind  the  middle  of  the  body  and  opens  to  the  exterior 
on  the  ventral  surface.  I  have  not  detected  a  permanent  opening  in  the  pellicle. 

The  macronucleus  is  typically  sausage-shaped  and  is  situated  in  the  posterior 
half  of  the  body  with  its  longitudinal  axis  placed  obliquely  to  the  longitudinal  axis 


FIGURE  4.     Enerthccoma  propcrans   Jarocki.     Distribution   of   ciliary   rows,    somewhat 

diagrammatic.     Ventral  aspect. 

of  the  body.  In  specimens  stained  with  iron  hematoxylin  the  chromatin  appears 
to  be  more  or  less  homogeneous,  but  in  preparations  stained  by  the  Feulgen  re- 
action it  appears  to  be  organized  into  a  dense  reticulum  enclosing  vacuole-like  clear 
spaces  of  varying  size.  In  ten  individuals  fixed  in  Schaudinn's  fluid  and  stained 
by  the  Feulgen  reaction  the  macronucleus  ranged  in  length  from  10 /A  to  19  ^  and 
in  width  from  4  ^  to  7  p. 

The  micronucleus  is  situated  anterior  to  or  to  one  side  of  the  macronucleus. 
In  most  of  the  individuals  of  E.  properans  which  I  examined,  the  micronucleus  is 
elongated  and  more  or  less  fusiform.  I  have  observed  very  few  specimens  to  have 
a  round  micronucleus  such  as  that  described  by  Jarocki.  The  micronucleus  does 
not  stain  readily  with  iron  hematoxylin  and  it  is  possible  that  Jarocki  may  have 
mistaken  food  inclusions  for  micronuclei.  In  specimens  stained  by  the  Feulgen 
reaction  the  chromatin  of  the  micronucleus  appears  to  be  concentrated  in  peripheral 
granules  or  strands.  In  ten  individuals  fixed  in  Schaudinn's  solution  and  stained 


CILIATES  OF  THE  FAMILY  ANCISTROCOMIDAE.    IV  209 

by  the  Feulgen  reaction  the  micronucleus  ranged  in  size  from  0.8  p.  by  2.3  /*  to  1  /* 
by  3.8  n. 

Enerthecoma  properans  was  abundant  on  the  gills  of  nearly  all  specimens  of 
Viviparus  mallcatus  which  I  collected  in  Stow  Lake,  San  Francisco,  California, 
and  in  Evans  Lake,  Riverside,  California.  It  is  undoubtedly  a  common  parasite 
of  this  introduced  snail  wherever  the  latter  has  become  established. 

Enerthecoma  properans  Jarocki 

Diagnosis:  Length  32/^-56^  (according  to  Jarocki  33^-60^),  average  about 
44/.I. ;  width  13  /x-21  p.  (according  to  Jarocki  15/x-22/x),  average  about  18  /x;  thick- 
ness 10fi-13/x,  average  about  11.5/x.  The  ciliary  system  is  composed  of  eight  ap- 
proximately equal  rows  about  two-thirds  the  length  of  the  body  which  originate 
close  to  the  base  of  the  suctorial  tentacle  and  occupy  a  narrow,  relatively  flat  area 
on  the  ventral  surface.  The  first  five  row's  from  the  right  are  more  widely-spaced 
than  the  remaining  three  rows,  and  in  living  specimens  appear  to  be  segregated 
from  the  latter  by  an  inconspicuous  longitudinal  eminence.  The  macronucleus  is 
elongated ;  the  micronucleus  is  typically  elongated  and  more  or  less  fusiform  (ac- 
cording to  Jarocki,  spherical).  Parasitic  on  the  gills  of  Viviparus  jasciatus  Miiller 
(Warsaw  [Jarocki])  and  Viviparus  mallcatus  (Reeve)  (San  Francisco,  California; 
Riverside,  California). 

LITERATURE  CITED 

JAROCKI,  J.,  1934.  Two  new  hypocomid  ciliates,  Heterocineta  janickii  sp.  n.  and  H.  Iwoffi 
sp.  n.,  ectoparasites  of  Physa  fontinalis  (L.)  and  Viviparus  fasciatus  Miiller.  Mem. 
Acad.  Cracovic,  Cl.  Sci.  math,  not.,  B,  1934:  167. 

JAROCKI,  J.,  1935.  Studies  on  ciliates  from  fresh-water  molluscs.  I.  General  remarks  on  pro- 
tozoan parasites  of  Pulmonata.  Transfer  experiments  with  species  of  Heterocineta 
and  Chaetogaster  limnaei,  their  additional  host.  Some  new  hypocomid  ciliates.  Bull, 
int.  Acad.  Cracovic,  Cl.  Sci.  math,  nat.,  B  (II),  1935:  201. 

JAROCKI,  J.,  AND  Z.  RAABE,  1932.  Uber  drei  neue  Infusorien-Genera  der  Familie  Hypocomidae 
(Ciliata  Thigmotricha),  Parasiten  in  Siisswassermuscheln.  Bull.  int.  Acad.  Cracovie, 
Cl.  Sci.  math,  nat.,  B  (II),  1932:  29. 

KOZLOFF,  E.,  1945.  Heterocineta  phoronopsidis  sp.  nov.,  a  ciliate  from  the  tentacles  of  Phoro- 
nopsis  viridis  Hilton.  Biol.  Bull.,  89 :  180. 

MAVRODIADI,  P.,  1923.  "Kosoe"  delenie  u  infuzoril.  Pratsy  Bclaruskaga  dziarshafinaga  univer- 
sytctu  H  Mcuskn,  4-5  :  166. 


PROGRAM  AND  ABSTRACTS  OF  SCIENTIFIC  PAPERS  PRESENTED 
AT  THE  MARINE  BIOLOGICAL  LABORATORY,  SUMMER  OF  1946 

JULY  9 
DR.  J.  E.  KINDRED.     No  abstract  submitted. 

The  cyanide  sensitivity  of  the  unfertilized  sea  urchin  egg.     W.  A.  ROBBIE. 

Reinvestigation  of  the  cyanide  sensitivity  of  unfertilized  eggs  of  Arbacia  punctulata,  using 
recently  devised  methods  for  the  control  of  HCN  concentration  in  manometric  experiments, 
showed  that  there  was  a  definite  inhibition  of  respiration.  The  respiration  is  depressed  by 
concentrations  of  HCN  as  low  as  10~5  M.,  and  for  a  four-hour  period  in  KT4  M.  it  is  only  40 
per  cent  of  the  control  value.  There  is  complete  inhibition  for  the  first  hour  or  more.  In  4  per 
cent  CX-96  per  cent  Nj  mixture  there  is  no  depression  of  the  respiration  of  the  control  egg,  but 
on  the  addition  of  10~l  M.  HCN  the  oxygen  consumption  is  reduced,  for  a  four-hour  exposure, 
to  20  per  cent  of  the  control  level. 

At  concentrations  of  cyanide  higher  than  10  4  M.  there  is  apparently  a  stimulation  in  oxygen 
uptake.  This  is  increased  with  high  and  reduced  with  low  oxygen  tensions.  It  is  possibly 
associated  with  oxidations  proceeding  through  a  cyanide-hemin  system,  or  with  the  metabolism 
of  a  carbohydrate  intermediate  catalyzed  by  HCN. 

Inhibition   of  fertilisation    in   sea    urchins   by   means   of   univalent   antibodies  vs. 
antifertilisin.     ALBERT  TYLER. 

In  order  to  obtain  further  information  as  to  the  role  of  the  specific  interacting  substance 
of  eggs  and  sperm  in  fertilization,  antisera  were  prepared  against  them  by  immunization  of 
rabbits,  and  the  antibodies  tested  for  their  ability  to  interfere  with  fertilization.  The  present 
report  concerns  tests  with  antibodies  prepared  against  purified  anifertilizin  derived  from  sperm 
of  the  sea  urchin  Lytcchinus  pictus  and  the  gephyrean  worm  Urcchis  caupo.  The  antisera  ag- 
glutinate the  species  sperm  to  high  titer,  but  cannot  be  used  directly  to  test  for  specific  action  on 
fertilization,  since  the  mechanical  effect  of  tying  up  the  sperm  would  itself  constitute  a  block  to 
fertilization.  However,  by  a  previously  described  method,  namely  photo-oxidation,  antibodies 
can  be  converted  into  a  non-agglutinating  form,  termed  "univalent."  This  treatment  was, 
therefore,  applied  to  the  anti-antifertilizin  sera  and  the  "univalent"  antibodies  thus  obtained 
were  tested  for  possible  action  on  the  ability  of  the  sperm  to  fertilize  eggs  of  the  same  species. 
The  results  showed  a  considerable  reduction  in  fertilizing  power  of  the  sperm,  ranging  in  dif- 
ferent tests  from  32-fold  to  greater  than  128-fold.  At  the  same  time,  the  motility  of  the  treated 
sperm  was  found  to  be  quite  as  high  as  the  controls. 

JULY  16 
Intermediate  steps  in  the  visual  cycle.     A.  F.  BLISS. 

The  primary  functions  of  a  visual  pigment  are  absorption  of  radiant  energy  and  its  transfer 
to  the  stimulatory  mechanism  of  the  visual  cell.  At  present  four  such  pigments  are  known  : 
rhodopsin  and  porphyropsin,  the  photosensitive  pigments  of  vertebrate  night  vision;  iodopsin, 
the  corresponding  pigment  of  daylight  vision;  and  cephalopsin,  the  photostable  red  pigment  of 
cephalopods  and  probably  other  invertebrates  (/.  Gen.  Physiol,  1943).  Instability  in  the  light 
has  generally  been  accepted  as  a  diagnostic  test  for  a  visual  pigment.  The  existence  of  a  light- 
stable  visual  pigment  in  the  squid  however  throws  doubt  on  the  validity  of  this  assumption. 

The  bleaching  by  light  of  vertebrate  visual  pigments  is  nevertheless  an  interesting  and 
complex  process  which  has  not  been  adequately  analyzed  into  its  component  steps.  The  first 
known  product  of  bleaching  visual  purple  is  a  thermally  unstable  complex  lipid,  called  Tran- 

210 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  211 

sient  Orange  by  Lythgoe  and  Provisual  Red  by  Krause.  Its  sluggish  reaction  to  base  and  tem- 
perature near  0°  C.  suggest  that  it  is  the  acid  tautomer  of  the  first  relatively  stable  product  of 
bleaching,  appropriately  named  Indicator  Yellow  by  Lythgoe.  Acid  Indicator  Yellow  is  a  red 
lipid,  becoming  reversibly  decolorized  in  base,  and  irreversibly  converted  in  chloroform  to  the 
greenish  yellow  carotenoid  retinene,  extracted  by  Wald  from  freshly  bleached  frog  retinas.  If 
bleached  retinas  are  allowed  to  stand  an  hour  before  extraction,  retinene  is  no  longer  found. 
Instead  an  equivalent  amount  of  vitamin  A  is  extracted.  Retinene,  however,  is  not  the  precursor 
to  vitamin  A,  but  is  due  to  the  irreversible  side  reaction  described  above.  In  the  normal  retina 
and  in  fresh  neutral  solution  Indicator  Yellow  forms  vitamin  A  under  the  influence  of  a  labile 
protein,  the  reaction  being  presumbaly  enzymatic  in  nature.  In  the  dark,  dissolved  rhodopsin 
is  reformed  in  part  from  Indicator  Yellow.  In  the  living  animal  the  vitamin  A  released  by 
bleaching  is  reincorporated  into  rhodopsin  by  unknown  means. 

MR.  W.  H.  PRICE.     No  abstract  submitted. 

The  dependence   of   the   resting   potential   of   nerve   on   potassium,    calcium,    and 
hydrogen  ions.*    ABRAHAM  M.  SHANES. 

On  the  basis  of  new  as  well  as  recent  -experimental  results  it  now  appears  possible  to  de- 
scribe a  specific  mechanism  necessary  and  sufficient  to  account  for  the  relationships  between  the 
resting  potential  and  metabolic  processes  in  frog  nerve.  Cellular  hydrogen  ion  production  ac- 
companied by  an  exchange  with  extracellular  potassium  ions  is  apparently  involved ;  under  the 
conditions  of  study  this  process  contributes  about  50  per  cent  of  the  total  resting  potential. 
Calcium  reduces  the  rate  of  ionic  exchange,  an  effect  of  possible  importance  in  the  energy  ex- 
penditure necessary  to  maintain  concentration  gradients  and  associated  potentials. 

The  evidence  consists  of  demonstrating  first  that  hydrogen  ions  from  (1)  CO,  produced 
by  nerve,  (2)  CO2  applied  to  nerve,  and  (3)  lactic  acid  and  possibly  other  sources  of  acid  within 
the  fibers  are  directly  concerned  with  the  production  and  maintenance  of  the  potentials.  This 
has  been  possible  chiefly  with  the  aid  of  inhibitors  of  carbonic  anhydrase — sulfanilamide  and 
thiophene-2-sulfonamide — by  means  of  which  the  role  of  hydrogen  ions  can  be  followed  during 
anoxia,  upon  return  to  oxygen  following  anoxia,  and  to  some  extent  during  relatively  normal 
aerobic  conditions.  The  changes  of  potential  associated  with  the  application  of  CQ..-O2  mix- 
tures and  related  experiments  show  that  the  effectiveness  of  the  hydrogen  ions  is  dependent  on 
the  ionic  gradients  established. 

The  involvement  of  extracellular  potassium  is  demonstrated  by  the  suppression  in  its  ab- 
sence of  the  changes  in  potential  normally  produced  by  CO2.  This  effect  is  used  to  show,  fur- 
ther, that  the  potassium  in  the  extracellular  spaces  is  reduced  by  the  rapid  large  increase  in 
potential  induced  by  the  return  of  the  anoxic  nerves  to  oxygen.  A  small  secondary  decline  of 
potential  which  follows  the  rise  in  CO2  and  which  is  independent  of  extracellular  buffering  is 
also  dependent  on  extracellular  potassium,  which  suggests  that  the  dense  connective  tissue  or 
the  other  sheathing  materials  of  nerve  are  interfering  with  the  potassium  exchange  between  the 
extracellular  space  immediately  adjacent  to  the  fibers  and  that  more  remote. 

Calcium  slows  the  rate  of  potential  rise  upon  application  of  CO2  and  this  effect  is  directly 
related  to  calcium  concentration ;  in  view  of  the  above  results  and  available  evidence,  this  is 
interpreted  as  an  effect  on  ionic  exchange.  At  lower  concentrations  calcium  depresses  to  almost 
the  same  degree  the  potential  changes  in  response  to  oxygen  following  anoxia  and  to  CO2 ; 
higher  calcium  concentrations,  known  to  suppress  the  metabolic  processes,  exert  a  more  marked 
effect  on  the  former. 

These  results  therefore  focus  attention  on  factors  important  in  the  production  and  modifica- 
tion of  the  resting  potential.  The  action  of  any  agent  on  the  potential  must  be  considered  from 
several  possible  standpoints:  (1)  inhibition  or  activation  of  metabolism  or  of  carbonic  anhy- 
drase, (2)  production  of  hydrogen  ions,  (3)  production  of  a  membrane  diffusion  potential,  (4) 
modification  of  equilibrium  or  membrane  diffusion  potentials.  The  experimental  procedures 
which  have  been  applied  provide  means  of  distinguishing  these  possibilities.  In  view  of  the 
conclusions  reached,  these  methods  should  also  prove  useful  in  studies  of  the  "potassium  pump" 
and  of  the  biochemical  processes  concerned  with  CO2  production  and  fixation  in  relatively  intact 
cells,  both  are  problems  of  considerable  interest  at  the  present  time. 

*  Aided  by  a  grant  from  the  Penrose  Fund  of  the  American  Philosophical  Society. 


212  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

DR.  J  T.  BONNER.     No  abstract  submitted. 

JULY  23 

Oxidation-reduction  studies  as  a  clue  to  the  mechanism  of  fertilisation  of  marine 
eggs.     MATILDA  M.  BROOKS. 

Eggs,  sperm,  and  larvae  at  stages  up  to  pluteus  of  three  marine  animals  (Arbacia  punctu- 
lata,  Asterias  Forbesii  and  Chactopterus  pcrgcmentaceus)  were  measured  for  Eh  and  Ph.  The 
egg,  sperm  or  larvae  were  centrifuged  and  1  cc.  of  the  mass  used  for  measurement  in  a  glass 
vessel  in  the  Coleman  electrometer.  It  was  found  that  there  is  a  definite  correlation  between 
the  rate  of  Q..  consumption  and  the  redox  potential  of  these  cells.  It  was  also  found  that  the 
redox  potential  of  sea  water,  as  diluted  by  hypertonic  NaCl,  CaCU,  MgCl,.,  butyric  acid  or  su- 
crose, became  more  negative  than  that  of  sea  water  alone.  These  facts  were  used  as  a  basis 
for  the  hypothesis  that  a  proper  redox  potential  or  ratio  of  oxidants  to  reductants  of  the  respira- 
tory enzymes  was  necessary  for  producing  fertilization  of  the  egg.  The  hypothesis  as  presented 
states  that  the  redox  potential  of  the  external  solution  or  sperm  as  compared  with  that  of  the 
exterior  of  the  egg  itself  is  an  important  factor  in  producing  fertilization  of  the  egg. 

From  the  results  with  KCN  in  sea  water  which  produced  fertilization  membranes  but  not 
cleavage,  it  was  concluded  that  the  formation  of  the  fertilization  membrane  is  not  associated 
with  oxidations,  and  appears  rather  to  be  due  to  change  in  the  physical  aggregation  of  some 
proteins  at  the  surface  of  the  egg  or  to  a  denaturation  process  occurring  as  the  redox  potentials 
is  changing. 

DR.  C.  L.  YNTEMA.     No  abstract  submitted. 

JULY  30 

The  action  oj  napthoquinone  antimalarials   on   respiratory  systems.     CHRISTIAN 
B.  ANFISEN  AND  ERIC  G.  BALL. 

In  confirmation  of  the  findings  of  Wendel  (unpublished  reports)  a  series  of  2-hydroxy-3- 
alkyl-naphthoquinones  have  been  found  to  exert  a  powerful  inhibitory  effect  on  the  respiratory 
metabolism  of  the  malarial  parasite.  The  most  powerful  tested  to  date  is  the  compound  2- 
hydroxy-3(2-methyl-octyl)-naphthoquinone-l,4  (M-285)  which,  at  a  level  of  1  mg./liter  in- 
hibits Plasmodium  kiwzvlcsi  respiration  60  per  cent.  In  experiments  to  localize  the  site  of  ac- 
tion of  M-285  in  the  main  respiratory  chain  of  enzymes  it  was  found  that  p-phenylene-diamine 
oxidation,  requiring  only  the  cytochrome  system,  was  not  inhibited  by  the  drug,  while  the  oxida- 
tion of  succinate  to  fumarate  by  succinic  oxidase  prepared  from  beef  heart  was  completely  in- 
hibited at  about  1  mg./liter.  The  drug,  therefore,  appears  to  inhibit  at  an  oxidation-reduction 
potention  level  below  that  of  cytochrome  C.  Succinic  dehydrogenase  activity,  as  measured  by  the 
Thunberg  methylene  blue  technique,  was  only  very  slightly  diminished  even  at  high  drug  con- 
centrations. Similarly,  the  respiration  of  both  fertilized  and  unfertilized  eggs  of  Arbacia  punc- 
tulata,  neither  presumably  containing  succinic  dehydrogenase,  was  inhibited  strongly  at  levels 
as  low  as  0.1  mg./liter  (2  X  10"7  M.).This  enzyme,  therefore,  does  not  seem  to  be  the  inhibited 
system.  A  number  of  flavoproteins  including  d-amino  acid  oxidase  and  xanthine  oxidase,  as 
well  as  several  systems  involving  the  mediation  of  the  pyridine  nucleotides,  showed  no  decrease 
in  activity  in  the  presence  of  the  drug. 

It  appears  possible  that  the  naphthoquinones  under  study  are  inhibiting  a  hitherto  un- 
detected enzyme  or  enzyme  group  in  the  main  chain  of  oxidative  metabolism  having  an  E0  below 
that  of  cytochrome  C  and  above  that  of  the  flavoproteins. 

Chemical  sense  and  taste  in  the  Sea  Robin,  Prionotus.     ERNST  SCHARRER. 

The  differentiation  of  taste  and  chemical  sense  is  partly  based  on  the  concept  that  chemical 
sensitivity  can  evoke  only  negative  or  defensive  reactions ;  positive  reactions  to  food  are  medi- 
ated by  the  sense  of  taste  (Kappers,  Huber,  Crosby,  1936,  p.  347).  Observations  in  the  sea 
robin,  Prionotus,  do  not  support  this  conclusion.  Prionotus  possesses  three  free  fin  rays.  Their 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  213 

epithelium  is  innervated  by  spinal  nerves  ;  taste  buds  are  absent.  The  afferent  fibers  end  in 
accessory  lobes  on  the  dorsal  surface  of  the  cephalic  end  of  the  spinal  cord.  Secondary  fibers 
from  these  lobes  which  represent  the  greatly  enlarged  dorsal  horns,  ascend  cephalad  to  the 
funicular  nucleus  from  which  fibers  pass  ventromedially,  crossing  in  the  ventral  commissure, 
and  ending  in  the  contralateral  ventral  horn.  When  the  free  fin  rays  of  blinded  and  sufficiently 
hungry  sea  robins  are  stimulated  with  extracts  of  clams  or  crabs  the  animals  react  positively  by 
turning  to  and  snapping  in  the  direction  from  where  the  juice  comes.  Positive  reactions  to 
chemical  stimuli  are  mediated  in  this  case  by  spinal  nerves  and  in  the  absence  of  taste  buds. 
The  differentiation  between  chemical  sense  and  taste  can,  therefore,  be  based  only  on  the  inner- 
vation  and  the  presence  or  absence  of  taste  buds.  The  reaction  of  the  animal  cannot  be  used 
as  a  criterion. 

Studies  o\  the  respiration  of  the  iniaginal  discs  of  Drosophila  using  the  Cartesian 
diver  ultramicrorespiromctcr.     CLAUDE  A.  VILLEE. 

A  determination  of  the  effects  of  a  mutant  gene  on  the  metabolic  activity  of  a  particular 
group  of  cells  provides  a  basic  approach  to  the  analysis  of  gene  action  in  development.  In 
most  animals  it  is  impossible  to  locate  exactly  the  cells  which  will  give  rise  to  a  particular 
structure,  but  in  Drosophila  each  organ  develops  from  a  discrete  group  of  cells,  an  imaginal 
disc,  which  can  be  dissected  out  of  the  larva.  The  rate  of  respiration  of  wing  and  leg 
discs  from  wild,  "miniature"  wing,  and  "vestigial"  wing  stocks  were  determined  by  the 
Cartesian  diver  ultramicrorespirometer  and  their  weights  measured  by  the  quartz  fiber  balance 
of  Lowry.  The  legs  of  the  adults  of  all  three  stocks  are  normal,  the  wings  of  adult  "miniature" 
flies  are  about  two-thirds  normal  size  but  of  normal  shape,  and  the  wings  of  adult  "vestigial" 
flies  are  small  misshapen  stumps,  less  than  one-quarter  the  size  of  the  normal  wing.  At  each 
of  several  stages  studied,  before,  at  and  after  pupation,  the  Qo2  of  wild  type  discs  and  the  leg 
discs  of  all  stocks  used  varied  only  slightly  from  20  cu.  mm.  O2  per  hour  per  milligram  of 
tissue.  The  Qo2  of  "miniature"  wing  discs  was  18  and  of  "vestigial"  wing  discs  9  cu.  mm.  O,. 
per  hour  per  milligram  of  tissue.  The  weights  of  "vestigial,"  "miniature"  and  wild  type  wing 
discs  are  the  same  at  corresponding  developmental  stages  in  the  larvae  and  early  (1-2  hour) 
pupae.  The  discs  contain  considerable  reserves  of  substrate  and  will  respire  in  the  divers 
twelve  hours  or  more.  The  mutant  genes  "vestigial"  and  "miniature"  produce  their  effects 
by  altering  the  rate  of  some  chemical  reaction  in  the  wing  disc  of  the  larva  which  is  reflected 
by  a  lowered  rate  of  oxygen  consumption.  These  results  do  not  mean  that  the  "miniature" 
and  "vestigial"  genes  affect  the  same  chemical  reaction  in  development  to  a  different  extent 
but  rather  that  in  affecting  different  processes  they  each  lower  the  overall  metabolic  rate  of  the 
disc.  The  metabolism  of  the  leg  discs,  and  probably  of  the  other  discs  as  well,  is  not  changed, 
although  the  cells  contain  the  mutant  gene.  The  "vestigial"  and  "miniature"  genes  therefore 
produce  their  physiological  as  well  as  their  morphological  effects  only  in  certain  cells  of  the 
body,  presumably  due  to  the  interaction  of  the  gene  or  gene  products  with  specific  components 
of  the  cytoplasm  of  those  cells. 

AUGUST  6 

The  specificity  of  chlorine  est erase.     PHILIP  B.  ARMSTRONG. 

The  relative  rates  of  hydrolysis  of  choline  esters  acting  at  the  nerve  terminations  in  the 
sphincter  pupillae  of  the  turtle  could  be  inferred  by  determining  the  relative  potentiations  by 
eserine  of  threshold  concentrations  for  pupillary  constriction  of  the  choline  esters.  A  com- 
parison of  the  potentiations  in  vivo  with  the  relative  hydrolysis  rates  of  the  choline  esters 
by  the  purified  specific  choline  esterase  in  vitro  indicates  that  the  enzyme  as  it  functions  in  vivo 
is  as  specific  if  not  more  so  than  in  vitro.  The  choline  ester  substrate  concentrations  in  vivo 
at  which  eserine  was  effective  were  much  lower  than  those  for  effective  substrate  hydrolysis 
in  vitro. 

DR.  T.  H.  BULLOCK.     No  abstract  submitted. 


214  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  endocrine  role  of  the  corpora  allata  in  insects.     BERTA  SCHARRER. 

In  Lcncophaca  wadcrac  (Orthoptera)  extirpation  of  the  corpora  allata  at  nymphal  stages 
earlier  than  the  last  causes  an  abbreviation  of  development  (suppression  of  molts)  which  results 
in  animals  with  adult-like  characters  ("adultoids").  In  operated  seventh  instars  the  following 
nymphal  molt  is  suppressed,  and  the  animals  emerge  as  adultoids,  resembling  normal  adults 
except  for  their  smaller  size  and  comparatively  shorter  wings.  Allatectomized  sixth  or  fifth 
instars  result  in  "pre-dultoid"  stages  which  show  less  adultoid  differentiation  and  require  an 
additional  molt  before  becoming  adultoids.  In  the  adult  insect  the  corpora  allata  are  necessary 
for  the  development  of  the  eggs.  In  females  allatectomized  shortly  after  the  beginning  of  a 
reproductive  cycle  the  eggs  do  not  develop  appreciably  beyond  the  stage  typical  of  the  ovary 
at  the  time  of  operation.  The  accessory  sex  glands  in  these  operated  females  show  little  or  no 
sign  of  secretion  in  contrast  to  normal  control  glands.  Reimplantation  of  the  corpora  allata 
into  allatectomized  females  causes  the  eggs  and  the  nymphs  hatching  from  them  to  develop 
as  normally  as  those  of  unoperated  animals.  In  a  series  of  experiments  in  which  the  time  of 
allatectomy  is  varied  it  can  be  demonstrated  that  the  corpora  allata  are  necessary  throughout 
the  period  of  growth  and  yolk  deposition  which  constitutes  about  the  first  third  of  the  total 
period  required  for  the  development  of  the  eggs.  The  corpora  allata  are  apparently  not  essential 
for  the  reproductive  activity  of  male  Leucophaea.  Allatectomized  males  when  mated  with 
normal  virgin  females  are  capable  of  fertilizing  the  eggs. 

Contrasts  between  visible  and  dominant  lethal  mutation  rates  in  x-rayed  Habro- 
bracon  eggs.     ANNA  R.  WHITING  AND  H.  C.  GEORGE. 

Senior  author  has  previously  reported  that  eggs  x-rayed  in  late  metaphase  I  have  lethal  dose 
about  2,000  r  and  one-hit  dose-hatchability  curve.  Death  appears  to  be  due  to  terminal  deletions. 
Eggs  x-rayed  in  prophase  I  have  lethal  dose  about  45,000  r  and  complex  dose-hatchability  curve. 
Death  appears  to  be  due  to  several  factors,  including  translocations  and  inversions.  Majority  of 
lethal  effects  in  both  stages  are  dominant.  Recently,  two  groups  of  females  were  treated  with 
doses  giving  about  90  per  cent  mortality,  one  with  1,120  r  for  metaphase  I  and  the  other  with 
28,000  r  for  prophase  I.  They  were  then  crossed  with  untreated  males  and  their  daughters  were 

F,  5?  heterozygous 

tested  for  heterozygosis  for  visible  mutations.         -p — ~T—  -  was  2.11  per  cent  lor  eggs 

.b,  Y¥  tested 

treated  in  metaphase  I,  12.69  per  cent  for  eggs  treated  in  prophase  I.  By  x2  test  there  is  less 
than  one  chance  in  one  hundred  that  these  stages  belong  to  same  class  in  respect  to  visible  muta- 
tion rate  although  they  have  same  dominant  lethality  rate.  This  strengthens  theory  of  terminal 
deletions  (which  would  not  produce  visibles)  as  most  common  response  to  x-rays  of  metaphase 
I.  Visibles  produced  in  metaphase  I  are  probably  genie  and  their  low  percentage  is  what 
would  be  expected  at  low  doses  tolerated  by  this  stage.  Most  visibles  from  treated  prophase 
I  are  probably  also  genie  although  a  few  may  be  due  to  position  effects  of  translocations  or 
inversions.  Their  high  percentage  is  possible  because  of  high  doses  tolerated. 

AUGUST  13 

A  new  factor  from  the  adrenal  influencing  fat  deposition  in  the  liver.     KATHERINE 
A.  BROW  NELL. 

Starvation  in  the  normal  mouse  leads  to  a  large  deposition  of  fat  in  the  liver.  This  fails 
to  occur  after  adrenalectomy.  With  these  facts  as  a  basis  we  have  developed  a  test  for  a  fat 
factor  in  various  fractions  prepared  from  ox  adrenals. 

The  method  is  briefly  as  follows  :  Adrenalectomized  mice  are  fed  for  24  hours  then  fasted 
for  24  hours.  During  this  48-hour  period  they  are  injected  every  6  hours  with  0.2  cc.  of  the 
preparation  to  be  tested.  Two  to  3  hours  after  the  final  injection  the  livers  are  removed  and 
the  total  lipid  determined  gravimetrically. 

Over  30  fractions  from  the  adrenal  gland  including  crystalline  compounds  have  been  tested 
by  this  method.  The  table  shows  results  on  adrenalectomized  untreated  animals;  two  fractions, 
a  whole  extract  from  which  these  fractions  were  taken  and  three  crystalline  compounds  already 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 


215 


proven  to  have  glyconeogenic  potency.  Both  fractions  are  crude,  being  specific  in  only  one 
respect — namely,  that  the  carbohydrate  factor  fraction  has  no  electrolyte  potency  and  the  sodium 
factor  fraction  no  glyconeogenic  potency.  The  only  fraction  that  gave  a  highly  significant 
response  was  that  containing  the  carbohydrate  factor.  The  low  response  given  by  whole 
extract,  we  attribute  to  inhibiting  substances,  three  of  which  have  been  tested. 

Since  the  liver  fat  response  was  given  almost  exclusively  by  the  carbohydrate  factor 
fraction,  some  of  the  crystalline  compounds  having  glyconeogenic  properties  were  tried  to  de- 
termine whether  or  not  they  were  responsible.  The  table  shows  that  the  only  one  used  which 
gave  a  significant  response  was  dehydrocorticosterone ;  a  25  per  cent  increase  over  the  control 
level  and  in  order  to  obtain  this  response  two  and  one  half  times  as  much  pure  substance  (0.96 
mgm.)  was  used  as  that  estimated  to  be  present  in  our  carbohydrate  factor  fraction  (0.35 
mgin.).  The  other  two  compounds,  corticosterone  and  17-hydroxy- 11 -dehydrocorticosterone, 
gave  liver  fat  responses  only  on  the  borderline  of  significance  and  to  obtain  even  these  small 
responses  two  to  two  and  one  half  times  as  much  material  was  used  as  that  estimated  to  be 
present  in  the  carbohydrate  factor  fraction.  The  fourth  known  glyconeogenic  compound, 
hydroxycorticosterone,  we  were  unable  to  test  on  account  of  lack  of  material. 

There  remain  two  possibilties :  (1)  that  hydroxycorticosterone  is  the  fat  factor.  If  so, 
the  effect  on  fat  metabolism  is  a  new  property.  (2)  There  is  in  the  carbohydrate  factor  fraction 
a  new  factor  regulating  fat  deposition  in  the  liver. 

Effect  of  adrenal  fractions  on  deposition  of  fat  in  the  liver 


Treatment 

No.  of  animals 

Total  lipid  per  cent 

Increase  per  cent 

Adect.  untreated 

29 

6.31 

— 

Carbo.  factor  fraction  * 

15 

8.42 

33 

No  factor  fraction  * 

7 

6.74 

9 

Whole  extract  * 

7 

7.13 

13 

Dehydrocorticosterone  f 

8 

7.87 

25 

Corticosterone  f 

8 

7.11 

13 

1  7-hydroxy-  1  1  -dehydrocorticosterone  t 

7 

6.87 

9 

*  The  extracts  represent  300  gm.  of  tissue  per  cc. 

t  The  solutions  of  crystals  represent  0.6  mgm.  solid  per  cc. 

Hyper  activity  of  the  adrenal  cortex.     FRANK  A.  HARTMAN. 

• 

At  rest  or  under  conditions  of  minimal  activity  there  is  a  basal  secretion  of  adrenal  cortical 
hormones.  In  response  to  various  stresses  such  as  exercise,  exposure  to  cold,  trauma,  anoxia, 
and  poisons,  there  is  an  increase  in  output  of  the  hormones  which  subsides  after  the  stimulus  dis- 
appears. After  removal  of  a  large  proportion  of  both  adrenals  by  enucleation,  in  the  mouse,  a 
considerable  rise  in  the  basal  secretion  occurs.  This  higher  level  of  secretion  is  maintained  for 
months.  The  following  table  illustrates  these  changes.  Fat  and  glycogen  (as  sugar)  in  the 
liver  were  determined  after  24  hours'  starvation. 


Values  indicating  changes  in  hormone  production  after 
enucleation  of  both  adrenals 


Normal 

Adrenalectomized 
Enucleated  2  days 
Enucleated  7  days 
Enucleated  15  days 
Enucleated  29  days 
Enucleated  99  days 


Total  lipid 
per  cent 

8.5 
6.3 
6.6 

11.8 

10.0 

10.0 


Glycogen 
per  cent 

0.12 

0.04 
0.24 
0.58 


216  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  wide  difference  in  time  at  which  the  peaks  for  the  production  of  the  fat  factor  and  car- 
bohydrate factor  occur,  is  evidence  that  the  two  factors  are  not  identical. 

By  enucleation  we  removed  an  average  of  75  per  cent  of  the  adrenal  tissue.  Less  than  25 
per  cent  of  the  active  tissue  remained  since  the  circulation  was  disturbed  and  this  25  included  the 
capsule.  Thirteen  days  after  enucleation  the  adrenals  averaged  0.69  per  cent  of  the  body  weight 
which  is  one-half  the  normal  weight.  Removal  of  cortical  tissue  probably  reduces  the  in- 
hibitory effect  on  the  adrenotrophic  hormone  production  by  the  pituitary  so  that  after  a  lag 
of  three  or  four  days  there  is  sufficient  recovery  of  the  remaining  cortices  to  respond  to  the  in- 
creased output  of  adrenotrophic  hormone.  However,  the  new  level  of  cortical  hormone  pro- 
duction .does  not  return  the  adrenotrophic  output  to  the  old  level.  Thus  a  higher  basal  level  is 
established.  The  performance  of  a  relatively  small  number  of  cortical  cells  indicates  a  large  fac- 
tor of  safety.  This  capacity  of  cortical  cells  for  sustained  activity  in  disease  where  a  large 
proportion  of  cortical  tissue  is  destroyed  is  important  in  prolonging  life. 

There  is  now  evidence  for  three  mother  hormones  secreted  by  the  adrenal  cortex ;  the  fat 
factor,  the  carbohydrate  factor,  and  the  sodium  factor. 

Studies  on  the  mechanism  of  allo.nin  action.     ARNOLD  LAZAROW  AND  STANLEY 
LEVEY. 

A  number  of  compounds  related  to  alloxan  were  synthesized  and  tested  for  their  diabetogenic 
effect.  These  compounds  were  injected  intraperitoneally  into  rats  in  high  doses  and  the  blood 
sugar  was  determined  at  0.  1.  3,  8,  24,  48,  and  72  hours  after  injection.  Alloxan,  N-methyl 
alloxan,  and  alloxantin  which  dissociates  into  alloxan  all  produced  diabetes.  N-N-dimethyl 
alloxan  was  toxic  and,  therefore,  could  not  be  injected  in  doses  equivalent  to  that  required  for 
the  production  of  diabetes  with  alloxan.  Since  alloxan  is  a  ureid  of  mesoxalic  acid,  some  deriva- 
tives were  prepared  in  which  the  urea  or  mesoxalic  acid  portions  of  the  molecule  were  intact. 
None  of  these  (mesoxalamide,  mesoxalic  acid,  dimethyl  mesoxylate,  or  diacetyl  urea)  produced 
diabetes  in  the  doses  used.  Freshly  prepared  dialuric  acid,  alloxanic  acid,  and  barbituric  acid 
did  not  produce  diabetes  ;  whereas,  dialuric  acid  which  was  allowed  to  stand  overnight  was  di- 
abetogenic. (This  is  interpreted  as  oxidation  of  dialuric  acid  to  alloxan  by  molecular  oxygen.) 
Slight  alterations  in  the  structure  of  alloxan  abolish  its  diabetogenic  effect. 

It  has  been  reported  by  other  investigators  that  alloxan  combines  with  sulfhydryl  groups  of 
proteins  and  that  on  injection  it  produces  a  rapid  drop  in  the  blood  and  tissue  glutathione.  Since 
one  of  us  has  shown  that  injection  of  glutathione  or  cysteine  immediately  preceeding  a  diabetogenic 
dose  of  alloxan  completely  protected  the  animals  from  diabetes ;  and  since  others  have  shown  that 
pancreas  contains  less  glutathione  than  do  other  tissues ;  it  was  suggested  that  variations  in 
tissue  glutathione  may  determine  the  selectivity  of  alloxan.  Studies  are  now  being  carried  out 
to  determine  the  glutathione  content  of  the  beta  cells  of  the  pancreas  which  are  selectively  de- 
stroyed by  alloxan. 

Biological  specificity  and  the  synthesis  of  native  proteins.     DOROTHY  WRINCH. 

A  common  starting  point  for  the  discussion  of  biological  specificity  today  is  the  assumption 
that  biological  function  is  an  outward  and  visible  sign  of  atomic  pattern.  Furthermore  indications 
from  many  fields  reinforce  the  old  assumption  that  the  native  protein  is  the  dominant  structure 
type  in  all  living  systems.  A  vast  number  of  physiological  problems  turn  upon  questions  of 
atomic  pattern,  particularly  such  matters  as  (1)  local  stereochemical  features  and  (2)  the  pres- 
ence of  internal  OH...O,  NH...O  and  NH...N  bridges  and  of  linkages  dependent  upon  the 
presence  of  a  foreign  ion. 

Of  all  these  problems,  the  most  fundamental  is  the  synthesis  of  native  proteins.  We  must 
presume  that  the  power  of  native  proteins  to  produce  replicas  of  themselves  depends  in  some 
basic  way  upon  their  structure,  and  that  it  is  intimately  related  to  the  presence  on  native  protein 
surfaces  of  'active  patches'  to  use  Warburg's  term,  each  of  which  functioning  as  a  template  or 
mold  permits  the  laying  down  on  itself  of  a  complementary  constellation. 

It  is  useful  to  notice  that  the  associations  of  simple  molecules  within  crystals  offer  many  ex- 
amples of  such  complementary  constellations,  e.g.,  (1)  hexamethylene  tetramine,  with  pairs 
which  are  not  identical  associated  about  tetrahedrally  related  planes  with  a  common  three-fold 
axis  and  (2)  the  phosphotungstic  acid  29-hydrate  with  identical  (i.e.,  self  complementary)  con- 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  217 

stellations  associated  about  such  planes  with  a  common  three-fold  axis  and,  in  addition,  self-com- 
plementary constellations  associated  about  cube  planes  with  a  common  two-fold  axis. 

Visualizing  the  formation  of  new  'active  patches'  on  the  surfaces  of  an  already  existing 
species  of  native  protein  molecules,  we  see  that  such  new  constellations  comprise  the  material  re- 
quired for  the  formation  of  a  new  and  identical  molecule  if  (1)  the  species  carries  complementary 
patches  (which  may  be  but  need  not  be  individually  self-complementary)  and  (2)  the  molecule 
is  wholly  made  up  of  such  patches,  i.e.,  is  a  surface  structure. 

In  order  to  have  a  mechanism  whereby  these  isolated  constellations  on  several  different 
molecules  may  be  integrated  so  as  to  interlock  in  the  same  spatial  pattern  as  in  the  original  mole- 
cule, something  has  to  be  postulated  as  to  the  capacity  of  the  original  molecules  to  form  a  crystal. 
Thus  for  example,  let  us  visualize  a  body-centered  cubic  lattice  with  molecules  placed  a:  the  8 
body-centers  and  the  6  cube  corners  nearest  the  origin,  with  the  molecule  at  the  origin  missing. 
With  the  complementary  constellations  in  position  on  each  of  the  8+6  faces  of  the  molecules 
turned  to  the  origin,  we  have  a  situation  in  which  interlocking,  possibly  in  a  number  of  distinct 
steps,  could  take  place,  the  resulting  molecule  being  a  replica  of  the  original  molecule.  This  is 
but  one  example  of  a  number  of  such  possibilities,  with  the  original  molecules  characterized  by 
antipodal  pairs  of  complementary  patches.  All,  however,  have  in  common  the  dependence  upon 
the  capacity  of  the  original  molecules  to  crystallize,  an  outstanding  characteristic  and  most  re- 
markable property  of  unnumbered  native  proteins.  Similarly,  all  theories  as  to  the  formation 
of  new  native  protein  molecules  by  autocatalysis  must,  it  would  seem,  have  in  common  the  picture 
of  such  molecules  as  surface  structures,  i.e.,  atomic  fabric  cages. 

AUGUST  20 

Naturally  occurring  polyploidy  in  the  development  of  Allium  cepa  L.     Dr.  C.  A. 
BERGER. 

One  of  the  factors  in  the  developmental  pattern  of  Allium  cepa  is  the  formation  of  some 
tetraploid  cells  and  their  division  as  tetraploids.  These  cells  are  found  throughout  the  cortex  of 
the  cotyledon  and  of  the  intermediate  region  between  root  and  shoot.  They  are  found  in  seed- 
lings between  20  and  40  mm.  in  length.  They  are  never  found  in  the  root.  During  prophase  of 
mitosis  in  tetraploid  cells  the  chromosomes  are  closely  paired  and  relationally  coiled.  The  two 
members  of  each  pair  are  united  at  a  single  undivided  SA-region.  These  cytological  details 
show  that  the  chromosomes  have  not  separated  since  the  time  ofxtheir  formation.  Since  the  pair- 
ing and  relational  coiling  is  present  from  earliest  prophase  the  double  chromosome  reduplica- 
tion must  have  taken  place  during  the  resting  stage  immediately  preceding  the  4n  division.  At 
metaphase  the  tetrachromosomes  undergo  two  successive  divisions  of  the  SA-region  and  ana- 
phase  is  normal.  Since  no  tetraploid  division  figures  were  found  with  unpaired  chromosomes  it 
was  concluded  that  only  one  division  of  these  tetraploid  cells  occurs. 

Chick  embryology  at  the  medical  schools  of  Ancient  Greece.     TAGE  U.  H.  EL- 
LINGER. 

Of  the  seventy  titles  comprising  the  Hippocratic  Corpus,  the  most  significant  work,  from 
a  biological  standpoint,  is  the  lecture  on  embryology  represented  by  the  two  texts  On  Semen  and 
On  the  Development  of  the  Child.  It  deals  with  human  embryology  from  the  formation  of  the 
semen  to  the  birth  of  the  child.  The  author  is  unknown,  but  he  was  not  Hippocrates  nor  one 
of  his  followers.  His  work  reflects  the  teachings  of  the  medical  school  at  Cnidos  and  of  that 
of  Empedocles  whose  influence  is  evident  in  doctrine  as  well  as  in  scientific  method  and  in  the 
choice  of  vocabulary.  This  pre-Aristotelian  author,  who  wrote  in  the  last  quarter  of  the  fifth 
century  B.C.,  at  the  time  of  Socrates,  was  indeed  a  very  great  scientist  and  a  great  teacher  as  well. 

To  the  modern  reader  perhaps  the  most  amazing  revelation  is  the  use  made  of  observations 
on  chick  embryology  in  explaining  to  the  students  the  development  of  the  human  embryo.  The 
following  quotations  are  in  the  author's  translation. 

In  chapter  13,  the  Greek  physician  after  describing  a  "semen  which  had  stayed  six  days  in 
the  womb  and  which  fell  out,"  adds  "A  little  later  I  will  describe  another  test  in  addition  to  this 
one,  that  will  enable  anyone  who  seeks  knowledge  to  see  this  for  himself,  as  well  as  a  proof  that 
my  whole  discourse  is  correct,  as  far  as  that  is  possible  for  a  mortal  discussing  such  a  matter." 


218  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

He  returns  to  this  topic  in  chapter  29 :  "Now  I  shall  recount  the  crucial  test,  that  I  promised 
a  little  while  ago  to  make  known,  which  is  as  clear  as  possible  to  a  human  intelligence  and  makes 
plain  to  anyone  who  wants  to  be  informed  about  it,  that  the  semen  is  in  a  membrane  and  that  the 
navel  is  in  the  middle  of  it,  and  that  it  first  draws  air  in  and  expels  it  outward,"  (according  to 
the  Empedocles*  pneuma  theory  of  differentiation)  "and  that  there  are  membranes  from  the  navel. 
You  will  also  find  the  further  growth  of  the  child,  as  I  have  described  it,  to  be  from  beginning 
to  end,  such  as  it  is  in  my  account,  if  you  will  apply  the  method  of  inquiry  that  I  am  about  to 
describe.  Take  twenty  eggs  or  more  and  give  them  to  hatch  to  two  hens  or  more ;  then  on  every 
day  from  the  second  to  the  last,  that  of  hatching,  remove  an  egg,  break  it  and  examine  it.  You 
will  find  that  everything  in  it  conforms  with  my  statements,  in  so  far  as  one  can  compare  the 
growth  of  a  bird  with  that  of  man.  That  there  are  membranes  extending  from  the  navel,  and 
all  my  other  statements  about  the  child,  you  will  find  illustrated  from  beginning  to  end  in  the 
hen's  egg ;  and  he  who  has  not  yet  made  these  observations  will  be  surprised  that  there  is  a 
navel  in  a  hen's  egg.  Such  are  the  facts,  and  such  is  my  account  of  them." 

Again  in  chapter  30,  the  Greek  author  advances  chick  observations  to  illustrate  and  explain 
conditions  in  man.  He  states :  "Now  in  proof  of  my  theory,  that  it  is  the  lack  of  nourishment 
that  causes  the  child  to  come  forth,  provided  it  suffers  no  violence,  I  offer  the  following  evidence. 
The  bird  develops  from  the  yolk  of  the  egg  in  the  following  way.  Under  the  brooding  mother 
the  egg  is  heated  and  the  content  of  matter  inside  receives  the  impulse  to  development  from  the 
mother.  When  the  content  of  the  egg  is  heated,  it  forms  air  and  attracts  other  cold  air  from 
the  atmosphere  through  the  egg;  for  the  egg  is  porous  enough  to  admit  the  attracted  air  in 
sufficient  quantity  to  the  matter  inside.  The  bird  grows  in  the  egg  and  is  differentiated  in  the 
same  or  in  a  similar  way  to  the  child,  as  I  have  already  said  above.  It  develops  from  the  yolk, 
but  it  receives  its  nourishment  and  material  for  growth  from  the  white  that  is  in  the  egg.  This 
was  at  once  apparent  to  all  those  who  have  given  attention  to  it.  Whenever  nourishment  from  the 
egg  is  insufficient  for  the  chick,  then,  not  having  sufficient  nourishment  to  live  on,  it  moves  vio- 
lently in  the  egg  seeking  more  nourishment,  and  the  membranes  about  it  burst.  When  the  mother 
notices  that  the  chick  has  moved  violently,  she  pecks  and  removes  the  shell.  And  this  happens 
in  twenty  days.  And  it  is  evident  that  this  is  so,  for,  when  the  mother  pecks  the  shell  of  the  egg, 
there  remains  in  it  no  liquid  worth  mentioning,  since  it  has  been  expended  on  the  chick." 

Reproductive  economy  in  closccrossed  species  zvith  haploid  males.     P.  W.  WHIT- 
ING AND  RUDOLPH  G.  SCHMEIDER. 

According  to  the  multiple-allele  theory  of  sex  determination,  proved  true  for  the  wasp 
Habrobracon,  every  mating  must  involve  either  three  or  two  sex  alleles.  The  three-allele 
matings  produce  only  females  (sex  heterozygotes)  and  normal  (haploid)  males  (azygotes)  ;  but 
the  two-allele  matings  produce  also  sex-homozygotes  which  either  develop  into  sterile  (diploid) 
males  or  are  inviable.  Outcrossing  reduces  the  chance  for  two-allele  crosses  with  their  attend- 
ant reproductive  wastage.  The  Habrobracon  theory  has  been  tentatively  applied  to  the  six  or 
seven  invertebrate  groups  characterized  by  male  haploidy.  Since  many  species,  however,  re- 
produce with  much  inbreeding,  this  theory  would  imply  loss  approximating  half  of  the  fertilized 
eggs.  It  has  now  been  shown  that  in  the  wasp  Melittobia  over  90  per  cent  of  the  eggs  from  close- 
crosses,  including  selfcrosses  (mother  X  haploid  son),  may  develop  into  females.  If  Melittobia 
females  are  sex-heterozygotes,  some  method  must  therefore  have  been  evolved  other  than  multiple 
alleles  for  avoiding  production  of  sex-homozygotes  equal  in  number  to  the  females.  Although 
the  method  of  sex  determination  in  Melittobia  is  not  yet  understood,  it  has  now  for  the  first  time 
been  shown  that  reproductive  economy  is  high  in  a  closecrossed  species  with  haploid  males. 

A  comparative  study  of  the  lipids  in  some  marine  annelidcs.     CHARLES  G.  WILBER. 

Studies  on  the  metabolism  of  lipids  have  been  in  the  past  confined  to  observations  made  on 
vertebrates.  Very  few  studies  have  been  made  on  the  lipids  in  the  invertebrates;  consequently 
a  detailed  investigation  seems  justified. 

The  following  marine  annelides  were  studied:  Nereis  pelagica,  Amphitrite  ornata,  Arcnicola 
marina,  Phascolosoma  gouldii,  Lepidonotus  squamatus,  Glycera  americana,  and  Chactoptcrns 
variopedatus. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  219 

Whole  worms  or  individual  tissues  were  prepared  by  grinding  or  in  the  Waring-blendor. 
Lipids  were  extracted  with  boiling  alcohol.  Phospholipids  were  precipitated  with  acetone  and 
magnesium  chloride  and  estimated  by  oxidation-titration  method  of  Bloor.  Fatty  acids  were 
estimated  by  oxidation-titration  and  cholesterol  colorimetrically  using  the  acetic  anhydride-sul- 
furic  acid  reagent.  The  ratios,  cholesterol/fatty  acid  (lipocytic  index)  and  cholesterol/phos- 
pholipid,  were  calculated. 

It  was  found  that  the  absolute  values  of  the  various  lipids  in  the  same  species  and  in  different 
species  were  not  always  the  same.  On  the  other  hand,  the  lipocytic  index  and  the  relation, 
cholesterol/phospholipid,  were  constant  for  a  given  species  and  tissue.  If  the  lipocytic  index  of 
each  worm  were  plotted  against  the  phospholipid  of  the  same  worm  the  points  representing  the 
various  species  fell  along  a  straight  line ;  a  similar  straight  line  was  obtained  when  the  cholesterol 
was  plotted  against  phospholipid. 

There  is,  therefore,  an  apparent  relationship  between  cholesterol  and  phospholipid  and  be- 
tween phospholipid  and  lipocytic  index  in  marine  annelides.  Tissues  with  a  high  lipocytic  index 
or  high  cholesterol  content  have  a  high  phospholipid  content.  These  results  indicate  that  in 
the  marine  annelides,  just  as  Bloor  found  in  the  vertebrates,  since  cholesterol  is  associated  with 
and  in  constant  relation  to  phospholipids,  it  is  probably  a  normal  protoplasmic  constituent.  These 
results  confirm  in  part  the  results  of  analyses  made  on  vertebrate  tissues  and  agree  with  the  con- 
tention of  Mayer  and  Schaeffer  that  the  lipocytic  index  is  characteristic  of  the  organ  of  an  animal 
in  a  given  species. 

GENERAL  SCIENTIFIC  MEETINGS 
AUGUST  23 

Vascular  reactions  to  ergonovine  maleate  *  as  seen  directly  with  the  microscope 
in  the  living  mammal.1     RICHARD  G.  ABELL. 

Erognovine  was  injected  intravenously  in  amounts  varying  from  0.005  mgm.  to  0.2  mgm., 
and  its  effect  upon  the  arterioles,  capillaries  and  venules  observed  directly  with  the  microscope 
in  transparent  'moat'  chambers  (Abell  and  Clark,  '32)  in  rabbits'  ears.  The  clinical  intravenous 
dose  of  ergonovine  is  0.1  mgm.  The  equivalent  dose  in  the  rabbit  is  approximately  0.005  mgm. 
Injections  of  0.005  mgra.  caused  constriction  of  arterioles  to  approximately  0.7  to  0.9  of  their  con- 
trol diameters,  and  a  slight  reduction  in  velocity  of  flow.  The  arterioles  returned  to  their  con- 
trol diameters  and  the  flow  to  its  control  rate  within  3  to  5  minutes.  Daily  injections  of  0.005 
mgm.  made  for  a  period  of  2  weeks  caused  similar  results.  One  hundredth  mgm.  (twice  the 
clinical  dose)  caused  constriction  of  the  arterioles  to  approximately  0.6  to  0.8  of  their  control 
diameters,  and  a  slightly  greater  reduction  in  rate  of  flow  than  0.005  mgm.  One  tenth  mgm. 
(20  times  the  clinical  dose)  caused  arterioles  15  to  30  microns  in  diameter  to  constrict  to  the  point 
of  obliterating  their  lumens  and  stopping  the  blood  flow  for  approximately  30  seconds  to  one 
minute.  The  vessels  relaxed  to  their  control  diameters  within  approximately  12  minutes.  Two 
tenths  mgm.  (40  times  the  clinical  dose)  caused  more  vigorous  and  prolonged  arteriolar  con- 
striction, which  lasted  for  from  1  to  1%  minutes,  and  stopped  all  of  the  blood  flow  within  the 
chamber.  The  venules  constricted  to  approximately  0.6  to  0.7  of  their  control  diameters.  The 
arterioles  returned  to  their  control  diameters  in  approximately  15  to  20  minutes.  Four  injections 
of  0.2  mgm.  at  15  minute  intervals  made  the  small  arterioles  (15  to  30  microns  in  diameter)  un- 
responsive to  further  injections,  but  not  the  larger  arterioles  (80  to  90  microns).  Intravenous 
injections  of  0.025  mgm.  of  epinephrine  while  the  small  arterioles  were  still  unresponsive  to 
ergonovine,  caused  them  to  constrict  to  the  point  of  obliterating  their  lumens,  which  is  the  typical 
response  to  this  amount  of  epinephrine. 

None  of  the  above  injections  caused  any  sign  of  injury  to  the  blood  vessels,  or  any  abnormali- 
ties in  appearance  and  distribution  of  the  red  blood  cells,  the  white  blood  cells,  or  the  platelets. 
Thus  it  is  clear  that  ergonovine  maleate,  which  is  used  widely  to  prevent  post  partum  hemorrhage 
and  to  give  symptomatic  relief  of  migraine  headache,  does  not  cause  any  observable  injury  to  the 
blood  vessels  and  associated  structures  even  when  given  in  amounts  of  40  times  the  clinical  dose. 

*"Ergotrate"  (Ergonovine  Maleate,  U.S. P.,  Lilly). 

1  This  work  was  aided  by  a  grant  made  by  Eli  Lilly  and  Company  to  the  Department  of 
Anatomy  of  the  University  of  Pennsylvania  Medical  School. 


220  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  effect  of  halogcnated  alkyl  amines  on   the  respiration  of  Arbacia   eggs  and 
sperm.     E.  S.  GUZMAN  BARRON,  E.  G.  MENDES  AND  H.  T.  NARAHARA. 

Halogenated  alkyl  amines  at  0.001  M  concentration  produce  an  inhibition  of  the  respiration 
of  animal  tissues,  and  complete  inhibition  of  pyruvate  and  choline  oxidation  (Barron  et  al.1).  In 
smaller  concentrations  the  early  cleavage  of  the  fertilized  sea  urchin  egg  is  inhibited  or  retarded 
(Cannan  et  al.1).  There  is  also  inhibition  of  mitosis  in  the  corneal  epithelium  of  mammals 
(Friedenwald  and  Scholz  1)  and  a  high  incidence  of  sex-linked  lethals  as  well  as  a  significant 
number  of  translocations  and  inversions  in  the  chromosomes  of  Drosophila  inelanogastcr  ( Auer- 
bach  et  al.1). 

Dichloroethylmethylamine  HC1,  and  trichloroethylamine  HC1  at  a  concentration  of  0.001  M. 
and  dissolved  in  sea  water,  produced  a  definite  increase  in  the  respiration  of  sea  urchin  sperm 
(from  170  to  50  per  cent).  The  increase  of  respiration  could  be  noticed  even  with  1  X  10"5  M. 
The  respiration  of  sea  urchin  eggs,  fertilized  or  unfertilized,  was  slightly  inhibited  by  this  con- 
centration of  alkyl  amine  (14  to  17  per  cent).  Higher  concentrations  produced  inhibition  of 
respiration  probably  due  to  a  decrease  in  pH  as  a  result  of  the  hydrolysis  of  these  compounds. 
When  the  alkyl  amines  were  previously  neutralized  and  the  sperm  and  eggs  suspended  in  0.05  M 
citrate  buffer,  pH  6.8,  the  effect  of  the  alkyl  amines  was  erratic.  It  is  quite  possible  that 
penetration  of  the  alkyl  amines  into  the  cell  occurs  only  in  an  acid  milieu. 

The  experiments  of  Cannan  et  al.1  on  retardation  of  the  rate  of  cleavage  of  fertilized  Ar- 
bacia eggs  were  confirmed.  Eggs  treated  with  0.001  M  dichloroethylmethylamine  HCL  (dis- 
solved in  sea  water)  for  15  minutes  prior  to  insemination,  and  fertilized  eggs  treated  at  the  time 
of  the  first  cleavage  showed  a  definite  retardation  in  the  rate  of  cleavage.  Furthermore  none 
of  the  treated  eggs  reached  the  pluteus  stage. 

The  effect  of  uranyl  nitrate  on  the  respiration  of  Arbacia-  sperm.     D.  BENEDICT 
AND  E.  S.  G.  BARRON. 

Uranium,  like  other  heavy  metals,  is  quite  toxic  and  it  has  been  extensively  used  for  the 
production  and  study  of  experimental  nephritis.  Uranyl  nitrate  in  concentrations  varying  from 
10~2  to  5  X  10"5  M.  inhibited  the  respiration  of  Arbacia  sperm.  The  inhibition  was  complete  at 
5  X  10~4  M.  (92  per  cent  inhibition).  W4  M.  UO2(NOS)2  produced  partial  inhibition  (from  53 
to  15  per  cent),  5  X  10"5  M.  inhibited  15  per  cent,  and  10"5  M.  had  no  effect  at  all.  This  inhibi- 
tion must  be  due  to  combination  of  respiratory  enzymes  with  uranium,  a  combination  which  can  be 
reversed  completely  on  addition  of  a  citrate  at  a  ratio  of  U :  citrate  of  1 :  2.  Addition  of  phosphate 
at  a  ratio  of  1  :100  brought  only  partial  release  (25  per  cent).  The  experiments  were  performed 
in  acetate-sea  water  buffer  at  pH  6.4  to  avoid  precipitation  of  the  uranyl  salt.  Dry  weights  of 
sperm  were  obtained  after  centrifugation  of  the  sperm  at  16,000  g.  There  was  in  the  control 
experiments  a  rise  in  the  pH  value  of  about  0.6  units  at  the  end  of  one  hour,  probably  due  to  the 
formation  of  NH:!. 

Some  properties  of  purified  squid  visual  pigment.     ALFRED  F.  BLISS. 

The  photostable  red  visual  pigment  of  the  squid  (Bliss,  1943,  Jour.  Gen.  Pliysiol.)  was 
found  to  become  reversibly  light  sensitive  in  the  presence  of  formalin.  A  method  was  devised 
for  the  extraction  of  this  pigment  in  a  state  of  purity  approximating  that  of  the  best  prepara- 
tions of  vertebrate  rhodopsin.  The  principal  impurity  of  previous  extracts,  melanoprotein,  was 
rendered  insoluble  by  the  following  procedure.  Retinas  were  rinsed  in  distilled  water  and  kept 
frozen  until  use.  They  were  then  homogenizd  with  0.2  M  NaL.  HPO4  and  centrifuged.  The  resi- 
due was  washed  with  pH  4.5  buffer  and  distilled  water.  The  visual  pigment  was  extracted  with 
3  per  cent  digitonin  at  6°  C.  for  2  minutes  and  centrifuged  5  minutes.  The  absorption  spectrum 
of  the  extracted  pigment  did  not  differ  significantly  from  that  of  rhodopsin.  In  its  chemical  prop- 
erties it  differed  significantly  from  rhodopsin,  since  it  was  rapidly  destroyed  by  digitonin  even  at 
6°  C.  The  primary  breakdown  product  in  cold  acetone  was,  like  that  of  rhodopsin  (Bliss,  1946, 
Biol.  Bull.),  the  acid  tautomer  of  the  lipid  "Indicator  Yellow."  Because  of  the  distinctive  prop- 
erties of  the  squid  rhodopsin,  a  differentiating  name,  cephalopsin,  is  suggested. 

1  All  quoted  from  Gilman,  A.,  and  Philips,  F.,  Science  103:  409  (1946). 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  221 

Studies  on  the  viscosity  and  elasticity  of  striated  muscle.     MANFRED  BRUST. 

By  the  use  of  a  spring  vibrating  against  the  resistance  of  frogs'  (Rana  pipicns)  sartorius 
muscles — as  described  by  Gasser  and  Hill  (Proc.  Roy.  Soc.  B.  96:  398,  1924) — the  effects  of 
urea  and  iodoacetic  acid  (IAA)  on  the  viscosity  and  elasticity  of  these  muscles  were  studied. 
All  initial  slack  was  removed  from  the  system  by  stretching  the  muscles  17.5  per  cent  beyond 
their  resting  length  and  putting  them  under  3.5  gm.  tension. 

Thirty  minutes  immersion  in  solutions  of  2.5  M  urea  in  Ringer's  shortens  the  muscles  on 
the  average  by  26.1  per  cent.  When  extended  to  their  original  length  they  still  exert  the  ten- 
sion originally  exerted  at  that  length.  They  will  not  return  to  their  urea  induced  length  when  re- 
leased from  stretch.  Their  viscosity  is  reduced  on  the  average  of  53.6  +  13.0  per  cent  of  that 
in  the  untreated  muscles,  while  the  elasticity  is  similarly  reduced  to  60.4  +  18.6  per  cent. 

Sixty  minutes  immersion  of  Ringer's  equilibrated  muscles  in  1-80000  IAA  (6.72  X  10"5  M) 
in  Ringer's  sometimes  causes  a  rise  in  viscosity  and  elasticity  even  without  activity  by  the  poisoned 
muscles.  Summer  frogs  show  this  response  less  often  than  winter  frogs.  Measurements  made 
during  30  second  rest  periods  between  5  second  isometric  tetani  show  a  short  initial  decrease  fol- 
lowed by  a  gradual  increase  in  both  viscosity  and  elasticity.  Average  maximum  rigor  values  of 
181  per  cent  and  258  per  cent  respectively  of  the  untreated  muscle  values  are  attained. 

The  urea  results  would  agree  with  the  findings  by  other  authors  that  this  agent  disrupts  myosin 
and  other  protein  molecules  thus  transforming  them  into  disconnected  less  asymmetric  entities. 
Collagen  is  not  believed  to  be  markedly  affected  since  muscle  shape  is  maintained  while  tension 
remains  the  same  as  before  treatment  at  the  same  lengths.  The  IAA  results  would  agree  with 
the  progressively  diminishing  solubility  and  increase  in  hardness  of  actomyosin  in  gradually  de- 
creasing concentrations  of  adenosine  triphosphate  reported  by  the  Szent-Gyorgyi  group  (Acta 
I'liysiol.  Scand.  9:  Snppl.  xxv,  1945). 

Arterial  anastomoses.     ELIOT  R.  CLARK  AND  ELEANOR  LINTON  CLARK. 

This  study  represents  an  attempt  to  discover  factors  responsible  for  the  presence  or  absence 
of  arterial  anastomoses,  which  vary  so  greatly  in  different  organs. 

The  governing  factor  appears  to  be  the  histo-mechanical  principle  established  by  R.  Thoma 
in  1892,  corroborated  by  E.  R.  Clark  in  1918  in  studies  on  living  vessels  in  the  tadpole's  tail,  that 
the  size  of  the  lumen  of  an  artery  is  regulated  by  the  amount  of  blood  flow.  In  the  absence  of 
flow,  the  lumen  is  reduced  to  zero  and  the  artery  obliterated.  In  order,  then,  for  arterial  an^to- 
moses  to  survive,  conditions  must  be  such  as  to  provide  a  fldw  of  blood  through  the  .terminal 
connecting  portion. 

In  most  cases  this  requires  the  presence  of  factors  which  force  the  blood  to  flow  part  of  the 
time  in  one  and  part  of  the  time  in  the  reverse  direction.  Such  factors  are  present  in  the  periph- 
eral parts  of  the  body  in  the  form  of  varying  outside  pressures  that  are  exerted  irregularly  upon 
large  supplying  arteries  or  small  distributing  arterioles. 

A  study,  with  the  aid  of  artificial  chambers,  of  the  living  circulation  in  the  rabbit's  ear,  where 
anastomeses  are  abundant,  reveals  frequent  reversals  of  flow  in  connecting  portions  of  anasto- 
moses, but  controlled  by  an  unsuspected  factor,  namely,  the  irregular  contraction  of  the  arteries  or 
arterioles  themselves,  described  in  an  earlier  paper. 

In  types  of  artificial  chambers,  installed  in  rabbits'  ears,  which  are  invaded  by  new  tissue, 
there  are  often  arterioles  that,  for  weeks,  are  unprovided  with  nerves  and  hence  contract  little, 
if  at  all.  In  many  such  chambers  no  arterial  anastomoses  survive.  However,  in  this  type  of 
chamber,  occasionally  arterioles  receive  a  nerve  supply,  and  in  such  cases  arterial  anastomoses 
may  survive.  In  every  case  in  which  such  anastomoses  have  persisted  in  newly-formed  tissue, 
there  have  been  frequent  reversals  of  flow  in  the  connecting  portion. 

The  effects  of  the  ultra-violet  radiations  on  Styela  eggs.     A.  M.  DALCQ. 

The  M.D.L.  installation  for  microphotography  with  U.V.  rays  (2537  A)  may  be  used  for 
irradiating  part  of  the  Ascidian  egg  or  certain  of  the  various  blastomeres  up  to  the  Vlll-cell 
stage.  The  method  was  worked  out  with  the  aid  of  Dr.  G.  I.  Lavin.  The  egg  is  placed  in  a 
drop  of  sea  water  near  the  edge  of  a  thin  quartz  coverslip,  which  is  itself  put  on  the  transverse 
arm  of  the  mechanical  stage.  The  coverslip  is  adjusted  under  the  microscope  in  such  a  way 
that  the  part  of  the  egg  to  be  irradiated  protrudes  over  the  edge  of  the  metallic  stage  arm  which 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

acts  as  a  protection  screen  for  the  rest  of  the  egg.  Attention  should  be  paid  to  two  sources  of 
error:  (1)  the  effect  of  hypertony  due  to  evaporation  of  the  drop  and  (2)  to  the  reflexion  of 
the  rays  by  the  objective  lens  of  the  microscope,  which  is  easily  eliminated  by  interposing  some 
black  paper  during  the  irradiation.  In  exploring  a  considerable  range  of  exposure  no  favorable 
effect  of  the  irradiation  could  be  found.  If  feeble,  it  produces  a  delayed  disorganization  of  the 
embryonic  layers.  If  stronger,  it  stops  the  cleavage  with  rapidity  varying  according  to  the 
dosage.  In  order  to  obtain  stopping  of  the  next  cleavage,  exposures  of  at  least  10  minutes  are 
necessary.  When  a  division  is  suppressed,  the  cell  may  manifest  a  delayed  attempt  at  cleavage, 
but  this  is  always  abortive.  After  exposure  of  the  unsegmented  eggs,  deviations  of  the  first 
cleavage  plane  may  be  observed.  Observations  of  the  movements  of  yolk  and  yellow  pigment 
and  the  elongation  of  the  cell-body  indicate  that  the  effect  of  the  radiation  is  not  primarily  on 
the  nuclear  activity.  That  the  influence  of  the  rays  is  exerted  on  the  surface  protoplasm  is 
shown  by  the  transitory  appearance  of  alterations  of  the  surface  film  (small  protuberances, 
"blisters")  in  coincidence  with  attempts  at  cleavage. 

By  means  of  this  method,  the  division  of  one  or  more  blastomeres  of  the  II,  IV,  and  VIII 
cell  stages  has  been  inhibited.  The  non-irradiated  cells  exhibit  normal  development  with  respect 
to  mitotic  rhythm  and  arrangement.  Their  capacity  for  differentiation,  which  seems  rather  poor 
when  large  blastomeres  remain  undivided  in  the  germ,  must  still  be  studied  in  sections. 

A  correlation  betzveen  gill  surface  and  activity  in  marine  fishes.     I.  E.  GRAY. 

The  units  of  respiration  in  the  gills  of  fishes  are  the  numerous  microscopic  secondary  lamel- 
lae which  appear  as  thin,  leaf-like  plates  set  at  right  angles  to  the  main  axes  of  the  primary 
lamellae.  Within  each  plate  lies  a  capillary  network  through  which  the  interchange  of  gases 
takes  place.  Among  fishes  there  are  species  differences,  not  only  in  the  number  of  gills,  but 
also  in  the  number  and  length  of  the  gill  filaments  (primary  lamellae)  and  in  the  number  of 
respiratory  units  (secondary  lamellae).  By  determining  the  number  of  respiratory  units  per 
gram  of  body  weight  it  is  possible  to  obtain  an  estimate  of  the  relative  respiratory  ability  of 
different  fishes. 

There  is  a  marked  contrast  in  the  number  of  respiratory  units  per  gram  of  body  weight 
between  the  active,  surface,  migratory  fishes  (mackerel,  2550;  butterfish,  1725;  menhaden, 
1685)  and  the  sluggish  bottom  fishes  (flounder,  265;  toadfish,  135;  goosefish,  50).  The  number 
of  respiratory  units  of  fishes  of  medium  activity  fall  between  these  two  extremes  (scup,  1325; 
sea 'trout,  1250;  sea  bass,  1110;  eel,  900;  sea  robin,  800:  puffer,  505;  tautog,  440).  A  four 
hundred  gram  mackerel  has  a  total  of  nearly  three-fourths  million  respiratory  units  while  a 
toadfish  of  the  same  weight  has  only  fifty  thousand.  The  number  of  respiratory  units  is  also 
directly  correlated  with  the  amounts  of  sugar  and  hemoglobin  in  the  blood. 

The  distribution  of  lipid  between  the  light  and  heavy  halves  of  the  Arbacia  egg. 
F.  R.  HUNTER  AND  A.  K.  PARPART. 

Unfertilized  Arbacia  eggs  were  centrifuged  for  10-20  minutes  in  an  air  turbine  at  ap- 
proximately 16,000  X  G.  in  a  medium  of  graded  density  obtained  by  mixing  sea  water  and  0.95 
1  molal  sucrose.  The  light  and  heavy  halves  which  resulted  were  collected,  packed  in  an  air 
turbine,  frozen,  dried  in  a  vacuum  desiccator  and  weighed.  This  dried  material  was  then  ex- 
tracted with  ether,  dried  and  again  weighed.  The  loss  in  weight  was  taken  as  a  measure  of 
the  amount  of  free  fats  and  sterols.  This  material  was  then  extracted  with  alcohol-ether  and 
again  dried  and  weighed.  This  was  considered  to  give  a  value  for  the  bound  lipid.  In  order 
to  relate  the  amount  of  lipid  to  the  number  of  halves,  counts  were  made  on  suspensions  of  halves 
prior  to  drying.  The  following  values  expressed  as  nigs,  of  lipid  per  million  halves  were'  ob- 
tained:  heavy  halves — 6.6  (ether  fraction),  12.2  (alcohol-ether  fraction)  ;  light  halves — 2.2 
(ether  fraction),  9.6  (alcohol-ether  fraction).  Thus,  75.0', per  cent  of  the  free  fats  and  sterols, 
56.0  of  the  bound  lifids  and  61.6  per  cent  of  the  total  lipids  are  in  the  heavy  halves.  The  sum 
of  the  total  lipids  in  the  two  halves  is  equal  to  30.6  mgs.  per  10"' cells  which  compares  favorably 
with  the  value  34.1  mgs.  per  10"  cells  calculated  from  the  data  given  by  Parpart  (Biol.  Bull., 
81:  296,  1941)  for  unfertilized,  whole  eggs.  Similarly  a  comparison  can  be  made  between  the 
sum  of  the  bound  lipids  of  the  two  halves  and  of  the  whole  egg.  Their  values  are  71.3  per 
cent  and  77  per  cent,  respectively. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Evidence  for  enzymatic  participation  in  the  penetration  of  the  human  erythrocyte 
by  glyccrol.     PAUL  G.  LEFEVRE. 

Jacobs  and  his  associates  have  reported  that  an  amount  of  copper  sufficient  to  cover  only 
a  very  small  fraction  of  the  surface  of  the  cells  involved  markedly  inhibits  hemolysis  of  human 
red  cells  in  isotonic  glycerol.  This  report  concerns  the  extension  of  this  finding  to  the  effects 
of  other  substances  which  inhibit  the  same  types  of  enzymes  affected  by  traces  of  copper. 

Following  the  pattern  prescribed  by  Barren  and  Singer  for  identification  of  sulfhydryl 
activity,  iodine,  mercuric  ion,  the  arsenical  Mapharsen,  and  p-chloromercuribenzoate  were  shown 
to  inhibit  hemolysis  by  glycerol,  buffered  at  pH  7.1.  This  inhibition  failed  in  the  presence  of 
cysteine,  glutathione,  or  thioglycolic  acid ;  and  could  be  reversed  by  later  addition  of  these  sub- 
stances at  2-3  times  the  concentration  of  the  inhibitor,  except  with  Mapharsen.  These  relations 
indicate  strongly  that  active  •-  SH  groups  are  involved  in  carrying  glycerol  into  the  cell. 
Though  sensitive  to  the  inhibitors  mentioned,  the  hemolytic  process  was  not  affected  by  iodoace- 
tate ;  this  indicates  that  the  sulfhydryl  groups  involved  are  of  the  difficultly  available  type,  not 
inactivated  by  the  alkylating  agents. 

Since  phosphorylation  is  apparently  essential  in  transfer  of  sugars  and  other  substances 
across  the  membranes  of  the  kidney  tubule  and  the  intestinal  cell,  it  is  proposed  tentatively  that 
the  enzymatic  step  involved  in  the  present  studies  is  the  phosphorylation  of  glycerol.  Adenosine 
triphosphatase,  capable  of  this  step,  is  present  in  the  erythrocyte,  and  shows  the  same  pattern 
of  sensitivity  to  inhibitors  as  found  in  the  present  instance,  as  well  as  similar  relations  of  ac- 
tivity to  pH.  Further,  more  decisive  tests  of  the  proposed  identity  of  the  enzymatic  factor  are 
planned. 

"Accommodation"  and  opening   excitation   in   nerve   and   muscle.     PAUL   G.   LE- 
FEVRE. 

In  his  mathematical  analysis  of  electrical  excitation  in  1936,  Hill  pointed  out  that  the  accom- 
modative process  (recession  of  threshold  under  the  influence  of  a  stimulus)  itself  accounted  for 
the  phenomenon  of  excitation  at  the  anode  at  the  "break"  of  a  constant  current.  There  seems 
to  have  been  no  attempt  to  test  this  neglected  implication  of  Hill's  theory:  that  "accommodation" 
is  an  essential  prerequisite  for  "opening  excitation"  at  the  anode.  This  report  concerns  the 
occurrence  of  opening  excitation  in  tissues  showing  no  accommodation. 

Following  Solandt's  practise,  frog  sciatic  nerves  were  treated  with  citrate  until  they  no 
longer  showed  any  accommodation  :  their  threshold  was  independent  of  the  rate  of  increase  of 
the  excitatory  current  (delivered  with  Solandt's  condenser-charge  arrangement).  In  such 
preparations,  in  spite  of  the  absence  of  accommodation,  there  was  no  difficulty  in  eliciting  an 
anodal  response  at  the  cessation  of  a  steady  current.  The  same  result  was  readily  obtained  with 
exposed  sciatic  nerves  of  anesthetized  rats  treated  with  citrate. 

Frog  sartorii,  or  the  pharyngeal  retractors  of  Thyone,  if  stimulated  in  the  nerve-free  re- 
gions, or  following  neural  degeneration,  also  showed  no  accommodation.  But  all  attempts  to 
demonstrate  any  response  at  "break"  in  these  muscles  failed ;  this  is  in  accordance  with  the 
predictions  of  Hill's  theory.  Only  in  the  case  of  citrated  nerves  was  seen  the  troublesome  occur- 
rence of  opening  excitation  in  the  absence  of  any  accommodation. 

A  further  analysis  of  this  matter  is  planned,  to  determine  whether  the  results  may  be  ex- 
plained on  the  basis  of  a  postulated  fundamental  difference  between  accommodation  at  the 
cathode  and  that  at  the  anode ;  the  latter  persisting  in  the  absence  of  the  calcium  ion  required 
by  the  former. 

A  photometric  study  of  the  kinetics  of  fibrin  formation.     JOSEPH  LEIN. 

The  clotting  of  fibrinogen  solutions  by  thrombin  was  studied  by  measuring  the  optical 
density  and  light  scatter  as  the  process  occurred.  The  results  can  be  analyzed  kinetically  only 
when  purified  preparations  were  used.  If  other  plasma  proteins  are  present  the  degree  of  light 
scatter  also  depends  on  their  concentrations.  This  is  believed  due  to  a  trapping  effect  of  non- 
clottable  proteins  by  the  fibrin  as  it  is  formed.  The  light  scatter  studies  were  particularly  useful 
in  the  kinetic  analysis  of  the  clotting  process. 


224  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  reaction  was  considered  from  a  polymerization  viewpoint,  the  fibrin  representing  the 
polymer  formed  through  the  action  of  thrombin  on  the  monomer  fibrinogen.  First  order  re- 
action kinetics  were  employed.  The  following  assumptions  were  made.  (1)  With  constant 
thrombin  concentrations  the  rate  of  increase  of  the  polymer  size  is  proportional  to  the  fibrinogen 
concentration  (dN/dT  —  K,F~).  (2)  The  rate  of  decrease  of  the  fibrinogen  is  proportional  to 
the  fibrinogen  concentration  (—  dF/dT  -=  K~F).  (3)  The  light  scatter  under  the  conditions 
of  the  experiment  is  proportional  to  the  increase  in  particle  size  once  it  reaches  the  critical  size 
(N0)  which  first  scatters  light.  (LS  —  KS(N  —  N0).  From  these  formulas  a  relationship 
was  derived  that  included  light  scatter  (LS},  the  initial  concentration  of  fibrinogen  (Fn),  time 
(T),  and  time  (Ta)  for  the  polymer  to  reach  a  size  (AM  that  would  scatter  light.  The  for- 
mula may  be  expressed  as  : 

log  (K.KJK,  F.e-K^T,  -  LS)  =    -  0.434  K,T  +  log  KtKJK,  Fn 

The  relationship  was  tested  on  a  series  of  experiments  in  which  the  initial  concentration 
of  fibrinogen  was  altered,  the  thrombin  being  kept  constant.  The  calculated  values  of  the  con- 
stants agreed  with  the  experimental  values  within  a  6  per  cent  average  deviation.  It  thus  ap- 
pears that  the  course  of  the  reaction  may  be  considered  to  be  molecular  and  that  thrombin  acts 
as  a  true  catalyst,  not  forming  part  of  the  final  fibrin  product. 

The  effect  of  iodacetate  on  the  changes  in  muscular  latency  induced  by  activity. 
A.  SANDOW.     No  abstract  submitted. 

Formation  of  the  nuclear  membrane  and  other  mitotic  events  in  Chaos  chaos  Linn 
and  Chaos  ncos  (nczv  species).     A.  A.  SCHAEFFER. 

The  mitotic  stages  of  the  amebas  mentioned  are  easily  followed  in  the  living  animal.  The 
principal  stages  are  the  following:  1.  the  nucleus  about  to  divide  swells  up  to  about  6  times  its 
former  volume.  2.  The  chromatin  grains  (300  to  600  in  number)  gradually  disappear,  as  if 
going  into  solution.  Some  of  these  grains  coalesce  before  going  into  solution.  3.  A  new  mass 
of  small  grains  (about  2500)  appear,  before  all  the  larger  grains  of  the  so-called  resting  nucleus 
have  disappeared.  These  small  grains  arrange  themselves  first  as  a  lens-shaped  cloud,  then  as 
a  plate  of  about  2  grains  thickness.  At  this  stage  the  plate  of  grains  is  occasionally  seen  to  be 
indistinctly  divided  into  at  least  4,  possibly  as  many  as  8  or  12,  smaller  groups  of  equal  size. 
4.  This  plate  of  grains  then  separates  into  2  plates  which  rapidly  move  apart.  5.  Fibers  analo- 
gous to,  if  not  identical  with,  spindle  fibers,  appear  between  the  plates,  as  the  plates  separate. 
Fibers  also  appear  on  the  other  face  of  the  plates.  All  fibers  are  at  first  horizontal  and  parallel. 
6.  The  plates  separate  and  the  inter-plate  fibers  lengthen  until  the  plates  are  separated  to  about 
2  or  3  times  their  diameter  when,  because  of  the  streaming  of  the  protoplasm,  the  plates  are 
torn  apart.  During  this  time  the  nuclear  membrane  breaks  into  pieces  which  eventually  com- 
pletely disappear.  7.  The  separated  plates,  still  granular,  become  bent  like  a  concavo-convex 
lens,  with  polar  fibers  still  attached.  The  granules  soon  disappear,  leaving  a  very  thin,  per- 
fectly homogeneous  flat  disk  that  shows  a  brilliant  blue  green  color  when  seen  on  edge.  No 
refractory  edge  can  be  made  out.  This  stage  is  difficult  to  see.  8.  After  a  few  minutes  the 
disk  shrinks  in  diameter  and  is  thrown  into  rope-like  folds  around  the  periphery.  9.  Very  soon 
thereafter  a  refractory  edge  begins  to  appear  as  the  folds  disappear.  10.  Very  fine  grains  pres- 
ently begin  to  appear  until  about  1,400  are  formed.  Many  of  these  coalesce  to  form  larger 
grains  until  only  about  600  to  700  remain  in  the  newly  formed  daughter  nucleus.  (Further 
reduction  in  number  may  occur  during  the  next  few  hours.)  While  the  small  grains  are  ap- 
pearing, the  edge  of  the  nucleus  becomes  more  and  more  refractory  until  in  the  new  nucleus  it 
is  seen  as  the  new  nuclear  membrane.  The  steps  outlined  here  require  about  28  minutes. 

The  mitotic  events  of  Chaos  difflucns,  as  far  as  they  have  been  observed,  are  practically 
identical  with  those  of  the  above-mentioned  species,  except  for  size. 

Correlated  histories  of  individual  sense  organs  and  their  nerves,  as  seen  in  living 
frog  tadpoles.     CARL  CASKEY  SPEIDEL. 

In  the  living  frog  tadpole  it  is  possible  to  make  daily  observations  on  the  same  individual 
nerves  and  sense  organs  of  the  lateral-line  for  many  weeks  or  months.  By  suitable  operations 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  225 

some  sense  organs  may  be  deprived  of  their  nerve  supply  (nerveless  organs),  and  conversely, 
some  aberrant  lateral-line  branches  may  be  induced  to  grow  without  reaching  any  sense  organ 
(organless  nerves). 

Prolonged  observations  of  nerveless  organs  (1  to  21  months)  reveal  the  following:  (1) 
During  the  first  two  months  in  regenerating  or  growing  zones  the  sense  organs  are  largely 
independent  of  their  nerve  supply.  They  grow  and  divide  readily.  (2)  During  later  months, 
however,  regressive  changes  of  atrophy  and  degeneration  take  place.  The  organs  become 
smaller.  Some  degenerate  and  disappear.  Occasionally,  however,  a  nerveless  organ  may  per- 
sist for  more  than  a  year. 

Prolonged  observations  of  organless  nerves  reveal  the  following:  (1)  During  the  first  two 
months  they  are  largely  independent  of  the  sense  organs.  They  grow  and  become  provided 
with  both  neurilemma  and  myelin  sheaths.  (2)  During  later  months,  however,  regressive 
changes  ensue.  The  myelin  sheath  is  not  maintained  on  any  functionless  fiber.  It  becomes 
thinner  and  ultimately  disappears.  The  untnyelinated  fiber  resulting  may  then  itself  degenerate, 
leaving  only  a  collapsed  neurilemma  tube. 

Thus,  the  structural  integrity  of  both  lateral-line  sense  organ  and  nerve  fiber  is  definitely 
correlated  with  the  successful  establishment  of  a  functional  relationship  between  the  two. 

Sense  hairs  and  orange  granules  are  specialized  structures  of  lateral-line  organs.  The  be- 
havior of  both  of  these  under  various  experimental  conditions  indicates  their  relative  inde- 
pendence of  nerve  influence. 

Many  other  histories  involving  nerve  and  sense  organ  relations  in  wound  zones  have  also 
been  recorded.  Illustrative  cine-photomicrographs  have  been  made. 

The  effect  of  prolonged  starvation  on  the  lipids  in  Phascolosoma  gonldii.     CHARLES 
G.  WILBER. 

It  is  known  that  the  muscle  in  vertebrates  serves  as  a  storehouse  of  fats  and  that  during 
starvation  the  fats  in  muscle  decrease  whereas  the  fat  in  various  internal  organs  is  not  changed. 
Whether  this  is  true  for  invertebrates  is  not  known. 

In  order  to  throw  light  on  the  problem,  worms  (Phascolosoma  c/onldii)  were  starved  for 
one  month  and  then  the  whole  worm,  the  muscle,  and  the  perivisceral  fluid  respectively  were 
analyzed  for  phospholipid,  for  cholesterol,  and  for  fatty  acid.  These  results  were  compared 
with  the  results  of  similar  analyses  made  on  control  worms. 

It  was  found  that  in  the  whole  worm  there  was  a  loss  of  all  lipid  constituents.  In  the  peri- 
visceral  fluid,  phospholipid  and  fatty  acid  decreased  greatly,  but  cholesterol  did  not  decrease. 
In  the  muscle  there  was  an  apparent  increase  in  lipid  material  which  can  be  explained  on  the 
basis  of  absorption  of  some  of  the  tissue.  In  muscle  the  fatty  acid  is  decreased,  as  is  clear  from 
the  larger  lipocytic  coefficient  of  the  muscle  of  starved  worms. 

It  is  concluded  that  the  perivisceral  fluid  serves  as  a  storehouse  of  lipid  in  Phascolosoma 
and  that  the  muscle  does  not.  Moreover,  it  seems  that  phospholipid  and  fatty  acid  are  used 
during  starvation.  Whether  cholesterol  is  also  used  is  not  certain.  Phascolosoma  differs, 
therefore,  from  the  vertebrates  in  the  use  of  phospholipid  during  starvation  and  in  the  fact  that 
muscle  is  not  the  important  storehouse  of  fat. 

Protoplasmic  clotting  in  isolated  muscle  fibers.     ARTHUR  A.  WOODWARD. 

Isolated  muscle  fibers  provide  a  material  favorably  adapted  to  the  quantitative  study  of 
protoplasmic  clotting.  The  cut  ends  form  clots  which  pass  in  waves  over  the  length  of  the  fiber. 
The  rate  of  the  clotting  reaction  can  be  measured  and  is  expressed  as  mm.  of  fiber  converted  into 
clot  per  minute.  Single  fibers  are  teased  from  the  adductor  magnus  of  Rana  pipicus;  all  solutions 
used  are  kept  at  pH  7.1 — 7.4  with  glycine  buffer. 

In  Ringer's  solution,  used  as  a  standard  for  comparison,  the  rate  of  clot  formation  is  constant 
for  a  given  fiber  and  varies  only  moderately  from  fiber  to  fiber  within  a  muscle.  The  normal 
rate  is  about  0.050  mm./min. 

Ca  ion  causes  a  very  rapid  clot  formation ;  in  this  case  it  is  shown  that  the  rate  is  largely 
a  function  of  the  speed  with  which  Ca  diffuses  into  the  end  of  the  fiber,  the  protoplasm  clotting 
with  great  rapidity  once  it  is  exposed  to  free  Ca  ion. 


226  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The  clotting  process  is  relatively  insensitive  to  pH  changes  in  the  region  from  pH  5  to  pH  9 ; 
above  and  below  this  the  rate  increases  very  rapidly.  Near  the  regions  in  which  thrombin  is 
inactivated  liquefaction  has  been  observed  under  certain  conditions. 

Solutions  of  crystalline  trypsin  cause  clot  formation  at  a  rate  averaging  about  50  times  that 
of  the  control.  In  the  absence  of  Ca,  trypsin  produces  only  a  slight  increase  over  the  control. 
Crystalline  chymotrypsin  is  much  less  active  than  trypsin  and  also  has  very  little  effect  in  the 
absence  of  Ca.  Preparations  of  crude  papain  cause  clot  formation  at  a  moderately  high  rate ; 
addition  of  glutathione  increases  the  effect  to  the  magnitude  of  that  produced  by  trypsin.  Ab- 
sence of  Ca  has  no  effect  on  the  action  of  papain. 

AUGUST  24 

Some  aspects  of  the  histology  and  physiology  of  luminescence  in  "railroad  worms." 
JOHN  B.  BUCK. 

In  the  Uruguayan  "railroad  worm,"  Phrixothrix,  the  lateral  photogenic  organs  are  small 
compact  ovoid  masses  of  small  dense  cells  near  the  posterior  edges  of  the  segments  somewhat 
above  the  spiracular  level.  The  organ  is  apparently  supplied  by  one  trachea  ramifying  profusely 
between  the  cells.  There  are  no  end-cells.  Large  oenocyte-like  cells  are  present  near  some  of 
the  lateral  photogenic  organs  and  elsewhere. 

In  Phengodcs,  a  close  American  relative  of  Phrixothrix,  the  lateral  organs  are  in  the  pos- 
terior ends  of  horizontal  rolls  of  tissue  which  extend  along  the  segments  ventral  to  the  spiracles. 
Light  is  also  emitted  along  the  dorsal  posterior  edges  of  most  of  the  segments.  Both  lateral 
and  dorsal  organs  apparently  consist  of  loose  aggregations  of  very  large  oenocyte-like  cells  with- 
out end-cells  or  special  tracheal  supply.  Similar  cells  are  present  in  small  numbers  in  parts  of 
the  body  not  regarded  as  luminous  but  not  in  the  lateral  tissue  roll  except  in  the  region  which 
emits  light.  Further  evidence  is  furnished  by  the  observation  that  the  light  of  Phcnyodcs  can  be 
seen  microscopically  to  come  from  clusters  of  round  or  oval  spots  corresponding  in  shape,  posi- 
tion, number,  and  size  to  the  oencyte-like  cells. 

The  photogenic  organ  of  Phyri.vothrix  is  very  similar  to  that  in  the  larval  firefly  and  agrees 
with  the  generalization  that  luminous  beetles  which  produce  a  lingering  glow  rather  than  a  short 
flash,  have  organs  of  relatively  simple  structure  without  end-cells. 

The  photogenic  organs  of  Phcngodes  are  the  simplest  yet  known  in  insects  and  represent 
the  first  time,  that  bioluminescence  has  been  ascribed  to  eonocytes.  A  corresponding  physiological 
simplicity  may  be  the  fact  that  the  light  is  continuous. 

Phengodcs  dims  in  the  vapor  of  KT2  and  1CT3  M.  HCN  at  about  the  same  rate  as  luminous 
bacteria,  and  faster  than  fireflies. 

Effect  of  caffeine  concentration  upon  retardation  of  Arbacia  development.     RALPH 
HOLT  CHENEY. 

Sea  urchin  eggs  and  sperm  were  subjected  to  eight  different  concentrations  of  caffeine-in- 
sea-water  for  15  minutes,  then  mixed  for  fertilization  and  the  developmental  rates  in  S.W.  and 
S.W.C.  were  compared  with  the  normal  rate  of  untreated  ova  and  sperm.  Observations  were 
made  at  intervals  during  a  three-day 'period.  Normal  time  rates  were  accepted  as  stated  by  E.B. 
Harvey,  1940  (Biol.  Bull,  79,  (1)  "Plate  II,  photographs  16-32  inclusive). 

All  eggs  utilized  in  a  single  experiment  were  obtained  from  the  same  female  and  all  sperm 
from  one  male.  Eggs  and  sperm  were  shed  directly  into  S.W.  or  S.W.C.  prior  to  mixing.  The 
series  of  six  combinations  presented  in  1942  (Biol.  Bull.,  83)  were  repeated  and  extended  to  a 
full  three  day  period  as  follows :— N?  X  Nrf,'  N?  X  Crf,  C?  X  Nrf,  C?  X  Cd,  all  developed  in 
normal  sea  water  after  the  original  immersion  of  fifteen  minutes  after  shedding  as  indicated 
into  S.W.  or  S.W.C.  In  the  cases  of  C?  X  NJ1  and  C$  X  Cd1,  each  was  developed  also  in  S.W.C. 

Results  indicated  that  the  period  of  immersion  (15  min.)  in  the  caffeine  concentrations  em- 
ployed prior  to  mixing  the  gametes  did  not  render  the  ova  non-fertilizable  subsequently  nor 
destroy  the  ability  of  the  sperm  to  fertilize.  Eggs  and/or  sperm,  however,  were  not  unaffected 
at  least  by  higher  concentrations,  since  C?  X  Cd  cultures,  although  they  did  form  the  fertilization 
membrane  when  mixed  and  developed  in  uncaffeinized  S.W.,  the  fertilized  ova  never  survived 
longer  than  the  early  cleavage  stages. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  227 

Plutei  developed  in  normal  time  and  form  in  all  of  the  six  combinations  of  0.002  per  cent  and 
0.004  per  cent  S.W.C.  Gametes  shed  into  two  per  centum  S.W.C.,  in  each  of  the  four  combina- 
tions developed  in  S.W.  formed  the  P.M.  but  showed  retarded  development  and  in  no  case 
reached  the  pluteus  stage  before  death.  In  the  two  combinations  developed  in  S.W.C.,  the  P.M. 
was  not  formed.  Intermediate  percentages  used  between  these  extremes  of  concentration  gave 
intermediate  effects  indicating  that  in  general,  the  effects  were  directly  proportional  to  the  con- 
centration of  the  caffeine. 

Other  experimental  series  subjected  normally  fertilized  ova  (N?  X  N^1  in  S.W.)  which  had 
developed  to  a  desired  stage  of  development,  to  the  different  concentrations  of  caffeine-in-S.W. 
Results  here  indicated  similarly  that  the  retardation  effect  upon  the  development  time  was  pro- 
portional to  the  concentration. 

Shape  changes  in  flic  denuded  Nereis  egg  preceding  first  cleavage.     ALBERTA  T. 
JONES. 

•It  is  known  from  Hoadley  (1934)  that  Nereis  eggs  undergo  a  series  of  amoeboid  changes 
prior  to  first  cleavage.  Neither  the  reason  for  this  phenomenon  nor  the  exact  pattern  followed 
has  been  completely  described.  It  is  the  purpose  of  this  paper  to  report  the  principal  findings  on 
the  pattern  of  shape  changes  up  to  first  cleavage  in  the  egg  of  Nereis  limbata.  The  denuded  egg 
was  used  to  eliminate  the  complications  of  membrane  and  external  jelly. 

Gametes  were  taken  from  animals  caught  the  previous  night  in  Eel  Pond  (Woods  Hole)  and 
artificial  insemination  was  carried  out.  The  fertilized  eggs  were  denuded  by  treatment  with 
alkaline  0.53  molar  NaCl  solution  brought  to  pH  10.5  by  addition  of  NaL,CO3.  This  is  the  method 
used  by  Costello  (1939  and  1945).  Observations  began  when  the  denuded  eggs  were  rinsed  free 
of  alkali  and  the  first  polar  body  had  formed.  Outline  drawings  of  the  eggs  were  made  with  a 
camera  lucida  at  three  minute  intervals.  To  serve  as  reference  points,  the  position  of  polar 
bodies  and  oil  droplets  was  indicated. 

Hoadley  states: — "pulsations  of  the  (Nereis)  egg  are  of  two  sorts,  one  of  which  is  quite 
extensive  and  results  in  general  distortion  of  the  sphere,  and  the  other  of  which  results  in  surface 
irregularities  which  appear  more  or  less  localized."  The  shape  changes  discernible  in  denuded 
eggs  seem  to  correspond  to  Hoadley's  first  category.  A  consistent  pattern  of  sequences,  dif- 
ferent from  those  described  by  Hoadley  for  the  intact  egg,  has  been  found.  The  sequences  in- 
clude such  general  distortions  as :  ( 1 )  polar  flattening  followed  by  rerounding ;  (2 )  elongation 
in  the  polar  axis  followed  by  rerounding;  and,  (3)  elongation  in  the  equatorial  axis  followed  by 
formation  of  the  first  cleavage  plane.  The  magnitude  of  these  changes,  in  comparison  with  the 
pulsations  observed  by  Hoadley,  may  be  attributed  to  the  absence  of  jelly  mass  and  membrane. 

It  may  be  concluded  that  shape  changes  in  the  denuded  Nereis  egg  prior  to  first  cleavage 
proceed  (1)  according  to  a  definite  pattern;  and  (2)  always  with  a  particular  relation  to  the 
polar  axis  of  the  egg. 

'Hormone  control  of  dchydrogenasc  activity  of  Crustacean  tissues.     ELOISE  KUNTZ. 

Sea  water  extracts  of  the  sinus  glands  of  Libinia  cmarginata,  Honiants  americanus,  Uca 
pugilator  and  U.  pugnax  and  similarly  prepared  extracts  of  the  central  nervous  system  of  Libinia, 
Homarus  and  the  arachnoid,  Limulus  polyphcmus,  were  made.  These  were  boiled  and  centri- 
fuged  and  the  supernatant  fluid  was  used.  The  extracts  were  tested  for  their  effect  upon  de- 
hydrogenase  activity  of  gastric  gland  and  muscle  of  Libinia,  Honiants  and  Limulus,  which  were 
measured  in  Thunberg  tubes  with  methylene  blue  as  the  hydrogen  acceptor. 

The  effect  of  sinus  gland  extract  was  dependent  upon  the  concentration.  Half  of  a 
Libinia  sinus  gland  doubled  the  rate  of  methylene  blue  reduction,  but  the  reduction  rate  rapidly 
fell  to  slightly  above  that  of  the  controls  with  increasing  concentrations.  Half  of  a  Uca  pugilator 
sinus  gland  also  doubled  the  reduction  rate,  but  activity  remained  high  for  concentrations  of  I 
sinus  glands,  falling  to  the  level  of  the  controls  at  6  sinus  glands.  Here  it  remained.  Uca 
pugnax  showed  strong  inhibitory  action  in  concentrations  of  6  to  12  sinus  glands.  The  character 
of  the  curves  suggests  the  possibility  of  two  active  substances  which  vary  in  relative  proportions 
in  different  species. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Central  nervous  system  extracts  of  Homarus,  Libinia  and  Limuhts  in  concentrations  of  0.7 
mg.  tissue  per  cc.  strongly  stimulated  dehydrogenase  activity.  Extracts  of  other  tissues  were 
ineffective  at  several  times  this  concentration. 

Localization  of  hormone  production  within  the  nervous  system  was  demonstrated.  In 
Homarus  the  circumoesophageal  ganglia  and  second  ventral  ganglion  were  most  effective.  The 
brain  and  suboesophageal  ganglion  were  ineffective.  The  remainder  of  the  ventral  cord  had  a 
relatively  weak  action.  All  parts  of  the  circumoesophageal  ring  of  Limulus  were  effective. 

An  antagonistic  action  of  sinus  gland  and  nervous  system  extract  was  demonstrated.  The  ad- 
dition of  sinus  gland  hormone  to  a  system  stimulated  by  central  nervous  system  extracts  de- 
pressed dehydrogenase  activity  to  that  of  the  controls. 

A  comparative  study  of  cholinest erase  activity  in  normal  and  gcneticallv  deficient 
strains  of  Drosop/iila  melanogaster.     DR.  F.  POULSON  AND  E.  J.  ROELL. 

Cholinesterase  activity  has  been  determined  in  late  embryos  of  several  strains  of  Drosophila 
melanogaster  by  means  of  the  cartesian  diver  technique  which  measures  the  evolution  of  CO^ 
from  Ringer-bicarbonate  solution  following  hydrolysis  of  acetylcholine  in  the  presence  of  an 
atmosphere  of  95  per  cent  N2  and  5  per  cent  CO2.  Timed  eggs  from  a  stock  of  the  deficiency 
known  as  Notch8  crossed  to  Canton-S  wild  strain  (Ns/+)  were  dechorionated  by  Slifer's  hypo- 
chlorite  method  and  classified  as  normal  or  Notch-deficient.  At  24  hours  normals  are  larvae 
ready  to  hatch,  while  the  deficient  male  embryos  are  strikingly  abnormal  and  possess  a  nervous 
system  about  three  times  normal  size.  The  ratio  of  types  is  3  normal :  1  abnormal.  Embryos 
were  cut  up  and  placed  in  divers  containing  1  mm.:f  Ringer-bicarbonate  and  0.5  mm.3  of  1.5  per 
cent  acetylcholine.  Although  it  is  possible  to  carry  out  measurements  on  single  embryos,  two 
to  five  embryos  per  diver  were  used  in  most  experiments.  Readings  were  made  at  ten  minute 
intervals  for  one  hour  after  the  divers  had  reached  thermal  equilibrium. 

A  series  of  four  determinations  on  24-hour  normals  gave  an  average  of  12.8  m  ju.l.  CO2/em- 
bryo/hour.  A  series  of  five  determinations  on  Notch  8  deficient  male  embryos  gave  an  average  of 
34.0  m./z.l.  COo/embryo/hour.  The  cholinesterase  activity  of  Notch  embryos  is  2.7  times  that  of 
normal,  which  is  nearly  the  same  as  the  volume  ratio  of  Notch/normal  nervous  systems,  3.3  as 
determined  by  planimeter  from  camera  lucida  outlines  of  sections.  Thus  cholinesterase  activity 
is  proportional  to  volume  of  nervous  tissue.  Notch-deficient  embryos  of  other  strains  have  given 
similar  results.  Thus  the  Notch  male  nervous  system  while  abnormal  in  size  and  morphology 
is  biochemically  normal  with  respect  to  cholinesterase.  A  first  step  has  been  made  in  studying 
the  rate  of  increase  with  development  of  cholinesterase  activity  in  both  normal  and  deficient  em- 
bryos. At  18.5  hours  the  activity  of  the  Notch  embryo  is  8.3  m.ju.l.  CO2/hour,  that  of  normal  3.8 
m.ju.l.  COo/hour.  In  the  unhatched  Notch  embryo  at  48  hours  the  activity  increases  to  51.0 
m.fji.l.  COn/hour. 

As  checks,  cholinesterase  activity  of  unfertilized  eggs  and  methyl  butyrase  activity  of  normal 
and  Notch  embryos  were  measured  and  found  to  be  negligible. 

To  determine  the  location  of  cholinesterase  in  normal  embryos,  central  nervous  systems  were 
dissected  out  and  their  cholinesterase  activity  measured  separately  from  the  remaining  portion  of 
the  embryos.  One  determination  has  given  a  value  of  17.0  m.  .l./N.S./hour.  The  value  for  the 
remnant  is  2.6  m./i.l./hour.  Since  the  central  nervous  system  makes  up  not  more  than  one- 
sixth  the  embryonic  volume  the  cholinesterase  activity  there  is  roughly  forty  times  that  in  the 
remnant. 

Possible  metabolic  and  pliysical  chemical  factors  in  the  production  of  the  injury 
potential  in  spider  crab  nerve*     A.  M.  SHANES. 

In  contrast  to  frog  sciatic  nerve,  spider  crab  nerve  is  permeable  to  both  potassium  and 
chloride  ions  and  to  a  lesser  extent  to  sodium.  The  relationship  between  metabolism  and  the 
potentials  therefore  cannot  be  the  same  as  in  frog  nerve  which  is  highly  impermeable  to  chlo- 
ride and  other  small  anions  as  well  as  to  sodium.  This  is  confirmed  by  the  following  observa- 
tions:  (1)  Although  0.002  to  0.0006  M  iodoacetate  (IAA)  produces  a  continuous  slow  fall  in 

*  Aided  by  grants  from  the  Penrose  Fund  of  the  American  Philosophical  Society  and  from 
the  American  Academy  of  Arts  and  Sciences. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  229 

potential  in  oxygen,  0.02  M  pyruvate  is  unable  to  counteract  this  inhibition;  (2)  pyruvate 
accentuates  IAA  inhibition  of  the  post-anoxic  recovery  of  potential ;  (3)  the  decline  of  potential 
in  nitrogen  is  more  rapid  than  in  frog  nerve  and  is  not  hastened  by  IAA ;  (4)  glucose  does  not 
retard  the  fall  in  potential  during  anoxia  and  inhibits  a  recovery  in  oxygen.  The  inhibitory 
effects  of  pyruvate  and  glucose  may  be  the  result  of  acid  production,  for  5  per  cent  CO2  lowers 
the  potential  in  this  system  in  contrast  to  its  effect  in  frog  nerve. 

These  results  provide  a  basis  for  understanding  some  peculiarities  of  carbohydrate  metabo- 
lism in  spider  crabs.  For  example,  blood  sugar  levels  average  only  1  mg.  per  cent,  nerve  gly- 
cogen  ranges  from  500  to  2,000  mg.  per  cent  wet  weight  (Kleinholtz,  unpublished),  and  the 
breakdown  of  glycogen  is  reported  to  be  largely  to  simple  sugars  as  well  as  to  lactic  acid. 

The  potassium  content  of  these  fibers  is  known  to  be  high  and  the  potential  is  inversely 
related  to  the  extracellular  potassium  concentration.  Consequently  the  injury  potential  is 
probably  a  potassium  concentration  potential  as  in  frog  nerve.  In  yeast  (Rothstein  and  Haege, 
1943)  potassium  retention  is  stoichiometrically  related  to  hydrogen  ions  lost  to  the  medium  when 
glucose  is  assimilated  to  form  reserve  carbohydrate.  This  mechanism  may  explain  the  high 
levels  of  both  glycogen  and  potassium,  and  hence  the  metabolism-potential  relationship,  in  spider 
crab  nerve. 

Some  effects  of  tannic  acid  on  osmotic  hemolysis.     T.  H.  WILSON  AND  M.  H. 
JACOBS. 

Human  erythrocytes  are  less  easily  hemolyzed  in  hypotonic  solutions  of  NaCl  in  the  pres- 
ence of  tannic  acid  than  in  its  absence.  The  salt  solution  employed  in  the  present  experiments 
ranged  from  0.091  to  0.069  M  and  those  of  tannic  acid  from  1/800  to  1/51,200  per  cent.  Even 
at  the  lowest  of  these  concentrations  of  tannic  acid  there  was  a  marked  protective  effect.  In 
hypotonic  solutions  of  Na2SO4  ranging  from  0.045  to  0.031  M  and  with  the  same  concentrations 
of  tannic  acid  as  before,  the  effect  was  the  exact  opposite,  hemolysis  invariably  being  increased 
except  at  the  lowest  concentrations  of  tannic  acid.  Very  similar  effects,  somewhat  complicated 
by  the  permeability  of  the  erythrocyte  to  ammonium  salts,  were  obtained  with  NH4C1  and 
(N,H4)2SO4,  respectively. 

In  the  light  of  results  obtained  with  molecular  films  of  proteins  by  Schulman  and  others, 
the  action  of  the  tannic  acid  in  the  chloride  solutions  might  be  explained  either  by  a  strengthen- 
ing of  the  cell  surface  or  by  a  decrease  in  its  permeability  to  hemoglobin.  Such  an  action  in  the 
case  of  the  sulfate  solutions  is  not  necessarily  excluded,  but  it  seems  to  be  overshadowed  by  an- 
other effect  of  a  different  nature,  namely,  the  decreased  permeability  to  anions  produced  by 
tannic  acid,  described  elsewhere  by  Jacobs,  Stewart  and  Butler.  Since  swelling  of  the  erythro- 
cyte is  known  to  be  opposed  by  the  exchange  of  bivalent  sulfate  ions  from  the  outside  for  uni- 
valent  anions  from  the  inside,  tannic  acid,  by  hindering  this  exchange,  might  in  this  particular 
case  indirectly  favor  hemolysis,  despite  its  more  direct  protective  effect  on  the  cell  surface. 

The  effect  of  roentgen  radiation  on  protoplasmic  viscosity  changes  during  mitosis. 
WALTER  L.  WILSON. 

Roentgen  radiation  has  a  marked  effect  on  the  protoplasmic  viscosity  of  the  dividing  sea- 
urchin  egg.  If  the  eggs,  or  sperm,  or  both  the  eggs  and  sperm  of  Arbacia  punctulata  are  ir- 
radiated before  fertilization,  then  the  normal  pattern  of  viscosity  change  is  altered.  In  the 
control  the  viscosity  was  low  shortly  after  fertilization,  then  increased  to  a  peak  at  15  minutes 
(23°  C.).  It  remained  high  for  6-10  minutes  and  then  decreased.  This  decrease  was  markedly 
retarded  by  irradiation  of  the  sperm  or  eggs  (11,300  r  at  6400  r/m),  or  both  the  sperm  and 
eggs  (5,000  r  at  6400  r/m)  before  fertilization.  In  these  experiments  the  viscosity  remained 
high  two  or  three  times  longer  than  in  the  controls.  In  three  experiments  out  of  ten  in  which 
irradiated  eggs  were  fertilized  with  normal  sperm,  the  viscosity  increased  to  a  value  almost 
twice  that  of  the  controls. 

Biological  specificity  and  the  synthesis,  oj  native  proteins.     D.  WRINCH.     No  ab- 
stract submitted. 


230  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

PAPERS  READ  BY  TITLE 

The  effects  of  massive  doses  of  ergonovine  male  ate  *  upon  the  smaller  blood 
vessels  as  seen  directly  with  the  microscope  in  the  living  mammal.1  RICHARD 
G.  ABELL. 

In  these  experiments,  as  in  those  described  above,  the  blood  vessels  were  studied  in  trans- 
parent 'moat'  chambers  in  rabbits'  ears.  All  injections  were  of  3.0  mgm.,  and  all  were  made 
intravenously.  Three  mgm.  in  a  rabbit  corresponds  to  60  mgm.  in  a  man,  which  is  600  times 
the  clinical  intravenous  dose.  In  these  experiments  the  injection  of  3.0  mgm.  caused  complete 
arteriolar  constriction  for  3  to  4  minutes.  The  larger  arterioles  (80  to  90  microns  in  diameter) 
remained  narrowed  to  approximately  one-half  of  their  control  diameters  for  3  to  4  hours.  A 
temporary  constriction  of  the  venules  to  from  0.5  to  0.8  of  their  control  diameters  occurred. 
In  addition,  this  amount  of  ergonovine  caused  thicking  of  leukocytes  to  the  walls  of  the  arte- 
rioles, capillaries  and  venules.  The  degree  of  sticking  varied  widely  in  different  rabbits  ;  in 
some  cases  it  was  slight ;  in  others  large  numbers  of  leukocytes  stuck  to  the  walls  of  the  capil- 
laries and  venules,  and  emigrated  into  the  surrounding  tissue.  In  one  rabbit  injections  of  3.0 
mgm.  were  followed  by  the  formation  of  leukocytic  emboli,  which  blocked  many  of  the  capil- 
laries and  venules  and  formed  thrombi.  The  reaction  was  reversible  and  the  thrombi  usually 
disappeared  within  approximately  4  hours  following  the  injections.  This  is  in  accord  with  the 
flow  toxicity  of  ergonovine,  and  its  failure  to  produce  gangrene  on  repeated  injections. 

As  shown  by  the  work  of  numerous  investigators,  two  other  ergot  alkaloids,  ergotoxine  and 
ergotamine,  do  produce  gangrene  on  .repeated  injection.  Such  gangrene  also  occurs  in  ergot 
poisoning  and  is  due  to  obliterative  endarteritis  and  thrombosis.  The  formation  of  these 
thrombi  is  usually  attributed  to  prolonged  constriction  of  the  small  arteries,  and  interruption  of 
the  blood  flow,  but  this  is  entirely  hypothetical. 

In  the  present  experiments  thrombi  were  formed  due  to  the  increase  in  stickiness  of  the 
endothelium  toward  leukocytes  and  of  the  leukocytes  toward  each  other. 

Perhaps  gangrene  produced  by  ergot  and  its  more  toxic  alkaloids  may  be  caused  by  a  more 
severe  and  prolonged  reaction  of  the  type  described  above. 

Secretory  cells  in  the  branchial  epithelium  of  fislics.     GERRIT  BEVELANDER. 

It  was  shown  by  Smith  (1930,  1931,  1932)  that  the  osmotic  regulation  of  the  body  fluids  in 
fresh  and  salt  water  teleosts  and  in  elasmobranchs  is  effected  considerably  by  the  extrarenal 
excretion  of  salt  (NaCl  and  KC1)  under  conditions  that  probably  involve  considerable  osmotic 
work.  It  was  further  inferred  that  this  exchange  occurred  in  the  gills.  A  previous  study  of 
the  branchial  epithelium  in  an  extensive  and  widely  divergent  group  of  fishes  (Bevelander 
(1935),  led  this  writer  to  conclude  that  the  only  specialization  occurring  in  the  branchial  epi- 
thelium of  fishes  consists  in  a  thicker  epithelium  in  the  elasmobranchs  than  in  teleosts  and  the 
presence  of  numerous  mucous  cells  in  all  species  examined.  The  cells  which  we  described  as 
mucous,  were  alleged  to  be  "chloride  secreting"  cells  in  Anguilla  and  in  some  fresh  water  tele- 
osts, but  not  in  elasmobranchs  by  Keyes  and  Willmer  (1932). 

A  re-examination  of  this  problem  included  the  experimental  stimulation  of  secretion  of  the 
cells  in  dispute  in  representative  teleosts  and  elasmobranchs.  These  cells  were  then  subjected 
to  a  number  of  histochemical  tests  and  were  shown  to  be  positive  for  mucin.  Further,  the  oral 
and  opercular  membranes  were  also  examined  and  it  appears  unlikely  on  the  basis  of  structure 
that  they  are  concerned  with  osmotic  regulation. 

In  order  to  comply  with  the  observed  physiological  data,  the  cells  which  are  responsible 
for  extrarenal  excretion  must  be  in  intimate  relation  with  the  blood  supply  and  the  external 
milieu,  they  must  be  very  extensive  to  account  for  the  considerable  work  performed,  and  finally 
they  must  be  present  in  teleosts  and  elasmobranchs.  Our  observations  reaffirm  the  absence  of 
any  specialized  structures ;  the  only  cells  which  comply  with  the  three  criteria  required  are  the 

* 'Ergotrate'  (Ergonovine  Maleate,  U.S. P.,  Lilly). 

1  This  work  was  aided  by  a  grant  made  by  Eli  Lilly  and  Company  to  the  Department  of 
Anatomy  of  the  University  of  Pennsylvania  Medical  School. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  231 

respiratory  epithelial  themselves.  It  is  further  suggested  that  the  observed  conservation  of 
urea  in  the  elasmobranch  gills  may  be  affected  by  the  relatively  thick  respiratory  epithelium 
which  covers  the  gill  filaments. 

A  modified  Crompton  formula  for  the  latent  heat  of  vaporisation.     ALBERT  P. 
MATHEWS. 

The  general  formula  for  the  latent  heat,  L,  which  I  have  found  is 

(1)  L  =  CR'Thie(d/D) 

R'  is  the  actual  value  of  the  gas  constant  in  the  liquid  phase,  constantly  falling  as  molecular  co- 
aggregation  increases  with  falling  temperature,  d  is  the  liquid  density  and  D  that  of  the  vapor, 
C  is  a  constant  peculiar  to  the  substance,  often  not  far  from  2. 

The  values  of  C  and  R'  are  obtained  from  the  following  general  formulas  which  apply  to  all 
non-associating  substances  with  the  possible  exceptions  of  hydrogen  and  helium. 

(2)  C  =C(R/R'e)(L/(L-E)-)a 

) 

(3)  C  -  1.1292-  (3/8)5-+  (9/64  )  S2  =  0.8792  +  (  (3/8)5  -  0.5)2 

5  in  (3)  is  the  critical  coefficient:  RTc/pcVc  in  which  R  has  its  ideal  value.  5  may  be  com- 
puted by  (4)  : 


(4)        S  =  [(dmRTc/Mpc)  +  16((7c-D/ro)  -12((TC-T)/TCY] 

/[I  +  5.158((7\,  -  T)/Te)  -  3.158  ((r.  -  T)/Tcy] 


dm  is  the  mean  density  of  saturated  vapor  and  liquid  at  temperature,  T  ;  pc  and  Tc  the  critical 
pressure  and  temperature. 


(5)  (L/(L-£))«= 

L-E  is  the  internal  latent  heat  of  vaporization.     (5)  is  obtained  from  (6). 

(6)  ((T/p)(dp/dT))e  =  (L/£)c=l  +  (27S2tf'c)/64  R") 

(7)  R'c/R  =  (512  -  64S  +  216S2  -  27S3)/512.S 
R'T  in  (1)  is  obtained  from  (8). 

(8)  (L/(L-E)  )r  =  R'T/R'.  =  1  +  (  (L/(L-E)  )„  -  1)  (  (9/16)  (T/TC)  +  7/16)  (7YTC)2 

(9)  R',  =  R 


At  absolute  zero  L/  (L-E)  is  1  and  it  advances  with  temperature  as  co-aggregation  diminishes. 
In  the  ideal  state  C,  5  and  (L/£)c  are  1.  When  5  has  its  highest  value  of  4  in  a  normal  sub- 
stance (L/E)C  will  be  7.3346  and,  when  5"  has  its  lowest  value  of  3/8,  (L/E)C  will  be  4.  5  may 
be  calculated  also  from  the  latent  heat  of  vaporization  at  any  temperature,  C'  being  equal  to 
(L-E)/RTln.(d/D)  by  (10): 

(10)  5=  (8/3)  (0.5  +  V  (C-  0.8792)) 

Obtained  from  (3)  above;  E  being  taken,  with  small  error  usually,  as  equal  to  p(V-v). 

Formula  (1)  above  is  an  easier  and  more  accurate  way  of  computing  the  latent  heat  of 
vaporization  than  by  the  thermodynamic  equation:  L—  (Tdp/dT)  (V-v).  The  results  by  (1) 
agree  usually  within  1  per  cent  with  the  experimental  determinations  at  the  normal  boiling  point 
as  made  by  J.  H.  Mathews  and  others.  The  derivation  of  all  the  foregoing  formulas  will  be 
given  in  the  full  papers  together  with  examples  of  application  to  specific  cases  and  also  the  general 
formula  for  the  Cailletet  and  Mathias  law  (11)  : 

(11)  rfm  =  rf.[l  +  (5.158-  16/5)  ((T,-T)/Tc)  +  (12/5  -  3.158)  ((7.  - 


232  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

Heat  Death.     PAUL  R.  ORR. 

I.  Time-temperature  relationships  in  marine  animals. 

Temperature  as  an  intrinsic  ecological  factor  which  determines,  to  a  great  extent,  the  abun- 
dance, life  cycle,  and  distribution  of  marine  organisms,  is  a  well-established  fact.  However,  the 
duration  of  exposure  required  to  produce  death  at  each  temperature  in  the  effective  series  has 
not  been  taken  into  consideration.  Thus,  in  order  to  state  accurately  the  conditions  of  heat 
death,  it  is  necessary  to  plot  a  curve  in  which  both  variables,  temperature  and  time,  are 
represented. 

Heat  death  curves  have  been  plotted  for  Uca  pugilator,  Asterias  forbesi,  Ophiodenna 
brevispuium,  Arbacia  punctulata,  Nassa  obsolcta,  Fiindulus  hctcrnclitus. 

All  of  the  curves  have  approximately  the  same  shape.  For  a  relatively  slight  rise  in  tem- 
perature there  is  a  marked  drop  in  the  length  of  exposure  necessary  to  cause  death.  This  rela- 
tionship is  not  one  of  direct  proportionality. 

II.  Differential  response  of  the  entire  animal   (Rana  pificns)   and  several  of  its  organ  systems. 

Whether  we  are  dealing  with  cells  or  multicellular  organs  and  tissues,  or  the  organism  as 
a  whole,  we  are  confronted  with  the  fact  that  not  all  of  the  cells,  organs,  etc.,  have  the  same 
sensitivity  to  heat.  An  animal  exposed  to  excessive  heat  for  a  length  of  time  to  cause  complete 
loss  of  excitability  might  well  be  pronounced  dead,  for  it  never  again  will  show  any  signs  of 
life  as  a  complete  organism.  Yet  there  are  parts  of  the  complex  animal  that  are  "alive." 

The  animal  as  a  whole,  the  tadpole,  sciatic  nerve,  sartorius  and  gastrocnemius  muscles,  and 
heart  were  separately  studied,  and  curves  were  plotted  for  the  heat  death  points  of  each.  The 
data  show  that  in  the  adult  animal  the  order  of  death  is:  (1)  the  organism  as  a  whole;  (2)  the 
muscular  system;  (3)  heart,  and  (4)  nervous  tissue. 

All  heat  death  curves  plotted  are  of  the  same  shape,  showing  a  sudden  drop  followed  by  a 
gradual  approach  to  a  constant  level. 

III.  The  effect  of  high  temperatures  on  heart  rate  in  Venus  mercenaria. 

In  the  clam  heart  (Venus  mercenaria}  we  have  an  automatic  mechanism  by  which  the  effect 
of  heat  can  be  studied.  By  subjecting  excised  hearts  to  a  series  of  high  temperatures  and  noting 
the  heart  rate  it  was  possible  to  determine  the  lethal  point  for  each  temperature  and  thus  plot 
a  curv.e  showing  time/temperature  relationship. 

For  the  clam  heart  the  same  general  type  of  curve  was  found  as  shown  in  previous  studies 
on  marine  animals  and  frogs.  That  is,  there  is  a  point  at  which  the  hearts  will  beat  for  a  rela- 
tively long  period  of  time;  then  as  they  are  subjected  to  higher  temperatures  there  is  a  rapid 
decrease  in  heart  rate,  followed  by  a  leveling  off  to  a  constant  rate. 

Penetration  glands  in  tapeworm  Onchosphcres.     \Y.  MALCOLM  REID. 

Although  various  types  of  cystogenous  and  penetration  glands  have  long  been  figured  and 
studied  as  a  part  of  the  internal  structure  of  trematode  cercariae  and  miracidia,  they  have  not 
been  recognized  in  the  onchosphere  stage  of  cestodes.  A  pair  of  such  glands  has  been  found  in 
the  fowl  cestodes  Raillietina  cesticillus  (Molin),  and  Choanotaenia  infundibulum  (Bloch)  and 
in  a  herring  gull  cestode  Hymcnolepis  sp.  Although  these  glands  may  be  seen  under  favorable 
conditions  without  special  stains,  they  respond  in  the  same  manner  to  vital  stains  as  do  trema- 
tode glands,  showing  up  best  with  Nile  blue  sulfate  and  neutral  red.  The  gland  stretches  to  the 
posterior  end  of  the  larva,  where  it  appears  to  be  anchored.  The  secretion  pores  are  located 
near  the  anterior  and  slightly  to  the  side  and  above  the  middle  pair  of  hooks  when  these  hooks 
are  oriented  with  the  points  directed  anteriorly  and  downward.  The  granular  contents  may  be 
seen  to  move  about  as  the  general  contour  of  the  glands  is  changed  by  the  violent  contractions 
associated  with  hook  movements  and  at  times  some  of  the  secretion  may  be  seen  exuding  from 
the  pores.  A  single  nucleus  is  located  near  the  middle  of  each  gland,  and  the  two  glands  are 
connected  by  a  narrow  isthmus  near  the  posterior  end. 

The  nature  of  the  secretion  has  not  been  determined  but  it  is  possible  that  it  assists  the 
larva  in  penetration  since  this  granular  substance  is  given  off  at  a  time  in  the  life  cycle  when 
the  six-hooked  embryo  must  break  out  of  the  covering  membranes  of  the  egg  and  penetrate  the 
gut  of  an  arthropod  intermediate  host. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  233 

Intensity-duration  relation  in  stimulation  by  light.     F.  J.  M.  SICHEL  AND  P.  B. 
ARMSTRONG. 

The  excised  sphincter  pupillae  of  many  vertebrates  will  respond  by  constriction  to  stimula- 
tion by  visible  light  of  suitable  wave-lengths.  In  these  experiments  the  sphincter  pupilla  of  the 
eel,  Anguilla  rostrata,  was  used.  The  sphincter  was  excised  from  small  adults,  15  to  18  inches 
in  length. 

The  sphincter  was  pinned  out,  anterior  surface  uppermost,  on  white  beeswax.  It  was 
illuminated  for  observation  by  transmitted  red  light,  to  which  the  preparation  is  insensitive. 
The  source  of  light  for  stimulation  was  a  tungsten  filament  lamp  maintained  at  constant  voltage. 
This  was  focussed  on  the  preparation  obliquely  from  above.  The  intensity  of  the  stimulating 
light  was  varied  by  Wratten  neutral  niters  and  a  neutral  wedge.  The  duration  of  the  stimu- 
lating flash  was  controlled  by  a  shutter  manually  operated  and  timed  by  a  stop  watch.  The 
criterion  of  threshold  was  the  smallest  contraction  visible  through  a  low-power  microscope. 
An  eyepiece  filar  micrometer  was  used  to  advantage  in  determining  the  threshold  stimulus. 
The  preparation  was  bathed  in  a  Ringert's  fluid  and  permitted  to  become  dark-adapted  before 
each  experiment. 

The  threshold  was  found  to  be  a  function  of  the  duration  and  of  the  intensity  of  the  stimu- 
lating flash.  The  intensity-duration  relation  conforms  with  Hill's  theory  of  excitation  for  rec- 
tangular stimuli.  The  chronaxies  averaged  about  12  seconds,  the  range  being  from  about  6 
seconds  to  20  seconds.  In  terms  of  the  reciprocity  law  this  would  mean  that  the  law  holds 
reasonably  well  for  flashes  shorter  than,  say,  10  seconds.  At  longer  durations  the  deviation  is, 
in  direction  and  amount,  what  would  be  expected  on  the  basis  of  Hill's  equation  for  excitation. 
There  is  a  definite  rheobase,  or  minimal  intensity  of  the  stimulating  flash  below  which  excitation 
is  never  produced,  even  for  very  long  exposure  times. 

The  pattern  of  flic  intrinsic  palmar  musculature.     WILLIAM  L.  STRAUS,  JR. 

The  intrinsic  palmar  musculature  of  tetrapod  vertebrates  comprises  two  fundamental  series : 
(1)  a  superficial,  arising  from  fascia  or  tendon,  and  showing  variable  tendency  toward  strati- 
fication, and  (2)  a  deep,  arising  from  bone  and  always  arranged  in  two  layers  separated  by  the 
deep  palmar  nerves  and  vessels.  Between  the  two  series  lies  the  mid-palmar  space. 

In  urodeles  (Nectunts  inaculosus,  Cryptobranchus  alleghaniensis),  the  superficial  series  is 
a  single  layer  (flexores  breves  superficiales)  arising  from  the  dor  sum  of  the  long  flexor  tendon. 
The  deep  series  is  composed  of  a  superficial  (contrahentes  or  adductores)  and  a  deep  (flexores 
breves  profundi,  intermetacarpales,  interphalangeus  III?;  in  Cryptobranchus  also  flexores  breves 
minimi)  layer. 

In  lizards  (Sceloporus  spinosus,  Ctenosaura  siinilis},  the  superficial  series  tends  to  form 
two  layers — a  superficial  (flexores  breves  superficiales,  marginal  abductors),  arising  from  the 
transverse  carpal  ligament,  and  a  deep  (lumbricales),  arising  from  the  long  flexor  tendon;  in 
Sccloporus,  however,  such  lamination  is  incomplete,  for  fibers  of  the  superficial  layer  also  arise 
from  the  long  flexor  tendon.  The  deep  series  again  exhibits  superficial  (contrahentes)  and 
deep  (flexores  breves  profundi)  layers. 

In  mammals  (Didclphis  virginiana,  Macaco  mulatto,  Homo},  the  superficial  series  forms 
two  distinct  layers — a  superficial  (abductor  pollicis  brevis,  flexor  pollicis  brevis,  opponens  pol- 
licis?,  palmaris  brevis,  flexor  V  brevis,  abductor  V;  in  Didclphis  also  a  flexor  brevis  manus), 
largely  from  palmar  aponeurosis  and  transverse  carpal  ligament,  and  a  deep  (lumbricales),  from 
the  deep  long  flexor  tendon — separated  by  the  superficial  palmar  vessels  and  nerves.  The  deep 
series  again  has  superficial  (contrahentes  ;  only  adductor  pollicis  in  man)  and  deep  (interossei, 
opponens  V)  layers. 

Muscular  homologies,  at  least  between  vertebrate  classes,  cannot  be  reasonably  extended 
beyond  comparison  of  entire  palmar  layers.  Direct  homology  of  individual  muscle  units  is 
profitless  and  probably  invalid. 


234  PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY 

The   toxicity   of  a  mixture   of  high   molecular   alkyl-dimethyl-benzyl   ammonium 
chlorides  to  Fundulus.     CHARLES  H.  TAFT. 

The  mixtures  of  high  molecular  alkyl-dimethyl-benzyl  ammonium  chlorides  used  is  sold  by 
the  Winthrop  Chemical  Company  under  the  trade  name  Zephiran  Chloride  *  for  use  as  an  anti- 
septic or  disinfectant. 

Taft  and  Strandtmann  (1945.  Fed.  Proc.,  4:  136)  showed  that  under  laboratory  conditions 
this  material  is  an  efficient  larvicide  for  the  mosquito  Culex  quinquefasciatus  and  Aedes  aegypti 
in  dilutions  up  to  1  :  250,000.  It  seems  desirable  to  determine  its  toxicity  to  some  of  the  animals 
it  might  be  brought  in  contact  with  if  used  for  this  purpose.  Taft  (1946.  Texas  Rpts.  on  Biol. 
and  Mcd.,  4:  25)  has  reported  its  toxicity  for  various  invertebrates. 

To  determine  the  toxicity  by  injection  fundulus  were  injected  intraperitoneally  with  different 
doses  of  one  per  cent  solution;  0.25  cc.  killed  17  out  of  22,  0.05  cc.  killed  15  out  of  17  while  0.1 
cc.  killed  24  out  of  24  fundulus.  When  these  fish  died  they  were  darker  than  the  controls  and 
in  many  of  them  the  abdomen  was  red  about  the  site  of  injection.  When  the  abdomen  was 
opened  there  was  frequently  a  greenish  fluid  present  and  the  viscera  had  the  appearance  of 
having  been  cooked.  The  liver,  gall  bladder,  heart,  kidneys,  and  gills  appeared  normal. 

Other  fundulus  were  placed  in  finger  bowls  containing  225  cc.  aerated  sea  water  with  dif- 
ferent concentrations  of  the  drug.  When  the  fish  were  placed  in  dilutions  of  from  approxi- 
mately 1 :  2,500  to  1 :  100,000  all  the  fish  died  in  from  35  to  105  minutes.  On  autopsy  there 
were  no  significant  gross  changes.  A  dilution  of  1  :  225,000  killed  25  per  cent  of  the  fish  while 
1  :  500,000  did  not  kill  any  of  the  fish  exposed  to  it. 

To  determine  the  effects  of  longer  exposure  to  the  drug  several  fundulus  were  placed  in 
battery  jars  in  aerated  sea  water  solution  of  from  1  :  100,000  to  1 :  400,000  and  observed  at  the 
end  of  24  hours.  All  the  fish  exposed  to  1  :  100,000  and  1 :  200,000  were  found  dead.  Twenty- 
five  per  cent  of  those  exposed  to  1 :  300,000  died  while  the  1  :  400,000  solution  failed  to  kill  any 
fish.  It  is  evident  that  the  effective  range  of  this  drug  when  employed  as  mosquito  larvicide 
might  be  deleterious  to  fundulus. 

Further  evidence  of  polypoidy  in  the  conjugation  of  green  and  colorless  Paramecium 
bnrsaria.     RALPH  WICHTERMAN. 

In  a  study  of  the  time-relations  of  the  nuclear  events  in  living  and  Feulgen-stained  prepara- 
tions through  conjugation,  instances  of  polyploidy  were  encountered.  Polyploidy  was  first  re- 
corded in  Paramecium  by  Chen  (1940,  Proc.  Nat.  Acad.  Sci.  V:  26)  for  P.  bursaria  and  this 
represents  the  second  report  of  the  phenomenon.  Pure-line  races  of  the  colorless  (255)  and 
green  (B9)  paramecia  were  mated.  The  individuals  of  each  race  have  well-defined  micronuclei 
of  approximately  equal  size. 

The  three  pregamic  divisions  were  found  to  be  remarkably  constant  in  respect  to  time  and 
micronuclear  behavior  at  a  given  temperature.  However,  in  the  cytological  examination  of 
many  hundreds  of  joined  pairs,  approximately  2  per  cent  were  observed  in  which  the  micro- 
nuclear  behavior  resulted  in  the  polyploid  conditions  only  after  the  pregamic  divisions.  The 
crucial  stage  where  polyploidy  occurs  is  found  during  the  period  of  pronuclear  transfer,  ap- 
proximately 16-18  hours  after  the  animals  have  been  mated.  It  follows  the  third  suggestion 
made  by  Chen  in  accounting  for  polyploidy ;  namely,  the  failure  of  a  migratory  pronucleus  in 
one  of  the  conjugants  to  migrate  to  the  other  conjugant.  The  result  is  an  individual  with  one 
small  pronucleus  (the  "stationary")  which  is  haploid,  and  the  conjugant  with  three  pronuclei 
(two  "migratory"  and  one  "stationary")  which  fuse  and  form  a  larger  triploid  synkaryon. 

What  is  the  fate  of  each  nuclear  body  that  is  now  comparable  to  the  normal  synkaryon  ?  The 
subsequent  micronuclear  stages  show  a  conspicuous  and  persisting  size  difference  in  all  later 
stages  and  hence  are  recognized  easily.  In  the  haploid  conjugant,  late  anaphase  stages  (com- 
parable to  postgamic  ones)  measure  10.8 /z  in  length  and  are  very  narrow;  similar  stages  in 
the  triploid  co-conjugant  measure  27  /JL  in  length  and  are  proportionately  wider.  Their  division 
products  measure  8  (j.  in  the  haploid  and  15.5  fj.  in  the  triploid  individuals  respectively. 

While  polyploidy  occurs  in  only  2  per  cent  of  the  cases  in  this  material,  it  nevertheless  cre- 
ates variation  in  micronuclear  composition  and  is  therefore  of  evolutionary  significance. 

*  Kindly  furnished  by  the  Winthrop  Chemical  Company. 


PRESENTED  AT  MARINE  BIOLOGICAL  LABORATORY  235 

The  Lipids  in  Pelomyxa  carolinensis.     CHARLES  G.  WILBER. 

In  1942  the  author  demonstrated  that  the  cytoplasm  of  Pelomyxa  carolinensis  contains  lipid 
material,  that  this  lipid  comes  from  digested  food,  and  that  it  is  composed  of  a  high  proportion 
of  fatty  acid.  In  the  latter  respect  the  stored  fat  differs  from  that  in  Amoeba  proteus  in  which 
fat  is  stored  in  the  neutral  form.  In  the  previous  work,  Nile  blue  sulfate  was  used  to  distinguish 
neutral  fat  from  fatty  acid.  This  dye  has  been  criticized  as  a  reagent  for  fat  tests.  Conse- 
quently it  seemed  desirable  to  use  specific  chemical  procedures  to  ascertain  the  nature  of  the 
lipid  material  in  Pelomyxa. 

Ninety  mg.  (wet  weight)  of  pelomyxae  were  thoroughly  washed  in  boiled  culture  fluid. 
By  repeated  centrifugation  the  cells  were  broken  up  and  then  the  lipids  were  extracted  in  hot 
alcohol.  The  quantities  of  phospholipid,  cholesterol,  and  fatty  acid  were  ascertained  by  the 
Bloor  method.  It  was  found  that  in  the  amount  of  cellular  material  used  there  was  no  meas- 
urable phospholipid  or  cholesterol.  The  total  weight  of  fatty  acid  in  54  mg.  of  cells  was  2.05  mg. 
or  3.8  per  cent  fatty  acid. 

These  results  are  in  agreement  with  the  results  previously  obtained  using  Nile  blue  sulfate. 
It  seems  that  in  Pelomy.ra  carolinensis  the  lipid  material  occurs  chiefly  as  fatty  acid  and  that 
the  amount  of  other  lipids  is  very  small. 

The  presence  of  lip  as  c  in  Pdomyxa  carolinensis.     CHARLES  G.  WILBER. 

The  digestion  of  fat  in  rhizopods  has  been  demonstrated  by  several  investigators.  More- 
over, it  has  been  shown  that  the  fats  digested  are  incorporated  into  the  cytoplasm.  However, 
none  of  the  investigations  so  far  has  given  direct  evidence  for  the  presence  of  lipase  in  the  cyto- 
plasm of  rhizopods. 

Pelomyxae  were  starved  and  then  ground  up  in  a  drop  of  water.  A  drop  of  this  solution 
was  added  to  a  drop  of  0.2  per  cent  emulsion  of  castor  or  olive  oil  and  a  drop  of  pure  water 
was  added  to  another  drop  of  the  emulsion  as  a  control.  After  30  minutes  both  drops  were 
treated  with  hydroxylamine  hydrochloricle  and  potassium  hydroxide.  Then  after  acidification 
each  drop  was  treated  with  1  per  cent  ferric  chloride  solution.  In  each  case  a  violent  brown 
color  was  produced  in  the  control,  whereas  no  color  was  produced  in  the  drop  containing  the 
ground  up  pelomyxae. 

The  above  reaction  is  a  test  for  esters.  Lipases  are  known  to  be  ester  ferments.  Since  the 
oil  emulsions  mixed  with  ground  pelomyxae  did  not  give  the  characteristic  ester  reaction,  it  can 
be  concluded  that  the  esters  were  broken  down  by  something  in  the  cytoplasm.  We  therefore 
have  direct  evidence  for  the  presence  of  lipase  in  Pelomyxae  carolinensis. 


PAPERS  PRESENTED  AT  THE  MEETING  OF  THE  SOCIETY 
OF  GENERAL  PHYSIOLOGISTS 

SEPTEMBER  5  AND  SEPTEMBER  6 
FIRST  SESSION — S.  C.  BROOKS,  CHAIRMAN 

The   effect   of  cold   on   capillary   permeability   and  fluid   movement   in   the   frog. 
ELLEN  BROWN,  M.D.*  AND  EUGENE  M.  LANDIS,  M.D. 

Micro-manipulative  methods  were  used  to  study  the  relationship  between  capillary  blood 
pressure  and  the  rate  of  fluid  movement  through  the  walls  of  single  capillaries  in  the  frog's 
mesentery  (a)  at  ordinary  room  temperatures  of  22.5°  to  25.5°  C.  and  (b)  when  the  mesentery 
was  cooled  to  between  —  2°  and  +  2°  C.  Cooling  the  mesentery  decreased  capillary  perme- 
ability, reduced  the  observed  rates  of  nitration  and  increased  the  observed  rates  of  absorption. 
The  nitration  constant  of  the  capillary  wall  was  reduced  from  the  control  value  of  0.0070 
i"-r'/i".2/sec./cm.  water  pressure  to  0.0019,  a  decrease  of  73  per  cent.  The  effective  osmotic  pres- 
sure of  the  blood  within  the  capillaries  was  elevated  from  10.5  to  13.8  cm.  water,  an  increase 
of  31  per  cent. 

Four  possible  causes  for  the  increase  in  apparent  or  effective  osmotic  pressure  were  con- 
sidered. (1)  An  increase  of  absolute  colloid  osmotic  pressure  was  excluded  because  plasma 
protein  concentrations,  calculated  from  specific  gravities  of  plasma  samples,  were  the  same  in 
the  two  series  of  frogs.  (2)  An  increase  in  effective  colloid  osmotic  pressure  due  to  greater 
retention  of  plasma  protein  during  cooling  could  also  be  excluded  because  the  control  experi- 
ments showed  that  plasma  proteins  were  already  retained  completely,  or  almost  completely,  even 
at  room  temperature.  (3)  It  is  possible,  however,  that  the  effective  non-protein  osmotic  pres- 
sure might  rise  if  the  passage  of  smaller  molecules,  e.g.,  glucose,  amino  acids,  urea  or  certain 
electrolytes,  was  impeded  more  than  that  of  water  as  the  permeability  of  the  capillary  wall 
decreased  during  chilling.  (4)  Tliermosmosis  might  also  be  responsible  because  relatively  warm 
blood  was  circulating  through  capillaries  surrounded  by  cooler  tissues.  Studies  are  in  progress 
to  determine  whether  or  not  this  factor  modifies  the  movement  of  fluid  through  membranes  in 
•vitro  using  a  schema  which  simulates  the  conditions  existing  in  vivo. 

The  effects  of  cold  on  the  capillaries  of  the  frog  differ  from  those  observed  in  mammalian 
capillaries  because  the  former  become  less  permeable  at  2°  C.,  whereas  the  latter  become  more 
permeable  as  temperature  falls  below  10°  C.  However,  actual  freezing  of  the  frog's  capillaries 
at  temperatures  of  —  5°  to  —  10°  C.  increased  capillary  permeability  conspicuously,  as  shown 
by  the  appearance  of  stasis  during  thawing.  If  the  duration  of  actual  freezing  was  brief  this 
stasis  usually  disappeared  within  a  few  minutes  as  the  capillary  wall  regained  its  normal  relative 
impermeability  to  protein. 

Bubble  formation  within  single  cells. ~f     E.  NEWTON  HARVEY,  K.  W.  COOPER,  A. 
H.  WHITELEY,  D.  C.  PEASE,  AND  W.  D.  MCELROY. 

Normal  living  isolated  cells  (Amoeba  sp.,  Chaos  chaos,  Paramoecium,  Arbacia  and  Aste- 
rias  eggs  and  Nitella)  do  not  form  internal  gas  bubbles  if  saturated  with  nitrogen  gas  at  80  to 
120  atmospheres  pressure  and  then  suddenly  decompressed.  Bubbles  may  form  on  the  outside 
of  the  cells  due  to  contamination  with  gas  nuclei  (minute  gas  phases  sticking  to  hydrophobic 
spots).  Cells  which  have  been  killed  by  chloroform  or  formalin  likewise  form  no  bubbles  within 
but  sometimes  spontaneously  dead  cells  or  those  previously  injured  by  twisting  before  subjec- 

*  Research  Fellow  in  Physiology,  Commonwealth  Fund. 

t  Part  of  the  work  described  in  this  Abstract  was  done  under  a  contract  recommended  by 
the  Committee  on  Medical  Research  between  the  Office  of  Research  and  Development  and 
Princeton  University. 

236 


PRESENTED  AT  THE  SOCIETY  OF  GENERAL  PHYSIOLOGISTS          237 

tion  to  the  high  gas  pressures  do  form  bubbles  within  after  decompression.  A  living  Nitella 
cell  just  after  decompression  from  high  gas  pressures  and  still  free  of  bubbles,  will  immediately 
form  a  bubble  inside  if  the  cell  wall  is  gently  pinched  (not  enough  to  penetrate  the  wall)  or 
twisted.  Such  bubbles  are  believed  to  result  from  local  decreased  tensions  that  tear  the  liquid, 
forming  a  space  or  cavity  (a  vapor  phase)  into  which  gas  diffuses,  forming  a  gas  nucleus 
that  immediately  grows  to  a  bubble.  Such  cavities  can  form  inside  or  outside  of  cells  even  at 
atmospheric  pressures.  They  are  believed  to  be  formed  during  muscular  exercise  in  man, 
when  the  incidence  of  aviator's  bends  is  greatly  increased. 

The  action  of  various  cations  of  muscle  protoplasm.     L.  V.  HEILBRUNN  AND  F.  J. 
WIERCINSKI. 

There  are  two  ways  to  study  the  colloidal  behavior  of  muscle  protoplasm.  One  way  is  to 
isolate  pure  proteins  and  follow  their  reactions  in  test  tubes;  the  other  way  is  to  subject  the 
protoplasm  itself  to  reagents  and  observe  the  results.  In  our  studies,  we  injected  solutions  of 
various  salts  into  the  interior  of  isolated  muscle  fibers  of  the  frog.  We  then  noted  the  degree 
of  shortening  of  the  constituents  of  the  muscle.  With  the  aid  of  a  micrometer  eyepiece,  we  were 
able  to  determine  the  effect  of  the  injections  on  the  length  of  the  fiber.  In  numerous  experiments 
we  found  that  rather  dilute  calcium  chloride  solutions  invariably  caused  an  immediate  and  pro- 
nounced shortening  of  the  protoplasmic  constituents  of  the  muscle.  On  the  other  hand,  potassium 
and  sodium  chloride  had  very  little  effect.  Even  when  injected  in  concentrations  isotonic  with 
the  muscle,  they  ordinarily  caused  no  shortening  whatsoever.  Rarely,  a  shortening  did  follow 
injection  of  isotonic  sodium  or  potassium  chloride.  This  we  believe  was  due  to  the  release  of 
calcium  ion.  Magnesium  ion  likewise  causes  no  shortening  of  the  protoplasmic  constituents. 
Barium  acts  like  calcium.  The  results  support  the  calcium  ion  theory  of  stimulation  and  they  are 
opposed  to  Szent-Gyorgyi's  opinion  that  potassium  is  the  ion  primarily  responsible  for  the  con- 
traction of  muscle. 

Further  observations  on  an  oligodynamic  action  of  copper  and  mercury  on  eryth- 
rocytes.     M.  H.  JACOBS  AND  DOROTHY  R.  STEWART. 

The  specific  effect  of  copper  in  decreasing  the  permeability  of  erythrocytes  to  glycerol  seems 
to  be  absent  in  all  species  whose  erythrocytes  show  a  low  degree  of  permeability  to  this  solute. 
It  is  also  lacking  in  a  number  which  show  a  very  high  permeability  both  to  glycerol  and  to  other 
hydrophilic  solutes  of  comparable  molecular  volume.  It  has  so  far  been  found  only  in  those 
species  whose  erythrocytes  show  a  disproportionately  great  permeability  to  glycerol,  thus  sug- 
gesting that  some  special  mechanism  of  penetration  may  be  involved,  which  is  reversibly  in- 
activated by  copper.  This  generalization  is  supported  by  the  behavior  of  the  erythrocytes  of  a 
number  of  birds  in  which  the  specific  permeability  to  glycerol  is  particularly  great. 

The  effects  of  HgCL  in  some  ways  resemble  and  in  others  differ  from  those  of  CuCL.  One 
of  the  most  important  differences  is  that  HgCU  forms  a  double  salt  with  NaCl,  and  its  activity 
is  therefore  greatly  reduced  by  the  presence  of  any  considerable  quantities  of  the  latter  salt.  A 
second  difference  is  that  HgCL  readily  enters  the  erythrocyte,  while  CuCU  does  not.  These  two 
fundamental  differences  are  responsible  for  a  number  of  secondary  ones. 

That  copper  may  hinder  the  escape  of  glycerol  from  human  erythrocytes,  as  well  as  its  en- 
trance into  them,  is  suggested  by  the  following  experiment.  To  a  suspension  of  erythrocytes  in 
an  isotonic  salt  solution,  small  amounts  of  copper  and  of  concentrated  glycerol  are  added,  and 
the  resulting  mixture  is  then  diluted  with  a  properly  chosen  hypotonic  salt  solution.  If  the  copper 
is  added  before  the  glycerol,  it  decreases  hemolysis  by  preventing  the  entrance  of  glycerol  into  the 
cells.  If  the  copper  is  added  30  seconds  or  more  after  the  glycerol,  it  increases  hemolysis  by 
preventing  the  escape  of  the  glycerol  that  has  entered  the  cells. 

The  prolytic  loss  of  K  from  red  cells.     ERIC  PONDER. 

The  prolytic  loss  of  K,  i.e.,  the  loss  of  K  which  takes  place  from  red  cells  exposed  to  hypolytic 
concentrations  of  lysin,  has  been  measured  by  means  of  the  flame  photometer  in  systems  containing 
distearyl  lecithin,  sodium  taurocholate,  sodium  tetradecyl  sulfate,  saponin,  and  digitonin.  The 


PRESENTED  AT  THE  SOCIETY  OF  GENERAL  PHYSIOLOGISTS 

lysins  are  added  in  various  concentrations  to  washed  red  cells  from  heparinised  human  blood, 
and  the  K  in  the  supernatant  fluids  is  determined  after  various  intervals  of  time  at  various  tem- 
peratures. This  prolytic  loss  of  K,  KP,  is  compared  in  every  experiment  with  the  loss  Ks  into 
standard  systems  containing  one  per  cent  NaCl  alone,  without  lysin. 

The  losses  K,,  and  Ks  increase  with  time,  so  that  new  steady  states  are  approached  logarith- 
mically. The  values  of  Kp  which  correspond  to  the  new  steady  state  depends  on  the  lysin  used, 
being  greatest  with  taurocholate  and  smallest  with  powerful  lysins  such  as  digitonin  (confirming 
an  observation  of  Davson  and  Danielli).  The  extent  and  course  of  the  K  losses  seem  to  have  no 
simple  relation  to  the  prolytic  phenomenon  of  the  disk-sphere  transformation. 

Just  as  the  prolytic  loss  of  K  occurs  without  the  loss  of  any  Hb,  so  in  concentrations  of  lysin 
sufficient  to  produce  hemolysis  the  loss  of  K,  expressed  as  a  percentage  of  the  total  red  cell  K, 
increases  much  more  rapidly  with  lysin  concentration  than  does  the  loss  of  Hb,  expressed  as  a 
percentage  of  the  total  Hb.  The  explanation  of  these  relations  depends  on  whether  the  loss  of  K 
is  treated  as  being  all-or-none  in  the  case  of  the  individual  cell,  or  as  being  the  result  of  the  loss 
of  part  of  the  K  from  all  the  cells.  This  point  has  yet  to  be  decided. 

SECOND  SESSION — L.  MICHAELIS,  CHAIRMAN 

Effect  of  fluoroacetate  on  the  metabolism  of  baker's  yeast.     E.  S.  GUZMAN  BARRON 
AND  GEORGE  KALNITSKY. 

• 

Among  the  organic  halogen  compounds,  those  containing  fluorine  occupy  a  special  position 
regarding  their  chemical  and  physiological  properties.  Because  of  the  high  value  of  the  energy 
of  the  C-F  bond  and  of  the  electro-negativity  of  F,  the  introduction  of  F  into  the  C  atom  pro- 
duces a  greater  stability,  specially  in  alyphatic  compounds.  This  is  shown  on  measuring  the  rate 
of  combination  of  cysteine  with  halogen  acetates.  At  23°,  half-reaction  with  iodoacetate  took 
place  in  4.4  minutes ;  with  bromoacetate  in  6.2  minutes ;  with  chloroacetate,  in  125  minutes. 
With  fluoroacetate  it  did  not  react  at  all.  There  is  a  certain  relationship  between  the  rates  of 
reaction  and  the  bond-energy  values  of  the  C-halogen  bonds  as  well  as  the  electronegativity 
values  of  the  halogens.  On  studying  the  effect  of  these  halogen  acids  on  the  rate  of  oxidation 
of  acetate  by  baker's  yeast  it  was  found  that  0.001  M  of  fluoroacetate  inhibited  it  90  per  cent; 
bromoacetate,  17  per  cent;  iodoacetate,  chloroacetate,  and  trifluoroacetate,  none  at  all.  On  com- 
paring the  interatomic  distances  between  C  and  the  halogen  it  can  be  seen  that  the  C-F  bond 
with  a  distance  of  1.41  A  approaches  most  closely  the  distance  of  the  C-H  bond,  1.09  A.  By  in- 
creasing the  size  of  the  fluoroacetate  molecule  through  the  replacement  of  the  other  two  hydrogens 
with  fluorine  (trifluoroacetate)  the  inhibiting  effect  was  destroyed.  This  inhibition  is  a  sub- 
strate competitive  inhibition,  the  fluoroacetate  occupyng  the  place  of  acetate  in  the  protein  moiety 
of  the  acetate  metabolism  enzyme.  Increase  of  the  length  of  the  molecule  as  in  fluoropropionate, 
fluorobutyrate,  and  fluorocrotonate  destroyed  the  inhibition.  Inhibition  was  partially  reversed 
on  addition  of  large  amounts  of  acetate  (0.08  M).  Inhibition  occurs  in  the  first  step  of  acetate 
metabolism,  namely,  condensation  with  oxaloacetate  to  give  citrate.  The  formation  of  citrate 
from  acetate  was  completely  inhibited  with  0.005  M  fluoroacetate.  In  the  presence  of  ethanol, 
the  rate  of  O2  uptake  was  not  affected  by  fluoroacetate  up  to  42  per  cent  of  the  total  O?  uptake, 
when  the  inhibitory  effect  appeared.  This  is  indication  that  ethanol  oxidation  occurs  in  three  suc- 
cessive steps :  -oxidation  of  ethanol  to  aldehyde ;  and  of  aldehyde  to  acetate,  both  unaffected  by 
fluoroacetate ;  and  oxidation  of  acetate,  inhibited  by  it.  At  the  end  of  the  experiment  there  were 
in  the  control  1572  cmm.  O2  used,  and  150  cmm.  of  acetate  formed  from  940  cmm.  of  ethanol;  in 
the  presence  of  fluoroacetate  there  were  975  cmm.  O...  used  and  890  cmm.  of  acetate  formed. 

The  effect  of  sodium  aside  on  Parameciuwi  calkinski.     E.  J.  BOELL. 

Sodium  azide  is  generally  regarded  as  an  inhibitor  of  respiration  by  virtue  of  its  inactivation 
of  cytochrome  oxidase.  In  Parameciiim  calkinsi,  experiments  have  shown  that  this  compound,  in 
a  concentration  of  0.001  to  0.01  molar,  reversibly  depresses  respiratory  activity  by  50  to  60  per 
cent.  Under  certain  circumstances,  however,  azide  instead  of  inhibiting  respiration  serves  as  a 
powerful  respiratory  stimulant.  The  stimulating  effect  of  azide  seems  to  depend  primarily  upon 
the  pH  of  the  medium.  For  example,  a  0.01  molar  solution  of  NaN3  at  pH  6.02  will  depress 
respiration  to  a  value  about  30  per  cent  of  normal ;  at  pH  6.24  respiration  is  70  per  cent  of  nor- 


PRESENTED  AT  THE  SOCIETY  OF  GENERAL  PHYSIOLOGISTS          239 

mal,  while  at  pH  6.59  the  same  concentration  of  azide  stimulates  respiration  of  238  per  cent  of 
normal.  Calculation  of  the  hydrazoic  acid  concentration  at  these  pH  values  shows  that  the  ef- 
fect produced  depends,  within  certain  limits,  upon  the  concentration  of  undissociated  HN3. 

A  study  has  been  made  of  the  mechanism  of  azide  stimulation.  It  has  been  found  that  the 
respiratory  quotient  of  normal  animals  averages  0.99 ;  that  of  animals  in  the  presence  of  a 
stimulating  dose  of  azide  averages  1.05.  The  increased  oxidation  thus  involves  the  metabolism 
of  organic  substrate.  It  is  also  apparently  mediated  by  the  normal  enzymic  mechanisms  for  it 
is  sensitive  to  cyanide.  Carbon  monoxide,  however,  exerts  only  a  slightly  depressing  effect. 

The  metabolism  of  Paramecia  under  normal  circumstances  is  accompanied  by  the  production 
of  large  quantities  of  ammonia  nitrogen.  On  the  assumption  that  such  ammonia  production 
represents  protein  breakdown,  approximately  75  per  cent  of  the  total  oxygen  consumption  of 
control  animals  can  be  accounted  for  in  this  way.  Although  Paramecia  treated  with  azide  show 
increased  ammonia  production,  only  22  per  cent  of  the  extra  oxygen  uptake  induced  by  azide  can 
be  accounted  for  as  protein  breakdown  with  ammonia  as  the  end  product. 

In  addition  to  the  effects  already  noted,  azide  interferes  with  the  ability  of  Paramecium 
calkinsi  to  maintain  normal  water  balance.  The  activity  of  the  contractile  vacuoles  is  greatly 
reduced  and  supernumerary  vacuoles  are  frequently  formed. 

The  oxygen  consumption  concerned  with  growth  in   bacterium  coli.     KENNETH 
FISHER.     No  abstract  submitted. 

Enzymatic  acetylation  and  the  co enzyme  of  acetylation.     FRITZ  LIPMANN. 

The  mechanism  of  enzymatic  acetylation  of  aromatic  amines  has  been  studied  in  pigeon  liver 
homogenates  and  extracts  (Lipmann,  F.,  1945.  /.  Biol.  Chcm.,  160:  173).  In  this  enzymatic 
system  the  condensation  of  an  aromatic  amine,  like  sulfanilamide,  with  acetate,  is  effected 
through  a  transfer  of  phosphate  bond  energy  from  adenylprophosphate.  (Cf.,  Nachmansohn, 
D.  and  Machado,  A.  L.,  1943.  J.  Neurophysiology,  6:  397  for  a  similar  system  of  choline  acety- 
lation in  brain). 

A  heat  stable  and  dialysable  coenzyme  was  recently  found  necessary  in  this  reaction,  besides 
the  energy  donor  adenylpyrophosphate.  The  characterization  of  this  new  coenzyme  is  now  in 
progress  in  this  laboratory  in  collaboration  with  Dr.  Nathan  O.  Kaplan.  We  find  the  same 
coenzyme  necessary  to  complement  dialyzed  brain  extracts  for  acetylation  of  choline,  although 
the  brain  enzyme  is  specific  for  choline  and  the  liver  enzyme  specific  for  amines.  The  coenzyme 
is  present  in  largest  amounts  in  brain,  liver,  and  kidney.  Appreciable  amounts  are  present  in 
all  tissues  tested,  including  carcinoma.  Therefore  its  action  must  be  a  very  general  one  and 
probably  not  merely  restricted  to  acetylation. 

The  coenzyme  is  destroyed  by  intestinal  phosphatase  with  liberation  of  phosphate.  It  is 
inactivated  by  a  rather  general  tissue  enzyme  without  liberation  of  phosphate.  The  link  at- 
tacked by  the  latter  enzyme  is  unknown.  The  compound  follows  the  general  pattern  of  nucleo- 
tide  precipitation.  Our  most  active  preparations  showed  sporadic  crystals  on  microscopic  ex- 
amination. This  quite  uniform  fraction  contained  adenine,  ribose,  and  phosphate  in  the  propor- 
tion 1  to  1  to  2.  Acid  hydrolysis  showed  the  second  phosphate  not  to  be  in  pyrophosphate 
linkage.  If  we  assume  the  presence  of  some  crystals  to  indicate  near  purity,  which  it  not  neces- 
sarily does,  then  the  content  of  approximately  50  per  cent  of  adenylic  acid  in  our  best  prepara- 
tions should  mean  the  coenzyme  to  be  of  dinucleotide  structure.  Therein  the  adenylic  acid 
should  be  linked  through  the  second  phosphate  to  an  as  yet  unidentified  part.  Electrotitration 
and  cleavage  experiments  seem  to  support  the  outlined  constitution. 

Penetration  and  action   of  cholincsterasc   inhibitors.     DAVID   NACHANSOHN.     No 
abstract  submitted. 

The  metamorphosis  of  visual  systems  in  amphibia.     GEORGE  WALD. 

In  the  rods  of  the  vertebrate  retina  two  visual  systems  are  found.  One  is  based  upon  the 
red  photosensitive  pigment  rhodopsin,  engaged  in  a  cycle  with  vitamin  A, ;  the  other  involves 
the  purple  photopigment,  porphyropsin,  bound  in  a  similar  cycle  with  vitamin  A2. 


240          PRESENTED  AT  THE  SOCIETY  OF  GENERAL  PHYSIOLOGISTS 

The  porphyropsin  system  appears  to  be  the  more  primitive  in  vertebrate  evolution.  The 
cyclostome,  Petromyzon  marinus,  possesses  only  this  system.  The  same  is  true  of  all  types  of 
freshwater  fish  so  far  examined. 

Vertebrates  have  followed  two  pathways  out  of  fresh  water,  one  into  the  sea,  the  other 
to  land.  Both  have  led  them  to  the  use  of  vitamin  A,  in  vision.  Thus  all  marine  fishes  which 
have  been  examined,  with  the  single  exception  of  certain  Labridae,  have  the  rhodopsin  system 
alone ;  so  also  do  all  the  birds  and  mammals  investigated. 

Interpolated  between  freshwater  and  marine  fishes  are  euryhaline  forms,  which  can  exist 
as  adults  in  either  environment.  Among  them,  the  salmons  and  the  "freshwater"  eel  have  mix- 
tures o'f  the  rhodopsin  and  porphyropsin  systems ;  while  the  alewife  and  white  perch  have  only 
the  latter.  In  all  these  forms  the  visual  system  is  predominantly  or  exclusively  that  normally 
associated  with  the  environment  in  which  the  fish  develops  embryonically,  and  is  relatively  inde- 
pendent of  the  environment  in  which  it  is  found  as  an  adult. 

Interpolated  between  freshwater  fishes  and  true  land  vertebrates  are  the  amphibia.  Their 
life  histories  for  the  most  part  are  closely  analogous  with  those  of  euryhaline  fishes,  amphibian 
migrations  to  land  replacing  fish  migrations  into  the  sea. 

Adult  frogs  possess  the  rhodopsin  system  and  vitamin  A,  alone.     The  tadpole  of  the  common 
bullfrog,  Rana  catesbiana,  however,  has  exclusively  the  porphyropsin-vitamin  An  system  just. 
prior  to  metamorphosis.     During  metamorphosis  it  transfers  completely  to  the  rhodopsin  sys- 
tem, which  is  found  alone  in  the  new  emerged  frog.     Partly  metamorphosed  animals  have  mix- 
tures of  both  systems,  such  as  have  been  found  otherwise  only  in  euryhaline  fishes. 

The  common  newt,  Triturus  viridescens,  begins  its  life  as  a  gilled  larva  in  fresh  water. 
After  several  months  it  metamorphoses  to  the  land-living  red  eft;  then  after  1-2  years  of  growth 
it  undergoes  a  second  metamorphosis  to  the  sexually  mature  newt,  returning  to  the  water  for 
the  remainder  of  its  life.  The  eye  of  the  red  eft  contains  a  mixture  of  vitamins  Aj  and  A2, 
predominantly  the  former;  while  that  of  the  water-phase  adult  presents  just  the  reverse  propor- 
tions of  both  vitamins.  This  is  a  change  opposite  in  direction  to  that  in  the  frog,  but  associ- 
ated in  the  same  way  with  the  chemical  metamorphosis  of  visual  systems. 

Amphibia,  therefore,  like  euryhaline  fishes  possess  as  a  group  both  the  rhodopsin  and 
porphyropsin  systems ;  but  in  amphibia  these  systems  succeed  one  another  as  the  animal  goes 
through  its  basic  metamorphoses. 

THIRD  SESSION — J.  H.  BODINE,  CHAIRMAN 

X-ray  effects  in  mixtures  of  compounds.     RUBERT  S.  ANDERSON. 

It  has  been  reported  previously  that  ascorbic  acid,  as  shown  by  experiments  in  plasma,  has 
a  preferential  ability  to  react  with  the  materials  produced  in  water  by  x-rays.  Much  of  the 
ascorbic  acid  reaction  is  not  observed  in  irradiated  muscle.  Non-uniform  distribution  of  the 
ascorbic  acid  in  muscle  would  tend  to  make  the  observed  result  too  low.  Another  possibility 
is  that  other  compounds  are  present  in  muscle  which  take  some  of  the  reactive  material  away 
from  the  ascorbic  acid. 

Evidence  has  been  obtained  that  the  ascorbic  acid  reaction  consists  in  part  of  a  reversible 
oxidation,  presumably  to  dehydroascorbic  acid.  When  present  during  the  irradiation,  glutathi- 
one  and  cysteine  gave  substantial,  although  variable,  protection  of  ascorbic  acid  against  x-rays. 
Alanine  was  much  less  effective,  suggesting  that  the  sulfhydryl  grouping  is  largely  responsible, 
whether  it  is  a  true  competitive  protection  or  a  reversal  of  oxidation.  Glutathione  and  cysteine 
and  possibly  protein  sulfhydryl  groups  could  thus  account  for  a  part  of  the  protective  effect  of 
muscle  on  ascorbic  acid. 

There  is  no  evidence  that  the  destruction  of  a  small  amount  of  these  compounds  woulc 
damage  a  cell.  However,  the  work  shows  that,  in  principle,  a  compound  through  which  the 
water  reaction  might  damage  the  cell  could  exist. 

Ascorbic  acid,  glutathione  and  cysteine  partially  protect  pepsin  from  the  inactivating  effect 
of  x-rays.  Alanine  is  much  less  active. 

If  at  least  a  part  of  the  reaction  in  water  is  distributed  randomly  throughout  the  proteins 
of  the  cell  and  nucleus,  the  occasional  loss  of  a  molecule  or  two  from  compounds  represented 
by  hundreds  of  molecules  need  have  little  effect  on  the  cell  although  the  products  formed,  such 
as  denatured  proteins,  might  secondarily  be  harmful  to  the  cell.  However,  if  there  are  in  the 


PRESENTED  AT  THE  SOCIETY  OF  GENERAL  PHYSIOLOGISTS          241 

cell  or  nucleus  some  thousands  of  different  protein  compounds  or  structures  each  form  of  which 
is  essential  to  the  cell  and  each  one  of  which  is  represented  by  but  one  or  two  molecules  or 
particles,  then  randomly  distributed  products  of  irradiated  water  might  destroy  one  of  these 
entities  and  so  damage  or  kill  the  cell.  This  is  essentially  the  argument  used  by  Lea  in  reaching 
the  conclusion  that  his  theory  led  to  the  expectation  of  gene  and  chromosomal  effects  from 
irradiation. 

Electrical  studies  of  acetylcholine  and  choline  estcrase.     T.  C.  BARNES. 

Acetylcholine  passes  through  the  thin  oil  layer  of  a  bubble  of  guaiacol-resin-cholesterol  so 
rapidly  that  the  spike  potential  must  be  recorded  by  an  oscillograph.  First  the  acetylcholine 
produces  a  negative  phase  boundary  potential  on  one  side  of  the  oil  layer  but  on  reaching  the 
opposite  side  of  the  oil  a  new  potential  is  established  which  produces  the  descending  part  of  the 
spike  (no  esterase  required).  At  the  suggestion  of  Osterhout,  solutions  were  shaken  5  hrs. 
with  guaiacol  with  these  results :  oil  with  saline  5  X  1(T7  mhos ;  same  with  0.002  M  acetylcholine 
35  X  10"7  mhos  (conductivity  was  determined  of  oil  separated  from  aqueous  solution).  Apply- 
ing the  Nernst  equation,  0.058  times  log  conductivity  difference  (6)  gives  40  mv.  (observed 
phase  boundary  potential).  At  the  suggestion  of  Loewi,  tetramethylammonium  iodide  was 
found  to  give  no  potential  on  nitrobenzene  but  0.05  per  cent  gave  25  mv.  negative  on  guaiacol. 
The  type  of  oil  and  not  the  tertiary  or  quarternary  nature  of  the  compound  determines  electro- 
genie  effects.  Thus  prostigmine  produces  85  mv.  negative  on  guaiacol  compared  with  35  mv. 
generated  by  acetylcholine  (both  0.002  M).  Prostigmine  inhibits  the  cord  in  spasticity  by 
flooding  with  high  negative  potential  which  may  also  act  as  a  stimulus  on  muscle  in  myasthenia. 
Dialantin  and  phenobarbital  produce  positive  potential  (20  mv.  at  concentration  of  0.05  per  cent) 
which  probably  neutralizes  the  excess  negativity  of  acetylcholine  in  the  brain  in  epilepsy. 
Lyovac  plasma  reduces  the  phase  boundary  potential  of  0.05  per  cent  acetylcholine  from  35  to 
15  mv.  (residual  potential  is  produced  by  choline).  Potential  of  benzyol  choline  is  destroyed 
by  serum  and  part  of  the  mecholyl  potential  by  one  per  cent  ground  cat  brain.  Eserine  and 
DFP  preserve  the  potential  of  acetylcholine  in  the  oil-cell  in  the  same  manner  as  in  the  nerve. 

One  per  cent  DFP  increases  the  specific  conductance  of  guaiacol  100  per  cent  which  explains 
part  of  its  blocking  action  on  nerve  and  muscle. 

The  action-current  in  cholinergic  nerve  is  probably  a  phase-boundary  potential  of  acetyl- 
choline (sympathin  is  the  electrogenic  amine  in  adrenergic  nerve). 

Two  schools  of  thought  in  electrophysiological  theory.     R.  BEUTNER  AND  T.  C. 
BARNES. 

The  older  school,  entrenched  as  the  hypothesis  of  sieve  membranes  retaining  negative  but 
not  positive  ions,  explains  everything  but  solves  no  problems.  The  newer  school  omits  hypotheses 
and  proposes  searches  for  electrogenic  materials  in  tissues  by  setting  up  artificial  battery  systems 
composed  of  lipoid  layers  (oils)  inserted  between  aqueous  salt  solutions.  Some  of  these  resemble 
analogous  battery  systems  containing  a  tissue  in  place  of  the  lipoid.  One  type  of  system  studied 
is  : — concentrated  saline/tissue  or  lipoid  (oil) /diluted  saline  +.  Tissue,  in  such  a  set-up,  may  pro- 
duce the  maximum  e.m.f.  of  58  millivolts  if  the  concentrated  solution  is  1/10  mol. ;  the  diluted  one, 
1/100  mol.  Only  few  oils  show  such  an  effect,  as  e.g.,  fatty  acid  dissolved  in  a  phenol-derivative, 
but  not  neutral  fats,  gelatin,  etc. 

The  production  of  bio-electricity  does  not  depend  on  such  aqueous  salt  concentrations  but  on 
metabolic  processes  in  tissues,  chiefly  oxidation.  A  search  for  suitable  electrogenic  systems  has 
led  to  the  following  one  (Beutner,  Loznerea,  1930)  : — saline/reduced  substance  e.g.,  a  higher 
alcohol  or  lower  fatty  acid  as  in  dying  tissue/oxidized  substance  e.g.,  corresponding  acid  or  cor- 
responding higher  fatty  acid  as  in  respiring  tissue  saline  +.  For  the  action  current  one  possible 
electrogenic  substance  is  acetylcholine  since  even  dilute  solutions  produce  an  e.m.f.  in  contact 
with  oils  in  a  system  such  as :  +  saline  without  addition/oil/saline  with  acetylcholine  added 
1 :  100,000  to  1  :  several  million— (Beutner  and  Barnes,  1941). 

'Tissue  extract  can  be  used  in  the  place  of  the  oil,  also  frog's  nerve  by  the  Netter  technique. 
Adrenergic  amines  produce  similar  negative  potentials  but  on  different  oils  which  are  inactive  in 
contact  with  choline  esters.  A  difference  in  chemical  composition  may  therefore  be  responsible 
for  the  specific  function  of  cholinergic  and  adrenergic  fibers.  The  rapid  disappearance  of  the 


242          PRESENTED  AT  THE  SOCIETY  OF  GENERAL  PHYSIOLOGISTS 

negative  potential,  which  occurs  even  in  the  absence  of  choline  esterase,  may  be  explained  by  a 
penetration  of  acetylcholine  through  a  thin  lipoid  layer  (membrane)  creating  a  potential  dif- 
ference in  the  opposite  direction  on  the  other  side.  Physico-chemical  studies  are  not  needed  for 
the  search  for  electrogenic  substances,  but  when  performed  on  oil  cells,  they  show  the  existence 
of  phase  boundary  potentials  depending  on  electrolyte  distribution ;  the  charged  pore  theory  fails 
to  explain  the  phenomena  and  is  contradictory. 

The  frequency  of  x-ray-induced  chromated  breaks  in  Tradescantia  as  modified  by 
near  infrared  radiation.     C.  P.  SWANSON  AND  ALEXANDER  HOLLAENDER. 

The  frequency  of  x-ray-induced  chromatid  breaks  in  Tradescantia  can  be  significantly  in- 
creased by  treatment  of  the  inflorescences  with  near  infrared  radiation.  Pretreatment  with  near 
infrared  radiation  for  seven  hours  prior  to  x-radiation  inceased  the  frequency  of  single  deletions, 
double  (isochromatid)  deletions,  and  translocations  between  and  within  chromosomes;  post-treat- 
ment increased  only  single  deletions  and  translocations.  A  delay  of  21  hours  between  treatment 
with  infrared  and  x-rays  did  not  appreciably  decrease  the  effectiveness  of  the  infrared,  suggesting 
that  the  changes  within  the  cell  induced  by  the  infrared  were  of  a  relatively  permanent  nature. 
At  the  present  time,  the  nature  of  the  effect  of  infrared  is  not  clearly  understood. 


Vol.  91,   No.  3  December,   1946 

THE 

BIOLOGICAL  BULLETIN 

PUBLISHED   BY  THE   MARINE  BIOLOGICAL  LABORATORY 


A   STRONGLY   INTERSEXUAL   FEMALE   IX    HABROBRACON 


P.  W.  WHITING 

University  of  Pcmisyk-ania,  Philadelphia,  and  the  Marine  Biological  Laboratory,  Woods  Hole 

In  the  parasitic  wasp  Habrobracon  fuglandis  (Ashmead),  diploid  males  have 
never  shown  any  tendency  toward  intersexuality ;  they  are  as  definitely  male  as 
their  normal  haploid  brothers.  When  a  "diploid  male  with  female  genitalia"  was- 
found,  it  was  therefore  regarded  with  especial  interest.  The  specimen,  designated 
freak  994,  developing  from  a  heavily  x-rayed  ( 29,300  r)  egg,  occurred  among  the 
offspring  of  a  treated  wild  type  (stock  33)  female  crossed  with  an  untreated  lemon 
honey  male  (Experiment  by  A.  R.  Whiting.  1945). 

Freak  994  shows  the  heterozygous  condition  of  the  semidominant  body  color 
gene  lemon  inherited  from  its  father.  (Note  light  base  of  antennae  in  Figure  1.) 
The  number  of  its  antennal  segments  and  its  large  ocelli  are  male  characteristics. 
It  was  to  be  expected,  therefore,  that  male  reproductive  reactions  would  occur. 
Several  tests  at  different  times  failed  to  evince  any  response  toward  females  although 
the  specimen  appeared  healthy,  drank  honey  water  and  lived  for  several  days  until 
fixed  in  Carnoy  fluid.  Since-  it  likewise  failed  to  give  any  response  (female)  to 
caterpillars,  its  indifference  was  probably  not  due  to  its  sex  type  but  to  some  un- 
known factor. 

Because  of  the  small  "feminized"  genitalia  on  the  "male"  body,  freak  994  was 
at  first  recorded  as  a  "diploid  gynandroid  male."  ( 1\  nandroids,  however,  have 
always  been  haploids.  They  are  mosaic  males  in  which  the  two  sexually  different 
types  of  male  tissue  react  in  a  complementary  way  to  feminize  the  external  genitalia 
(Whiting,  Greb,  and  Speicher,  1934).  Their  mosaicism  is  shown  by  their  asym- 
metry, not  only  in  body  color,  in  number  of  antennal  segments,  in  mutant  traits, 
and  often  in  wing  length,  but  especially  in  the  external  genitalia  which  are  a  mixture 
of  normal  male  and  feminized  male  structures  with  much  reduplication  and  irregu- 
larity. In  freak  994  there  are  no  male  genital  structures  and  the  female  genitalia, 
consisting  of  a  pair  of  sensory  gonapophyses  with  no  visible  sting,  are  symmetrical 
and  larger  than  in  gynandroids.  They  are  much  smaller,  however,  than  the  female 
genitalia  found  in  gynanders  which  are  male-female  mosaics  with  clearly  separated 
male  and  female  regions.  That  freak  994  is  not  a  sex  mosaic  is  shown  by  its 
symmetry  in  body  coloration,  in  antennal  flagella  with  nineteen  segments  in  each 
and  in  length  of  wings  and  legs. 

Two  types  of  intersexes  have  hitherto  been  reported  in  Habrobracon.  (1) 
Gynoid,  dependent  upon  a  single  mutant  gene,  is  a  weakly  intersexual  male,  func- 
tioning normally  as  a  male,  but  having  certain  external  traits,  including  antennae, 
feminized.  (2)  Nine  intersexual  females  were  reported  (Whiting.  1943)  occurring 
in  a  single  fraternity.  "Superficially,  these  appear  to  be  the  reverse  of  the  gynoid 

243 


244 


P.  W.  WHITING 


males,  being  more  masculine  anteriorly,  feminine  posteriorly."  They  resemble 
freak  994  in  head  and  thorax  and  in  the  anterior  part  of  the  abdomen  which  are 
altogether  like  those  of  the  male.  In  the  posterior  region,  however,  the  sclerites  are 
thickened,  there  is  a  normal  sting  and  the  sensory  reproductive  appendages  are  of 
full  leneth  characteristic  of  the  female.  "The  nine  intersexual  females  must  be 


FIGURE  1 


regarded  as  UK  ire  strongly  intersexual  than  gynoid  males  since  antennae,  ocelli  and 
instincts  are  completely  sex  reversed."  Freak  994  is  an  intersexual  female,  com- 
parable to  these  nine  but  still  more  strongly  intersexual  because  of  greater  restric- 
tion of  the  "female"  region  and  reduction  of  the  genitalia. 

In  Habrobracon,  normal  haploid  males  have  cells  almost  as  large  as  the  corre- 
sponding cells  of  diploid  females  and  in  some  stocks  they  are  actually  larger  (Grosch, 


INTERSEX  IN  HABROBRAO  )\  245 

1945).  Cells  of  diploicl  males  are  much  larger  than  are  those  of  females  or  of 
haploicl  males.  These  relationships  have  heen  determined  by  counts  of  micro- 
chaetae  within  a  given  area  on  the  upper  surface  of  the  wings,  each  microchaeta 
corresponding  to  a  single  cell.  Study  of  the  dispersion  of  microchaetae  in  freak 
994  showed  its  cell  size  to  be  within  the  range  for  the  female  or  haploid  male  and 
therefore  much  smaller  than  that  characteristic  of  the  diploid  male.  The  marked 
shift  of  the  intersex  in  the  male  direction  does  not  then  affect  the  size  of  its  cells. 
It  may  be  fundamentally  female,  heterozygous  for  the  sex  factor.  This  condition 
perhaps  prevents  the  abnormal  expansion  of  cell  size  while  permitting  development 
of  antennae  and  ocelli  of  normal  male  type. 

The  nine  intersexual  females  previously  reported  had  internal  abdominal  struc- 
tures as  in  the  female  with  normal  poison  sac  and  glands  and  seminal  receptacle. 
Each  ovary,  however,  appeared  to  be  a  pair  of  sacs  of  oogonia  showing  no  differen- 
tiation of  nurse  cells  and  ova.  Serial  sections  were  made  of  the  abdomen  of  freak 
994  and  the  internal  structures  were  studied.  The  digestive  tract  is  entirely  normal 
with  the  crop  greatly  distended  from  honey  water  feeding.  A  poison  apparatus  is 
present  but  imperfectly  developed  and  situated  near  the  median  plane,  directly 
dorsal  to  the  compound  posterior  nerve  ganglion  instead  of  being  shifted  laterad  to 
the  digestive  tract.  The  poison  glands  are  normal  although  of  somewhat  small  size. 
Their  ducts  converge  to  a  common  duct  connecting  distally  with  an  imperfect  poison 
"sac"  and  proxiinally  traversing  the  very  short  distance  to  the  region  where  nor- 
mally lies  the  root  of  the  sting.  The  poison  ''sac,"  of  approximately  normal  length, 
is  reduced  in  diameter  to  an  irregularly  scleroti/.ed  strand.  It  is  surrounded  In- 
longitudinal  muscles  as  in  a  normal  female.  Nothing  corresponding  to  a  seminal 
receptacle  could  be  located,  nor  were  any  gonads  to  br  found.  The  tat  body  appears 
normal,  surrounding  the  digestive  tract  and  the  poison  apparatus  dorsally  and 
laterally. 

DISCUSSION 

In  the  report  on  the  nine  intersexual  females,  it  was  suggested  that  they  might 
be  accounted  for  by  a  dominant  mutation  in  a  sex  allele  changing  .vb  to  xbm.  The 
intersexes  would  then  be  modified  females,  .ni  .vb"1.  A  similar  hypothesis  would 
cover  freak  994.  but  here  the  mutation  may  have  been  x-ray  induced  and  more 
potent  than  in  the  previous  case  so  that  the  intersexuality  would  be  more  extreme 
with  turning-point  earlier  in  development. 

Failure  to  find  gonads  in  freak  994  does  not  necessarily  mean  that  they  were 
lacking  from  the  beginning  for  they  may  have  begun  development  and  then  disin- 
tegrated. 

Comparison  may  be  made  between  freak  994  and  certain  types  of  "deficient" 
individuals  previously  reported  in  Habrobracon  (Whiting,  1926).  Some  of  the 
"deficient"  had  external  genitalia  lacking  but  gonads  present.  Others  had  testes 
of  reduced  size,  or  present  on  one  side,  lacking  on  the  other.  Some  of  the  "de- 
ficient" females  with  no  trace  of  poison  apparatus  had  well  differentiated  ovaries 
with  eggs  and  nurse  cells.  This  is  just  opposite  to  the  condition  found  in  the 
intersexual  female,  freak  994.  There  was  no  intersexuality  among  the  "deficient." 


24f)  P.  W.  WHITING 

SUMMARY 

An  inter  sexual  female  developed  from  a  heavily  x-rayed  egg  fertilized  by  an 
untreated  sperm.  The  specimen  is  more  strongly  intersexual  than  a  group  of  nine 
previously  reported,  for  its  external  female  genitalia  are  much  reduced,  its  poison 
apparatus  defective  and  its  ovaries  altogether  lacking.  Externally,  it  appears  like 
a  diploid  male  with  small  female  genitalia. 

It  is  suggested  that  the  x-radiation  may  have  caused  a  change  within  a  sex- 
differentiating  allele,  so  that  the  heterozygote  would  develop  into  an  intersex  rather 
than  a  normal  female. 

LITERATURE  CITED 

GROSCH,  D.  S.,  1945.     The  relation  of  cell  size  and  organ  size  to  mortality  in  Habrobracon. 

Growth,  9:  1-17. 
WHITING,  P.  W.,  1926.     Influence  of  age  of  mother  on  appearance  of  an  hereditary  variation 

in  Habrobracon.     Biol.  Bull..  51 :  371-385. 
WHITING,  P.  W.,   1943.     Tntersexual   females  and   inUTst-xuality   in   Habrobracon.     Biol.   Bull,. 

85:  238-243. 
VYnnrxr,,  P.  W..  RAYMOND  J.  GREB  AND  B.  I\.  Sri K  HKR,  1934.     A  nr\v  type  of  sex-intergrade. 

Biol  Bull..  66:  152-165. 


LOCI  OF  ACTION  OF  DDT  IN  THE  COCKROACH 
(PERIPLANETA  AMERICANA) 

J.  M.  TOBIAS  AND  J.  J.  KOLLROS  * 
University  of  CJiicago  To.ricity  Laboratory^  and  the  Department  of  Physiology 

In  the  cockroach.  DDT  produces  symptoms  which  clearly  reflect  involvement 
of  the  neuromuscular  apparatus.  These  are  qualitatively  much  the  same  in  all 
arthropods  which  have  been  studied,  though  there  are  important  quantitative  dif- 
ferences. Thus,  in  any  given  animal  the  time  course  of  the  poisoning  is  a  function 
of  dose,  and  for  a  dose  of  comparable  toxicity  in  terms  of  final  mortality,  the  symp- 
toms unfold  and  death  occurs  much  more  rapidly  in  some  insects  (the  fly)  than  in 
others  (the  roach)  (Tobias,  Kollros,  and  Savit,  1946a).  In  the  roach,  the  sequence 
of  symptoms  is  initiated  by  hyperextension  of  the  legs,  elevation  of  the  center  of 
gravity  and  development  of  postural  instability.  The  hyperextension  then  decreases 
and  is  superseded  by  increasing  and  generalized  tremulousness,  involving  the  head, 
body,  and  all  appendages ;  the  gait  becomes  ataxic,  and  minor  stimuli  of  sound  or 
touch  result  in  great  hyperactivity,  exhibited  mainly  in  running  and  climbing.  The 
animal  falls  on  its  back  time  after  time  until  finally  it  can  no  longer  right  itself.  Leg 
movements  continue  in  the  supine  insect  with  two  components,  a  high  frequency 
intermittent  tremulousness  and  a  slower  incoordinated  flexion  and  extension.  These 
two  types  of  activity  possibly  reflect  the  double  innervation  which  has  been  described 
for  cockroach  muscle  (Pringle,  1939),  one  fiber  type  producing  relatively  slow  tonic 
contractions,  the  other  producing  relatively  fast  twitches.  It  will  be  seen  later  that 
after  poisoning  these  two  types  of  movement  can  be  independently  altered.  Activity 
finally  diminishes  progressively.  The  fast  tremors  disappear  first  and  finally  there 
remain  only  occasional  isolated  movements  of  body  wall,  tarsi,  palpi,  cerci,  or  an- 
tennae. When  no  further  somatic  movement  can  be  detected,  the  heart  usually 
continues  to  beat  for  some  time,  and  electrical  stimulation  of  the  nerve  cord  may  still 
evoke  muscle  responses.  The  animal  may  live  in  this  condition  for  a  day  or  so  and 
finally  die. 

Mammals  exhibit  a  similar  symptomatology  up  to  a  point.  In  the  rat  and  dog, 
given  DDT  intravenously  or  orally,  muscular  fibrillations  and  excessive  blinking  are 
followed  by  tremulousness,  ataxia,  falling  and  gross  convulsive  seizures.  The  ani- 
mal may  have  a  number  of  convulsions  and  die  in  the  tonic  phase  of  one  or  recover 
after  gradual  subsidence  of  symptoms.  There  is  no  period  of  prostration  and  nearly 
complete  immobility  as  in  the  insect,  because  death  occurs  when  systematic  respira- 
tory movements  cease.  In  the  insect,  the  small  amount  of  body  movement  and 
twitching  sufficiently  augment  diffusion  for  respiratory  exchange.  Then  too,  the 
insect  is  far  more  resistant  to  anoxia  than  is  the  mammal  (Wigglesworth.  1939). 

*  Department  of  Zoology  and  The  College.  Present  address.  Zoology  Department,  Uni- 
versity of  Iowa. 

t  This  work  was  carried  out  under  contract  with  the  Medical  Division  of  the  Chemical 
Warfare  Service. 

247 


248  J.  M.  TOBIAS  AND  J.  J.  KOLLROS 

The  frog,  as  might  be  expected,  responds  more  like  the  insect  than  the  mammal 
(Tobias,  Kollros,  and  Savit,  1946b).  Respiratory  exchange  through  the  skin  can 
sustain  life,  and,  after  a  period  of  hyperirritability,  the  animal  lies  prostrate  and  more 
or  less  immobile.  Such  symptomatology  has  prompted  a  number  of  investigations 
designed  to  discover  a  locus  of  action  of  DDT.  As  will  be  seen,  there  probably  are 
a  number  of  sites  of  action  depending  largely  on  dosage,  but  this  point  of  view  was 
only  gradually  attained. 

In  mammals  (Crescitelli  and  Oilman,  1946),  DDT  apparently  does  not  act  di- 
rectly on  either  muscle,  myoneural  junction  or  spinal  cord.  Since  tremors  persist 
after  decerebration  and  mesencephalic  transection,  and  since  abnormal  cerebral  and 
cerebellar  electrical  activity  persists  after  atlanto-occipital  transection,  neither  cere- 
bral cortex  nor  basal  ganglia  can  be  a  critical  site  of  action,  and  intact  spinal  afferents 
are  obviously  not  necessary  lor  the  central  effect.  The  cerebellum  is  considered,  by 
these  authors  to  be  the  most  likely  critical  site  of  action  in  the  mammal.  Locus  of 
action  has  also  been  investigated  in  insects.  In  Drosophila  (Bodenstein,  1946). 
DDT  seems  not  to  act  on  muscle  or  myoneural  junction,  but  does  act  on  peripheral 
nerve  and  may  act  on  the  central  nervous  system.  In  the  cockroach  (Periplaneta 
americana},  DDT  has  been  found  to  act  on  nerve  in  high  concentrations  (Yeager 
and  Munson,  1945),  and,  in  low  concentrations,  on  peripheral  receptors  (probably 
proprioceptors)  (Roeder  and  Weiant,  1946).  The  latter  workers  also  have  evi- 
dence which  they  interpret  to  mean  that  high  concentrations  may  act  directly  on 
either  the  myoneural  junction  or  muscle  itself.  In  the  crab  (Cancer  irroratus)  there 
is  evidence  for  action  on  motor  nerves  (Welsh,  1946). 

It  was  the  purpose  of  this  study  to  further  investigate  loci  of  action  of  DDT  in 
an  insect.  Because  of  its  large  size  and  ready  availability,  the  cockroach  (Peri- 
planeta americana}  was  used  throughout. 

METHODS 

Cockroaches  were  immobilized  by  exposure  to  100  per  cent  CO2  for  20-60  sec- 
onds or  by  etherization.  After  CO2,  anesthesia  seldom  lasted  over  a  minute.  Once 
anesthetized,  the  roach  was  fastened  to  a  bit  of  cardboard  by  pins  passed  through 
either  side  of  the  pronotum.  Appendages  were  held  in  any  desired  position  by 
pins  crossed  over  the  body. 

Decapitation  was  easily  achieved  by  simply  cutting  the  neck  with  a  small  scissors. 
The  exposed  stump  was  sealed  with  low  melting-point  paraffin.  Ligation  of  the 
neck  prior  to  decapitation  to  prevent  loss  of  hemolymph  did  not  prolong  survival 
time.  Such  animals  live  about  60  hours  (Table  I). 

To  expose  a  thoracic  ganglion,  the  spinasternum  just  caudal  to  the  ganglion 
was  cut  through,  and  the  incision  extended  along  the  sides  of  the  sternal  plate.  After 
the  plate  was  reflected  forward,  removal  of  superficial  fatty  tissue  and  tracheal  tubes 
fully  exposed  the  ganglion.  The  connectives  anterior  to  the  ganglion  were  held  in 
a  jeweler's  forceps  and  cut  with  iridectomy  scissors.  Traction  on  the  connectives 
exposed  the  lateral  nerves,  which  were  sectioned.  Finally,  the  posterior  connectives 
were  cut  and  the  ganglion  removed.  Simple  isolation  of  a  ganglion  from  the  rest 
of  the  nerve  cord  can  be  achieved  without  excising  it  by  cutting  the  connectives 
through  slits  in  the  cuticle.  Complete  transection  of  the  entire  roach  between  sets 


LOCI  OF  ACTION  OF  DDT 


249 


TABLE  J 

Effect  of  lesions  of  the  central  nervous  system  on  symptoms  of  DDT  poisoning  in  the  cockroach 


Operation 

No.  roaches 

Aver- 
age 
sur- 
vival, 

hours 

General  results 

Legs  which  showed 
DDT 
effects 

Con- 
trols, 
no 
DDT 

Operated 

Nature 

Site 
(See  Fig.  1  ) 

Before 
DDT 

After 
DDT 

Hyperactivity,  tremors 
and  convulsions 

Decapitation 

At  A 

14 

14 

14 

61 

56 
56 

Rare  tremors  in  3  ani- 
mals —  no  convulsive 
activity 
Typical    DDT    effects 
in  all  animals 
Typical    DDT    effects 
in  all  animals 

None 

All 
All 

Transaction  of 
ventral  nerve 
cord 

At  C  and  D  (both 
anterior  and  pos- 
terior to  thoracic 
ganglion  no.  2) 

16 

15 

— 

104 
60 

None  in  any  animals 

Typical    DDT    effects 
in  all  animals 

None 

All    in    13    animals;    2nd 
and   3rd   pairs   in   two 
animals 

Destruction  of 
ganglion 

Th.  2  (thoracic 
ganglion  no.  2) 

15 

IS 

— 

77 
71 

None  in  any  animals 

Typical    DDT    effects 
in  all  animals 

Leg  2  paralyzed  in  all 

1    and  3   in    13   animals. 
1,  2  and  3  in  two  ani- 
mals 

of  legs  results  in  an  isolated  segment  containing  a  ganglion,  nerves  and  the  attached 
legs.  Such  a  preparation,  if  kept  moist,  is  viable  for  at  least  6  to  8  hours. 

Excision  of  the  heart  largely  prevents  circulatory  removal  of  substances  applied 
to  structures  to  elicit  a  local  effect.  Longitudinal  incisions  through  the  cuticle,  on 
either  side  of  the  heart  tube  along  its  entire  length,  isolate  a  strip  whose  removal 
carries  the  heart  with  it.  The  heart  may  be  cauterized  with  equal  ease  (Yeager  and 
Munson,  1945). 

Methods  for  the  administration  of  measured  doses  of  DDT  to  insects  have  been 
described  elsewhere  (Tobias,  Kollros,  and  Savit,  1946a). 

RESULTS 
Localisation  experiments  with  uncontrolled  DDT  doses 

Except  where  otherwise  specified,  contact  poisoning  was  carried  out  by  confining 
the  roach  for  5-15  minutes  within  a  glass  cylinder  coated  with  DDT  previously  pre- 
cipitated from  acetone  solution. 

Roaches  decapitated  before  or  after  such  contact  with  DDT  behaved  like  intact 
poisoned  animals  (Table  I).  Therefore,  neither  the  supra-  nor  the  sub-oesophageal 
ganglia  are  essential  for  the  development  or  maintenance  of  DDT-induced  motor 
activity  in  the  legs  or  body.  Ventral  nerve  cord  connectives  were  transected  both 
anterior  and  posterior  to  the  mesothoracic  ganglion  (Fig.  1,  levels  C  and  D).  Ani- 
mals so  prepared  but  given  no  DDT  showed  incoordination  of  the  mesothoracic  legs 
when  walking,  but  there  were  no  symptoms  which  could  be  confused  with  those 
of  DDT  poisoning.  When  such  animals  were  subsequently  poisoned,  however,  the 
mesothoracic  as  well  as  the  other  legs  exhibited  typical  abnormal  activity  (Table  I). 
After  complete  transection  of  the  whole  body  of  the  poisoned  roach,  at  both  these 
levels  (excised  segment  Fig.  1),  leg  tremulousness  and  hyperactivity  continued  un- 


250. 


J.  M.  TOBIAS  AND  J.  J.  KOLLROS 


abated  in  the  isolated  segment.  The  application  of  DDT  emulsion  or  DDT  in  ace- 
tone to  the  cut  surface  of  such  segments  obtained  from  normal  roaches  evoked  typical 
DDT  effects  in  the  attached  legs  within  a  few  minutes.  The  same  was  true  of  DDT 
applied  directly  to  the  exposed  ganglion  in  the  otherwise  intact  animal.  Emulsion 
or  acetone  without  DDT  had  no  such  effect. 

The  cells  of  origin  of  the  leg  nerves  lie  within  the  lateral  halves  of  the  thoracic 
ganglia,  each  ganglion  in  the  adult  being  formed  by  the  midline  fusion  of  two  em- 
bryonic ganglion  masses.  Median  sagittal  section  of  the  ganglion  in  a  poisoned 
roach  (Fig.  1.  level  F)  did  not  stop  hyperactivity  in  either  of  the  legs  innervated 


FIGURE  1.     Levels  of  section  in  cockroach  nervous  system. 

from  the  resulting  ganglion  halves.     Therefore,  even  half  a  segment  contains  all  the 
structures  necessary  for  the  maintenance  of  DDT  symptoms  in  a  leg. 

If,  however,  the  entire  ganglion  was  removed  the  results  were  generally  quite 
different.  Mesothoracic  ganglia  were  removed  from  thirty  normal  roaches.  The 
corresponding  legs  of  all  were  paralyzed  and  failed  to  respond  to  touch  or  pressure. 
Shortly  after  the  operation,  fifteen  of  the  animals  were  contact  poisoned.  All 
showed  typical  DDT  effects  in  the  pro-  and  metathoracic  legs,  but  the  ganglionecto- 
mized  mesothoracic  legs  remained  entirely  quiet  in  thirteen  and  showed  only  occa- 
sional tarsal  twitching  and  some  slight  movement  of  the  other  joints  in  two.  Simi- 
larly, ganglionectomy  after  the  development  of  hyperactivity,  rather  than  before 


LOCI  OF  ACTION  OF  DDT 


251 


TABLE  II 

Experiments  on  isolated  roach  segments  containing  local  ganglion,  nerves,  and  legs 


No.  of 
segments 


6 
3 

7 


4 


Material  applied 


Nothing 

Emulsion*  without  DDT 

Acetone  without  DDT 


1  Per  cent  DDT  emulsion* 
10  Per  cent  DDT  in  acetone 

DDT  powder 


Route 


On  cut  surface 
Injected 


On  ganglion 
Injected  into  vicinity 

of  ganglion 
On  ganglion 


Number  of  segments  in  which 

there  was  persistent  DDT 

leg  activity 


None 
None 
None 


Occurred  in  all 
Occurred  in  all 

Occurred    questionably    in 
one 


*  Emulsion — 1  per  cent  DDT,  10  per  cent  peanut  oil,  1  per  cent  lecithin  and  88  per  cent 
0.90  percent  NaCI  (5). 

poisoning,  either  stopped  or  markedly  reduced  symptoms  in  the  corresponding  legs 
(Table  IV).  As  was  to  be  expected  from  these  experiments,  section  of  leg  nerves 
lateral  to  the  ganglion  stopped  or  markedly  reduced  activity  in  many  (65  per  cent) 
of  the  legs  (Table  III). 

These  experiments  tentatively  suggested  that  the  ventral  cord  ganglion  was 
critically  involved  in  the  motor  action  of  DDT  and  might  itself  be  a  site  of  action. 
Conflicting  data,  however,  were  also  obtained.  It  was  possible,  as  also  reported  by 
others  (Yeager  and  Munson,  1945;  Roeder  and  Weiant.  1946).  to  produce  motor 


TABLE  III 

Visible  effect  of  DDT  on  amputated  legs 
(Dose  not  controlled) 


No.  of 
legs 


30 


58 


12 
22 
35 


Source  of  legs 


Normal  roaches 


DDT  poisoned 
roaches  tremulous 
and  hyperactive 


Normal  roaches 
Normal  roaches 
Normal  roaches 


Treatment  after  amputation 


Normal  controls 


Emulsion  without  DDT  in- 
jected into  cut  end 

Emulsion  with  1  per  cent 
DDT  applied  to  cut  end 

Emulsion  with  1  per  cent 
DDT  injected  into  cut  end 


Results  after  amputation 


No  spontaneous  movement 


Continued  activity  in  20.      No 
movement  in  38 


No  movement  in  any 
Movement  in  1,  others  all  quiet 
Movement  in  25,  other  10  quiet 


9 
13 


Normal  roaches 
Normal  roaches 


Acetone  without  DDT  in- 
jected into  cut  end 

Acetone  with  DDT  injected 
into  cut  end 


Movement  in  1,  other  8  quiet 
Movement  in  3,  other  10  quiet 


252 


J.  M.  TOBIAS  AND  J.  J.  KOLLROS 


activity  in  a  large  percentage  of  amputated  legs  by  the  injection  of  DDT  emulsion 
(1  per  cent  DDT,  1  per  cent  lecithin,  10  per  cent  peanut  oil,  and  88  per  cent  0.9 
per  cent  NaC!  solution).  It  will  also  he  recalled  that  ganglionectomy  failed  to  en- 
tirely quiet  the  legs  in  two  of  fifteen  experiments  (Table  T). 

Such  conflicting  data  were  difficult  to  interpret.  Ganglionectomy  or  denerva- 
tion  usually  stopped  leg  activity,  but  this  was  not  invariably  the  case,  and  it  was 
possible  to  produce  activity  in  the  amputated  legs  by  injection  of  DDT.  It  was 
suspected  that  such  results  might  be  resolved  in  terms  of  DDT  dose.  Further 
experiments  were  then  done  with  measured  doses  of  DDT. 

Localization  experiments  with  controlled  doses  of  DDT 

It  was  immediately  found  that  the  effectiveness  of  ganglionectomy  in  abolishing 
motor  effects  was  inversely  related  to  dose  (Table  IV).  That  is,  as  the  dose  of 
DDT  was  increased  ganglionectomy  stopped  movement  in  progressively  fewer  cases. 

TABLI-:   IV 

Effect  of  ganglionectomy  on  symptoms  after  various  doses  of  DDT 


Results  of  ganglionectomy 

No. 

experi- 

DDT* 

ments 

Number  resulting  in  complete 

Number  resulting  in  a 

cessation  of  activity 

reduction  of  activity 

10 

Usual  moderate  contact  dose  (5-10  mins. 

70% 

30% 

in  DDT  coated  tube) 

5 

Excessive  contact  dose   (approximately 

20% 

80% 

2  hours  in  DDT  coated  tube) 

25 

5-30  mg.   DDT  per  kg.  injected  intra- 

68% 

32% 

abdominally  in  emulsion*" 

12 

60-70  mg.  DDT  per  kg.  injected  intra- 

50% 

50% 

abdominally  in  emulsion** 

15 

130  mg.    DDT   per   kg.   injected   intra- 

7% 

93% 

abdominally  in  emulsion** 

*  LD-50  for  DDT  injected  intra-abdominally  in  emulsion  is  20  mg.  per  kg.  (Tobias,  Kollros, 
and  Savit,  1946a). 

**  Emulsion — 1  per  cent  DDT,  1  per  cent  lecithin,  10  per  cent  peanut  oil,  88  per  cent  of 
0.9  per  cent  NaCl. 

In  all  cases,  however,  even  when  movements  were  not  entirely  stopped  they  were 
both  qualitatively  and  quantitatively  changed.  The  high  frequency  tremulousness 
was  always  markedly  reduced  or  entirely  abolished  and  the  slower  movements 
were  much  diminished. 

Nicotine,  in  low  concentrations,  is  known  to  block  synaptic  transmission  cen- 
trally as  well  as  peripherally  (Libet  and  Gerard,  1938;  Pringle,  1939)  but  not 
axonal  transmission.  When  applied  to  the  cockroach  ganglion  there  is  an  initial 
burst  of  electrical  hyperactivity  (100-800  impulses  per  sec.)  followed  by  electrical 
silence  (Pringle,  1939).  As  would  be  expected,  such  application  of  nicotine  to  a 
ganglion  also  produces  great  motor  hyperactivity  in  the  attached  leg  which  can  be 
abolished  by  amputating  the  leg  (Yeager  and  Munson,  1945). 

Now  then,  if  nicotine  applied  to  a  ganglion  in  a  concentration  which  did  not 


LOCI  OF  ACTION  OF  DDT 


253 


affect  peripheral  nerve  were  to  stop  DDT  symptoms,  this  would  be  added  evidence 
for  the  importance  of  the  ganglionic  cell  bodies  or  synapses  in  the  development  and 
maintenance  of  such  symptoms.  After  poisoned  roaches  became  hyperactive  the 
heart  was  excised.  This  did  not  decrease  activity,  but  served  to  greatly  diminish 
circulatory  transport  of  solutions  applied  for  local  effects.  Solutions  were  then 
applied  as  small  droplets  to  the  ganglion  or  a  region  of  leg  nerve  exposed  by  cuticle 
excision. 

Dilute  nicotine  solutions  (0.01  per  cent  in  insect  Ringer)  applied  to  the  leg 
nerves  of  the  normal  or  poisoned  roach  did  not  paralyze  the  leg.  Typical  DDT 
induced  activity  could  not  be  stopped  in  this  fashion.  This  was  almost  surely  not 
due  to  failure  of  nicotine  to  reach  the  nerve  since  spontaneous  movement  as  well 
as  that  following  electrical  stimulation  of  the  ganglion  could  be  stopped  by  a  similar 

TABLE  V 

Effect  of  locally*  applied  nicotine  and  novocaine  on  motor  symptoms  of 
DDT  poisoning  after  various  doses  of  DDT 


No. 
experi- 
ments 

DDT** 

Number  of  experiments  in  which  activity  was  modified 

1.0%  novocaine 

0.01%  nicotine 

Injected  into  tibia 

Injected  into  tibia 

Applied  to  ganglion 

Complete  inactivity 

Complete  inactivity 
or  reduced  activity 

Activity 
stopped 

Activity 
reduced 

4 

10-30  mg.  per  kg.  applied 
to  body  surface  18  hours 
before 

100% 

o% 

100% 

o% 

9 

100  mg.  per  kg.  applied  to 
body  surface  18  hours  be- 
fore 

100% 

0% 

77% 

22% 

13 

4 

500  mg.  per  kg.  applied  to 
body  surface 
1000  mg.  per  kg.  applied  to 
body  surface 

23% 
0% 

77% 
100% 

*  All  experiments  on  cardiectomized  roaches  to  prevent  circulatory  removal  of  substances 
applied  for  local  effect. 

**  DDT  applied  to  surface  in  acetone.  LD-50  for  DDT  so  applied  is  10  mg.  per  kg.  (Tobias, 
Kollros,  and  Savit,  1946a). 

administration  of  1  per  cent  novocaine.  When,  however,  this  nicotine  solution  was 
applied  to  a  ganglion  (in  the  same  normal  or  poisoned  animal  in  which  it  was  in- 
effective on  peripheral  nerve)  there  was  a  short-lived  burst  of  great  activity  in  the 
legs  of  the  segment,  followed  by  complete  immobility  or  markedly  decreased  activity. 
Since  the  nicotine  was  effective  in  concentrations  which  did  not  block  peripheral 
nerve,  it  was  concluded  that  it  was  acting  by  blocking  ganglionic  synapses  and  not 
by  spill-over  to  the  emerging  nerve  roots.  It  is  clear  (Table  V)  that,  as  in  the 
case  of  ganglionectomy,  the  immobilizing  effect  of  nicotine  decreased  as  the  dose 
of  DDT  increased,  and,  as  was  also  true  after  ganglionectomy,  if  nicotine  did  not 
stop  activity  it  considerably  decreased  and  modified  it. 


254  J.  M.  TOBIAS  AND  J.  J.  KOLLROS 

DISCUSSION 

It  is  clear  that  DDT  can  produce  motor  symptoms  l>y  effects  peripheral  to  the 
ganglion.  It  is  equally  clear,  however,  that  the  ganglion  plays  a  role  in  the 
initiation  and  maintenance  of  symptoms  and  that  this  role  is  to  some  extent 
dependent  upon  DDT  dose. 

Roecler  and  Weiant  (1946)  found  that,  in  the  cockroach,  very  low  concentra- 
tions of  DDT  can  initiate  centripetally  directed,  high  frequency  (300-400  per  sec.), 
temporally  irregular  bursts  of  nerve  impulses,  presumably  excited  by  action  of 
DDT  on  the  campaniform  sensilla  (presumptive  proprioceptors).  There  was  no 
evidence  of  any  muscle  movement  which  might  have  initiated  such  centrally  directed 
impulses.  Welsh  (1946)  has  demonstrated  that  DDT  in  very  low  concentrations 
can  also  favor  repetitive  response  of  motor  fibers  (Cancer  irroratus]  to  a  stimulus 
normally  evoking  single  responses,  and  Yeager  and  M  tin  son  (1945)  have  concluded 
that  high  concentrations  can  produce  similar  changes  in  the  cockroach. 

The  results  of  ganglionectomy,  in  the  cockroach  (surgical  or  nicotine  inacti- 
vated), after  various  doses  of  DDT,  support  the  view  that  the  initiation  and  con- 
tinuation of  the  hypermotor  symptoms  of  DDT  poisoning  after  low  doses  of  DDT 
require  an  intact  sensori-motor  reflex  arc,  and  that  random  afferent  impulses  in 
sensory  nerves  may  indeed,  as  suggested  by  Roeder  and  Weiant,  excite  motor 
neurones  in  the  ganglion  to  initiate  incoordinated  muscular  activity.  From  the 
experiments  here  reported,  this  would  appear  to  be  a  part  of  the  common  sequence 
of  changes  in  the  roach  poisoned  by  uncontrolled  contact  doses.  The  fact  that 
ganglionectomy  becomes  less  and  less  effective  as  the  dose  of  DDT  is  progressively 
increased  would  suggest  that  larger  amounts  of  DDT  may  act  directly  on  motor 
nerves.  Obviously,  these  data  do  not  rule  out  a  possible  direct  action  on  muscle. 
Within  the  dose  ranges  which  have  been  used  there  is,  however,  no  conclusive  evi- 
dence for  a  direct  action  on  muscle.  This  is  not  to  say  that  such  action  could  not 
occur  at  some  sufficient  dosage  level.  In  addition,  ganglionectomy  is  seen  to  have 
stopped  the  rapid  tremulousness  after  any  dose  which  was  tried,  suggesting  that 
the  high  frequency  movements  may  be  initiated  reflexly  rather  than  by  direct  action 
on  motor  fibers  at  large  as  well  as  at  low  doses  of  DDT.  This  general  picture  is 
compatible  with  the  subsidence  of  high  frequency  tremulousness  before  subsidence 
of  slower  muscular  activity.  If  the  former  is  reflex  and  the  latter  due  to  direct 
nerve  action  one  might  expect  this  order  of  dropping  out  on  the  basis  of  much 
greater  fatigability  for  the  reflex  arc  than  for  the  nerve  trunk. 

Pattern  development  of  symptoms 

•  It  has  been  claimed  (Laiiger,  Martin,  and  Miiller,  1944)  that  if  DDT  be  put 
on  one  leg  of  a  fly  the  development  and  progression  of  symptoms  follow  a  definite, 
orderly  and  reproducible  path  from  leg  to  leg.  Such  a  phenomenon  might  be  very 
important  indeed  for  an  understanding  of  the  mechanism  of  DDT  action.  The 
authors  have  not  been  able  to  confirm  this  finding. 

CONCLUSIONS 

1.  Neither  decapitation,  section  of  one  or  several  nerve  cord  connectives  nor 
complete  transection  of  the  entire  insect  body  at  one  or  several  levels  between  nerve 
cord  ganglia  prevents  the  development  of  the  typical  motor  effects  of  DDT  in  any 


LOCI  OF  ACTION  OF  DDT  255 

of  the  legs  of  the  cockroach.  After  combined  antero-posterior  isolation  of  a  nerve 
cord  ganglion,  even  median  sagittal  section  of  the  ganglion  does  not  prevent  motor 
symptoms  in  the  legs  still  attached  to  the  lateral  ganglionic  cell  masses.  There- 
fore, the  anatomical  elements  necessary  for  development  of  the  motor  symptoms  of 
DDT  are  contained  within  the  lateral  half  of  a  body  segment  which  contains  the 
lateral  half  of  a  ganglion,  leg  nerves  and  peripheral  structures. 

2.  Since  the  motor  symptoms  of  DDT  poisoning  can  occur  in  amputated  legs, 
in  legs  whose  nerves  have  been  cut,  and  in  legs  whose  segmental  ganglia  have  been 
destroyed,  it  is  possible  for  DDT  to  produce  its  motor  effects  by  action  on  some 
structure  or  structures  peripheral  to  the  segmental  ganglion. 

3.  The  motor  symptoms  of  DDT  poisoning  can  be  stopped  or  diminished  in  a 
leg  by  ganglionectomy,  leg  nerve  section,  or  ganglion  synaptic  block  with  nicotine. 
The  effectiveness  of  these  procedures  is  in  inverse  relation  to  the  dose  of  DDT 
administered.     These  findings  suggest  that,  in  the  cockroach,  low  doses  of  DDT 
may  excite  motor  fibers  reflexly  by  impulses  fired  into  the  ganglion  over  afferent 
nerve  fibers,  whereas  high  doses  may  act  on  elements  on  the  motor  side  of  the 
ganglion  and  thus  not  require  an  intact  reflex  arc.     Since  ganglionectomy  stops  the 
fast  component  of  the  hypermotor  activity,  however,  equally  well  after  large  or 
small  doses  of   DDT,  this  component  may  be   reflexly   initiated   and   maintained 
after  all  doses  of  DDT. 

LITERATURE  CITED 

BODENSTEIN,   D.,   1946.     Investigation  on  the  locus  of  action  of  DDT   in   flies    (Drosophila). 

Biol.  Bull,  90:  148. 
CRESCITELLI,  F.  AND  A.  OILMAN,  1946.     Electrical  manifestations  of  the  cerebellum  and  cerebral 

cortex  following   DDT   administration   in   cats   and   monkeys.     Amer.   Jour.   Physiol., 

147:  127. 
LAUGER,  P.,  H.  MARTIN  AND  P.  MULLER,  1944.     The  constitution  and  toxic  effect  of  botanicals 

and  new  synthetic  insecticides.     Hclv.  Chim.  Acta,  27. 
LIBET,  B.  AND  R.  W.  GERARD,  1938.     Automaticity  of  central  neurones  after  nicotine  block  of 

synapses.     Proc.  Soc.  Exp.  Biol.  and  Mcd.,  38 :  886. 

PRINGLE,  J.  W.  A.,  1939.     The  motor  mechanism  of  the  insect  leg.    Jour.  Exp.  Biol.,  16 :  220. 
ROEDER,  K.  D.  AND  E.  A.  WEIANT,  1946.    The  site  of  action  of  DDT  in  the  cockroach.    Sci- 
ence, 103 :  304. 
SAVIT,  J.,  J.  J.  KOLLROS  AND  J.  M.  TOBIAS,  1946.     The  measured  dose  of  gamma  hexachloro- 

cyclohexane  (y  666)  required  to  kill  flies  and  cockroaches  and  a  comparison  with  DDT. 

Proc.  Soc.  Exp.  Biol.  and  Mcd.,  62 :  44. 
TOBIAS,  J.  M.,  J.  J.  KOLLROS  AND  J.  SAVIT,  1946a.     Relation  of  absorbability  to  the  comparative 

toxicity  of  DDT  for  insects  and  mammals.     Jour.  Pharm.  and  Exp.  Ther.,  86 :  287. 
TOBIAS,  J.  M.,  J.  J.  KOLLROS  AND  J.  SAVIT,   1946b.     Acetylcholine  and  related  substances  in 

the  cockroach,  fly  and  crayfish  and  the  effect  of  DDT.    Jour.   Cell.   Comp.  Physiol. 

(In  press.) 

WELSH,  J.  H.,  1946.     Personal  communication. 
WIGGLESWORTH,  V.  B.,  1939.     The  principles  of  insect  physiology.     Dutton  and  Co.,  Inc..  New 

York. 
YEAGER,  J.  F.  AND  S.  C.  MUNSON,  1945.     Physiological  evidence  of  a  site  of  action  of  DDT 

in  an  insect.     Science,  102 :  305. 


TILLINA  MAGNA:  MICRONUCLEAR  NUMBER,  ENCYSTMENT 

AND  VITALITY  IN  DIVERSE  CLONES;  CAPABILITIES 

OF  AMICRONUCLEATE  RACES 

C.  D.  BEERS 

Department  of  Zoology,  University  oj  North  Carolina,  Chapel  Hill 

It  is  well  established  that  the  number  of  micronuclei  in  Tillina  magna  is  highly 
variable.  For  example,  Gregory  (1909)  found  6-10,  and  Ilowaisky  (1921),  in  a 
ciliate  which  he  called  Pseudocolpoda  cochlearis  cicnkoivskii,  reported  2-6.  An  ex- 
amination of  Ilowaisky's  text  and  figures  shows  conclusively  that  his  ciliate  was  T. 
magna  Gruber  (1879)  as  Kahl  (1931,  p.  282)  pointed  out.  Kahl  apparently  re- 
garded six  as  the  typical  number.  Bresslau  (1922)  observed  the  nuclei  in  sufficient 
detail  to  note  the  extrusion  of  macronuclear  material  at  division  and  the  presence  of 
several  micronuclei,  though  he  reported  no  counts  of  their  actual  number.  The 
writer  (1946),  in  a  study  dealing  chiefly  with  the  history  of  the  nuclei  during  divi- 
sion and  encystment,  counted  the  number  of  micronuclei  in  100  individuals  (50  active 
and  50  encysted)  of  each  of  three  clones,  and  found  that  it  varied  from  6  to  11  in  one 
clone  and  from  4  to  6  in  the  other  two.  Thus  the  number  was  found  to  vary  in  dif- 
ferent individuals  of  the  same  clone,  and  the  mean  number  was  found  to  vary  in  dif- 
ferent clones.  Active  specimens  and  resting  cysts  of  any  particular  clone  had  on  the 
average  like  numbers  of  micronuclei.  Contrary  to  statements  in  the  literature,  it 
was  shown  that  the  micronuclei  divide  at  the  time  of  cell  division,  and  not  indiscrimi- 
nately or  without  regard  to  cell  division.  The  mechanism  by  which  two  daughter 
cells  may  receive  unlike  numbers  of  micronuclei  at  division,  thus  accounting  for  vari- 
ations in  number  within  a  clone,  was  described. 

The  significance  of  the  wide  variation  in  micronuclear  number  is  unexplained. 
Structurally  and  physiologically  an  individual  having  only  4  micronuclei  does  not 
appear  to  be  fundamentally  unlike  one  having  11  micronuclei.  The  same  condition 
prevails  in  the  closely  related  species,  T.  canalijcra,  which  I  was  formerly  disposed 
to  regard  (1945)  as  identical  with  T.  magna.  However,  on  the  basis  of  informa- 
tion furnished  me  by  Dr.  George  W.  Kidder,  of  Amherst  College,  it  appears  that  T. 
canalifera  merits  recognition  as  a  valid  species,  chiefly  because  of  the  very  conspicu- 
ous nature  of  its  canal  system.  In  T.  canalifera,  Turner  (1937)  reported  4—14 
micronuclei,  though  Burt,  Kidder,  and  Gaff  (1941),  in  specimens  obtained  from  the 
late  Dr.  Turner,  found  only  one.  Hence,  it  is  clear  that  the  micronuclear  number 
may  vary  from  1  to  14,  yet  the  evidence  indicates  that  the  uninucleate  and  multi- 
nucleate  races  were  equally  cultivable,  vigorous,  and  capable  of  producing  normal 
resting  cysts. 

The  present  study  of  T.  magna  was  undertaken  in  order  to  obtain  additional  in- 
formation concerning  two  points :  ( 1 )  the  normal  variation  in  micronuclear  number 
in  various  natural  races  and  (2)  the  significance  of  such  variation.  The  investiga- 
tion of  the  first  point  is  readily  feasible,  in  that  the  micronuclei  may  be  counted  with 
absolute  certainty  in  Feulgen  preparations  of  favorably  oriented  resting  cysts  or 

256 


MICRONUCLEI  OF  TILL1NA  MAGNA  257 

medium-sized  trophic  specimens.  The  investigation  of  the  second  point,  though  less 
suited  to  direct  approach,  is  not  impracticable.  A  number  of  questions  arise,  some 
of  which  submit  to  experimental  analysis.  For  example,  is  the  number  of  micro- 
nuclei  related  in  any  way  to  size,  whether  of  trophic  specimens,  division  cysts  or 
resting  cysts ;  to  division  rate ;  to  vitality,  meaning  capacity  to  endure  with  undimin- 
ished  vigor  as  generations  pass ;  to  ability  to  produce  resting  cysts ;  to  the  viability 
of  such  cysts ;  or  to  ability  to  excyst  ?  Of  the  foregoing  measurable  characters,  the 
following  were  selected  as  being  most  readily  amenable  to  experimental  investiga- 
tion :  ability  to  produce  resting  cysts,  size  and  viability  of  such  cysts,  capacity  to 
excyst,  division  rate  and  vitality.  These  then,  will  be  considered  in  relation  to 
micronuclear  number,  though  not  all  of  them  will  receive  equal  consideration.  The 
study  assumed  unlooked-for  interest  when  it  became  evident  that  three  of  the  races 
were  amicronucleate.  Thus  a  comparison  of  the  potentialities  of  micronucleate  and 
amicronucleate  clones  became  possibile. 

MATERIALS  AND  METHODS 

Twenty  clones  of  T.  inagna,  to  be  designated  numerically,  were  used  in  the  study. 
The  progenitors  of  these  respective  clones  were  collected  in  a  meadow,  known  locally 
as  Sparrow's  Pasture,  in  the  vicinity  of  Chapel  Hill,  North  Carolina.  Comparisons 
with  clones  from  other  sources  were  desirable,  but  unfortunately  attempts  to  collect 
Tillina  elsewhere  in  the  Chapel  Hill  region,  and  in  the  vicinity  of  Stanford  Univer- 
sity, California,  and  Woods  Hole,  Massachusetts,  were  unavailing.  In  this  study 
a  clone  refers  to  all  the  progeny  which  were  derived  asexually  from  a  single  resting 
cyst  or  trophic  specimen.  The  intervention  of  encystment  and  subsequent  excyst- 
ment  is  not  considered  to  be  a  valid  reason  for  changing  the  clonal  designation,  since 
there  is  no  evidence  that  encystment  in  Tillina  involves  a  sexual  process  which 
might  change  the  genetic  constitution  of  the  clone.  (It  should  perhaps  be  recalled 
that  Tillina,  like  its  near  relative  Colpoda,  reproduces  within  a  thin-walled  tempo- 
rary cyst,  from  which  usually  four  progeny  emerge  shortly  as  a  result  of  two  succes- 
sive divisions.  The  term  encystment,  as  used  in  this  study,  does  not  refer  to  these 
temporary  division  cysts,  but  to  the  protective  or  resting  cysts.)  It  is  not  defi- 
nitely established  that  all  of  the  twenty  clones  were  genetically  different,  since  their 
histories  prior  to  their  period  of  laboratory  life  were  unknown. 

The  progenitors  of  clones  1,  2,  6,  8,  9,  11,  12,  13,  15,  17,  18,  and  19  were  taken 
as  active  specimens  on  Sept.  10,  1945,  and  these  clones  were  therefore  cultured 
simultaneously  in  the  early  part  of  the  study.  Eight  of  the  foregoing  clones,  namely, 
6,  11,  13,  18,  15,  19,  1,  and  17,  have  already  been  reported  on  briefly  under  the 
numerical  designations  1  to  8,  respectively  (Beers,  1946a).  In  the  present  paper 
my  original  numerical  designations  of  all  clones  have  been  changed  for  the  con- 
venience of  the  reader  in  using  the  accompanying  tables.  The  progenitors  of  clones 
3,  14,  16,  and  20  were  isolated  on  Feb.  4,  1946,  when  dried  leaves  and  debris,  after 
8  months  of  storage  at  19°  C.,  were  immersed  in  weak  hay  infusion;  these  clones 
were  therefore  cultured  simultaneously.  It  is  evident  that  they  were  derived  from 
dried  cysts.  The  progenitors  of  clones  4,  5,  7,  and  10  were  isolated  on  April  8, 
1946,  when  moist  leaves  and  debris,  which  had  recently  washed  against  the  bases  of 
willow  saplings  in  the  meadow,  were  immersed  in  hay  infusion;  these  clones  were 


258  C.  D.  BEERS 

maintained  in  culture  simultaneously  toward  the  end  of  the  study.  They  were  un- 
doubtedly derived  from  wet  cysts. 

An  attempt  was  made  to  maintain  each  of  the  clones  in  pure-line  culture  for  a 
period  of  60  days.  Sixteen  of  the  clones  were  readily  cultivable  and  continued  with 
undiminished  vigor  throughout  the  period ;  four  were  intractable  in  that  their  divi- 
sion rates  declined  and  the  lines  encysted  well  before  the  end  of  the  period.  Thus 
the  laboratory  histories  of  the  clones  varied,  although  the  conditions  of  culture -were 
uniform.  The  details,  in  relation  to  micronuclear  number,  will  follow. 

Each  clone  was  cultured  in  depression  slides  in  the  form  of  four  sub-lines. 
These  were  maintained  at  23°  C.  in  0.05  per  cent  lettuce  infusion  to  which  suitable 
quantities  of  Psciidomonas  fluorescent,  grown  on  nutrient  agar,  were  added  as  food. 
Previous  experience  has  shown  that  this  general  procedure,  combined  with  daily 
isolations  and  transfers  to  fresh  environments,  meets  adequately  the  cultural  needs 
of  Tillina  (Beers,  1944,  1945).  Records  were  made  daily  of  fission  rates  and  other 
points  of  interest. 

Surplus  animals  from  the  lines  were  stained  on  cover  glasses  by  the  Feulgen 
method  in  order  to  make  micronuclear  counts  of  active  specimens.  Small  stock 
cultures  of  each  clone  furnished  precystic  specimens  when  the  food  supply  neared 
depletion.  These  specimens  were  removed  and  allowed  to  encyst  on  cover  glasses 
in  the  manner  described  by  Beers  (1946).  Thus  convenient  preparations  were 
available,  first  for  making  measurements  of  living  cysts,  and  then  for  Feulgen  stain- 
ing. All  measurements  and  micronuclear  counts  of  cysts  were  made  on  single 
resting  cysts.  These  are  the  common  type.  They  are  practically  spherical  and 
therefore  well  suited  for  making  accurate  measurements. 

NORMAL  VARIATION  IN  NUMBER  OF  MICRONUCLEI 

The  data  bearing  on  diversity  in  micronuclear  number  in  the  twenty  clones  are 
summarized  in  Table  I,  in  which  the  clones  are  arranged  and  numbered  in  the  order 
of  decreasing  mean  numbers  of  micronuclei.  The  data,  ignoring  for  the  present 
the  mean  diameters  of  resting  cysts,  are  largely  self-explanatory.  A  few  points 
deserve  special  mention. 

In  any  particular  clone  both  active  specimens  and  resting  cysts  showed  practi- 
cally the  same  extremes  of  variation  (range)  in  micronuclear  number  and  had  essen- 
tially the  same  mean  number  of  micronuclei. 

In  different  clones  the  mean  numbers  of  micronuclei  were  extremely  variable. 
Some  clones  (e.g.,  1  and  2)  had  consistently  high  mean  numbers;  others  (e.g.,  16 
and  17),  consistently  low  numbers,  with  many  intergrades  between  these  extremes. 

Clones  18,  19,  and  20  were  amicronucleate.  This  statement  is  not  based  on 
casual  observation,  but  on  an  intensive  study  of  these  clones.  In  trophic  specimens 
and  resting  cysts  the  micronuclei  of  Tillina  are  not  disposed  toward  secretiveness. 
They  are  never  imbedded  in  the  macronucleus.  Each  has  an  endosome  which  stains 
intensely  and  conspicuously  by  the  Feulgen  method.  In  mature  resting  cysts  only 
the  rod-shaped  or  ellipsoid  macronucleus  and  the  micronuclei  stain  to  any  appre- 
ciable extent;  there  is  nothing  in  the  cytoplasm  to  conceal  the  micronuclei.  In 
trophic  specimens  it  is  true  that  the  food  vacuoles  also  stain,  but  the  micronuclei 
always  lie  in  the  clear  peri-macronuclear  space  and  are  not  in  a  position  to  be  con- 
cealed by  the  vacuoles.  Moreover,  considerable  numbers  of  individuals  of  clones 


MICRONUCLEI  OF  TILLINA  MAGNA 


259 


18,  19,  and  20  were  stained.  These  included  not  only  the  usual  resting  cysts  and 
medium-sized  trophic  specimens,  but  also  young  cysts,  cysts  in  the  process  of  excyst- 
ment,  and  individuals  just  excysted.  None  showed  a  micronucleus,  whereas  indi- 
viduals of  the  remaining  seventeen  clones,  stained  at  the  same  time  by  the  same 
method,  invariably  showed  micronuclei. 

The  individuals  of  some  clones  (e.g.,  1,  3,  5,  12)  showed  great  diversity  in 
micronuclear  number  within  the  clone.  This  fact  is  brought  out  clearly  by  the 
range  which  is  cited  for  these  clones,  and  it  is  further  emphasized  by  the  high 
standard  deviations  in  the  clones.  Clone  12  showed  the  greatest  degree  of  hetero- 
geneity in  that  the  range  in  micronuclear  number  extended  from  2  to  11,  with  all 
intervening  numbers  being  represented.  On  the  other  hand,  some  clones  (e.g., 
2,  4,  8;  13,  15,  16,  17)  were  relatively  homogeneous,  with  narrow  ranges  and  low 
standard  deviations.  Other  clones  lay  between  these  extremes.  Only  the  amicro- 
nucleate  clones  showed  complete  homogeneity. 

Thus,  it  is  seen  that  individuals  of  a  clone  exhibit  varying  numbers  of  micro- 
nuclei,  that  clones  differ  with  respect  to  their  mean  number,  and  that  amicronncleate 
clones  exist  in  nature. 

The  mean  number  of  micronuclei  in  the  850  micronucleate  active  specimens  of 
Table  I  (representing  17  clones)  was  7.06;  the  mean  number  in  the  850  micro- 
nucleate  cysts  was  7.08.  Unfortunately,  the  number  in  the  progenitor  of  each  clone 

TABLE  1 

Tillina  magna.  Variation  in  number  of  micronuclei  in  twenty  clones;  relation  of  micronuclear 
number  to  size  of  cysts.  The  clones  are  numbered  and  arranged  as  the  mean  number  of  micronuclei 
(average  of  means  for  fifty  active  specimens  and  fifty  resting  cysts}  decreases. 


Numerical 
designation 
of  clone 

Range  in  number  of  micronuclei 

Mean  number  of  micronuclei 
±  standard  deviation 

Mean  diameter  of 
50  resting  cysts 
in  microns 
±  standard 
deviation 

50  active 
specimens 

50  resting 
cysts 

50  active 
specimens 

50  resting 
cysts 

1 

10-16 

9-16 

12.90±1.87 

12.32±1.93 

85.76±  9.65 

2 

8-12 

9-12 

10.48±0.94 

10.96±0.87 

82.94±  7.65 

3 

7-14 

6-13 

9.62±2.30 

9.24±2.08 

79.68±  8.72 

4 

7-10 

7-10 

8.52  ±0.82 

8.60±0.95 

88.62±  7.25 

5 

6-14 

6-12 

8.17±1.95 

8.28±1.66 

88.36±  9.61 

6 

6-10 

6-10 

7.56±1.28 

7.78±1.24 

93.60±  6.21 

7 

5-10 

5-10 

7.25±1.28 

7.38±1.36 

92.44±  7.08 

8 

6-  9 

6-  9 

7.16±0.85 

7.06±0.92 

78.50±  7.82 

9 

5-  9 

4-  9 

6.52±1.15 

6.74±1.21 

84.16±  8.62 

10 

4-  8 

4-  8 

5.98±1.52 

5.94±1.46 

85.42±  5.28 

11 

5-  8 

5-  9 

5.90±1.03 

5.76±0.96 

86.14±  5.34 

12 

2-10 

2-11 

5.74±2.41 

5.90±1.97 

85.64±  5.32 

13 

4-  6 

4-  6 

5.12±0.42 

5.10±0.45 

80.64±  8.92 

14 

4-  8 

4-  8 

5.12±1.51 

4.98±1.42 

84.18±  4.76 

15 

4-  6 

4-  6 

4.86±0.53 

5.20±0.57 

81.60±  8.41 

16 

4-  6 

4-  6 

4.92  ±0.63 

4.98±0.75 

80.92±  7.37 

17 

3-  5 

3-  5 

4.28±0.75 

4.14±0.74 

91.42±  6.87  ' 

18 

— 

— 

0 

0 

86.28±  5.63 

19 

— 

— 

0 

0 

84.32  ±10.36 

20 

— 

— 

0 

0 

81.52±  5.34 

260  C.  D.  BEERS 

is  unknown,  since  the  micronuclei  cannot  be  identified  in  living  specimens.  It 
seems  reasonable  to  assume  that  the  progenitor  of  each  had  a  number  approximately 
equivalent  to  the  mean  which  was  determined  for  the  clone,  and  that  it  produced 
some  offspring  having  fewer,  and  some  having  more,  than  its  own  number. 

It  is  well  known  that  the  number  of  micronuclei  is  variable  in  many  species  of 
ciliates.  Thus,  Paramecium  multimicronucleatwn  has  2  to  7  (Powers  and  Mitchell, 
1910),  though  usually  4  (Wenrich,  1928)  ;  Spathidium  spathula,  6  to  9  (Maupas, 
1888,  p.  247)  ;  Urostyla  grandis,  10  to  more  than  40  (Tittler,  1935)  ;  and  S  tent  or 
coeruleus,  10  to  42  within  a  single  clone  (Schwartz,  1935).  On  the  whole,  such 
variations  within  a  species  appear  to  have  little  effect  on  the  structure  or  behavior 
of  the  individuals  and  to  be  without  functional  significance.  This  general  conclu- 
sion is  supported  by  the  observations  on  T.  umgiia  which  follow  immediately. 

NUMBER  OF  MICRONUCLEI  IN  RELATION  TO  VARIOUS  ASPECTS  OF  CYSTMENT 

All  the  clones  produced  normal  resting  cysts  upon  the  depletion  of  the  food 
supply  in  small  stock  cultures  prepared  with  surplus  animals  from  the  lines.  Fur- 
thermore, all  the  specimens  in  such  cultures  encysted ;  none  persisted  in  prolonged 
swimming,  thereby  to  perish  of  starvation.  Hence,  it  is  clear  that  the  ability  to 
encyst  is  not  dependent  on  the  presence  of  the  micronucleus,  since  amicronucieate 
as  well  as  micronucleate  clones  were  able  to  encyst.  Moreover,  the  cysts  of  all 
the  clones  remained  viable  for  many  months.  They  could  be  activated  at  any  time 
after  the  fourth  day  by  immersion  in  distilled  water  or  0.05  per  cent  lettuce  infusion. 
From  2  to  2.5  hours  were  required  for  emergence  at  23°  C.,  and  practically  100  per 
cent  of  the  specimens  excysted.  No  precise  figures  are  given  here,  since  the  per- 
centage of  excystment  under  various  conditions  has  been  dealt  with  in  a  previous 
paper  (Beers,  1945)  and  the  present  study  contributes  nothing  new  on  this  point. 
'However,  the  present  results  show  clearly  that  the  viability  of  resting  cysts  and 
their  capacity  to  excyst  are  in  no  wise  related  to  the  presence  of  a  micronucleus,  or 
to  the  number  of  micronuclei.  Well  over  90  per  cent  of  the  cysts  produced  in  the 
various  clones  were  single  ones ;  double  cysts  appeared  sporadically,  some  in  ami- 
cronucieate clones,  some  in  micronucleate.  Amicronucieate  cysts  undergo  the  usual 
colpodid  type  of  macronuclear  reorganization,  involving  the  extrusion  of  a  portion 
of  the  macronuclear  substance  into  the  cytoplasm  (Taylor  and  Garnjobst,  1941 ; 
Burt,  Kidder  and  Claff,  1941;  Beers,  1946). 

The  size  of  the  cysts  in  different  clones  was  made  the  subject  of  special  study, 
for  it  was  thought  that  the  number  of  micronuclei  might  affect  the  size  of  the  cysts. 
The  diameters  of  fifty  living  single  cysts  of  each  clone  were  measured,  each  measure- 
ment extending  from  the  outer  surface  of  the  ectocyst  of  one  side  to  the  correspond- 
ing surface  of  the  other.  The  results  of  these  measurements  are  included  in  Table 
I.  An  inspection  of  the  table  shows  at  once  that  the  calculation  of  coefficients  of 
correlation  between  micronuclear  number  and  cyst  size  would  be  of  little  value, 
since  cyst  size  is  independent  of  micronuclear  number.  For  example,  if  we  con- 
sider certain  extremes  in  micronuclear  number,  it  is  seen  that  the  cysts  of  clone  1 
had  a  mean  diameter  of  about  85  /A,  and  those  of  clone  19  a  diameter  of  84  p,  with 
approximately  equivalent  standard  deviations.  Clones  2  and  20  and  clones  4  and 
18  constitute  other  examples  of  extreme  disparity  in  micronuclear  number  with 
close  agreement  in  cyst  size.  Among  the  micronucleate  clones,  other  examples  of 


MICRONUCLEI  OF  TILLINA  MAGNA  261 

wide  divergence  in  micronuclear  number,  yet  with  general  uniformity  in  cyst  size, 
are  furnished  by  clones  3  and  16  and  by  clones  6  and  17.  On  the  other  hand,  some 
clones  having  widely  divergent  micronuclear  numbers  produced  cysts  of  dissimilar 
sizes,  e.g.,  clones  3  and  17  and  clones  6  and  13.  Thus  it  is  seen  that  clones  having 
widely  different  micronuclear  numbers  may  produce  cysts  of  equivalent  mean  sizes 
or  of  dissimilar  mean  sizes.  It  must  be  concluded  that  there  is  no  relation  between 
the  number  of  micronuclei  and  the  size  of  the  cysts. 

The  same  conclusion  is  reached  if  we  adopt  another  approach  and  consider  cyst 
size  in  clones  which  had  similar,  or  only  slightly  different,  micronuclear  numbers. 
For  example,  clones  6  and  7  had  similar  mean  numbers  of  micronuclei  and  they 
produced  cysts  of  equivalent  mean  sizes;  clones  15  and  16  constitute  a  second  ex- 
ample. On  the  other  hand,  clones  7  and  8  had  similar  micronuclear  numbers,  but 
they  produced  cysts  which  differed  significantly  in  mean  size;  clones  16  and  17 
furnish  another  example.  Hence,  clones  having  similar  mean  micronuclear  num- 
bers may  produce  cysts  of  equivalent  mean  sizes  or  of  different  mean  sizes.  Again, 
it  is  evident  that  there  is  no  relation  between  number  of  micronuclei  and  size  of 
cysts.  The  mean  diameter  of  the  850  micronucleate  cysts  of  Table  I  was  85.23  p.; 
that  of  the  150  amicronucleate  cysts  was  84.04  p.. 

A  word  concerning  the  size  of  individual  cysts  may  be  of  interest.  In  any 
particular  clone  of  T.  mayna,  whether  micronucleate  or  amicronucleate,  there  is 
usually  wide  variation  in  cyst  size,  even  though  the  cysts  under  immediate  consid- 
eration are  all  produced  in  the  same  small  stock  culture — meaning  a  Columbia 
culture  dish  containing  1  cc.  of  fluid.  Cysts  in  such  a  culture  often  vary  in  size 
from  75  ^  to  95  p. ;  extremes  of  64  /*,  and  104  /A  have  been  noted.  The  factors  which 
affect  cyst  size  appear  to  be  of  a  complex  physiological  nature  and  are  therefore  not 
readily  identifiable. 

The  point  of  greatest  interest  in  this  consideration  of  various  aspects  of  cystment 
is  the  fact  that  amicronucleate  and  micronucleate  clones  behaved  alike ;  clearly  the 
micronucleus  of  T.  magna  plays  a  negligible  role,  if  any,  with  reference  to  encyst- 
ment,  viability  of  cysts,  excystment,  macronuclear  reorganization  within  the  cysts, 
and  cyst  size. 

NUMBER  OF  MICRONUCLEI  IN  RELATION  TO  DIVISION  RATE  AND  VITALITY 

The  cultural  histories  of  the  twenty  clones  are  presented  in  Table  II.  Clones 
1,  2,  3,  and  5  could  not  be  maintained  in  culture  for  the  arbitrary  period  of  60  days. 
The  remaining  clones  were  maintained  with  undiminished  vigor  and  were  discon- 
tinued at  the  end  of  the  period. 

First,  the  division  rates  of  the  sixteen  vigorous  clones  will  be  considered  in 
relation  to  micronuclear  number.  Clones  4,  6,  7,  and  8,  as  has  been  seen,  had  rela- 
tively high  micronuclear  numbers  (means,  about  7  to  8.5).  The  total  average 
number  of  generations  produced  by  the  four  sub-lines  of  these  respective  clones 
varied  from  149  to  174.  Thus  the  clones  themselves  varied  with  respect  to  mean 
division  rate.  Clones  9-12  had  intermediate  micronuclear  numbers  (means,  6  to 
6.5)  ;  these  clones  produced  from  149  to  167  generations.  Clones  13-17  had  low 
micronuclear  numbers  (means,  4  to  5)  ;  they  produced  from  160  to  176  generations. 
Thus  clones  having  intermediate  or  low  micronuclear  numbers  produced  in  general 
as  many  generations,  and  had  therefore  the  same  division  rates,  as  clones  having 
relatively  high  micronuclear  numbers. 


262  C.  D.  BEERS 

Amicronucleate  clones  18-20  produced  from  154  to  172  generations.  Thus  the 
amicronucleate  clones  had  approximately  the  same  division  rates  as  the  micronu- 
cleate  clones;  e.g.,  clone  19  produced  almost  as  many  generations  as  clone  4;  clone 
18  produced  more  than  clone  10 ;  clone  20  produced  about  as  many  as  clone  9  or 
13.  Many  kinds  of  comparisons  are  possible,  and  the  reader  may  choose  to  make 
other  comparisons  between  amicronucleate  and  micronucleate  clones.  For  example, 
clones  18-20  had  higher  division  rates  (i.e.,  produced  more  generations)  than  clones 
8,  10,  and  14;  but  clones  18—20  had  lower  division  rates  than  clones  4,  7,  and  16. 
Thus  the  general  conclusion  that  amicronucleate  clones  have  the  same  division  rates 
as  micronucleate  clones  is  not  invalidated.  Sections  of  amicronucleate  division  cysts 
showed  that  the  macronucleus  undergoes  the  usual  reorganization  after  each  of  its 
divisions,  as  in  normal  micronucleate  cysts  (Burt,  Kidder  and  Gaff,  1941;  Beers,, 
1946). 

Next,  the  vitality  of  the  sixteen  vigorous  clones  must  be  considered.  An  ex- 
amination of  the  number  of  divisions  produced  in  the  successive  5-day  periods  in 
any  particular  clone  shows  that  the  clone  was  dividing  as  rapidly  at  the  end  of  the 
experiment  as  at  the  beginning.  Within  the  time  limits  of  the  experiment,  the 
clones  showed  no  decrease  in  vitality  as  measured  by  the  division  rate.  How  long 
the  sixteen  clones  would  have  continued  without  diminution  in  vitality  is  a  question 
that  cannot  be  answered  on  the  basis  of  the  available  data.  The  important  findings 
are  these :  Some  clones  which  have  relatively  high  micronuclear  numbers  are  as 
vigorous  as  those  which  have  low  numbers ;  amicronucleate  clones  are  fully  as 
vigorous  as  many  micronucleate  clones. 

Clones  1,  2,  3,  and  5  must  receive  special  consideration.  As  has  been  said,  these 
clones  could  not  be  maintained  in  culture  for  the  duration  of  the  60-day  experi- 
mental period.  Clone  1  showed  a  rapid  decrease  in  fission  rate  and  encysted  on 
the  fourth  clay.  Some  of  the  cysts  were  activated  and  new  lines  were  established. 
These  in  turn  declined  shortly  and  encysted.  Three  additional  attempts  were  made 
to  culture  clone  1 ;  each  time  the  lines  encysted  after  3-5  days.  Indeed,  clone  1 
was  so  refractory  that  without  these  repetitions  it  would  have  been  impossible  to 
obtain  sufficient  specimens  for  the  usual  number  of  micronuclear  counts.  Clones 
2,  3,  and  5  likewise  could  not  be  maintained  in  culture  for  60  days.  Their  histories 
are  presented  in  sufficient  detail  in  Table  II.  Following  the  encystment  of  the 
original  lines  of  these  clones,  new  lines  were  established  with  excysted  specimens, 
but  they  also  declined  and  encysted  after  3-4  weeks  of  culture.  It  may  be  main- 
tained that  the  decline  and  encystment  of  these  four  clones  resulted  from  a  failure 
to  meet  their  cultural  needs.  However,  the  conditions  of  culture  were  adequate  for 
a  total  of  sixteen  clones,  and  it  seems  not  unreasonable  to  assume  that  they  were 
likewise  adequate  for  clones  1,  2,  3,  and  5  and  to  conclude  that  these  clones  declined 
as  a  result  of  intrinsic  factors. 

It  has  been  shown  that  clones  1,  2,  and  3  had  higher  micronuclear  numbers  than 
any  of  the  other  clones.  Clone  5  likewise  had  a  high  number,  though  slightly  lower 
than  clone  4,  which  was  cultivable.  The  evidence  indicates  that  a  large  number  of 
micronuclei  may  be  detrimental  to  the  welfare  of  the  organism  and  incompatible 
with  high  vitality,  but  the  number  of  such  clones  studied  was  too  small  to  justify 
a  general  conclusion.  On  the  whole,  the  results  show  that  in  T.  inagna  the  rate 
of  division  and  the  vitalitv  of  the  race  are  in  no  wise  related  to  the  number  of  micro- 


MICRONUCLEI  OF  TILLINA  MAGNA 


263 


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Tillina  ma, 

sixty  days.  F 
diminished  mg( 

Successive 

>.  VI 

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Total  nuniber 
of  generations 

264  C.  D.  BEERS 

nuclei,  or  even  to  the  presence  of  a  micronucleus,  since  amicronucleate  clones  proved 
to  be  as  vigorous  as  micronucleate  clones. 

The  origin  of  the  amicronucleate  races  cannot  be  accounted  for  with  certainty. 
It  is  agreed  that  such  races  may  arise  at  any  of  four  periods  in  the  life  of  ciliates 
generally :  at  endomixis,  by  the  transformation  of  all  the  reconstruction  micronuclei 
into  macronuclei ;  at  autogamy  or  conjugation,  by  an  analogous  transformation  of 
all  the  derivatives  of  the  synkaryon  or  amphinucleus  into  macronuclei ;  or  at  division, 
by  an  unequal  distribution  of  the  micronuclei  to  the  daughter  cells.  By  studying 
dividing  individuals  of  an  unusual  race  of  Paramecium  caudatum,  Wichterman 
(1946)  observed  the  simultaneous  production  of  bimicronucleate  and  amicronu- 
cleate daughters.  In  T.  inagna  conjugation  is  of  rare  occurrence,  ahd  endomixis 
and  autogamy  are  unknown.  Therefore,  it  is  likely  that  amicronucleate  races  usu- 
ally take  their  origin  in  an  unequal  distribution  of  the  products  of  division.  How- 
ever, it  should  not  be  concluded  from  the  present  results  that  15  per  cent  of  all 
Tillina  clones  are  amicronucleate ;  actually  such  clones  appear  to  be  very  exceptional. 
I  have  stained  many  specimens  during  a  period  of  8  years;  all  these  specimens  had 
micronuclei,  except  for  the  members  of  clones  18-20. 

DISCUSSION 

The  functional  significance  of  the  nuclear  dimorphism  of  the  Euciliata  has  long 
held  the  attention  of  protozoologists.  In  general,  the  dimorphic  condition  has  been 
viewed  as  representing  a  segregation  of  idiochromatin  and  trophochromatin,  the 
former  in  the  micronucleus.  the  latter  in  the  macronucleus.  It  was  originally  as- 
sumed, since  ciliates  normally  possess  both  types  of  nuclei,  that  both  are  necessary 
for  the  continued  survival  of  the  individual.  Although  the  precise  functions  of  the 
respective  nuclei  are  difficult  to  determine,  two  lines  of  investigation  have  supplied 
pertinent  findings,  namely,  a  study  of  the  capabilities  of  amicronucleate  ciliates  of 
natural  occurrence  and  a  study  of  regenerative  capacity,  survival,  and  reproduction 
in  ciliates  which  have  been  deprived  experimentally  of  either  nucleus,  or  of  both, 
whether  by  merotomy  or  other  operative  procedures. 

The  existence  of  naturally  occurring  amicronucleate  races  has  long  been  conceded 
by  such  authorities  as  Woodruff  (1921),  Calkins  (1930),  and  Reichenow  (1929, 
p.  29)  and  is  now  accepted  as  a  fact.  The  potentialities  of  these  races,  as  revealed 
by  intensive  laboratory  study,  have  demonstrated  that  the  micronucleus  is  not  at  all 
necessary  for  the  maintenance  of  the  essential  vital  processes  of  the  individual, 
whereas  the  macronucleus  is  indispensable.  Aside  from  the  absence  of  a  micro- 
nucleus  and  the  manifest  inability  to  carry  to  completion  such  processes  as  endo- 
mixis, autogamy  and  conjugation,  amicronucleate  races  of  many  ciliates  do  not  differ 
structurally  or  physiologically  from  micronucleate  ones. 

Thus,  Dawson  (1919;  1920)  maintained  an  amicronucleate  race  of  Oxytricha 
liyinenostonia  in  pure-line  culture  for  289  generations  (4  months)  and  in  small  mass 
cultures  for  5  months  longer.  The  absence  of  micronuclei  did  not  prevent  the  ani- 
mals from  attempting  to  conjugate,  but  these  attempts  were  abortive.  Woodruff 
(1921)  cultured  amicronucleate  races  of  Oxytricha  jallax  and  Urostyla  grandis  for 
246  and  128  generations,  respectively,  and  maintained  a  race  of  Paramecium  can- 
datum  in  pure-line  culture  long  enough  to  determine  that  it  was  definitely  amicro- 


MICRONUCLEI  OF  TILLINA  MAGNA  265 

nucleate.     A  few  pairs  of  conjugants  occurred  in  mass  cultures  of  O.  falla.v,  but 
they  failed  to  live  when  isolated. 

Amicronucleate  races  of  other  ciliates  have  likewise  been  isolated  a,nd  cultured 
long  enough  to  demonstrate  not  only  their  viability  but  their  sustained  vigor  and 
good  health.  Among  these  are  the  following:  (1)  Spathidmm  spathula.  Moody 
(1912)  was  unable  to  find  micronuclei  in  her  specimens,  though  she  was  able  to 
culture  them  for  218  generations.  It  is  evident  that  they  were  amicronucleate,  since 
the  micronuclei  of  Spathidium  were  observed  and  counted  by  Maupas  and  were 
found  regularly  by  Woodruff  and  Spencer  (1922).  (2)  Didimuin  nasutum.  Pat- 
ten (1921)  cultured  an  amicronucleate  race,  which  was  derived  from  an  exconjugant 
of  a  normal  micronucleate  race,  for  652  generations.  Conjugation  occurred  in  the 
amicronucleate  race,  but  the  exconjugants  invariably  died.  The  resting  cysts  were 
likewise  inviable.  It  is  evident  that  the  race  arose  through  the  faulty  reorganiza- 
tion of  the  exconjugant.  (3)  Paramecium  bursaria.  Woodruff  (1931)  cultured 
a  race  characterized  by  micronuclear  instability  for  7  years.  Neither  endomixis  nor 
conjugation  was  observed.  The  race  was  originally  bimicronucleate ;  later  it  was 
variable,  exhibiting  from  1  to  4  micronuclei ;  then  for  about  4  years  it  was  unimicro- 
nucleate ;  finally,  a  derived  race  showed  no  micronucleus,  although  this  race  was 
apparently  as  healthy  and  vigorous  as  its  bimicronucleate  ancestors  which  were  iso- 
lated 7  years  earlier.  Woodruff  (p.  543)  aptly  points  out  that  "whatever  function 
the  micronuclear  apparatus  plays,  the  somatic  life  of  the  animals  is  not  obviously 
influenced  by  profound  variations  in  volume  or  in  distribution  of  micronuclear 
material."  (4)  Urostyla  grandis.  Tittler  (1935)  found  amicronucleate  individuals 
in  stock  cultures  which  previously  contained  only  micronucleate  specimens.  They 
were  indistinguishable  externally  from  their  micronucleate  progenitors,  and  they 
flourished  in  mass  cultures  for  2  years.  Their  macronuclear  divisions  followed  the 
usual  complex  pattern  characteristic  of  the  species.  The  race  produced  resting 
cysts,  some  of  which  could  be  excysted,  although  endomixis,  which  usually  occurs 
in  the  precystic  forms,  was  absent.  Evidently  some  of  the  cysts  \vere  not  entirely 
normal,  since  they  showed  a  tendency  to  disintegrate,  perhaps  because  of  the  omis- 
sion of  endomixis.  (5)  Colpoda  steini.  Piekarski  (1939)  studied  comparatively 
the  structure  and  reproduction  of  a  micronucleate  and  an  amicronucleate  race  and 
was  able  to  culture  the  latter  for  approximately  6  years.  Both  races  were  equally 
cultivable  and  vigorous.  They  reproduced  within  division  cysts  from  which  four 
progeny  regularly  emerged  and  they  produced  normal  resting  cysts.  They  showed 
the  same  Sequence  of  events  in  the  division  of  the  macronucleus.  These  events  are 
of  special  interest,  in  that  eight  chromatic  (Feulgen-positive)  bodies  appear  in  the 
macronucleus  of  the  young  division  cyst.  Ultimately  each  of  the  daughters  receives 
two  of  them,  and  thus  the  behavior  of  these  bodies  suggests  an  equational  distribu- 
tion of  chromosomes.  Piekarski  concludes  that  the  absence  of  a  micronucleus  had 
no  recognizable  effect  on  the  activities  of  the  animals. 

The  present  study  of  amicronucleate  races  of  Tilliim  inagna  likewise  demon- 
strates the  adequacy  of  the  macronucleus,  not  only  for  long-continued  reproduction 
accompanied  by  sustained  vigor,  but  also  for  encystment  and  excystment.  Thus 
endowed  with  the  ability  to  produce  viable  resting  cysts,  these  races  would  seem  to 
be  capable  of  indefinite  survival,  even  under  the  changeable  conditions  of  a  natural 
environment. 


266  C.  D.  BEERS 

Still  more  remarkable,  in  connection  with  the  capabilities  of  amicronucleate  races, 
are  the  observations  of  Sonneborn  (1940)  and  Kimball  (1941)  on  mating  types. 
In  certain  races  of  Paramecmm  aurelia  Sonneborn  found  a  small  percentage  of  ani- 
mals which,  upon  undergoing  autogamy  or  conjugation,  developed  a  new  macro- 
nucleus  from  a  fragment  of  the  old  macronucleus.  Since  the  micronuclei  commonly 
disappeared  in  clones  produced  by  these  animals  and  since  the  mating  type  never 
changed  at  macronuclear  reorganization,  Sonneborn  concludes  that  "hereditary 
characters  (including  mating  type)  of  clones  from  macronuclear  regenerates  cannot 
be  directly  determined  by  micronuclei,  for  they  persist  in  the  absence  of  micronuclei. 
Mating  type  must  be  determined  by  the  macronucleus.  .  .  ."  Kimball  was  able  to 
assign  amicronucleate  specimens  of  Euplotcs  patella  to  definite  mating  types,  since 
they  paired  readily  with  individuals  of  known  mating  type.  He  concludes  that  "the 
micronucleus  is  thus  unnecessary  for  an  animal  to  be  of  a  definite  mating  type." 

With  reference  to  the  role  of  the  nuclei  in  the  regeneration  of  ciliates  following 
merotomy,  the  results  obtained  with  different  species  are  not  always  in  complete 
accord.  The  physical  properties  of  the  cytoplasm  constitute  an  experimental  vari- 
able ;  a  fluid  cytoplasm  or  a  rigid  pellicle  may  interfere  with  the  closure  of  an  injury 
and  thus  affect  regeneration  adversely.  Balamuth  (1940)  has  presented  an  ex- 
cellent review  of  the  extensive  literature  on  this  subject.  For  the  present  only  a 
few  representative  examples  of  regeneration  in  ciliates  will  be  considered,  with  spe- 
cial reference  to  the  nuclear  components  of  the  merozoa  (cell  fragments). 

It  is  well  known  that  enucleate  fragments  of  ciliates  can  neither  regenerate  nor 
continue  to  live,  whereas  nucleate  fragments  regenerate  successfully  and  pursue 
normal  lives.  These  conclusions  are  particularly  evident  in  Balamuth's  six-page 
tabular  summary  of  the  findings  on  regeneration  in  the  ciliates.  As  a  rule  the 
macronucleus  and  micronucleus  cannot  be  separated,  and  a  nucleate  fragment,  as  in 
Dembowska's  work  (1925)  on  Stylonychia  mytilus,  usually  means  one  having  both 
macronuclear  and  micronuclear  material. 

However,  some  investigators  have  succeeded  in  obtaining  nucleate  merozoa  of 
the  two  types  desirable  for  an  experimental  analysis  of  the  role  of  the  individual 
nuclei  in  regeneration;  namely,  macronucleate  (without  micronuclei)  and  micro- 
nucleate  (without  any  part  of  the  macronucleus).  Reynolds  (1932),  for  example, 
in  work  on  an  amicronucleate  Oxytricha  jallax,  found  that  various  types  of  macro- 
nucleate  merozoa  could  regenerate  their  missing  parts  and  resume  their  normal 
physiological  activities.  Schwartz,  using  microdissection  techniques,  was  able  to 
remove  the  entire  macronucleus  from  Stcntor  and  yet  leave  a  number  of  micro- 
nuclei  in  the  specimens.  These  micronucleate  individuals  never  survived.  By 
means  of  successive  operations,  he  was  also  able  to  remove  all  the  micronuclei  from 
a  few  specimens,  leaving  a  portion  of  the  beaded  macronucleus  in  place.  These 
macronucleate  individuals  regenerated  and  could  be  cultured  as  pure  lines.  Thus 
he  produced  experimentally  an  amicronucleate  race,  which  as  regards  size  and  divi- 
sion rate  was  not  different  from  the  normal  controls.  Bishop  (1943),  employing 
the  ultra-centrifuge  as  a  means  of  obtaining  merozoa  of  Oxytricha  jallax,  obtained 
sixty-seven  macronucleate  fragments,  all  of  which  regenerated,  and  seven  micro- 
nucleate  fragments,  none  of  which  regenerated.  Twelve  of  the  regenerated  macro- 
nucleate  individuals  were  cultured  as  amicronucleate  pure  lines.  Bishop  concluded 
(p.  451)  that  "the  lack  of  micronuclear  material  makes  no  difference  in  the  regen- 
erative capacity,  division  rate,  motility  or  morphology  of  Oxytricha  jallax" 


MICRONUCLEI  OF  TILLINA  MAGNA  267 

On  the  other  hand,  there  is  evidence  that  in  some  forms  the  micronucleus,  as 
well  as  the  macronucleus,  is  necesesary  for  regeneration  and  survival.  Thus  Rey- 
nolds found  that  both  nuclei  are  necessary  for  the  regeneration  of  merozoa  of  Eu- 
plotcs  patella.  This  observation  is  in  accord  with  the  results  of  Taylor  and  Farber 
(1924),  who,  by  means  of  a  micro-pipette,  removed  the  micronucleus  from  fifty 
specimens  of  E.  patella,  all  of  which  died  within  a  few  days.  None  produced  more 
than  four  progeny.  Hence,  these  authors  conclude  that  "the  micronucleus  plays 
more  than  a  purely  germinal  role  in  the  life  history  of  Eiiplotes  patella."  However, 
the  situation  in  E.  patella  is  confused,  for  some  of  Kimball's  unimicronucleate  double 
animals  produced  viable  amicronucleate  individuals  at  division.  Some  of  them  sur- 
vived as  clones,  though  with  a  low  division  rate ;  one  such  amicronucleate  clone  sur- 
vived for  341  days.  The  results  show,  according  to  Kimball  (p.  30),  "that  the 
micronucleus  is  not  essential  for  continued  life  in  at  least  some  clones  of  Euplotes 
patella,  though  its  absence  results  in  a  marked  decrease  in  vigor."  In  various  spe- 
cies of  Uronycliia  (Young,  1922),  and  in  Uroleplus  mobilis  (Tittler,  1938),  Spa- 
thidium  spathula  and  Blepharisma  undulans  (Moore,  1924)  both  types  of  nuclei  ap- 
pear to  be  necessary  for  the  complete  regeneration,  growth  and  division  of  merozoa. 

Thus  the  evidence  afforded  by  the  long-continued  culture  of  a  number  of  natu- 
rally occurring  amicronucleate  races  demonstrates  conclusively  that  the  macro- 
nucleus  alone  suffices  for  the  maintenance  of  the  vegetative  life  of  the  organism- 
meaning  by  vegetative  life  such  diverse  activities  as  locomotion,  food  capture,  diges- 
tion, assimilation,  growth,  excretion,  respiration,  reproduction,  and  maintenance  of 
cell  proportions  and  form.  On  the  basis  of  this  evidence  the  normal  role  of  the 
micronucleus  in  vegetative  life  appears  to  be  one  of  relative  passivity.  The  evidence 
from  operative  procedures  shows  that  in  many  ciliates  the  macronucleus  alone  is 
adequate  for  complete  regeneration,  as  well  as  for  subsequent  growth  and  division, 
whereas  in  other  ciliates  the  micronucleus  also  is  necessary.  There  is  no  authenti- 
cated case  on  record  in  which  the  micronucleus  alone  is  adequate  for  the  maintenance 
of  vegetative  functions  or  for  regeneration.  Balamuth's  thorough  survey  of  the 
literature  leads  him  to  make  the  following  comment  in  his  summary :  "Of  the  dual 
nuclear  apparatus,  only  the  macronucleus  can  be  shown  to  function  in  the  actual 
regenerative  process.  The  role  of  the  micronucleus  in  this  connection  is  as  yet 
unclear ;  apparently  it  is  more  important  in  the  viability  of  some  forms  than  in  oth- 
ers." On  the  whole,  the  evidence  tends  only  to  emphasize  the  importance  of  the 
macronucleus  and  to  attest  to  the  validity  of  Calkins'  comment  (1930,  p.  161)  on 
this  organelle :  "Far  from  being  negligible  it  is  on  the  contrary  probably  the  most 
important  element  of  the  cell  in  matters  of  metabolism,  reorganization,  and  continued 
cell  life." 

The  tendency  to  underestimate  the  importance  of  the  macronucleus  in  the  life 
of  the  organism  results  probably  from  its  apparent  monotony  of  structure.  Lacking 
chromosomes,  its  division  is  usually  unspectacular.  Nevertheless,  its  mode  of  origin 
is  not  an  incidental  phenomenon  in  the  life  of  the  ciliate,  for  both  macro-  and  micro- 
nucleus  almost  invariably  have  a  common  and  simultaneous  origin.  Usually  they 
develop  from  the  synkaryon  of  the  conjugant,  by  divisions  which  appear  to  be  equa- 
tional.  Again,  they  develop  from  the  synkaryon  of  autogamy  or  from  the  endo- 
mictic  micronucleus.  Thus  they  inherit  equally  from  a  common  nucleus  of  origin, 
and  each  receives  equivalent  chromatin  elements,  chief  among  which  are  presumably 
the  genes.  In  the  definitive  micronucleus  these  elements  retain  the  ability  to  or- 


268  C.  D.  BEERS 

ganize  periodically  as  chromosomes,  and  thereby  to  arrest  the  attention  of  the  ob- 
server. Once  in  the  definitive  macronucleus,  on  the  contrary,  they  never  again 
assemble  in  the  form  of  chromosomes.  However,  it  is  possible  that  they  are  dis- 
tributed at  macronuclear  division  by  a  mechanism  which  is  fully  as  effective  as  the 
mitotic  distribution  of  chromosomes,  though  less  conspicuous.  For  example,  it  is 
not  impossible  that  they  are  represented  in  multiplicate  in  the  macronucleus  and 
distributed  at  random  throughout  its  substance.  Thus  each  daughter  at  division 
would  be  reasonably  assured  of  receiving  representatives  of  every  type  of  chromatin 
element.  The  behavior  and  the  potencies  of  the  macronuclear  fragments  of  Sonne- 
born's  unusual  specimens  of  Paranieciuin  aurelia  indicate  a  multiplicate  representa- 
tion of  the  chromatin  elements.  In  these  specimens  the  forty  or  more  macronuclear 
fragments  grew  and  segregated  during  subsequent  cell  divisions,  until  there  was  only 
one  in  each  cell.  Thus  each  fragment  was  adequate  for  the  regeneration  of  a  com- 
plete macronucleus  and  for  the  continued  life  of  the  organism,  even  in  the  absence  of 
micronuclei.  Hence,  Sonneborn  concludes  that  "the  normal  macronucleus  must  con- 
tain at  least  forty  complete  and  discrete  genomes."  A  more  precise  mechanism  for 
the  distribution  of  the  chromatin  elements,  involving,  for  example,  a  differential 
streaming  of  genetically  equivalent  elements  toward  opposite  ends  of  a  polarized 
macronucleus,  may  be  postulated.  Regardless  of  the  type  of  mechanism  involved 
in  the  distribution  of  the  chromatin  elements  of  the  macronucleus  at  division,  the 
fact  remains  that  inheritance  in  an  amicronucleate  ciliate  is  no  less  precise,  to  judge 
by  the  structure  and  physiological  activities  of  the  offspring,  than  in  a  micronucleate 
ciliate.  The  fact  that  the  behavior  of  the  macronucleus  does  not  conform  to  the 
chromosome  theory  of  heredity  in  sensu  strict o,  in  that  chromosomes  are  absent, 
may  mean  simply  that  a  different  mechanism  for  the  distribution  of  the  genes  is 
involved. 

Whether  the  macronucleus  of  amicronucleate  ciliates  may  justifiably  be  regarded 
as  an  amphinucleus — one  containing  idiochromatin  as  well  as  trophochromatin,  as 
Woodruff  (1921),  Moore  and  others  have  suggested — seems  doubtful  in  the  light 
of  recent  investigations.  Thus  it  has  been  shown  by  Schwartz  and  by  Bishop  that 
viable  amicronucleate  races  of  S  tent  or  and  Oxytricha  may  be  derived  by  experi- 
mental means  from  normal  individuals  in  which  the  idiochromatin  and  trophochro- 
matin were  presumably  segregated  in  the  two  types  of  nuclei.  The  macronucleus 
of  these  individuals,  following  removal  of  the  micronuclei,  was  adequate  to  maintain 
all  the  usual  vegetative  activities  in  the  derived  amicronucleate  races. 

In  conclusion,  the  evidence  shows  that  the  macronucleus  is  the  essential  nuclear 
element  in  the  vegetative  life  of  ciliates.  The  micronucleus  functions  largely,  if  not 
solely,  in  the  periodic  replacement  of  the  macronucleus  and  in  the  production  of 
new  genetic  combinations,  some  of  which  undoubtedly  render  the  species  better 
adapted  to  survival.  The  nature  of  the  physiological  conditions  which  call  for  a 
renewal  of  the  macronucleus  is  not  clear;  that  such  renewal  meets  an  imperative 
physiological  need  is  shown  by  the  widespread  occurrence  of  the  phenomenon  in 
the  Euciliata. 

SUMMARY 

The  number  of  micronuclei  was  examined  in  50  trophic  specimens  and  50  resting 
cysts  of  each  of  20  clones  of  Tillina  magna,  three  of  which  were  amicronucleate. 


MICRONUCLEI  OF  TILLINA  MAGNA  269 

In  any  particular  clone  trophic  specimens  and  resting  cysts  contained  approxi- 
mately equivalent  mean  numbers  of  micronuclei.  In  different  micronucleate  clones 
the  mean  number  varied  from  4.21  to  12.61.  The  mean  for  1,700  specimens  of  17 
clones  was  7.07. 

The  number  in  the  individuals  of  any  particular  micronucleate  clone  was  vari- 
able ;  some  clones  showed  relatively  little  variation,  e.g.,  3  to  5  micronuclei ;  others, 
considerable  variation,  e.g.,  2  to  11  micronuclei.  The  smallest  number  observed  in 
any  micronucleate  individual  was  2;  the  largest,  16. 

All  the  clones  produced  normal  resting  cysts  upon  depletion  of  the  food  supply 
(Pseudomonas  fluorescent} .  The  cysts  of  different  clones  were  equally  viable  and 
capable  of  excystment.  Their  size  was  unaffected  by  the  number  of  micronuclei. 
Amicronucleate  cysts  showed  the  usual  macronuclear  reorganization.  Hence, 
neither  the  number  of  micronuclei  nor  the  absence  of  micronuclei  affected  encyst- 
ment,  viability  and  size  of  cysts,  excystment  or  macronuclear  reorganization. 

An  attempt  was  made  to  maintain  each  clone  in  pure-line  culture  for  60  days 
and  thereby  to  examine  the  division  rate  and  vitality.  Four  clones  were  refractory 
and  encysted  before  60  days  expired.  The  remaining  16  clones,  including  the  three 
amicronucleate  ones,  survived  with  undiminished  vigor  and  were  discontinued.  The 
13  micronucleate  clones  produced  from  149  to  176  generations  during  the  60-day 
period;  the  three  amicronucleate  clones  produced  154,  164,  and  172  generations, 
respectively.  Hence,  the  16  surviving  clones  showed  slight  differences  in  their 
average  daily  division  rates,  but  neither  the  divison  rate  nor  the  vitality  of  these 
clones  was  correlated  with  variations  in  micronuclear  number  or  with  the  absence 
of  micronuclei.  Division  cysts  of  amicronucleate  clones  showed  the  usual  macro- 
nuclear  reorganization  after  each  division  of  the  macronucleus.  The  four  refractory 
clones  had  high  micronuclear  numbers. 

Since  conjugation  is  rare  and  endomixis  and  autogamy  are  unknown  in  Tillina, 
it  is  probable  that  amicronucleate  races  arise  at  division  by  an  unequal  distribution 
of  the  daughter  micronuclei. 

Some  of  the  literature  on  amicronucleate  ciliates  and  on  the  regeneration  of  vari- 
ous types  of  nucleate  merozoa  is  reviewed.  The  evidence  shows  that  the  macro- 
nucleus  is  the  indispensable  nuclear  element  in  the  so-called  vegetative  life  of  the 
organism,  whereas  the  micronucleus  during  this  period  appears  to  be  a  relatively 
passive  organelle.  Its  chief  function  concerns  the  periodic  replacement  of  the  macro- 
nucleus  and  the  production  of  new  hereditary  combinations.  Special  attention  is 
directed  to  the  fact  that  inheritance  in  an  amicronucleate  race  is  no  less  precise  than 
in  a  typical  micronucleate  race,  although  the  division  of  the  macronucleus  is  amitotic 
and  usually  reveals  no  suggestion  of  true  chromosomes.  It  is  evident  that  the 
hereditary  mechanism  of  amicronucleate  races,  and  perhaps  of  ciliates  generally, 
differs  radically  from  the  conventional  chromosomal  mechanism  of  metazoa. 

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THE  EFFECT  OF  LOW  TEMPERATURE  AND  OF  HYPOTONICITY 
ON  THE  MORPHOLOGY  OF  THE  CLEAVAGE  FURROW 

IN  ARBACIA  EGGS  * 

ALLAN  SCOTT 

Department  of  Biology,  Union  College,  Schenectady,  N.  Y.  and  The  Marine  Biological 

Laboratory,  Woods  Hole,  Massachusetts 

When  Arbacia  punctulata  eggs  are  exposed  to  low  temperature  during  the  first 
cleavage,  a  pronounced  stalk  develops  between  the  daughter  blastomeres.  A  stalk- 
also  develops  at  room  temperature  if  the  eggs  are  made  to  divide  in  hypotonic  sea 
water  or  in  sea  water  lacking  calcium  ion.  The  development  of  a  conspicuous 
cleavage  stalk  is  not  a  normal  feature  of  the  first  cleavage  in  Arbacia,  although  it 
does  occur  regularly  in  some  cells ;  for  example,  when  fibroblasts  divide.  The 
object  of  the  work  reported  here  was  to  examine  the  conditions  under  which  the 
stalk  is  formed  in  Arbacia  and  to  relate  these  facts  to  current  theories  of  the  mech- 
anism of  cleavage.  These  particular  experimental  treatments  were  used  because 
they  were  found  to  affect  the  appearance  of  the  cleavage  stalk. 

METHODS 

Eggs  of  Arbacia  punctulata  in  the  first  cleavage  served  as  experimental  material. 
Ovulation  was  induced  by  the  removal  of  the  oral  half  of  the  test;  eggs  emerging 
from  the  genital  pores  were  collected  in  a  dish  of  sea  water.  The  eggs  were  al- 
lowed to  settle  and  the  sea  water  was  decanted  after  which  fresh  sea  water  was 
added.  Two  such  washings  were  carried  out  to  minimize  contamination  by 
coelomic  fluid.  Fertilization  was  effected  by  the  use  of  diluted  "dry"  sperm,  and 
the  sperm  were  never  more  than  one  hour  old. 

The  fertilization  membranes  were  removed  by  shaking.  A  heavy  suspension 
of  eggs  was  placed  in  a  five-inch  test  tube,  one-half  full  of  the  suspension,  and 
shaken  rapidly  thirty  times.  Eggs  so  treated  cleave  in  time  with  the  controls. 
The  best  time  for  treatment  is  at  2%  minutes  after  fertilization,  for  if  shaken  earlier, 
many  exovates  are  formed,  and  if  shaken  later,  many  eggs  retain  the  fertilization 
membrane.  The  alternative  method  of  removing  the  fertilization  membrane  by 
treatment  with  the  hatching  enzyme  (Ishida,  1936)  was  not  attempted. 

The  hyaline  layer  was  removed  in  a  few  experiments  by  washing  the  eggs  in 
calcium-free  artificial  sea  water.  This  was  accomplished  by  several  decantations 
and  additions  of  the  calcium-free  mixture.  It  was  found  that  the  hyaline  layer 
regenerates  somewhat  if  the  eggs  are  returned  to  a  solution  possessing  calcium 
ions ;  hence  if  eggs  are  to  lack  the  hyaline  layer,  they  must  be  allowed  to  cleave  in 
the  calcium-free  mixture. 

This   study  was  largely  accomplished  by  photographic   means.     Photomicro- 

1  This  investigation  was  aided  by  a  Grant-in-Aid  from  the  Sigma  Xi  Alumni  Research 
Fund. 

272 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS  273 

graphs  taken  at  intervals  with  Leica-Ibso  apparatus,  were  projected  as  negatives 
(1,000  X)  and  measurements  made  with  dividers. 

Temperature  was  maintained  by  means  of  a  thermostatically  controlled,  water 
jacketed,  glass  well,  mounted  on  the  microscope  stage  and  connected  through  a 
centrifugal  pump  to  a  water  bath.  By  this  means  temperature  could  be  maintained 
within  ±  0.2°  C.  at  or  about  20°  and  within  ±  0.4°  C.  at  or  about  10°  C. 

Artificial  sea  water  lacking  calcium  ions  was  compounded  according  to  the 
method  of  Shapiro  (1941).  This  solution  has  an  osmotic  pressure  and  pH  closely 
similar  to  that  of  normal  sea  water. 

The  hypotonic  solutions  were  prepared  either  by  the  dilution  of  normal  sea 
water  or  of  the  calcium-free  mixture. 

A  few  observations  are  presented  on  polyspermic  eggs  cleaving  to  three  or  to 
four  cells  in  one  division.  Polyspermic  development  was  induced  by  the  method 
of  Smith  and  Clowes  (1924)  which  involves  fertilization  in  pH  7.2  sea  water  and 
the  return  of  the  eggs  to  the  normal  pH  of  8.4  within  two  or  three  minutes. 

RESULTS 
Morphology  of  the  cleavage  furrow 

The  shape  of  the  deepening  furrow  is  markedly  different  under  different  con- 
ditions; it  is  influenced  by  temperature,  concentration  of  calcium  ion,  tonicity  and 
by  presence  or  absence  of  the  fertilization  membrane. 

Temperature 

At  temperatures  between  20°  C.  and  30°  C.  there  is  normally  no  stalk  in 
cleaving  eggs  whose  fertilization  membrane  has  been  removed  (free  cleavage). 
The  furrow  is  peaked  at  the  apex  (Figs.  1  and  2).  At  low  temperatures,  6°  to 
12°  C.,  a  real  stalk  is  formed  during  the  latter  part  of  the  furrowing.  This  occurs 
whether  the  egg  is  enclosed  in  the  fertilization  membrane  or  not.  At  these  low 
temperatures  eggs  undergoing  membrane-free  cleavage,  come  to  resemble  a  dumb- 
bell with  a  handle  (Figs.  3  and  4). 

Calcium  ion  or  urea 

Chambers  (1938)  described  the  short  cleavage  stalk  which  develops  when  Ar- 
bacia  punctulata  eggs  divide  in  calcium-free  solutions  at  room  temperature. 
He  used  isotonic  mixtures  of  sodium  chloride  and  potassium  chloride.  In  the  pres- 
ent study  also  a  short  stalk  occurred  when  the  eggs  were  exposed  to  calcium-free 
sea  water.  Similarly  a  short  stalk  was  figured  by  Moore  (1930a  and  b)  and  by 
Motomura  (1934),  after  treatment  with  urea  solutions. 

Fertilization  membrane 

It  is  a  common  practice  to  remove  the  fertilization  membrane  either  by  dis- 
solving it  in  urea  solutions  or  by  shaking  an  egg  suspension  rather  violently.  These 
techniques  allow  the  mitotic  axis  to  become  much  longer  and  the  furrowing  is  thus 
more  readily  followed.  If  the  eggs  are  confined  in  the  fertilization  membrane  at 
10°  C.,  the  blastomeres  tend  to  stay  apart  and  the  walls  of  the  furrow  are  almost 
vertical  (Figs.  11  and  12).  At  the  end  of  the  cleavage  a  stalk  connects  the  two 
blastomeres.  If  this  same  experiment  is  varied  so  that  the  eggs  divide  within  their 


274  ALLAN  SCOTT 

fertilization  membranes  at  10°  C.  and  in  calcium-free  sea  water,  a  cleavage  stalk  like- 
wise develops.  In  this  case,  however,  the  stalk  moves  eccentrically  until  it  is  close 
to  the  fertilization  membrane  (Figs.  30  through  34).  The  difference  is  presumably 
due  to  the  fact  that  the  hyaline  layer  is  dissolved  in  solutions  lacking  calcium  ion 
and  when  the  hyaline  layer  is  missing  the  egg  is  able  to  slide  around  inside  the 
fertilization  membrane. 

Membrane-free  cleavage  in  polyspermic  eggs 

Polyspermic  eggs  may  undergo  free  cleavage  to  form  four  cells  in  the  first  divi- 
sion. In  such  cases  they  frequently  divide  so  that  a  symmetrical  figure  is  seen  from 
above.  In  this  circumstance  the  four  blastomeres  each  rest  upon  the  bottom  of  the 
glass  container  (Figs.  15,  16,  17,  and  18).  Frequently  one  blastomere  rests  upon 
the  other  three  at  the  end  of  the  cleavage  (Fig.  21).  The  former,  more  symmetrical 
type  of  cleavage  is  more  readily  followed.  When  such  an  egg  begins  to  cleave  it 
first  flattens  like  a  biscuit;  at  this  stage  it  resembles  a  balloon  around  which  two 
rubber  bands  have  been  placed  at  right  angles.  Such  a  balloon  is  flattened  on  the 
two  surfaces  where  the  rubber  bands  cross.  Perhaps  the  egg,  like  the  balloon,  is 
subject  to  greater  elastic  tension  in  the  region  where  the  incipient  furrows  cross, 
and  therefore  flattens  on  these  surfaces. 

As  seen  from  above,  the  egg  periphery  is  roughly  square,  with  corners  rounded 
(Fig.  15)  ;  the  wide  furrows  (at  10°  C.)  gradually  sink  towards  the  center  with  the 
apices  of  the  furrows  approaching  one  another.  The  upper  and  lower  surfaces 
meanwhile  remain  relatively  flat  although  the  two  flat  surfaces  slowly  come  together. 
The  whole  figure  at  the  stage  illustrated  in  Figure  16  resembles  a  balloon  stretched 
closely  over  four  tennis  balls  with  two  rubber  bands  placed  at  right  angles.  Finally 
a  definitive  stalk  is  formed  (Fig.  18). 

When  the  furrows  first  appear,  the  egg  is  to  be  considered  as  having  two  equa- 
torial furrows;  that  is,  two  constricting  rings  (Fig.  29a),  which  cross  each  other. 
The  quasi-independence  of  the  furrows  is  demonstrated  by  some  eggs  which  cleave 
in  a  similar  way  but  in  zvhich  the  furrows  incise  at  different  rates  (Figs.  19  and  20). 
In  Figure  29b  the  furrow  separating  ab  from  cd  is  well  in  advance  of  the  furrow 
separating  ad  from  be.  This  type  of  cleavage  leads  to  a  figure  like  Figure  20. 

It  appears  that  this  curious  type  of  cleavage  is  brought  about  by  the  develop- 
ment of  two  new  ring-like  tensions  which  develop  around  the  necks  of  the  indi- 
vidual blastomeres  after  the  deeper  furrow  is  well  established.  As  a  result  of  the 
deep  primary  furrow,  four  new  isthmuses  are  established  about  the  necks  of  the 
four  incipient  blastomeres  (cf.,  Fig.  29b).  Perhaps  the  most  significant  feature  of 

PLATE  I 

FIGURES  1  AND  2.     Egg  cleaving  at  20°  C.  in  sea  water,  fertilization  membrane  removed. 

FIGURES  3  AND  4.     Egg  cleaving  at  10°  C.  in  sea  water,  fertilization  membrane  removed. 

FIGURES  5  AND  6.  Egg  cleaving  at  20°  C.  in  65  per  cent  sea  water,  fertilization  membrane 
removed. 

FIGURE  7.     Egg  cleaving  within  the  fertilization  membrane  at  20°   C.  in  sea  water. 

FIGURE  8.     Late  cleavage  at  20°  C.  in  65  per  cent  sea  water,  fertilization  membrane  removed. 

FIGURES  9  AND  10.  Polyspermic  egg  fertilized  in  sea  water  at  pH  7.2,  transferred  to  nor- 
mal sea  water  at  room  temperature  until  cleavage  began.  Cleaving  at  10°  C.  in  sea  water. 

FIGURES  11  AND  12.     Eggs  cleaving  within  fertilization  membrane  at  10°  C. 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS 


275 


I 


276 


ALLAN  SCOT  I 


this  type  of  cleavage  is  the  bridge-like  stalk  which  results  (Figs.  23  and  24).  In 
these  latter  figures  note  that  one  circumferential  furrow  deepened  symmetrically 
and  more  rapidly  than  the  other.  The  furrow  which  started  later  is  very  asym- 
metrical, being  much  deeper  on  one  side  (cf.,  at  the  arrow)  than  the  other.  Egg^ 
cleaving  to  three  cells  show  a  similar  behavior  (Fig.  22)  and  when  cleavage  is  com- 


FIGURE  13. 
in  sea  water. 


Series  showing  late  cleavage  and  development  of  the  cleavage  stalk  at  10°  C. 


plete  they  may  have  a  Y-shaped  stalk,  or  if  one  furrow  deepens  more  rapidly  than 
the  others,  two  stalks  may  connect  to  one  blastomere  (Figs.  9  and  10). 

The  speed  of  furrowing  in  polyspermic  eggs  cleaving  to  four  cells  may  be  as  rapid 
as  when  two  cells  are  being  formed,  yet  it  should  be  remembered  that  the  amount 
of  new  surface  formed  is  much  greater  when  a  sphere  divides  into  four  equal  smaller 
spheres.  The  surface  of  a  sphere  divided  into  two  spheres  increases  26  per  cent, 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS 


277 


f 


l/V 


FIGURE  14.     Series  showing  late  cleavage  and  continued  activity  of  cleavage  stalk,  during 
fourteen  minutes  in  calcium-free  sea  water  at  11°  C. 


ALLAN  SCOTT 

if  divided  to  four  spheres  the  surface  increases  58  per  cent.  A  polyspermic  egg 
cleaving  to  four  cells  forms  about  26  per  cent  more  surface  than  the  normal  first 
cleavage  but  it  may  do  so  in  the  same  amount  of  time. 

fifiembrane-jree  cleavage  in  hyputonic  sea  water  and  in  hypotonic  calcium-free  sea 
water 

Dilution  of  the  sea  water  causes  a  swelling  of  the  egg ;  it  also  causes  an  unusually 
wide  furrow  to  develop  during  the  cleavage  and  leads  to  the  formation  of  a  stalk 
at  the  end  (Figs.  5.  6,  and  8).  This  effect  occurs  at  room  temperature  (20°  C.). 
The  stalk  may  become  very  long  if  the  sea  water  has  been  diluted  sufficiently.  In 
mixtures  of  65  parts  sea  water  and  35  parts  distilled  water,  for  example,  the  stalk 
may  finally  be  30  micra  long.  This  effect  occurs  either  in  the  presence  or  absence 
of  calcium  ion.  The  stalk  region  is  certainly  a  relatively  rigid  gel,  for  it  has  suffi- 
cient rigidity  to  push  the  daughter  blastomeres  far  apart.  Figure  8  and  Figures  40 
through  42  show  the  process  of  elongation  in  these  extreme  cases.  Enlarged  photo- 
graphs of  the  stalk  at  these  stages  are  shown  in  Figures  43,  44,  and  45  with  dimen- 
sions noted.  In  Figure  43  the  stalk  is  only  4.4  micra  in  diameter  at  one  point.  In 
Figure  44  its  minimum  width  is  about  2  micra  and  it  is  over  22  micra  long.  In 
Figure  45  the  constriction  is  completed.  The  stalk  is  still  5  micra  wide  at  some 
points,  but  it  is  less  than  3  micra  in  diameter  for  a  third  of  its  length.  Chambers 
(ibid.)  relates  that  a  spherical  oil  drop  lying  within  the  egg  in  the  furrow  region  is 
not  deformed  until  the  "external  surface  of  the  advancing  furrow  is  4  to  5  \n  from 
the  surface  of  the  oil."  If  the  egg  pictured  in  Figure  43  has  a  cortex  comparable 
in  thickness,  then  the  stalk  must  certainly  be  all  gel  by  the  time  its  diameter  is  re- 
duced to  7  or  8  fj..  One  blastomere  sometimes  ruptures  when  eggs  cleave  in  65 
per  cent  sea  water.  Xo  endoplasm  escapes  if  the  stalk  has  closed.  One  such  closed 
stalk  is  shown  in  Figure  28;  it  is  5  micra  in  diameter.  The  conclusion  that  the  stalk 
is  all  gel  (and  yet  it  continues  to  constrict)  is  a  most  important  conclusion  for  it 
strongly  supports  the  contraction  theory  of  cleavage  of  W.  H.  Lewis.  Close  in- 
spection at  high  magnification  fails  to  show  any  movement  of  granules  located  in  the 
stalk.  The  active  constriction  of  a  5  /A  stalk  is  recorded  in  Figures  26  and  27. 

PLATE  II 

FIGURES  15,  16,  17,  AXI>  18.  Cleavage  of  a  dispermic  egg,  cleaving  in  calcium-free  sea 
water  at  10°  C.  Egg  fertilized  in  pH  7.2  sea  water,  transferred  to  sea  water  at  room  tem- 
perature until  beginning  of  cleavage.  Time  after  fertilization:  Figure  15,  72  min. ;  Figure  16, 
74  min.;  Figure  17,  88  min.;  Figure  18,  190  min. 

FIGURES  19  AND  20.  Egg  snowing  dispermic  cleavage.  Treatment  as  in  Figures  15-18. 
Time  after  fertilization:  Figure  19,  86  min.;  Figure  20,  88  min. 

FIGURE  21.  Dispermic  egg.  Treatment  as  in  Figures  15-18.  One  blastomere  out  of  the 
horizontal  plane. 

FIGURE  22.     Diagram  illustrating  two  types  of  cleavage  to  three  cells. 

FIGURES  23  AND  24.  Egg  in  70  per  cent  sea  water  at  25°  C.,  after  accidental  polyspermy. 
Time  after  fertilization:  Figure  23,  50  min.;  Figure  24,  52  min. 

FIGURE  25.  Dispermic  egg  cleaving  in  sea  water  at  11°  C.,  following  fertilization  in  pH 
7.2  sea  water. 

FIGURES  26  AND  27.  Late  cleavage  of  egg  in  65  per  cent  sea  water.  Room  temperature. 
Time  after  fertilization  :  Figure  26,  83  min. ;  Figure  27,  85  min. 

FIGURE  28.  Closed  stalk  following  rupture  of  one  blastomere ;  65  per  cent  calcium-free 
sea  water. 

FIGURE  29.     See  text. 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS 


279 


DIAM.     if 


b 

29 


PLATE  II 


280  ALLAN  SCOTT 

The  stalk 

The  mitotic  axis  (greatest  length)  of  eggs  undergoing  free  cleavage  becomes 
progressively  longer  at  10°  than  at  20°  C.  (compare  Figs.  1  and  2  with  3  and  4)  ; 
moreover  the  early  furrow  at  10°  C.  is  much  more  blunt  in  contour  than  is  the 
furrow  of  eggs  at  higher  temperatures.  A  study  of  the  final  phase  of  cleavage  under 
high  power  (Fig.  13)  shows  how  the  wide  furrow  is  transformed  into  a  stalk. 

In  Figure  13a  the  deepening  furrow  is  still  blunt  with  a  diameter  of  about  14 
micra.  In  Figure  13/>,  however,  the  stalk  is  beginning  to  square  off.  The  arrows 
(Figs.  13d  and  r)  indicate  the  region  where  the  constriction  is  most  active.  The 
details  are  similar  and  are  very  clear  in  eggs  cleaving  in  calcium-free  sea  water  at 
10°  C.  The  series  of  diagrams  shown  in  Figure  14,  a  to  /.  again  show  that  the 
broad  furrow  first  deepens  until  the  diameter  of  the  waist  is  about  7  or  8  micra  (a 
and  b),  then  the  stalk  is  elongated  by  the  constriction  of  the  subequatorial  cortex 
(c  and  d,  see  arrows)  ;  meanwhile  the  entire  stalk  is  diminishing  in  diameter.  The 
minimum  diameter  of  the  stalk  is  about  4  micra  at  10°  C.  and  in  calcium-free  sea 
water ;  in  hypotonic  solutions  the  diameter  is  often  less.  When  the  diameter  of  the 
stalk  diminishes  below  4  micra,  it  does  so  in  local  areas  only  (cf.,  Fig.  14<y,  h,  /). 

Amoeboid  activity  and  cleavage  activity 

Many  workers  have  noted  that  the  polar  surface  of  the  cell  bubbles  actively  dur- 
ing cytokinesis  (Bowen,  1920 — in  Eucliistits  spcnnatocytcs;  and  Lewis  1942 — in 
tissue  culture  fibroblasts).  This  is  not  the  case  with  the  egg  of  the  sea  urchin  dur- 
ing the  first  cleavage,  instead  the  polar  surface  remains  smooth  and  inactive.  How- 
ever, a  variety  of  agents,  will  cause  the  formation  of  blebs  in  the  sub-furrow  region. 
One  such  agent  is  hypotonic  calcium-free  sea  water.  The  blebs  usually  begin  to 
form  after  the  completion  of  the  major  furrowing  and  they  give  rise  to  sizable 
spherules  which  are  cut  off  by  a  process  very  much  like  cleavage  (Figs.  46a,  b,  c}. 
The  inactivity  of  the  polar  surface  may  indicate  that  the  cortex  there  is  different 
from  the  equatorial  cortex  in  Arbacia." 

Eggs  that  have  been  in  the  hypotonic  medium  for  some  time  may  show  a  sud- 
den rush  of  endoplasm  from  one  blastomere  to  the  other,  often  causing  the  blasto- 
meres  to  become  very  unequal  in  size  (Fig.  47  and  Figs.  35  to  39). 3  This  endo- 
plasmic  flow  is  a  very  rapid  one,  usually  lasting  only  two  or  three  seconds.  It  is 
remarkable,  however,  that  the  flow  is  accompanied  toy  a  rapid  deepening  of  the  fur- 
row, appearing  as  though  a  tension  has  been  suddenly  overcome,  allowing  the  fur- 
row to  constrict  much  more  rapidly  than  usual.  The  sub-cortical  flow  in  such  eggs 
may  be  down  one  side  of  the  furrow,  through  the  constricted  stalk  and  up  the  other 
side  of  the  furrow,  yet  the  furrowing  continues  normally  to  completion  (Fig.  47). 

-  The  view  that  there  is  a  special  substance  (a  special  type  of  plasmagel)  located  around 
the  equator  has  been  espoused  by  a  number  of  workers.  Marsland  (1942)  and  Lewis  (1942) 
among  others.  Beams  and  King  (1937)  are  of  the  opinion  that  they  have  removed  the  "surface 
active  material"  of  Ascaris  eggs  by  centrifugation  at  150,000  X  gravity. 

3  The  rush  of  endoplasm  described  is  in  this  case  related  to  cleavage.  It  resembles  the 
amoeboid  activity  described  by  Moser  (1940)  after  urea  treatment.  Moser,  p.  77,  cites  other 
cases  from  the  literature. 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS 


281 


lit 


HO 


PLATE  III 

FIGURES  30,  31.  32,  33,  AND  34.  Eccentrically  placed  cleavage  stalk.  Eggs  in  fertilization 
membrane  at  10°  C,  in  calcium-free  sea  water. 

FIGURES  35,  36,  37,  38,  AND  39.  Volume  changes  of  individual  blastomeres.  Calcium-free 
sea  water. 

FIGURES  40,  41,  AND  42.  Elongation  of  the  cleavage  stalk;  in  65  per  cent  sea  water  at 
room  temperature.  Time  intervals:  Figures  40-41,  1  min.  and  40  sec.;  Figures  41-42,  1  min. 
and  35  sec. 

FIGURES  43,  44,  AND  45.  Enlargements  of  Figures  40,  41,  and  42.  The  edges  of  the  nearly 
transparent  stalk  have  been,  retouched  in  Figures  4,  8,  26,  27,  28,  43,  44,  and  45. 


282 


ALLAN  SCOTT 


DISCUSSION 


The  stalk 


The  occurrence  of  a  stalk  during  the  cleavage  of  the  Arhacia  egg  is  correlated 
.with  the  degree  of  gelation  of  the  furrow  cortex.     Both  the  observations  made  in 
this  paper  and  those  of  other  workers  who  have  concerned  themselves  with  the  de- 
gree of  gelation  of  the  egg  cortex  confirm  this.     The  results  of  several  workers  are 
summarized  below  : 

Brown  (1934)  :  Cortical  pigment  granules  are  especially  resistant  to  displace- 
ment by  centrifugation  during  the  division  phase. 

Chambers  (1938)  :  The  furrow  cortex  resists  disintegration  after  the  two  in- 
cipient blastomeres  have  been  punctured  at  the  poles. 


FIGURE  46 

Brown  and  Marsland  (1936)  :  There  is  a  quantitative  decrease  in  the  gel  value 
of  dividing  eggs  as  the  hydrostatic  pressure  is  increased.  Under  high  pressures  the 
furrow  regresses. 

No  one  has  yet  recorded  the  effect  of  temperature,  hypotonicity  and  lack  of 
calcium  ion  upon  the  cortex  of  the  dividing  Arbacia  egg,  although  these  observations 
have  been  made  upon  the  unfertilized  egg.  A  brief  summary  of  this  work  follows: 

Costello  (1938)  :  It  takes  progressively  longer  to  fragment  the  eggs  as  the  tem- 
perature is  lowered. 

Cole  (1932)  and  Harvey  (1943)  :  It  takes  longer  to  fragment  eggs  in  hypotonic 
than  in  isotonic  solutions. 

Harvey  (1945)  :  Arbacia  eggs  break  less  readily  in  solutions  possessing  calcium 
ions  than  in  solutions  lacking  calcium  ions. 

These  treatments  (low  temperature,  hypotonicity  and  calcium  ion)  are  pre- 
cisely the  ones  which  favor  the  development  of  a  cleavage  stalk.  It  is  possible  that 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS 


283 


these  treatments  may  increase  the  elastic  strength  of  the  egg  surface  by  toughening 
the  extra  cortical  structures,  but  it  is  probable  that  they  favor  cortical  gelation  as 
well. 

Hypotheses  concerning  the  mechanism  of  cleavage;  surface  tension 

Chambers  and  Kopac  (1937)  found  that  clean  oil  drops  of  the  proper  inter- 
facial  tension  with  sea  water,  will  coalesce  spontaneously  with  a  naked  egg  (Arbacia, 
Lytechinus,  and  Echinometra).  They  state:  "The  tendency  to  coalescence  in  the 
furrow  and  polar  zones  of  cleaving  eggs  (late  amphiaster  and  later)  was  investi- 
gated and  no  difference  was  found."  They  used  oils  whose  approximate  tensions 
in  contact  with  sea  water  were  30,  10,  and  3  dynes  per  cm.  The  fact  that  coalescence 
occurs  at  all  indicates  a  fluid  layer  around  the  egg  periphery.  Spontaneous  coales- 


FlGURE    47 


cence  does  not  occur  in  Amoeba  protcus  (Kopac  and  Chambers,  1937),  which  in- 
dicates a  non-fluid  surface.  In  view  of  these  observations  any  surface  tension 
hypothesis  is  untenable. 

Subcortical  currents 

Chambers  (1938)  has  hypothesized  that  cleavage  results  from  the  activity  of 
"the  sub-cortical  currents  (which)  sweep  around  the  two  asters  and  add  gelating 
material  to  the  inwardly  growing  cortex."  In  this  hypothesis  he  combines  his  own 
observations  with  those  of  Schechtman  (1937)  on  localized  cortical  growth  during 
the  cleavage  of  the  amphibian  egg.  It  was  shown  in  the  present  paper  (page  280) 
that  normal  furrowing  may  be  associated  with  abnormal  currents,  which  argues 
against  the  importance  of  such  currents  for  division;  moreover  Lewis  (1942)  found 
no  currents  in  the  dividing  fibroblast. 


284  ALLAN  SCOTT 

Astral  cleavage 

An  astral  theory  of  cleavage,  much  modified  from  Gray  (1931),  has  been  elabor- 
ated by  Katsuma  Dan  (1943).  He  believes  that  the  asters  are  composed  of  radiate 
fibers  with  intrinsic  rigidity ;  he  considers  them  to  be  anchored  to  the  cortex ;  he 
believes  that  the  rays  cross  at  the  equator ;  and  he  believes  that  the  spindle  elongates 
autonomously.  The  following  quotation  (Dan  1943)  summarizes  his  theory  of 
cytokinesis :  ".  .  .  it  was  also  shown  that  this  concept  of  the  mechanism  of  cell 
division  is  adequate  to  explain  the  stretching  phase  of  the  furrow  surface.  That  is, 
when  two  such  radiate  asters  are  pushed  apart,  they  can  in  turn,  push  the  cell  mem- 
brane of  the  polar  region  somewhat  as  a  paint  brush  would  push  some  object.  As 
they  travel  away,  howrever,  since  they  enclose  the  fluid  endoplasm  within  the  inter- 
spaces of  their  rays,  the  fluid  endoplasm  is  carried  away  from  the  equatorial 
region  and  the  cortex  there  is  sucked  in,  giving  rise  to  a  furrow.  The  cortex  is 
stretched  as  it  is  pulled  in  by  the  suction." 

The  strength  of  Dan's  hypothesis  lies  in  its  ability  to  explain  the  differential 
stretching  and  shrinkage  of  the  surface  which  he  and  his  coworkers  observed  (Dan, 
Yanagita,  and  Sugiyama,  1937;  Dan  and  Yanagita,  1938;  Dan,  1943)  and  for  which 
no  other  explanation  has  been  forthcoming.  It  appears  that  Dan's  hypothesis  will 
explain  such  unusual  cleavages  as  are  pictured  in  Figures  9  and  10  of  the  present 
paper.  It  could  be  assumed  that  one  element  of  the  tripolar  spindle  elongated  be- 
fore the  others  causing  the  asters  to  move  apart,  and  by  the  suction  mechanism,  caus- 
ing the  development  of  the  initial  furrow  (Fig.  9  at  a).  On  this  assumption  the  de- 
velopment of  the  other  furrows  (Figs.  9b  and  c )  begins  later,  presumably  because 
the  other  two  spindles  begin  their  elongation  later.  The  secondary  furrow  (Fig.  10 
at  a')  is  presumably  caused  by  the  suction  resulting  from  the  separation  of  the  lower 
two  asters.  Similar  explanations  would  doubtless  serve  for  the  tetra-astral  cleav- 
ages shown  in  Figures  23  and  24  of  the  present  study.  One  can  imagine  also  that 
the  crossing  rays  from  all  four  asters,  if  they  became  attached  to  the  cortex,  would 
explain  the  flattening  of  the  upper  and  lower  surfaces  of  the  egg  observed  in  Figure 
15. 

Dan's  hypothesis  is  not  in  accord  with  the  observations  presented  here  concerning 
the  continued  elongation  of  the  cleavage  stalk  in  hypotonic  sea  water  for  it  is  impos- 
sible to  see  how  the  astral  suction  mechanism  could  explain  the  further  constriction 
of  a  long,  completely  gelated  stalk. 

The  main  weakness  of  the  astral  suction  hypothesis  lies  in  its  limited  scope.  It 
fails  to  explain  undoubted  cases  of  anastral  cleavage  (tissue  culture,  for  example) 
frequently  noted  in  the  literature.  Dan's  easy  conclusion  that  all  of  these  anastral 
cases  are  explainable  by  his  astral  suction  hypothesis  (".  .  .  it  is  possible  to  imagine 
that  in  cells  of  the  anastral  type,  similar  gelation  systems  may  be  existing  although 
they  cannot  be  discerned  morphologically")  is  unconvincing. 

One  of  Wilson's  observations  is  discordant  with  Dan's  hypothesis.  Wilson  ob- 
served, in  a  form  which  normally  has  asters,  that  a  spindle  need  not  be  present  for 
complete  cleavage  to  occur.  He  found  that  a  cleavage  furrow  may  cut  in  around 
the  base  of  an  isolated  aster  and  result  in  a  complete  cleavage.  Compare  Wilson 
(1901),  page  376  and  Figure  11. 

In  one  of  Chamber's  microdissection  experiments  he  bisected  the  partially  cleaved 
egg  in  a  plane  at  45°  to  the  plane  of  the  furrow  (1924,  Fig.  36).  The  cut  resulted 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS 

immediately  in  two  cells.  However,  the  original  furrow  remained  on  each  artificially 
produced  blastomere  and,  on  each,  the  furrow  gradually  cut  through  forming  two 
small  "cells"  as  well  as  two  large  ones.  This  continued  cleavage  seems  to  be  quite 
unexplainable  by  Dan's  hypothesis  which  requires  crossed  astral  rays,  an  elongat- 
ing spindle  and  a  suction  produced  by  the  separation  of  the  asters. 

Cortical  grozt'th  or  cortical  contraction? 

Schechtman  has  proposed  another  theory  of  the  mechanism  of  cytokinesis.  He 
suggested  (1937)  that  the  furrow  cortex  grows  by  the  "intussusception  of  clear 
cytoplasm,"  but  simple  growth  of  the  equatorial  cortex  would  not  be  expected  to  cut 
the  egg  in  half.  Other  factors  must  account  for  the  inwardly  directed  furrow  and 
its  narrowing.  It  seems  clear  that  there  is  a  stretching  of  the  egg  cortex  at  the  time 
of  furrowing  as  concluded  by  Dan  et  al.  (1937,  1938),  by  Schechtman  (1937)  and 
by  Motomura  (1940),  but  whether  the  stretching  is  active  (the  result  of  growth) 
or  whether  it  is  passive  and  due  rather  to  a  contracting  ring  at  the  head  of  the  fur- 
row (Lewis,  1942),  is  not  easy  to  decide.  Schechtman  is  of  the  opinion  that 
"Cleavage  is  initiated  by  a  contraction  of  the  egg  cortex  at  the  site  of  the  future 
furrow."  And  he  notes  that  the  "'Cortex  becomes  thicker  and  bulges  toward  the 
egg  interior."  He  therefore  uses  both  contraction  and  cortical  growth  in  his  com- 
plete hypothesis.  The  observations  made  in  this  paper  on  the  continued  constric- 
tion of  small  stalks  after  they  consist  entirely  of  gelated  material  are  taken  as  strongly 
favoring  the  constricting  ring  theory  of  Lewis.  For  if  the  gelated  stalk  is  able  to 
contract  at  that  late  stage  of  cleavage,  it  seems  reasonable  to  suppose  that  it  possesses 
contractile  power  earlier.  The  direction  of  contraction  is  ringwise  about  the  equa- 
tor (Fig.  29c)  and  it  is  to  be  expected  that  such  contraction  would  draw  stained 
areas  out  into  fine  lines  as  Schechtman  observed,  if  such  areas  are  located  in  the  fur- 
row or  subfurrow  region. 

It  would  be  illuminating  to  know  whether  or  not  kaolin  particles  placed  around 
the  equator  would  be  brought  closer  together  during  the  furrowing  but  no  one  has 
made  these  observations. 

Amoeboid  activity  and  bleb  formation 

One  can  scarcely  observe  the  amoeboid  behavior  of  eggs  in  hypotonic  media  and 
particularly  the  "normal"  false  cleavages  which  occur  during  the  amoeboid  phase 
preceding  pronuclear  fusion  in  the  nematode  egg  (Spek,  1918),  without  being  con- 
vinced that  a  fundamental  similarity  exists  between  amoeboid  motion  and  cleavage. 
Moreover  the  abscission  of  blebs  is  strikingly  similar  to  cleavage.4  It  is  suggested 
that  any  deforming  force  which  establishes  an  isthmus  about  the  cell  or  a  part  of 
the  cell  will  result  in  the  development  of  a  contracting  ring  disposed  around  the 
isthmus,  provided  that  the  egg  is  in  the  cleavage  phase.  This  view  would  explain 
why  the  normal  egg,  deformed  by  the  elongating  spindle,  cleaves  at  the  equator.  It 
would  explain  why  cleavage  planes  cut  in  around  the  base  of  cytasters  which  are 
unconnected  to  a  spindle  (Wilson,  1901)  and  it  would  explain  why  blebs  formed  in 

4  Very  recently  Holtfreuter  (1946)  has  suggested  "that  in  normal  cytoplasmic  division  the 
activity  of  the  nucleus  and  of  the  endoplasm  are  of  a  mere  secondary  importance."  He  observed 
that  isolated,  embryonic  amphibian  cells  may  develop  annular  constrictions  which  lead  to  the 
fragmentation  of  the  cell.  He  considers,  however,  that  the  contraction  occurs  in  the  membrane 
rather  than  in  the  plasmagel  layer. 


286  ALLAN  SCOTT 

the  sub-furrow  region  may  cut  off  from  the  remainder  of  the  egg  as  reported  above. 
This  hypothesis  also  agrees  with  the  idea  that  the  enlarging  gelated  asters  play  a 
mechanical  role  in  localizing  the  furrow. 

SUMMARY 

1.  Under  certain  conditions  the  eggs  of  Arbacia  punctulata  develop  a  cleavage 
stalk  between  the  first  two  hlastomeres.    No  stalk  forms  in  sea  water  if  the  tempera- 
ture is  in  the  20°  C.  to  30°  C.  range;  low  temperature  (10°  C.)  causes  the  develop- 
ment of  a  stalk  in  sea  wrater;  a  short  stalk  develops  in  isotonic  calcium-free  sea 
water  at  20°  C. ;  a  very  long  stalk  develops  if  eggs  are  cleaving  in  hypotonic  sea 
water  (65  per  cent). 

2.  The  effect  of  the  above  treatments  on  the  appearance  of  cleaving  dispermic 
eggs  is  described. 

3.  Evidence  indicates  that  stalks  of  8  micra  diameter  are  all  gel,  yet  in  hypotonic 
sea  water  they  continue  to  constrict  and  elongate.     This  is  good  evidence  that  the 
cortical  gel  has  inherent  contractile  properties. 

4.  It  is  hypothesized  that  any  event  which  deforms  the  Arbacia  egg  (if  it  is  in  the 
"cleavage  phase")  leads  in  some  way  to  an  orientation  of  contraction  around  the 
isthmus.     The  deforming  force  may  be  an  enlarging  aster,  an  elongating  spindle,  or 
an  endoplasmic  flow. 

LITERATURE  CITED 

BEAMS,  H.  W.  AND  R.  L.  KING,  1937.     The  suppression  of  cleavage  in  Ascaris  eggs  by  ultra- 

centrifuging.     Biol.  Bull,  73:  99-111. 

BOWEN,  R.  H.,  1920.     Studies  on  insect  spermatogenesis.     I.     Biol.  Bull.,  39:   316-362. 
BROWN,  D.  S.,   1934.     The  pressure  coefficient  of  "viscosity"   in  eggs  of  Arbacia   punctiilata. 

Jour.  Cell.  Comp.  Physiol.,  5 :  335-346. 
BROWN,  D.   S.   AND  D.  A.   MARSLAND,   1936.     The  viscosity   of  Amoeba  at   high   hydrostatic 

pressure.    Jour.  Cell.  Comp.  Physiol.,  8:  159-165. 
CHAMBERS,  R.,  1924.     The  physical  structure  of  protoplasm  as  determined  by  micro-dissection 

and  injection.     General  cytology,  pp.  237-309.     The  University  of  Chicago  Press. 
CHAMBERS,  R.,  1938.     Structural  and  kinetic  aspects  of  cell  division.    Jour.  Cell  Comp.  Physiol., 

12:  149-165. 
CHAMBERS,  R.  AND  M.  S.  KOPAC,  1937.     The  coalescence  of  sea  urchin  eggs  with  oil  drops. 

Ann.  Report  Tortugas  Laboratory,  Carnegie  Inst.  of  Washington.     No.  36:  88. 
COLE,  K.  S.,  1932.     Surface  forces  on  the  Arbacia  egg.    Jour.  Cell.  Comp.  Physiol.,  1 :  1-9. 
COSTELLO,  D.,  1938.     The  effect  of  temperature  on  the  rate  of  fragmentation  of  Arbacia  eggs 

subjected  to  centrifugal  force.    Jour.  Cell.  Comp.  Physiol.,  11  :  301-307. 
DAN,  K.,  1943.     Behavior  of  the  cell  surface  during  cleavage.     VI.  On  the  mechanism  of  cell 

division.    /.  Fac.  Sci.,  Tokyo  Imp.  Univ.,  Sec.  IV,  6 :  323-368. 
DAN,  K.,  J.  C.  DAN  AND  T.  YANAGITA,  1938.     Behavior  of  the  cell  surface  during  cleavage. 

II.     Cytologia,  8:  521-531. 
DAN,  K.,  T.  YANAGITA  AND  M.  SUGIYAMA,  1937.     Behavior  of  the  cell  surface  during  cleavage. 

I.     Protopl.,  28  :  66-81. 

GRAY,  J.,  1931.    A  text  book  of  experimental  cytology.     Cambridge  University  Press. 
HARVEY,  E.  B.,  1943.     Rate  of  breaking  and  size  of  "halves"  of  the  Arbacia  punctulata  egg 

when  centrifuged  in  hypo-  and  hyper-tonic  sea  water.     Biol.  Bull.,  85:  141-150. 
HARVEY,  E.  B.,  1945.     Stratification  and  breaking  of  the  Arbacia  punctulata  egg  when  centri- 
fuged in  single  salt  solutions.     Biol.  Bull.,  89 :  72-75. 

HOLTFREUTER,  JOHANNES,  1946.     Structure  motility  and  locomotion  in  isolated  embryonic  am- 
phibian cells.     Jour.  Morph.,  79:  27-62. 
ISHIDA,   JURO,    1936.     An   enzyme   dissolving   the   fertilization   membrane    in    sea   urchin   eggs. 

Annotationes  Zoologicae  Japoncnses,  15 :  449-459. 


CLEAVAGE  FURROW  IN  ARBACIA  EGGS  287 

KOPAC,  M.  S.  AND  R.  CHAMBERS,  1937.     The  coalescence  of  living  cells  with  oil  drops.    Jour. 

Cell.  Comp.  Physiol,  9:  345-361. 
LEWIS,  W.  H.,  1942.     The  relation  of  the  viscosity  changes  of  protoplasm  to  amoeboid  motion 

and  cell  division.     The  structure  of  protoplasm,  pp.  163-197.     Iowa  State  College  Press. 
MARSLAND,  D.,   1942.     Protoplasmic  streaming  in  relation  to  gel   structure  in  the  cytoplasm. 

The  structure  of  protoplasm,  pp.  127-161.     Iowa  State  College  Press. 
MOORE,  A.  R.,  1930a.     Fertilization  and  development  without  membrane  formation  in  the  egg  of 

the  sea  urchin  Strongylocentrotus  purpuratus.     Protopl.,  9:  9-17. 
MOORE,  A.  R.,  1930b.     Fertilization  and  development  without  membrane  formation  in  the  egg  of 

Dendraster  eccentricut.     Protopl.,  9 :  18-24. 
MOSER,  F.,  1940.     Studies  on  a  cortical  layer  response  to  stimulating  agents  in  the  Arbacia  egg. 

III.    Biol.  Bull.,  78:  68-91. 
MOTOMURA,  I.,  1934.     On  the  mechanism  of  fertilization  and  development  without  membrane 

formation  in  the  sea  urchin  egg,  with  notes  on  a  new  method  of  artificial  partheno- 
genesis.    Science  Reports  of  the  Tohoku  Imp.  Univ.,  4th  scries,  Biol.,  9:  33-45. 
MOTOMURA,  I.,  1940.     Studies  of  cleavage.     I.     Science  Reports  of  the  Tohoku  Imperial  Unir., 

4th  series,  Biol.,  15:  123-130. 
SCHECHTMAN,  A.,   1937.     Localized  cortical  growth  as  the  immediate  cause  of  cell   division. 

Science,  85:  222-223. 
SHAPIRO,  H.  H.,  1941.     Centrifugal  elongation  of  the  cell  and  some  conditions  governing  return 

to  sphericity,  and  cleavage  time.    Jour.  Cell.  Comp.  Physiol.,  18 :  61-78. 
SMITH,  H.  W.,  AND  G.  H.  A.  CLOWES,  1924.     The  influence  of  hydrogen  ion  concentration  on 

the  fertilization  process  in  Arbacia,  Asterias  and  Chaetopterus  eggs.     Biol.  Bull.,  47: 

333-344. 
SPEK,  J.,  1918.     Die  amoboiden  Bewegung  und  Stromungen  in  die  Eizellen  einiger  Nematoden 

wahrend  der  Vereinigung  der  Vorkerne.    Arch.  f.  Entw.  Mcch.,  44:  217-254. 
WILSON,  E.  B.,  1901.     Experimental  studies  in  cytology  II  and   III.     Arch.  /.   £»/«'.  Mech., 

13 :  353-395. 


DEVELOPMENTAL  RELATIONS  BETWEEN  GENITAL  DUCTS 
AND  GONADS  IN  DROSOPHILA 

DIETRICH  BODENSTEIN 

Medical  Division,  Edge-wood  Arsenal,  Maryland 

While  studying  the  reproductive  system  of  Drosophila  sinmlans  gynandromorphs 
Dobzhansky  (1931)  made  the  following  interesting  observation:  If  female  genital 
ducts  and  testes  were  present  in  the  same  individual  and  if  the  female  ducts  were 
attached  to  the  testes,  the  latter  underwent  extreme  degeneration.  Yet,  the  attach- 
ment of  male  genital  ducts  to  ovaries  did  not  affect  the  development  of  these  organs. 

Now  we  know  that  in  normal  development  the  attachment  of  the  ducts  to  the 
gonads  takes  place  during  the  early  period  of  pupal  life.  We  know  further  that  by 
transplantation  of  gonads  from  one  individual  to  another  it  is  possible  to  obtain  at- 
tachment of  the  transplanted  organ  to  the  host  ducts.  This  knowledge  makes  it 
possible  to  attack  experimentally  the  question  whether  the  degeneration  of  testes 
when  attached  to  the  female  ducts,  as  observed  in  Drosophila  sinmlans  gynandro- 
morphs, is  a  peculiarity  of  this  special  case,  or  whether  the  phenomenon  is  a  general 
one  and  always  occurs  when  female  ducts  and  testes  are  brought  into  contact  with 
each  other. 

The  problem  can  be  approached  experimentally  in  two  ways :  the  larval  testes 
can  be  transplanted  into  female  host  larvae  or  the  female  genital  disc  from  which 
the  ducts  originate  can  be  transplanted  into  male  larvae.  By  transplanting  two  or 
three  testes  into  one  host,  the  chance  that  one  transplantal  will  attach  itself  to  the 
host  duct  is  quite  good.  The  chances  for  attachment  of  the  testes  to  the  female  duct 
are  even  better  when  the  six  oviducts  which  arise  by  outgrowths  from  the  three 
transplanted  imaginal  discs  compete  with  the  two  host  ducts  for  attachment.  In 
the  following  studies  both  these  methods  were  used. 

EXPERIMENTAL 

Transplantation  of  testes  into  female  ducts 

Two  or  three  testes  of  mature  virilis  larvae  were  transplanted  together  into  the 
abdominal  cavity  of  female  host  larvae  of  the  same  age.  After  the  hosts  had 
emerged,  the  condition  of  the  transplants  and  their  relationship  to  the  female  genital 
system  was  studied  in  careful  dissections.  This  series  consisted  of  ten  cases.  The 
following  was  found.  All  transplants  had  failed  to  assume  their  characteristic  spiral 
shape.  This  was  to  be  expected,  since  the  work  of  Dobzhansky  (1931)  and  Stern 
(1941a  and  b)  had  shown  that  the  testes  have  to  be  attached  to  the  vas  in  order  to 
accomplish  their  spiral  growth.  In  six  out  of  ten  cases  one  of  the  transplanted 
testes  had  attached  itself  to  one  of  the  oviducts  of  the  hosts.  The  attached  testis  was 
always  greatly  reduced  in  size  and  appeared  degenerate.  Figure  1  shows  camera 
lucida  drawings  of  five  representative  cases  of  this  series.  It  will  be  noted  that  the 
degenerative  reduction  occurs  only  when  the  testis  is  attached  to  the  oviduct  of 

288 


DEVELOPMENT  IN  DROSOPHILA 


289 


the  host  (Fig.  I  A,  B,  1)  and  E).  Testes  that  lie  free  in  the  body  cavity  (Fig.  IA 
and  B)  or  testes  that  have  been  attached  to  the  ovary  (Fig.  1C  and  B)  are  un- 
affected. Thus  the  principle  which  produces  degeneration  is  apparently  given  off 
only  by  the  oviducts  and  depends  for  its  action  on  a  close  cellular  contact  with  the 
testes.  This  principle,  moreover,  seems  unable  to  penetrate  larger  cell  barriers,  for 
testes  which  were  connected  to  ovaries  which  in  turn  had  their  normal  oviduct  con- 
nection remained  unaffected  (Fig.  1C).  Yet  two  testes  which  had  established  close 


FIGURE  1.     The  spatial  and  developmental  relations  of  transplanted  testes  to  the  reproductive 
system  of  their  female  hosts.     O,  ovary ;  OD,  oviduct :  7\  to  Tx,  transplanted  testes. 

contact  with  each  other  had  both  suffered  degenerative  reduction,  although  only  one 
of  these  fused  organs  has  actually  established  contact  with  the  oviduct  (Fig.  IA 
and  E). 

Transplantation  of  female  genital  discs  into  male  hosts 

In  a  second  series  of  experiments,  two  or  three  female  genital  discs  from  mature 
virilis  larvae  were  transplanted  together  into  the  body  cavity  of  hosts  of  the  same 
age.  The  condition  of  the  host  testes  and  their  relationship  to  the  transplanted 
female  structures  was  again  studied  by  dissection.  Several  of  the  affected  testes 


290 


DIETRICH  BODENSTFIN 


were  also  sectioned  and  studied  histologically.  Thirty  successful  cases  were  avail- 
able for  investigation.  In  seven  of  these  cases,  the  host  testes  were  not  connected 
to  the  transplanted  ducts,  although  the  latter  had  developed  well  and  were  found 
in  the  immediate  neighborhood  of  the  male  gonads.  The  testes  of  these  seven  hosts 
were  normal  in  size,  shape,  and  histology.  One  testis  in  each  additional  animal  was 
not  connected  with  the  vas  of  the  host,  nor  to  any  of  the  transplanted  ducts.  These 
testes  were  not  coiled  but  were  otherwise  normal.  The  other  testis  in  two  of  these 
individuals  was  connected  to  the  vas  of  the  host  and  was  normal,  while  the  other 
testis  of  the  third  individual,  although  connected  to  the  vas,  was  not  coiled.  The 
non-coiling  of  an  attached  testis  is  rare,  but  has  been  observed  at  times  in  otherwise 
normal  animals.  Whether  the  inabilitv  of  attached  testes  to  coil  is  a  result  of  faultv 

*•  * 

connections  with  the  vas  or  whether  the  vas  in  these  cases  has  lost  its  growth  in- 
ducing capacity  is  not  known. 

TABI.K  I 

Transplantation  of  female  genital  ducts  into  mule  hosts 


Number  of 
discs  trans- 
planted 

Testis  free  (round) 

Testis  (spiral). 
Normal  attached 

Testis  attached 
to  9  duct 

State  of  degeneration  of  testis 
when  attached  to   9  duct 

One  side 

Both  sides 

One  side 

Both  sides 

One  side 

Both  sides 

One  side 

Other  side 

3 

Yes* 

Yes 

+ 

3 

Yes 

Yes 

+  +  +  + 

3 

Yes 

Yes 

+  +  +  + 

3 

Yes 

Yes 

+  +  +  + 

3 

Yes 

3 

Yes 

3 

Yes 

Yes 

+  +  +  + 

2 

Yes 

Yes 

+  +  + 

2 

Yes 

Yes 

+  +  +  + 

2 

Yc-s 

+  +  +  +  + 

+  +  +  +  + 

2 

Yes 

Yes 

+ 

3 

Yes 

Yes 

+  + 

3 

. 

Yes 

3 

Yes 

Yes 

+  +  +  + 

3 

Yes 

Yes 

+  +  +  +  +.+ 

3 

Yes 

Yes 

+  +  +  +  + 

2 

Yes 

2 

Yes 

Yes 

+  +  + 

2 

Yes 

2 

Yes 

2 

Yes 

2 

Yes 

2 

Yes 

2 

Yes 

+  +  +  +  + 

+  +  +  +  + 

2 

Yes 

Yes 

+  +  +  + 

2 

Yes 

+  +  + 

+  +  +  +  + 

2  h. 

Yes 

2  h. 

Yes 

Yes 

+  +  +  + 

2  h. 

Yes 

Yes 

+  +  +  + 

2  hv. 

Yes 

Yes 

+  +  +  + 

h.  =  hydei  discs  into  hydei  hosts, 
vas,  but  not  spiral. 


hv.  =  hydei  discs  into  virilis  host.     *  =  testis  attached  to 


DEVELOPMENT  IN  DROSOPHILA 


291 


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DIETRICH  BODENSTEIN 

In  twenty  individuals  one  or  both  totes  were  connected  to  the  oviduct  of  the 
transplant.  In  some  cases  one  testis  was  even  found  to  have  connection  with  the 
oviducts  of  two  discs.  All  testes  that  were  attached  to  oviducts  showed  a  more  or 
less  pronounced  degree  of  reduction  and  appeared  degenerate.  Table  I  summarizes 
'the  results  of  this  experimental  series.  The  state  of  degenerative  reduction  of  the 
testes  in  this  table  is  indicated  by  crosses.  One  cross  signifies  slight ;  six  crosses, 
extreme  size  reduction.  Figure  2  shows  camera  lucida  drawings  of  four  representa- 
tive cases  of  this  series.  Figure  2A  is  a  case  listed  in  Table  I  having  one  cross. 
Figure  2C  is  listed  by  having  six  crosses  and  Figure  2D  and  B  are  listed  by  having 
five  crosses  each.  Figure  3  illustrates  by  microphotography  an  extremely  reduced 
testis. 


> 
t 


B 


FIGURE  3.     A,  normal  adult  testes.     B,  (arrow)   testis  of  the  same  individual  degenerated  under 

the  influence  of  a  transplanted  and  attached  oviduct. 

It  will  be  noted  from  Table  I  that  the  reduction  of  the  testes  is  in  most  cases 
very  pronounced.  Only  six  of  the  testes  attached  to  oviducts  were  reduced  to  state 
"3"  or  less,  while  sixteen  testes  were  degenerated  to  state  "4"  or  more. 

This  experimental  series  thus  confirms  strikingly  the  original  observation  that 
testes  attached  to  female  oviducts  suffer  degenerative  changes  and  that  it  is  the 
oviduct  that  elicits  the  principle  causing  degeneration.  The  observation  from  the 
previous  experiments  that  the  presence  of  oviducts  f>cr  sc  has  no  effect  on  testis 
development  is  also  confirmed,  for  testes  in  the  presence  of  as  many  as  three  pairs 
of  oviducts  in  the  immediate  organic  environment  remained  normal  if  they  were  not 
attached  to  the  oviduct.  In  comparing  the  attached  testes  of  the  two  series  with 
each  other,  a  difference  in  their  general  shape  was  noted.  While  the  transplanted 


DEVELOPMENT  IN  DROSOPHILA 


293 


testes  in  the  first  experimental  group  were  small,  roundish  bodies,  the  testes  in  the 
second  experimental  group  were  in  most  cases  thin  and  elongated  in  shape  (com- 
pare Fig.  1  with  Fig.  2).  Now  it  was  found  in  the  second  group  that  in  all  cases 
when  the  attached  testis  was  thin  and  elongated  it  was  attached  not  only  to  the  trans- 
planted oviduct  but  also  to  the  vas  efferens  of  its  host  (Fig.  2).  In  those  cases, 
however,  where  the  testis  attached  only  to  the  transplanted  oviduct,  had  not  estab- 
lished connection  with  the  vas,  it  was  roundish.  This  situation  is  well  illustrated 
in  Figure  2C.  The  left  testis  in  this  case,  a  small  roundish  degenerated  organ,  is 
attached  only  to  the  oviduct  while  the  right  testis.  which  is  attached  to  the  vas  ef- 
ferens of  the  host  and  to  one  transplanted  oviduct,  has  become  an  irregularly  elon- 
gated structure.  The  elongated  shape  of  such  a  degenerate  testis  is  thus  due  to  the 
stimulating  influence  of  the  vas  on  the  growth  of  the  testis,  which  in  normal  develop- 
ment leads  to  the  coiling  of  this  organ,  while  the  observed  degeneration  is  caused 
by  the  influence  of  the  oviduct. 


FH.IUE  4.     Sections  through  two  extremely  reduced  testes.     </,  degenerating  cells. 


Not  only  virilis  but  also  hydei  oviducts  cause  degeneration  of  hydei  testes  at- 
tached to  them.  This  was  shown  by  three  cases  in  which  larval  hydei  female  discs 
were  transplanted  into  hydei  male  larvae  (see  Table  I). 

The  factor  in  the  oviduct  causing  degeneration  of  the  testis  by  contact  is  not 
species  specific,  for  hydei  oviducts  will  cause  virilis  testes  to  degenerate  (see  Ta- 
ble I). 

Sections  of  reduced  testes  were  made  and  their  histology  studied.  It  was  found 
that,  depending  upon  the  degree  of  reduction  of  size,  the  testes  contained  various 
amounts  of  spermatogonia  and  spermatocytes  in  all  stages  of  degeneration.  The 
remnants  of  disintegrated  cells  in  the  form  of  granular  picnotic  masses  together  with 
quite  normal  appearing  cells  were  observed.  Figure  4  shows  the  condition  found 
in  an  extremely  reduced  testis. 

CONCLUSION  AND  SUMMARY 

By  transplanting  female  genital  discs  into  male  hosts,  attachment  to  the  host 
testes  of  oviducts  developed  from  transplanted  genital  discs  is  obtained.  In  these 
cases  the  attached  testes  suffer  extensive  degeneration.  Only  cellular  contact  of 


294  DIETRICH  BODENSTEIN 

the  oviducts  to  the  testes  brings  about  this  phenomenon.  Unattached  female  ducts 
do  not  affect  the  development  of  the  testis.  The  principle  causing  degeneration  is 
not  species  specific.  The  findings  indicate  that  the  phenomenon  encountered  is  no 
unique  instance,  but  representative  when  oviduct  and  testis  establish  cellular  contact 
during  pupal  development. 

LITERATURE  CITED 

BODENSTEIN,  D.,  1946.  The  post-embryonic  development  of  Drosophila.  In  Biology  of  Dro- 
sophila.  Pub!.  Carnegie  lust.  IT  ash.  (in  press). 

DOBZHANSKY,  T.,  1931.  Interaction  between  female  and  male  parts  in  gynandromorphs  of 
Drosophila  simulans.  Roux'  Arch.  Entzv.  Mech.,  123:  719-746. 

STERN,  C,  1941a.  The  growth  of  testes  in  Drosophila.  I.  The  relation  between  vas  deferens 
and  testis  within  various  species.  Jour.  E.\-p.  ZooL,  87:  113-158. 

STERN,  C.,  1941b.  The  growth  of  testes  in  Drosophila.  II.  The  nature  of  interspecific- 
differences.  Jour.  Exp.  ZooL,  87:  159-180. 


cV1 

r«^Xo*s  *; 

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A  HISTOLOGICAL  STUDY  OF  SYNDISYRINX  FRANCISCANUS. 

GEN.  ET  SP.  NOV.,  AN  ENDOPARASITIC  RHABDOCOEL 

OF  THE  SEA  URCHIN,  STRONGYLOCENTROTUS 

FRANCISCANUS  1 

H.  E.  LEHMAN 

Department    of   Zoology    of    the    I'nhrrsity    of    Xorth    Carolina  - 

I  NTRODUCTION 

Up  to  the  present  time  eight  genera  of  worms  endoparasitic  in  echinoderms  and 
sipunculids  have  been  described  that  belong  to  the  rhabdocoel  family  Umagillidae 
Wahl,  19101).  Schneider  described  the  first  species,  Anoplodinm  parasita,  in  1858. 
Since  then  six  questionable  and  three  valid  species  of  this  genus  have  been  re- 
ported from  widely  separated  localities  as  parasites  of  holothurians.  Their  distribu- 
tion extends  from  the  Mediterranean,  Ionian,  and  North  Seas  to  Japan  and  the 
Philippines  (Bock,  1926).  Syndesinis  ccliinoniin  Francois,  1886,  the  only  spe- 
cies of  the  genus,  is  found  in  echinoids.  It  has  been  collected  in  the  Mediterranean 
(Russo,  1895),  Norway  (Westblad.  1926),  and  the  English  Channel  (Braun.  1889). 
Three  species  of  the  genus  C'olUishuna  are  found  in  sipunculids  at  Roscoff  (Dorler, 
1900),  the  Gulf  of  Kola  (Beklemischev,  1916),  and  the  Bay  of  Naples  (Wahl, 
1910a).  The  genus  Dcsiuotc  is  represented  by  one  species,  D.  vora.v,  discovered  in 
a  crinoid  collected  in  the  Gulf  of  Kola  (Beklemischev,  1916).  A  single  species 
parasitic  in  holothurians  has  been  described  in  each  of  four  genera,  i.e..  a  Japanese 
form,  Xcnoiuctra  arbor  a  Ozaki,  1932,  and  three  reported  from  the'coa.st  of  Nor- 
way, Wahlia  tiiacrostylijera  Westblad.  1930,  Anoplodlcra  valuta  \Yesthlad.  1930. 
and  type  genus  Uiiiagilla  forskalensis  Wahl,  1909. 

The  only  reference  to  a  member  of  the  Umagillidae  from  the  Western  Hemis- 
phere was  made  by  Powers  in  1936.  He  reported  the  presence  of  a  Syndesmis- 
like  worm  in  the  coelomic  cavity  of  the  echinoid,  Ccntrccliinns  antillantin.  at  Tortu- 
gas.  A  complete  description  was  not  given;  however,  as  compared  with  Syndesinis. 
noticeable  differences  were  observed  in  details  of  the  copulatory  apparatus  and  the 
arrangement  of  the  shell  glands.  While  the  endoparasitic  rhabdocoel  of  Sirongylo- 
centrotiis  jranciscanns.  the  large  common  sea  urchin  of  the  California  coast,  is  well 
known  to  some  investigators  who  have  worked  at  Pacific  Grove,  a  description  of 
tli is  worm  has  not  been  recorded  in  the  literature  prior  to  the  present  account. 

1  This  work  was  done  at  the  Wilson  Zoological  Laboratory  of  the  University  of  North 
Carolina  in  partial  fulfillment  of  the  requirements  for  the  degree  of  Master  of  Arts.  The 
author  is  indebted  to  Professor  D.  P.  Costello  for  suggesting  the  problem,  for  the  slide  prep- 
arations upon  which  this  study  was  essentially  based,  and  for  the  invaluable  suggestions  and 
criticisms  rendered  during  the  preparation  of  this  paper.  The  author  wishes  to  acknowledge  his 
appreciation  to  Dr.  L.  H.  Hyman  for  many  valuable  recommendations  and  for  permission  to 
introduce  her  revised  and  hitherto  unpublished  terminology  relating  to  this  group.  To  Miss 
Catherine  Henley  the  .author  expresses  his  gratitude'  for  the  translation  of  a  number  of  the 
references  cited  herein. 

-  Now  in  the  School  of  Biological  Sciences  of  Stanford  University. 

295 


296  H.  E.  LEHMAN 

Systematic  position  :; 

Order  Rhalxlocoela 

Suborder  Lecithophora 

Section  Dalyellioida 

Family  Umagillidae  Wahl,  1910 

Subfamily  Umagillinae  Wahl.  1910 

Genus  Syndisyrinx.  gen.  nov. 
Genotype  Syndisyrinx  franciscanus,  sp.  nov. 

Holotypc.  A  whole  mount  in  the  United  States  National  Museum,  Washington 
D.  C. 

Repositories  oj  type  material.  In  each  of  the  following  repositories  a  whole 
mount,  a  transversely  sectioned,  and  a  sagittally  sectioned  preparation  selected  from 
the  type  material  have  been  deposited :  U.  S.  National  Museum,  Washington.  D.  C. ; 
American  Museum  of  Natural  History,  New  York  City ;  British  Museum.  London ; 
California  Academy  of  Science,  San  Francisco ;  Wilson  Zoological  Laboratory  of 
the  University  of  North  Carolina ;  and  Museum  of  Natural  History,  Stanford  Uni- 
versity. Additional  preserved  material  may  be  obtained  from  the  author  or  from  any 
of  these  institutions. 

Type  locality.  Mussel  Point.  Monterey  Peninsula.  California,  Lat.  36°.  37', 
20"  N.,  Long.  121°,  54',  15"  W. 

Collectors.     D.  P.  Costello,  1937  and  H.  E.  Lehman,  1945. 

Distinguishing  characteristics.  Umagillinae  with  a  single  intestine,  paired  and 
branched  ovaries,  cuticular  penis,  and  a  bursa  seminalis  connected  by  cuticular  ducts 
to  the  seminal  receptacle  and  bursal  canal. 

MATERIALS  AND  METHODS 

Fifty-four  rhabdocoel  parasites  were  obtained  from  two  specimens  of  the  sea 
urchin  Strongylocentrotus  franciscanus  (A.  Agassiz)  by  Dr.  D.  P.  Costello  in  Au- 
gust 1937  at  Pacific  Grove,  California.  These  specimens  were  fixed  in  Heath's, 
Boveri's,  Lillie's  and  Worcester's  solutions.  Five  of  the  individuals  were  sectioned 
serially  at  10/i  and  stained  with  Heidenhain's  iron  hematoxylin  and  orange  G. 
One  of  these  preparations  was  exceptionally  fine  and  the  majority  of  the  accompany- 
ing figures  \vere  made  from  it.  Unfortunately  this  preparation,  which  the  author 
intended  to  designate  as  the  holotype,  was  lost  when  a  microscope  was  stolen. 
This  material,  including  the  slide  preparations,  was  turned  over  to  me  by  Dr.  Cos- 
tello. The  morphological  study  was  based  on  this  material. 

In  the  summer  of  1945  during  June,  July,  and  August,  the  author  collected  sev- 
eral hundred  additional  specimens  from  the  same  locality.  Over  sixty  urchins  were 
examined  and  all  were  found  to  be  infested ;  frequently  three  dozen  or  more  parasites 
were  obtained  from  the  intestine  of  a  single  host.  These  worms  were  fixed  in 
Heath's  and  Beauchamp's  solutions.  Seventy  were  sectioned  serially  at  10  ,u  and 
stained  with  Mayer's  acid  hemalum  and  triosin.  Thirty  whole  mounts  stained 
with  paracarmine  were  also  made.  The  type  material  was  selected  from  these  prep- 

3  Classification  according  to  Bresslau  (1933),  with  the  exception  of  "Family  Anoplodiidae 
Graff,  1913,"  which  has  been  rejected  in  favor  of  "Family  Umagillidae  Wahl,  1910b,"  inasmuch 
as  no  reason  is  given  by  Graff  for  discarding  the  older  name  or  for  selecting  Anoplodium  as 
type  genus.  The  subfamily  Umagillinae  has  been  retained  as  designated  by  Wahl,  1910b. 


A  PARASITIC  RHABDOCOEL  297 

parations.  At  this  time  another  parasite  of  Str.  jranciscanus  was  discovered  which 
differed  from  Syndlsyrin.v  in  shape,  manner  of  locomotion,  and  color.  A  description 
of  this  worm  is  being  prepared  and  preliminary  examination  of  sectioned  material 
indicates  a  close  relationship  to  Syndesmis  cchinorum.  Upon  the  suggestion  of 
Prof.  A.  R.  Moore,  who  had  occasionally  observed  parasitic  worms  in  Str.  purpur- 
atus  (Stimpson),  forty-seven  of  these  urchins  were  examined.  In  twenty-nine  of 
them,  worms  that  are  very  similar  to,  and  may  be  identical  with  Syndisyrin.r  fran- 
ciscanits  were  present  in  small  numbers. 

GENERAL  MORPHOLOGY 

The  living  animals  are  bright  red  with  a  dark  brown  or  yellow  median  longi- 
tudinal line  which  marks  the  extent  of  the  intestine.  The  worms  are  flattened 
dorsoventrally  and  have  a  leaf-like  appearance,  being  rounded  at  the  anterior  end 
and  slightly  pointed  posterad.  Individuals  vary  in  size  from  2  to  3  mm.  long  and 
1.6  to  2.5  mm.  wide.  The  body  is  thickest  at  approximately  one-fourth  of  the  dis- 
tance from  the  anterior  end  and  at  this  level  measures  about  0.5  mm.  in  the  dorso- 
ventral  axis.  Laterally  and  posteriorly  the  thickness  of  the  body  diminishes  gradu- 
ally to  about  0.2  mm.  at  the  periphery.  A  ciliated  epithelium  covers  the  entire 
surface ;  rhabdites  and  cuticle  are  lacking. 

The  mouth  is  situated  on  the  ventral  surface  about  one-fourth  of  the  distance 
from  the  anterior  end  and  a  common  genital  pore  opens  ventrally  at  the  posterior 
extremity  of  the  body.  The  musculature  and  parenchyma  are  typical  of  other 
Umagillidae.  No  excretory  system  was  observed.  The  strongly  muscular  pharynx 
is  typically  doliiform  and  possesses  pharyngeal  glands ;  it  communicates  by  a  short 
oesophagus  with  the  gut.  The  intestine,  possessing  a  number  of  small  lateral  diver- 
ticula,  extends  posterad  under  the  dorsal  epidermis  along  the  mid-line  and  termi- 
nates one-quarter  of  the  distance  from  the  posterior  end  of  the  body.  The  gut  con- 
tains no  permanent  lumen  and  food  masses  lie  in  temporary  cavities  surrounded  by 
large  digestive  cells.  The  brain,  composed  of  two  cerebral  ganglia  connected  by  a 
wide  commissure,  lies  anterior  to  the  pharynx  and  gives  off  paired  anterior,  lateral, 
and  posterior  nerves. 

Lobed  testes  lie  lateral  to  the  mid-line  in  the  anterior  half  of  the  body.  Acces- 
sory glands  empty  into  the  sperm  duct  that  arises  from  each  testis  and  passes 
anteracl.  These  paired  tubes  unite  mesially  and  enter  a  small  spermiducal  vesicle 
that  is  continued  posterad  as  a  muscular  common  sperm  duct  which  lies  dorsal  to 
the  uterus  along  the  mid-line.  This  tube  terminates  in  an  elongated  cuticular  stylet, 
the  penis,  which  is  enlarged  and  funnel-like  at  the  base.  The  penis  stylet  enclosed 
in  the  male  antrum  extends  through  the  posterior  third  of  the  body  to  the  common 
genital  antrum  and  over  most  of  its  length  does  not  exceed  3  p.  in  diameter. 

Paired  vitellaria  are  found  immediately  posterior  to  the  testes ;  they  are  greatly 
ramified  and  fill  most  of  the  ventrolateral  spaces  in  the  middle  third  of  the  body. 
Posterior  to  the  vitellaria  a  pair  of  ovaries  is  located,  one  on  each  side  of  the 
mid-line.  Laterally  each  branches  into  five  or  more  finger-like  lobes.  Three  or 
four  collecting  ducts  from  the  vitellaria  empty  with  the  ovaries  and  seminal  recep- 
tacle into  the  anterior  end  of  the  ovovitelline  duct.  The  seminal  receptacle  is  oval 
and  filled  with  sperm.  Located  posterodorsad  to  this  organ  is  a  vesicular,  sperm- 
filled  bursa  seminalis  connected  to  the  seminal  receptacle  by  a  fine  cuticular  insemi- 


298 


H.  E.  LEHMAN 


ph.g. 


.5 


bur.  c. 


FIGURE  1.     Semidiagrammatic  median  sagittal  section. 


A  PARASITIC  RHABDOCOEL  299 

nation  canal.  Arising  in  close  association  with  this  tubule  is  a  similar  duct,  the 
cuticular  proximal  part  of  the  bursal  canal  that  passes  posterad  from  the  bursa 
seminalis  approximately  60  p,  before  widening  into  the  posterior  muscular  portion 
of  the  bursal  canal  (vagina).  A  cuticular  sheath  surrounds  the  openings  of  these 
two  ducts  into  the  bursa  seminalis.  The  composite  structure,  consisting  of  this 
sheath  and  the  canals  passing  through  it,  makes  up  the  bursal  valve. 

An  ovovitelline  duct,  into  which  accessory  glands  empty,  arises  ventrally  at  the 
anterior  end  of  the  seminal  receptacle.  It  passes  posterad  and  unites  with  the  female 
antrum.  The  uterus,  lying  close  to  the  ventral  epidermis,  extends  anteriorly  from 
the  female  antrum  almost  to  the  pharynx.  At  the  anterior  end  of  the  uterus  an  egg 
capsule  containing  from  one  to  five  ova  and  numerous  yolk  cells  is  generally  found. 
The  capsule  is  continued  posterad  as  a  long  coiled  whip  similar  to  those  found  in 
related  forms.  Most  of  the  ventrolateral  spaces  of  the  posterior  third  of  the  body 
are  filled  by  cement  glands ;  they  communicate  by  many  small  ducts  with  the  female 
antrum.  The  common  genital  antrum  is  an  elongated  cavity  at  the  posterior  end 
of  the  body  into  which  the  female  antrum  enters  ventrally,  the  male  antrum  and  penis 
open  mesially  and  the  bursal  canal  is  given  off  dorsally.  At  its  posterior  end  is  the 
common  genital  pore  which  opens  ventrally  to  the  exterior. 

HlSTOLOGICAL   STRUCTURE 

Epidermis 

A  ciliated  epithelium  covers  both  dorsal  and  ventral  surfaces  of  the  body.  No 
pigment  or  special  gland  cells  were  observed  in  this  layer  and  a  cuticle  and  rhabdites 
are  lacking.  The  cytoplasm  of  the  cells  in  the  epidermal  layer  is  granular  and  cell 
boundaries,  though  faintly  stained,  are  distinct.  The  cells  covering  the  dorsal  sur- 
face are  cuboidal  and  measure  10 /A  from  basement  membrane  to  external  surface. 
The  cytoplasm  of  these  cells  stains  moderately  with  hematoxylin.  On  the  ventral 
surface  the  cells  are  flattened  and  are  about  7 /A  thick  and  from  12  to  35  ^  wide; 
they  have  little  affinity  for  hematoxylin.  Cilia  of  the  ventral  epidermis  are  about 
6.5  p.  long  and  are  almost  twice  the  length  of  those  found  on  the  dorsal  surface. 
Cells  possessing  the  staining  properties  and  short  cilia  characteristic  of  the  dorsal 
layer  extend  for  a  short  distance  ventrally  around  the  lateral  edges.  A  zone  4  to  6 
cells  wide  of  intermediate  nature  accomplishes  the  transition  between  typical  dorsal 
and  ventral  epithelium. 

Musculature  and  parenchyma 

The  arrangement  of  the  musculature  is  essentially  the  same  as  that  described  for 
other  Umagillidae.  Under  the  basement  membrane  of  the  surface  epithelium  is 

Abbreviations  for  Figures  1  and  2. 

a.o.d. — accessory  glands  of  ovovitelline  duct,  a.s.d. — accessory  glands  of  sperm  duct,  br. — 
brain,  b.c. — buccal  cavity,  bur.  c. — bursal  canal,  bur.  c'. — cuticular  end  of  bursal  canal,  b.s. — 
bursa  seminalis,  b.v. — bursal  valve,  e.g. — cement  glands,  c.s.d. — common  sperm  duct,  e.c. — egg 
capsule,  f.a. — female  antrum,  g.a. — common  genital  antrum,  g.p.— genital  pore,  int. — intestine, 
i.e. — insemination  canal,  1.  int. — lumen  of  intestine,  m.a. — male  antrum,  oe. — oesophagus,  ov.— 
ovary,  ov'. — ovum,  o.d. — ovovitelline  duct,  p. — penis,  p'. — base  of  penis,  ph. — pharynx,  ph.  g. 
— pharyngeal  glands,  s.d. — sperm  duct,  s.r. — seminal  receptacle,  s.v. — spermiducal  vesicle,  te.— 
testis,  u. — uterus,  vit. — vitellaria,  vit.  d. — vitelline  ducts,  w. — whip  of  egg  capsule,  y. — yolk  cells. 


300 


H.  E.  LEHMAN 


c.s.d. 


a.s.d. 


vit.  d. 


(7.  O.  d. 


g.a 


m 


-9-P-  2 

FIGURE  2.     Semidiagrammatic  median   frontal   section,    intestine   omitted. 


A  PARASITIC  RHABDOCOEL  301 

found  a  thin  layer  of  suhepidermal  muscles  (Figs.  3-5,  7,  8).  The  superficial  mus- 
cles are  circular ;  these  overlie  a  longitudinal  sheet,  and  interposed  at  intervals  be- 
tween these  layers  are  well-developed  oblique  fibers.  In  addition  to  these,  bundles 
of  fibers  attached  to  the  internal  organs  or  the  basement  membrane  of  the  epidermis 
pass  dorsoventrally  through  the  parenchyma  (Figs.  1-3).  The  special  muscles  of 
the  reproductive  and  digestive  systems  will  be  described  in  connection  with  the 
organs  with  which  they  are  associated. 

A  parenchyma,  composed  of  large,  irregularly  shaped  cells  with  coarsely  granular 
or  vacuolated  cytoplasm,  fills  most  of  the  spaces  between  the  internal  organs  and 
epidermis.  A  histologically  distinct  parenchymatous  mass  of  cells  enclosed  in  a 
fibrous  capsule  extends  posterad  along  the  mid-ventral  line  from  the  posterior  level 
of  the  pharynx  to  the  region  in  which  the  female  antrum  enters  the  common  genital 
antrum.  The  flattened,  nonvacuolated  cells  of  this  tissue  possess  finely  granular 
cytoplasm  and  are  arranged  in  concentric  layers  around  the  reproductive  ducts,  most 
of  which  pass  through  the  mid-ventral  parenchyma  (Figs.  3-5,  7).  Nowhere  within 
the  parenchyma  were  flame  cells  or  collecting  ducts  of  an  excretory  system  observed. 

Nervous  system 

The  brain  is  similar  in  all  respects  to  those  described  in  other  members  of  the 
family.  It  is  located  just  anterior  to  the  pharynx  and  consists  of  two  ganglia  con- 
nected bv  a  wide  commissure.  Around  the  central  fibrous  mass  of  the  brain  are 

j 

numerous  ganglionic  cells  that  stain  quite  evenly  with  hematoxylin.  Poorly  devel- 
oped anterior,  lateral  and  posterior  pairs  of  nerves  leave  the  brain  and  can  be  traced 
for  short  distances  into  the  parenchyma.  No  theca  separates  the  brain  or  nerves 
from  the  parenchyma  and  no  special  sensory  organs  were  found. 

Digestive  system 

The  mouth  lies  on  the  ventral  surface  about  one-fourth  of  the  distance  from  the 
anterior  end  of  the  body.  It  opens  into  a  very  small  buccal  cavity  lined  by  flattened 
ciliated  cells  that  are  continuous  externally  with  the  ventral  epithelium  (Figs.  1,  8). 
A  sphincter  underlying  the  epithelium  regulates  the  size  of  the  oral  opening.  Lying 
immediately  dorsal  to  the  mouth  and  opening  into  the  buccal  cavity  is  the  doliiform 
pharynx  which  has  the  appearance  of  a  dorsally  compressed  sphere.  Its  dorso- 
ventral  axis  is  about  0.1  mm.  long  and  its  greatest  diameter  is  about  0.17  mm. 
Passing  dorsoventrally  through  the  pharynx  is  a  funnel-shaped  lumen  that  is  nar- 
rowest at  the  oral  or  ventral  end.  The  musculature  of  the  pharynx  is  similar  in 
most  details  of  its  organization  to  that  found  in  Syndcsuiis  as  described  by  Russo 
(1895).  A  thin  superficial  layer  of  vertical  fibers  overlies  the  well-defined  muscles 
encircling  the  lumen  of  the  pharynx.  In  addition  to  the  circular  and  vertical  mus- 
cles, radial  fibers  pass  from  the  lumen  to  the  peripheral  surface  of  the  pharynx. 
Nonmuscular  cells  with  heavily  staining  reticular  cytoplasm  fill  the  spaces  between 
the  radial  fibers  (Fig.  8).  Surrounding  the  pharynx  is  a  sharply  defined  basement 
membrane  to  which  are  attached  numerous  short,  radially  arranged,  protractor  mus- 
cles that  extend  to  the  basement  membrane  of  the  ventral  epidermis.  The  more 
oblique  of  these  fibers  serve  also  as  dilators  of  the  pharynx.  Poorly  developed  re- 
tractors are  attached  to  the  equator  of  the  pharynx  and  pass  to  the  dorsal  surface. 
Pharyngeal  glands  are  present  encircling  the  dorsal  end  of  the  pharynx.  The 


302  H.  E.  LEHMAN 

peripheral  contours  of  these  glands  are  lobular  and  a  thin  basement  membrane  sepa- 
rates them  from  the  parenchyma.  The  cells  which  make  up  these  glands  have  in- 
distinct cell  boundaries  and  dense  cytoplasm  containing  numerous  granules  that 
stain  darkly  with  hematoxylin.  Cytoplasmic  continuations  of  the  cells  extend  ven- 
trally  and  line  the  lumen  of  the  pharynx  (Fig.  8).  Leading  dorsad  from  the 
pharynx  is  a  short  oesophagus  which  passes  through  the  pharyngeal  glands  and 
opens  into  the  anterior  end  of  the  intestine. 

The  intestine  lies  along  the  mid-line  under  the  dorsal  epidermis  and  extends  pos- 
terad  from  the  level  of  the  brain  to  about  one-fourth  of  the  distance  from  the  pos- 
terior end  of  the  body  (Fig.  1).  The  width  of  the  gut  varies  from  0.1  to  0.2  mm. 
at  the  anterior  end  and  diminishes  gradually  posteriorly.  Short  diverticula  ex- 
tend laterally  on  each  side.  The  epithelium  of  the  intestine  is  made  up  of  large  ir- 
regularly shaped  cells  containing  moderately  granular  cytoplasm.  The  basal  end 
of  most  cells  reaches  the  fibromuscular  investing  sheath  of  the  intestine  that  sepa- 
rates it  from  the  parenchyma.  The  lumen  of  the  intestine  can  only  be  observed  when 
ingested  material  is  present ;  this  condition  is  similar  to  that  found  in  some  alloeo- 
coels.  In  an  animal  that  has  been  feeding,  food  masses  often  lie  in  cavities  that  have 
lost  all  direct  communication  with  the  oesophagus  (Fig.  1).  Food  vacuoles  of 
varying  sizes  are  generally  present  in  the  cells  surrounding  the  ingested  material 
and  digestive  cells  were  occasionally  observed  that  had  apparently  migrated  into 
the  food  masses  by  amoeboid  movement. 

Male  reproductive  system 

The  paired  testes  lie  lateral  to  the  mid-line  in  the  anterior  half  of  the  body. 
They  are  approximately  0.5  mm.  long  and  from  0.3  to  0.5  mm.  wide..  Each  is  made 
up  of  four  to  six  vesicular  lobes,  the  lumina  of  which  are  in  direct  communication 
with  one  another  (Fig.  2).  Separating  the  testes  from  the  parenchyma  is  a  fibrous 
sheath  that  pentrates  and  partially  subdivides  the  lobes.  The  chambers  so  formed 
are  filled  with  developing  germ  cells  and  tangled  masses  of  mature  spermatozoa 
(Fig.  3).  Mature  sperm  are  present  in  all  lobes  but  are  more  numerous  midway 
between  the  anterior  and  posterior  ends  of  the  testes  near  the  wide  openings  of  the 
sperm  ducts.  These  ducts  run  mesially  from  the  testes  and  enter  the  mid-ventral 
parenchyma,  whereupon  they  diminish  to  about  10  //,  in  diameter  and  generally  con- 
tinue their  course  anterad,  dorsolateral  to  the  uterus  (Figs.  1-3).  A  thin  epithelium 
surrounded  by  loose  fibromuscular  elements  makes  up  the  walls  of  the  sperm  ducts. 
Near  the  origin  of  these  ducts  from  the  testes,  glandular  cells  that  probably  possess 
some  accessory  function  are  found  in  the  mid-ventral  parenchyma  adjacent  to  the 
ventral  walls  of  the  tubes  (Fig.  2). 

At  varying  distances  posterior  to  the  pharynx  the  sperm  ducts  unite  mesially 
and  enter  the  anterior  end  of  a  common  sperm  duct  which  at  this  point  is  somewhat 
enlarged  to  form  a  small  spermiducal  vesicle  (Figs.  1-3).  The  slightly  coiled  com- 
mon sperm  duct  continues  posterad  from  the  vesicle  through  the  mid-ventral  paren- 
chyma. It  gradually  diminishes  in  diameter  from  45  ^  to  12  (j..  Its  walls  are  com- 
posed of  connective  tissue  cells  surrounded  by  a  sheath  of  circular,  oblique,  and 
longitudinal  muscle  fibers.  The  lumen  of  the  tube  is  lined  by  a  thin  squamous 
epithelium  that  is  separated  from  the  theca  by  a  thick  basement  membrane.  Pos- 
teriorly, the  common  sperm  duct  unites  with  the  enlarged  base  of  the  penis  at 


A  PARASITIC  RHABDOCOEL  303 

about  one-third  of  the  distance  from  the  posterior  end  of  the  body  (Figs.  1,  2,  4). 
The  penis  lies  in  a  muscular  sheath,  the  male  antrum,  which  is  a  diverticulum  of  the 
genital  antrum.  Histologically  this  sheath  is  similar  in  most  details  of  its  structure 
to  the  common  sperm  duct ;  however,  the  lining  epithelium  of  the  male  antrum  is 
thicker,  in  some  regions  almost  occluding  the  lumen,  and  a  thick  basement  mem- 
brane is  lacking  (Fig.  5).  The  copulatory  organ  is  a  cuticular  tubule  that  extends 
through  the  posterior  third  of  the  body  and  is  about  3  ^  in  thickness  over  most  of 
its  length.  The  lumen  of  the  stylet  does  not  exceed  2  /*  in  diameter  except  at  the 
anterior  end  of  the  penis  which  is  enlarged  to  12  p,  at  its  union  with  the  posterior  end 
of  the  common  sperm  duct  (Figs.  1—3).  The  rim  of  the  funnel-like  base  of  the 
penis  is  thickened  to  form  a  collar ;  longitudinal  muscles  in  the  walls  of  the  male 
antrum  and  common  sperm  duct  attach  to  this  collar  and  function  as  protractors 
and  retractors  of  the  penis. 

Female  reproductive  system 

The  paired  ovaries  lie  in  the  posterior  third  of  the  body.  Each  is  made  up  of 
from  five  to  ten  lobes  that  branch  dichotomously  from  common  trunks  arising 
near  the  anterior  end  of  the  seminal  receptacle.  The  lobes  of  the  ovaries  are  di- 
rected posterolaterad  and  are  separated  from  the  parenchyma  by  a  very  poorly  de- 
veloped theca.  The  branches  are  made  up  of  dovetailed  chains  or  rouleaux  of  com- 
pressed ova  that  are  proliferated  from  primordial  cells  at  the  distal  ends  of  the  lobes 
(Fig.  2).  Mature  ova  are  approximately  75 /A  in  diameter  and  vary  in  thickness 
from  20  to  60  \n.  The  cytoplasm  of  immature  eggs  is  at  first  homogeneous,  'but  as 
development  continues  many  small  peripherally  distributed  granules  appear  that 
are  probably  stored  nutrient  materials.  During  the  period  of  growth  the  nuclei  of 
the  ova  increase  from  7  to  25  p.  in  diameter  and  the  chromatin  granules  gradually 
lose  their  affinity  for  basic  dyes.  In  mature  ova  only  the  spherical  or  oval  nu- 
cleolus  stains  deeply  with  hematoxylin  (Fig.  4). 

A  pair  of  greatly  branched  vitellaria  lie  anterior  to  the  ovaries  and  fill  most  of 
the  ventrolateral  spaces  in  the  middle  third  of  the  body  (Figs.  1,  2).  Many  of  the 
dorsoventral  muscles  of  the  parenchyma  contribute  fibers  to  the  diffuse  sheath  that 
encloses  these  ducts.  Primordial  cells  at  the  distal  ends  of  the  branches  give  rise  to 
yolk  cells.  As  the  cells  increase  in  size,  the  cytoplasm  which  at  first  is  homogeneous, 
becomes  filled  with  refractile  granules  that  coalesce  to  form  amber-colored  droplets 
(Figs.  3,  4).  From  each  side  three  or  four  collecting  ducts  packed  with  mature 
yolk  cells  pass  posterad  from  the  vitellaria  and  unite  near  the  mid-line  shortly  be- 
fore emptying  into  the  anterior  end  of  the  ovovitelline  duct  (Fig.  2). 

The  seminal  receptacle  is  somewhat  oval  and  lies  ventral  to  the  intestine  within 
the  sheath  that  surrounds  the  gut.  Its  anterior  extremity  is  about  one-third  of  the 
distance  from  the  posterior  end  of  the  body.  The  posterior  part  of  this  organ  is 
thin  walled  and  masses  of  mature  spermatozoa  are  observable  in  its  extensive  lumen. 
Anteriorly  the  seminal  receptacle  opens  with  the  paired  ducts  of  the  vitellaria  and 
ovaries  into  the  ovovitelline  duct  which  arises  ventrally  in  this  region  (Figs.  1,  2). 
The  wall  of  the  anterior  third  of  the  seminal  receptacle  is  lined  by  large  gland-like 
cells  that  restrict  the  lumen  to  a  narrow  channel  6  to  10  /A  wide  which  connects  the 
posterior  vesicular  portion  to  the  ovovitelline  duct  (Fig.  4). 

The  bursa  seminalis  lies  dorsal  to  the  vesicular  portion  of  the  seminal  receptacle. 
It  is  enclosed  in  the  same  sheath  that  surrounds  the  seminal  receptacle  and  the  pos- 


304  H.  E.  LEHMAN 

terior  end  of  the  intestine  (Figs.  1,  2).  The  large  lumen  of  the  bursa  seminalis  is 
lined  by  an  epithelial  layer  very  similar  to  that  lining  the  posterior  part  of  the  seminal 
receptacle.  In  every  specimen  examined  spermatozoa  were  found  in  the  bursa ; 
frequently  they  were  aggregated  into  roughly  spindle-shaped  masses  in  which  degener- 
ating sperm  were  observable  (Figs.  5,  6).  Arising  ventrally,  or  in  some  cases  later- 
ally, from  the  wall  of  the  posterior  half  of  the  bursa  seminalis  is  the  insemination 
canal,  a  fine  cuticular  tubule  about  4  ^  in  diameter  connecting  the  lumina  of  the 
bursa  seminalis  and  seminal  receptacle.  In  close  association  with  the  insemination 
canal,  a  second  cuticular  tube  of  the  same  dimensions  arises  from  the  wall  of  the 
bursa  seminalis  and  connects  the  bursa  posteriorly  to  the  bursal  canal  (Figs.  1,  2, 
5,  6).  Surrounding  the  ends  of  the  ducts  as  they  penetrate  the  lining  epithelium  of 
the  bursa  is  a  cuticular  sheath,  7  /JL  in  diameter  and  10 /x  long.  The  inner  end  of 
this  sheath  is  involuted  and  fused  to  the  ends  of  the  two  ducts  (Fig.  6).  To  desig- 
nate this  composite  cuticular  structure  made  up  of  the  insemination  canal,  the  proxi- 
mal end  of  the  bursal  canal  and  the  sheath  surrounding  the  ends  of  these  ducts,  the 
term,  "bursal  valve,"  is  suggested. 

The  bursal  canal  (vagina)  is  a  tubular  structure  about  0.1  mm.  long  and  20  ^ 
in  diameter  that  arises  as  an  anterodorsal  continuation  of  the  common  genital  an- 
trum.  Its  wall  is  composed  of  an  inner  epithelial  layer  surrounded  by  a  strong 
fibromuscular  sheath.  At  the  posterior  end  of  the  canal  the  epithelium  possesses 
cilia-like  projections  characteristic  of  the  lining  of  the  common  genital  antrum. 
Anteriorly  the  lumen  of  the  canal  is  reduced  and  the  thin  basement  membrane  un- 
derlying the  epithelium  becomes  continuous  with  the  cuticular  wall  of  the  tubule 
leading  into  the  bursa  seminalis. 

A  flattened  muscular  ovovitelline  duct  (ductus  communis)  arises  ventrally  near 
the  anterior  end  of  the  seminal  receptacle  and  receives  the  ducts  of  the  ovaries  and 
vitellaria.  It  passes  posterad  through  the  mid-ventral  parenchyma  to  about  the  level 
of  the  posterior  end  of  the  bursa  seminalis  and  here  enters  the  anterior  end  of  the 
female  antrum  (Figs.  1,  2,  5).  The  ovovitelline  duct  is  approximately  35  ^  wide 
but  is  capable  of  considerable  expansion  to  allow  ova  and  yolk  cells  to  pass  into  the 
uterus.  Circular,  oblique  and  longitudinal  muscles  are  observable  in  contact  with 
the  thin  basement  membrane  that  underlies  the  lining  epithelium ;  no  fibrous  sheath 
separates  this  duct  from  the  cells  of  the  mid-ventral  parenchyma.  Running  parallel 
to  the  ovovitelline  duct  in  the  lateral  parenchyma  are  paired  accessory  glands  which 
enter  the  posterior  part  of  the  duct  prior  to  its  union  with  the  female  antrum  (Figs. 
1,  2,  5).  Generally  the  cytoplasm  of  these  gland  cells  stains  evenly;  however,  in 
some  cases  the  cells  were  observed  to  be  filled  with  eosinophil  granules. 

The  uterus  arises  ventrally  from  the  anterior  end  of  the  female  antrum.  It  ex- 
tends anterad  almost  to  the  pharynx  through  the  mid-ventral  parenchyma  and 

FIGURE  3.     Transverse  section  through  egg  capsule  and  spermiducal  vesicle   (X350). 
FIGURE  4.     Transverse  section  through  entrance  of  ovary  into  seminal  receptacle   (X500). 

Abbreviations  for  Figures  3  and  4. 

a.o.d. — accessory  glands  of  ovovitelline  duct,  e.g. — cement  glands,  e.c. — egg  capsule,  int.— 
intestine,  mu. — muscle  sheath,  mu'. — subepidermal  muscles,  mu".— dorsoventral  muscles  of 
parenchyma,  ov. — ovary,  ov'. — ovum,  p'. — base  of  penis,  pa. — mid-ventral  parenchyma,  s.d.— 
sperm  duct,  s.r. — seminal  receptacle,  s.v. — spermiducal  vesicle,  te. — testis,  u. — uterus,  \v. — whip 
of  egg  capsule,  y. — yolk  cells. 


A  PARASITIC  RHABDOCOEL 


305 


•.?s  -ii/**'      4  -'.. .  -  •• 


FIGURES  3-4. 


306  H.  E.  LEHMAN 

through  its  entire  course  lies  very  close  to  the  ventral  surface  of  the  body  (Figs.  1, 
2,  4).  The  anterior  end  of  the  uterus  is  enlarged  and  encloses  an  amber-colored, 
oval  egg  capsule  containing  numerous  yolk  cells  and  from  one  to  five  spherical  eggs 
.(Figs.  1,  2,  3).  The  egg  capsule  is  cuticular  and  possesses  a  whip-like  prolongation 
that  extends  posterad  through  the  entire  length  of  the  uterus  and  female  antrum. 
Over  most  of  its  length  the  whip  is  about  10 /A  thick.  In  the  middle  portion  of  the 
uterus  the  whip  is  often  coiled  back  upon  itself  a  number  of  times  so  that  its  total 
length  may  greatly  exceed  that  of  the  uterus  (Figs.  1,  2).  The  uterine  wall  is  very 
similar  in  structure  to  the  ovovitelline  duct  and  is  able  to  enlarge  greatly  to  accom- 
modate the  egg  capsule  and  the  folded  part  of  the  egg  whip  (Figs.  3,  4). 

The  female  antrum  extends  from  the  posterior  ends  of  the  uterus  and  ovovitelline 
duct  to  the  common  genital  antrum  (Figs.  1,  2).  The  walls  are  lined  by  columnar 
epithelial  cells  surrounded  by  a  thin  basement  membrane  and  a  muscular  layer  that 
is  continuous  with  the  fibers  enclosing  the  uterus  and  ovovitelline  duct.  The  lu- 
men is  about  12 /A  in  diameter  and  the  posterior  end  of  the  egg  whip,  when  present, 
almost  completely  fills  this  space  (Figs.  5,  7).  The  ventrolateral  spaces  of  the  pos- 
terior third  of  the  body  contain  numerous  unicellular  cement  glands.  The  cyto- 
plasm of  these  cells  is  generally  uniformly  filled  with  small  granules  that  have  a  strong 
affinity  for  hematoxylin.  Throughout  the  entire  length  of  the  female  antrum  many 
ducts  from  these  glands  enter  the  lateral  walls  (Figs.  1,  2,  5,  7).  The  secretions  of 
the  cement  glands  are  believed  to  be  associated  with  the  attachment  of  the  egg  cap- 
sules to  the  substrate  when  expelled.  Living  animals  compressed  under  a  cover 
glass  were  occasionally  observed  at  low  magnification  to  undergo  a  series  of  rapid 
contractions  which  resulted  in  the  extrusion  of  the  egg  capsule  and  whip.  How- 
ever, nothing  is  known  about  the  normal  deposition  and  attachment  of  the  capsules, 
nor  are  other  details  of  the  life  cycle  understood. 

The  common  genital  antrum  lies  at  the  posterior  end  of  the  body.  It  is  an 
elongated  tube  lined  by  flattened  cells  that  appear  to  have  cilia  about  20  //  long  which 
extend  into  the  lumen  (Figs.  1,  2,  7).  A  diffuse  fibrous  sheath  separates  this  or- 
gan from  the  parenchyma.  The  common  genital  antrum  receives  the  terminal 
ducts  of  both  male  and  female  reproductive  systems :  the  bursal  canal  arises  from  it 
as  a  dorsal  diverticulum ;  the  male  antrum  enclosing  the  penis  stylet  is  given  off  as 
a  mesial  evagination ;  and  the  female  antrum  enters  it  ventrally.  The  common  geni- 
tal pore  opens  on  the  ventral  surface  at  the  posterior  end  of  the  body.  At  this  point 

FIGURE  5.     Transverse  section  through  bursa  seminalis  and  bursal  valve   (X350). 
FIGURE  6.     Bursal  valve   (X  1,050). 

FIGURE  7.  Transverse  section  through  the  entrance  of  female  antrum  and  male  antrum 
into  the  common  genital  antrum  (X  350). 

FIGURE  8.     Transverse  section  through  pharynx   (X  200). 

Abbreviations  for  Figures  5  through  8. 

a.o.d. — accessory  glands  of  ovovitelline  duct,  a.o.d'. — ducts  of  accessory  glands  of  ovovitel- 
line duct,  b.c. — buccal  cavity,  bur.  c. — bursal  canal,  bur.  c'. — cuticular  end  of  bursal  canal,  b.s.— 
bursa  seminalis,  b.v. — bursal  valve,  cil. — cilia,  e.g. — cement  glands,  d.c.g. — ducts  of  cement 
glands,  f.a.— female  antrum,  g.a. — common  genital  antrum,  int. — intestine,  i.e. — insemination 
canal,  1.  int. — lumen  of  intestine,  m.a.— male  antrum,  mu. — muscle  sheath,  mil'. — subepidermal 
muscles,  mu". — pharyngeal  protractor  muscles,  n. — nerves,  oe. — oesophagus,  o.d. — ovovitelline 
duct,  pa. — mid-ventral  parenchyma,  p. — penis,  ph. — pharynx,  ph.  g. — pharyngeal  glands,  s. — sper- 
matozoa, s'. — degenerating  spermatozoa,  u. — uterus,  w. — whip  of  egg  capsule. 


A  PARASITIC  RHABDOCOEL 


307 


•    CIS&-V 

r  1  ^  '  7     '-•  A  v  •', 

J.    .    ..  >  •  :  .-'•-,!      '       : ,',          : 

6 


" 


7 


FIGURES  5-8. 


308  H.  E.  LEHMAN 

the  ciliated  ventral  epithelium  is  invaginated  and  forms  a  short  bulb-like  canal  which 
meets  an  outpocketing  of  the  common  genital  antrum.  Sphincters  encircle  both  ends 
of  this  canal  and  regulate  the  size  of  the  pore. 

DISCUSSION 
Comparison  of  genera 

Although  the  parasite  described  here  is  similar  in  many  respects  to  all  genera  in 
the  family  Umagillidae,  there  are  certain  structural  characteristics  that  do  not  cor- 
respond to  those  of  any  previously  reported  genus  of  this  family.  Therefore,  it  is 
considered  necessary  to  establish  a  new  genus  to  be  designated  by  the  name  Syndi- 
syrinx. This  name  is  intended  to  describe  the  complex  bursal  valve  which  is  not 
present  in  any  other  genus  of  the  family.  The  specific  name,  Syn.  franciscanus,  is 
given  to  designate  the  host,  Strongylocentrotus  franciscanus,  in  which  it  was  first 
found. 

For  the  sake  of  uniformity  in  the  following  comparison  of  genera  of  the  family 
Umagillidae,  the  morphological  nomenclature  used  by  the  various  authors  in  their 
original  descriptions  of  genera  and  species  has  been  altered  to  conform  with  the 
terminology  employed  in  the  preceding  analysis  of  Syndisyrinx. 

In  addition  to  the  fact  that  both  Syndisyrinx  and  Syndcsmis  are  found  in  the  in- 
testine of  echinoids,  the  morphological  characteristics  of  Syndisyrinx  indicate  a 
closer  relationship  to  Syndcsmis  than  to  the  other  genera  of  the  family.  The  loca- 
tion and  appearance  of  important  organs,  viz.,  muscular  pharynx,  lobed  testes,  small 
spermiducal  vesicle,  muscular  common  sperm  duct,  ramified  vitellaria,  dichotomously 
branched  ovaries,  elongated  uterus  and  egg  capsule  with  whip,  are  very  similar  in 
Syndcsmis  and  Syndisyrinx  and  strongly  suggest  a  close  relationship  between  these 
two  genera.  Syndisyrinx  differs  from  Syndcsmis  chiefly  in  the  structure  and  re- 
lationships of  the  bursa  seminalis  and  seminal  receptacle.  In  Syndcsmis  a  single 
vesicle  is  present  for  the  reception  of  sperm  and  cuticular  structures  such  as  the 
parts  which  make  up  the  bursal  valve  of  Syndisyrin.\-  are  lacking.  In  addition  to 
these  differences,  the  stucture  of  -the  penis  is  markedly  dissimilar  in  these  twro  forms. 
The  penis  of  Syndisyrinx  is  a  cuticular  hollow  stylet  attached  only  at  the  base, 
whereas  the  copulatory  organ  of  Syndcsmis  is  a  muscular  eversible  tube  with  a  cu- 
ticular lining  (Russo,  1895;  Fig.  16). 

Structures  corresponding  to  the  cuticular  canals  in  the  bursal  valve  of  Syndi- 
syrinx are  found  in  Anoplodicra  valuta,  Wahlia  macrostylifcra,  and  Dcsmote  vorax. 
In  A.  valuta  the  relationships  of  the  two  cuticular  canals  to  the  bursa  seminalis,  as 
described  by  Westblad  (1930),  are  very  similar  to  the  arrangement  of  these  struc- 
tures in  Syndisyrinx.  However,  the  cuticular  sheath  that  surrounds  the  entrance 
of  these  ducts  into  the  bursa  is  lacking  in  A.  valuta.  There  do  not  appear  to  be 
grounds  for  concluding  that  Syndisyrinx  and  Anoplodiera  are  closely  related  since 
the  appearance  and  location  of  the  testes  and  vitellaria,  the  presence  of  a  single  ovary, 
and  the  absence  of  a  female  antrum  connecting  the  ovovitelline  duct  and  uterus  to 
the  common  genital  antrum  in  A.  valuta  differ  strikingly  from  the  arrangement 
found  in  Syndisyrinx, 

In  W.  macrostylifcra,  described  by  Westblad  (1930),  and  D.  vorax,  according 
to  Beklemischev  (1916),  the  proximal  end  of  the  bursal  canal  is  cuticular  but  an  in- 
semination canal  is  lacking.  In  other  respects  W.  macrostylifcra  differs  from 


A  PARASITIC  RHABDOCOEL  309 

Syndisyrinx  chiefly  in  regard  to  the  morphology  of  the  male  reproductive  system. 
The  penis  stylet  is  greatly  elongated,  and  paired  sperm  ducts  arising  from  compact 
testes  unite  and  communicate  by  means  of  a  single  duct  with  the  large  spermiducal 
vesicle  situated  anterior  to  the  pharynx.  Many  points  of  difference  are  likewise 
found  by  comparing  the  morphology  of  Syndisyrinx  and  Dcsmotc.  The  most 
evident  of  these  are  the  bipartite  gut  and  the  presence  of  two  genital  pores,  the  an- 
terior pore  by  which  the  uterus  opens  to  the  exterior  and  the  posterior  pore  which 
serves  for  copulation  in  D.  vorax. 

The  other  genera  of  the  family  lack  cuticular  parts  in  the  copulatory  complex 
comparable  to  those  in  the  bursal  valve  of  Syndisyrinx  and  to  a  greater  or  less  de- 
gree exhibit  dissimilarities  in  the  location,  distribution,  number,  arrangement  and 
relationships  of  organs  in  the  body.  In  these  genera  the  most  conspicuous  differ- 
ences with  respect  to  Syndisyrinx  are :  the  single  ovary  and  absence  of  a  cuticular 
copulatory  stylet  in  the  genus  Anoplodiuin ;  the  unbranched  ovaries  and  double- 
walled  cuticular  penis  stylet  in  the  genus  Umagilla;  the  absence  of  a  cuticular  penis 
and  the  general  arrangement  of  testes  and  vitellaria  in  the  genus  Xcnoinctra;  and 
the  single  testis  in  the  genus  Collastoma.  A  manuscript  is  in  preparation  which  will 
deal  at  greater  length  with  the  structural  relationships  of  these  forms. 

Bursal  valve 

There  is  a  superficial  similarity  between  the  bursal  valve  of  Syndisyrinx  and  the 
cuticular  nozzle-like  mouthpieces  of  acoels.  In  the  acoel,  Amphichoerus,  described 
by  Graff  (1891),  and  many  allied  forms,  one  end  of  the  mouthpiece  is  generally  con- 
nected to  a  vesicular  sac  or  bursa  filled  with  sperm ;  the  other  end  is  directed  toward 
the  ovary.  L.  H.  Hyman  (1937)  points  out  that  the  function  of  these  mouthpieces 
is  apparently  to  direct  sperm  toward  the  ova  to  help  insure  fertilization.  This  func- 
tion can  hardly  be  ascribed  to  the  insemination  canals  of  Anoplodiera  and  Syndi- 
syrinx which  conduct  sperm  from  the  bursa  seminalis  to  the  seminal  receptacle  and 
not  directly  to  the  ova;  nor  does  it  seem  probable  that  the  insemination  canals  of 
Umagillidae  are  homologous  to  these  mouthpieces.  Noncuticular  ducts  connect  the 
bursa  seminalis  to  the  seminal  receptacle  and  bursal  canal  in  most  genera  of  Umagil- 
lidae,  which  suggests  that  cuticular  structures  are  probably  of  relatively  recent 
rather  than  primitive  origin.  In  an  analysis  of  the  existing  genera,  Wahl  (1910b) 
presents  evidence  which  leads  him  to  conclude  that  Umagilla  is  the  most  primitive 
and  least  modified  genus  of  the  family.  If  one  accepts  this  view,  it  lends  support 
to  the  opinion  expressed  above,  inasmuch  as  Umagilla  lacks  any  cuticular  structures 
that  might  be  considered  homologous  to  the  bursal  valve.  It  is  possible  that  the 
absence  of  cuticular  parts  in  some  of  the  species  is  due  to  a  greater  degree  of  simpli- 
fication associated  with  a  parasitic  existence.  However,  there  is  no  direct  evidence 
for  this  supposition,  since  in  the  most  closely  related  free-living  families,  Grarfillidae 
and  Dalyelliidae,  cuticular  structures  such  as  these  are  not  found.  This  suggests 
that  these  tubules  have  arisen  independently,  and  until  additional  information  is 
available,  the  insemination  canals  and  bursal  canals  of  Umagillidae  should  not  be 
considered  as  mouthpieces  in  a  true  sense. 

Although  copulation  has  not  been  observed  in  Syndisyrinx,  it  is  believed  that  the 
sperm  of  one  animal  are  injected  by  means  of  the  protrusible  penis  into  the  bursal 
canal  of  another.  Before  fertilization  can  take  place,  sperm  must  migrate  from  the 


310  H.  E.  LEHMAN 

bursal  canal  through  its  narrow  proximal  end  into  the  bursa  seminalis,  there  re- 
maining until  able  to  find  their  way  through  the  insemination  canal  into  the  seminal 
receptacle.  Evidently  many  sperm  are  unable  to  accomplish  this  migration  and  de- 
generate in  the  lumen  of  the  bursa  seminalis.  Sperm  that  do  reach  the  seminal  re- 
ceptacle must  then  pass  through  the  constricted  anterior  part  of  this  organ  to  fer- 
tilize the  mature  ova  that  enter  the  ovovitelline  duct  at  the  anterior  end  of  the 
seminal  receptacle. 

It  is  difficult  to  explain  any  selective  advantage  for  the  presence  of  the  fine  canals 
that  make  up  the  bursal  valve  of  Syndisyrinx.  It  was  thought  at  first  to  be  a  mech- 
anism for  the  prevention  of  polyspermy.  However,  this  explanation  is  negated  by 
the  presence  of  large  masses  of  spermatozoa  in  the  seminal  receptacle.  The  simplest 
explanation  for  the  presence  of  these  ducts  is  that  they  act  as  valves  which  regulate 
the  number  of  spermatozoa  entering  the  bursa  seminalis  and  seminal  receptacle.  If 
this  interpretation  is  correct,  it  is  probable  that  the  function  of  the  bursal  valve  is 
to  insure  a  necessary  aging  of  the  sperm  in  the  bursa  before  fertilization.  The  cu- 
ticular  walls  are  necessary  to  prevent  the  collapse  of  these  narrow  tubes.  It  is 
evident  that  the  bursal  valve  restricts  the  free  passage  of  sperm  from  the  bursa  sem- 
inalis and  therefore  as  the  result  of  a  single  copulation,  a  continuous  supply  of 
sperm  may  be  maintained  over  a  long  period  of  time. 

SUMMARY 

After  completing  a  histological  study  of  an  endoparasitic  rhabdocoel  from  the 
Pacific  Coast  sea  urchin,  Strongylocentrotus  jranciscanus,  the  following  conclusions 
have  been  reached : 

1.  This  parasite  belongs  to  the  rhabdocoel  family  Umagillidae  but  differs  in  cer- 
tain characteristics  from  the  eight  known  genera  of  the  family. 

2.  The  distinguishing  characteristics  are  a  single  intestine,  paired  and  lobed 
ovaries  and  testes,  a  tubular  single-walled  ctiticular  penis  stylet,  and  cuticular  ducts 
connecting  the  bursa  seminalis  to  the  bursal  canal  and  seminal  receptacle. 

3.  A  characteristic  structure  typical  of  this  parasite  and  not  present  in  other 
genera  of  the  family  is  the  bursal  valve  composed  of  two  cuticular  tubes,  the  in- 
semination canal  and  proximal  end  of  the  bursal  canal,  which  enter  the  bursa  sem- 
inalis through  a  cuticular  cup-like  sheath. 

4.  The  parasite  here  described  is  given  the  name  Syndisyrinx  frandscanus,  gen. 
et  sp.  nov. 

LITERATURE  CITED 

BEKLEMISCHEV,  W.,  1916.     Sur  les  Turbellaries  parasites  de  la  cote  Mourmanne,  II.     Rhabdo- 

coela.     Trav.  Soc.  Imp.  Nat.  Petrograd,  Zool.  et  Physiol.,  Sect.  4,  45:  1-59  (Resume, 

60-79). 
BOCK,   S.,   1926.     Anoplodium  stichopi,   ein  neuer   Parasit  von   der   Westkiiste   Skandinaviens. 

Zool.  Bidrag,  Uppsala,  10:  1-30. 
BRAUN,    M.,    1889.     Uber   parasitische    Strudehvurmer    in    Rostok.     Ccntralbl.    Bakt.    Parasit., 

Abt.  1,5:  41-44. 
BRESSLAU,    E.,    1933.     Turbellaria.     Kiikenthal    und   Kntmbach,    Handbnch    der   Zoologic,   2: 

264-269. 
DORLER,  A.,  1900.     Neue  und  wenig  bekannte  rhabdocole  Turbellarien.     Zcitschr.  zviss.  Zool., 

68 :  1-42. 


A  PARASITIC  RHABDOCOEL  311 

FRANCOIS,   P.  H.,   1886.     Stir  le  Syndesmis,  nouveau  type  de  Turbellaries  decrit  par,   W.   A. 

Silliman.     C.  R.  Acad.  Sci.  Paris,  103  :  752-754. 

GRAFF,  L.  v.,  1891.     Die  Organisation  der  Turbellaria  acocla,  Leipzig    (Engelmann),  p.  73. 
GRAFF,  L.  v.,  1913.     Turbellaria,  II.     Rhabdocoelida.     Das  Tierreich,  Berlin   (F.  E.  Schulze), 

35:  152-163. 

HYMAN,  L.  H.,  1937.     Reproductive  system  and  copulation  in  Amphiscolops  langerhansi  (Tur- 
bellaria acoela).     Biol.  Bull.,  72:  319-326. 
OZAKI,  Y.,  1932.     On  a  new  genus  of  parasitic  Turbellaria,  Xenometra,  and  a  new  species  of 

Anoplodium.     Jour.  Sci.  Hiroshima  Univ.  (Series  B,  Zoo/.),  1:  81-89. 
POWERS,   P.  B.  A.,   1936.     Studies  on  the  ciliates  of   sea  urchins.     A   general    survey   of   the 

infestations    occurring    in    Tortugas    echinoids.     Pap.    Tortugas    Lab.    Carnegie    hist. 

Washington,  29:  319-320. 
Russo,  A.,  1895.     Sulla  morfologia  del  Syndesmis  echinorum  Francois.     Ricerche  Lab.  Anat., 

Roma,  Fasc.  1,5:  43-68. 
SCHNEIDER,  A.,  1858.     Uber  einige  Parasiten  der  Holothuria  tubulosa.     I.  Anoplodium  parasita. 

Miillcr's  Arch.  f.  Anat.  Phys.  und  u-iss.  Mcd.,  Berlin:   324-329. 
WAUL,  B.,  1909.     Untersuchungen  liber  den  Bau  der  parasitischen  Turbellarien  aus  der  Familie 

der  Dalyelliiden  (Vorticiden).     II.  Teil,  Die  Genera  Umagilla  und  Syndesmis.     Wien, 

Sitz.-Bcr.  kais.  Akad.  u'iss.  Math.-nat.,  Abt.  1,  118:  943-965. 
WAHL,    B.,    1910a.     Untersuchungen    iiber    den    Bau    der    parasitischen    Turbellarien    aus    der 

Familie    der    Dalyelliiden    (Vorticiden).     III.    Teil,    Das    Genus    Collastoma.     Wien, 

Sitz.-Bcr.  kais.  Akad.  iviss.  Math.-nat.,  Abt.  1,  119:  363-391. 
WAHL,  B.,  1910b.     Beitrage  zur  Kenntnis  der  Dalyelliiden  und  Umagilliden.     Fcstschr.  f.  R. 

Hertivig,  Jena  (G.  Fischer),  2:  41-60. 
WESTBLAD,  E.,   1926.     Das   Protonephridium  der  parasitischen   Turbellarien.     Zoo!.   Anz.,  67 : 

323-333. 
WESTBLAD,  E.,   1930.     Anoplodiera  voluta  und   Wahlia  macrostylifera,   zwei   neue  parasitische 

Turbellarien  aus  Stichopus  tremulus.     Zcitschr.  f.  Morph.  u.  Okol.  Tierc,  19 :  397-426. 


A  QUANTITATIVE  STUDY  OF  THE  RELATIONSHIP  BETWEEN 
THE  ACTIVITY  AND  OXYGEN  CONSUMPTION  OF  THE 
GOLDFISH,  AND  ITS  APPLICATION  TO  THE  MEAS- 
UREMENT OF  RESPIRATORY  METABOLISM 

IN  FISHES 

W.  A.  SPOOR 

Department  of  Zoology,  University  of  Cincinnati 

INTRODUCTION 

The  fact  that  fish  consume  more  oxygen  when  active  than  when  quiescent  has 
been  observed  by  many  investigators  (Krogh,  1916;  Bowen,  1932;  Clausen,  1933, 
1936;  Wells,  1935;  Schlaifer,  1938;  Smith  and  Matthews,  1942),  but  apparently 
no  attempt  has  been  made  to  determine  the  exact  relationship  between  oxygen  con- 
sumption and  activity  in  fishes.  It  is  the  purpose  of  this  paper  to  present  data 
which  are  believed  to  provide  an  objective  and  quantitative  basis  for  the  relationship 
between  activity  and  oxygen  consumption  in  the  goldfish,  and  to  describe  a  method 
for  making  the  necessary  measurements.  The  method  is  based  on  the  use  of  a  re- 
cording activity  detector  (Spoor,  1941)  combined  with  a  continuous  flow  system  for 
measuring  oxygen  consumption. 

The  lack  of  definite  information  on  the  activity  of  fish  under  experimental  condi- 
tions has  been  one  of  the  chief  sources  of  difficulty  in  work  on  the  respiratory  metab- 
olism of  fishes,  and  attention  has  been  called  to  the  need  for  an  experimental  method 
which  would  make  it  possible  to  distinguish  between  "standard  metabolism"  and 
the  increased  metabolism  due  to  muscular  movements  (Wells,  1935).  In  view  of 
the  fact  that  the  oxygen  consumption  is  affected  by  changes  in  the  basal  metabolic 
rate  as  well  as  by  changes  in  activity,  the  importance  of  such  a  method  is  apparent. 
The  method  employed  in  the  present  work  seems  to  meet  this  need,  inasmuch  as 
the  state  of  activity  is  recorded  continuously  and  periods  of  inactivity  can  be  selected 
for  measuring  basal  oxygen  consumption. 

Szymanski  (1914)  and  Spencer  (1939),  using  other  types  of  activity  detectors, 
have  reported  that  goldfish  show  considerable  individual  variation  in  activity  and 
that  the  activity  pattern  is  affected  by  light.  Spencer  (1939)  also  found-  activity 
to  be  influenced  by  food.  Knowledge  of  the  behavior  of  the  fish  under  the  experi- 
mental conditions  is  of  importance  in  the  collection  of  data  on  oxygen  consumption 
in  the  method  to  be  described,  as  well  as  in  the  interpretation  of  these  data.  For 
this  reason  further  observations  on  the  patterns  and  rates  of  activity  and  on  the  ef- 
fects of  food,  light  and  disturbances  are  included  in  the  present  paper. 

THE  ACTIVITY  OF  THE  GOLDFISH  UNDER  EXPERIMENTAL  CONDITIONS 
Method 

Several  dozen  goldfish  (Carassius  auratus)  ranging  between  24  and  96  grams 
in  weight  were  selected  at  random  from  a  stock  obtained  from  a  local  goldfish  farm 

312 


ACTIVITY  AND  OXYGEN  CONSUMPTION 


313 


and  studied  individually  in  experimental  chambers,  each  of  which  was  equipped  with 
a  recording  activity  detector.  The  experimental  chambers  were  set  up  in  a  ground 
floor  aquarium  room  which  was  seldom  entered  except  for  the  purposes  of  this 
study,  so  that  the  fish  could  be  left  for  long  periods  with  relatively  little  disturbance. 
The  recording  apparatus  was  kept  in  another  room.  Records  of  the  activity  of  each 
fish  were  started  shortly  after  its  introduction  into  a  chamber  and  continued  for  pe- 
riods ranging  from  a  few  days  to  many  months  in  length,  during  which  the  patterns 
and  rates  of  activity  and  the  effects  of  food,  light  and  disturbances  upon  them  were 
studied.  With  a  few  exceptions,  oxygen  consumption  was  not  measured  in  this 
series  of  observations. 

The  experimental  chamber    (Fig.   1)    consisted  of  a  one-gallon  brown  glazed 


FIGURE   1.     Diagram  of  apparatus  for  measuring  oxygen  consumption  and  activity.  (1) 

paraffin  oil  (this  was  omitted  when  activity  alone  was  being  measured),   (2)   glass  plates,  (3) 

No.  44  copper  wire,  (4)  to  sensitive  relay,  (5)  resistor,  (6)  wire  screen,  (7)  glass  tube,  (8) 
wire  frame  protecting  paddle.  Explanation  in  text. 

crock  fitted  with  a  galvanized  iron  wire  screen  of  %  mcn  mesh  to  prevent  the  fish 
from  reaching  the  surface  of  the  water.  A  glass  tube  about  3  cm.  in  diameter  was 
fitted  into  an  opening  in  the  center  of  the  screen  so  that  it  extended  3.5  cm.  above 
and  3  cm.  below  the  screen ;  its  purpose  will  be  considered  in  a  later  section.  The 
surface  of  the  water  stood  about  3  mm.  above  the  screen,  the  total  volume  to  this 
level  being  2,600  cc.  Water  entered  the  chamber  from  a  constant  level  reservoir 
through  8  mm.  glass  tubing  and  left  by  way  of  a  siphon  of  8  mm.  glass  tubing  which 
dipped  into  a  constant  level  drain,  the  rate  of  flow  (between  70  and  100  cc.  a  minute) 
being  regulated  by  means  of  a  glass  stopcock  in  the  inlet.  The  intake  of  the  siphon 
was  placed  about  5  cm.  above  the  bottom  of  the  chamber,  so  that  feces  and  other 
debris  that  fell  to  the  bottom  did  not  enter  the  siphon  until  they  had  been  broken 
into  small  pieces  in  the  course  of  their  passage  upward  to  the  intake.  The  chamber 
was  practically  self  cleaning  under  these  conditions,  the  flow  of  the  water  and  the 
movements  of  the  fish  being  sufficient  to  move  debris  into  the  siphon.  The  fish 


314  W.  A.  SPOOR 

could  therefore  be  maintained  in  the  chamber  for  months  without  cleaning.  A 
thistle  tube  entering  the  inlet  provided  for  the  introduction  of  food,  being  closed  off 
at  all  other  times.  The  water  supply  consisted  of  tap  water  passed  through  an  ac- 
tivated charcoal  filter,  brought  to  the  desired  temperature  and  aerated  until  it  ap- 
proached equilibrium  with  the  atmosphere.  Most  of  the  observations  were  made  at 
temperatures  between  20  and  24°  C.  The  fish  seldom  extracted  more  than  one- 
third  of  the  oxygen  from  the  w^ater  at  the  rates  of  flow  employed,  and  they  usually 
took  less  than  this.  In  view  of  the  findings  of  Crozier  and  Stier  (1925),  Toryu 
(1927),  and  Schlaifer  (1938),  it  seems  unlikely  that  behavior  was  influenced  by 
the  oxygen  tension  of  the  water. 

The  chamber  was  enclosed  in  a  wooden  case  to  minimize  disturbances  and  to 
make  it  possible  to  control  the  light.  The  top  of  the  case  was  fitted  with  a  pane  of 
glass  for  natural  illumination,  and  with  a  wooden  cover  when  either  complete  dark- 
ness or  constant  light  was  desired.  A  ventilator  in  the  side  of  the  case,  with  baffles 
to  prevent  light  from  entering,  permitted  some  circulation  of  the  air.  The  water 
inlet,  outlet  siphon,  and  a  tube  leading  to  a  U  tube  indicating  the  water  level  in  the 
chamber  passed  through  the  wall ;  a  coat  of  black  paint  over  each  tube  prevented 
light  from  entering  the  chamber  through  these  openings. 

The  detector  consisted  of  a  light-weight  aluminum  paddle  suspended  in  the  water 
in  the  experimental  chamber  by  a  fine  copper  wire  in  such  a  way  that  a  silver  rod 
at  the  top  of  the  paddle  shaft  passed  through  a  small  hole  in  a  fixed  silver  plate. 
Water  currents  set  up  by  the  movements  of  the  fish  moved  the  paddle,  causing  the 
rod  to  make  and  break  contact  with  the  sides  of  the  hole  and  thus  to  activate  a  sen- 
sitive relay.  This  relay  operated  the  recording  apparatus.  The  blade  of  the  paddle 
consisted  of  aluminum  foil  (5  cm.  long  and  2.5  cm.  wide)  with  the  corners  bent  in 
at  right  angles  so  that  the  water  currents  struck  a  flat  surface  regardless  of  their 
points  of  origin.  The  shaft  (10  cm.  long)  consisted  of  no.  22  aluminum  wire  ce- 
mented to  the  blade  and  imbedded  at  its  upper  end  (7  cm.  above  the  blade)  in  a 
bakelite  insulating  rod  (2  cm.  long  and  0.2  cm.  in  diameter)  in  the  upper  end  of 
which  the  silver  rod  (1  cm.  long  and  about  0.04  cm.  in  diameter)  was  imbedded. 
This  silver  rod  was  soldered  to  a  14.5  cm.  length  of  no.  44  enameled  copper  wire 
held  in  an  insulated  binding  post  attached  to  a  wooden  supporting  shaft.  A 
wooden  bracket  rising  from  the  case  supported  this  shaft  in  a  vertical  position 
so  that  the  paddle  hung  in  the  water  through  the  glass  tube  in  the  center  of  the 
screen.  A  cylindrical  frame  of  galvanized  iron  wire  protected  the  paddle  from 
contact  with  the  fish.  A  small  lead  weight  (about  0.1  gm.)  clamped  to  the 
paddle  shaft  below  the  bakelite  helped  to  bring  the  paddle  back  to  the  resting 
position  after  displacement  by  the  water  currents.  The  silver  plate  (about  0.5 
cm.  square)  was  attached  to  the  supporting  shaft  and  held  in  a  horizontal  posi- 
tion about  6  cm.  above  the  screen.  The  hole  in  the  plate  was  between  0.08  and  0.1 
cm.  in  diameter.  The  current  to  operate  the  sensitive  relay  was  supplied  by  a  6  volt 
storage  battery;  the  coil  of  the  relay  had  a  resistance  of  1,000  ohms.  A  5,000  ohm 
resistor  across  the  detector  contacts  prevented  sparking  and  welding  without  caus- 
ing an  observable  reduction  in  the  sensitivity  of  the  detector.  The  plate  was  kept 
warm  by  means  of  a  small  insulated  heating  coil  in  order  to  prevent  water  from 
condensing  upon  it  from  the  humid  atmosphere  above  the  chamber. 

The  sensitivity  of  the  detector  could  be  controlled  somewhat  by  adjusting  the 
position  of  the  silver  rod  with  respect  to  the  sides  of  the  hole  in  the  plate.     The  nor- 


ACTIVITY  AND  OXYGEN  CONSUMPTION  315 

mal  movements  of  the  operculum  and  the  position-maintaining  fin  movements  of  a 
quiescent  25-  to  30-gram  goldfish  were  usually  sufficient  to  move  the  paddle  slightly, 
and  when  the  rod  was  close  to  the  plate  these  movements  were  recorded.  For  the 
observations  to  be  described,  however,  the  rod  was  centered  in  the  hole  so  that  the 
ordinary  respiratory  movements  did  not  move  the  paddle  enough  to  make  contact, 
these  movements  being  considered  as  among  the  basal  functions  of  the  fish.  Vigor- 
ous respiratory  movements  and  any  movement  that  resulted  in  a  change  in  the  posi- 
tion of  the  fish  moved  the  paddle  enough  to  make  and  break  contact,  slow  swimming 
movements  causing  few,  and  vigorous  activity  causing  many  impulses  to  be  recorded. 
At  the  flow  rates  used  in  these  experiments  the  flow  of  water  through  the  chamber 
did  not  move  the  paddle. 

The  sensitive  relay  activated  a  counter  which  in  turn  caused  signal  magnets  to 
record  every  tenth  and  hundredth  impulse  on  a  long  paper  kymograph  moving  about 
30  mm.  an  hour.  The  frequency  of  the  impulses  was  such  (ranging  up  to  6,000  an 
hour)  that  they  usually  could  not  be  counted  when  recorded  individually.  The 
counter  was  capable  of  following  and  recording  at  least  10  impulses  a  second.  Time 
was  recorded  in  hours  beneath  the  activity  record. 

Patterns  and  rates  of  activity 

In  agreement  with  the  results  of  Szymanski  (1914)  and  Spencer  (1939),  the 
goldfish  used  in  this  study  proved  to  be  quite  variable  in  their  patterns  and  rates  of 
activity,  even  when  they  were  maintained  under  almost  identical  conditions  of  light, 
feeding,  temperature,  water  supply  and  disturbance.  Three  general  types  of  be- 
havior appeared  when  the  fish  were  kept  under  natural  conditions  of  light:  (1) 
arhythmic  activity,  in  which  no  relation  to  clay  or  night  could  be  detected;  (2) 
rhythmic  activity,  in  which  the  fish  were  active  by  day  and  quiescent  at  night;  (3) 
rhythmic  activity,  in  which  they  were  quiescent  by  day  and  active  at  night. 

Fish  showing  the  first,  arhythmic,  type  of  behavior  were  extremely  variable. 
A  few  were  vigorously  active  da)'  and  night  for  periods  as  long  as  ten  days,  others 
were  moderately  active  throughout  the  24-hour  period  for  weeks  at  a  time,  and  still 
others  remained  practically  inert  for  similar  periods.  Some  of  these  arhythmic  fish, 
particularly  those  in  the  last  group,  showed  irregular  bursts  of  activity  now  and  then, 
with  no  apparent  relation  to  the  time  of  day,  feeding,  or  disturbance. 

An  example  of  the  second  type  of  behavior,  diurnal  activity  and  nocturnal  quies- 
cence, is  shown  in  Figure  2,  which  is  based  on  the  number  of  impulses  recorded  by 
a  35-gram  male  goldfish  during  each  hour  between  1  P.M.  January  11  and  1  P.M. 
January  13,  1946.  The  fish  was  fed  at  11:05  A.M.  on  January  12,  otherwise  the 
room  was  not  entered  between  2:45  P.M.  January  11  and  7:15  P.M.  January  13. 
Aside  from  the  feeding,  the  effects  of  which  are  discussed  below,  light  was  the  only 
known  variable,  the  temperature,  rate  of  flow  and  aeration  of  the  water  being  the 
same  at  the  end  as  at  the  beginning  of  the  period.  Most  of  the  fish  showing  rhythmic 
changes  in  activity 'followed  patterns  of  this  type,  although  the  active  phase  varied 
considerably,  sometimes  being  interrupted  by  several  hours  of  quiescence  during 
the  day,  sometimes  beginning  later  in  the  day,  and  occasionally  continuing  well  into 
the  night.  The  most  constant  period  of  quiescence  occurred  between  midnight  and 
4  A.M.,  which  is  in  agreement  with  Spencer's  (1939)  observations. 

The  third  type  of  behavior,  diurnal  quiescence  and  nocturnal  activity,  was  found 
less  frequently  than  the  second,  although  it  was  not  uncommon.  An  example  is 


316 


W.  A.  SPOOR 


shown  in  Figure  3,  which  is  based  on  records  of  the  activity  of  a  32-gram  male  gold- 
fish between  1  P.M.  July  3  and  1  P.M.  July  5,  1945.     The  room  was  not  entered  be- 
'tween  4  P.M.  July  3  and  8  A.M.  July  5,  and  aside  from  the  daily  changes  in  light  the 
environmental  conditions  apparently  remained  constant  throughout  the  period. 

The  patterns  of  rhythmic  fish  did  not  seem  to  be  fixed,  however,  even  when  the 
environmental  conditions  remained  unchanged.  After  several  weeks  of  rhythmic 
behavior  the  fish  frequently  became  arhythmic  for  several  weeks  or  months,  occa- 
sionally becoming  rhythmic  again  in  the  course  of  extended  periods  of  observation. 
This  suggests  that  those  fish  which  did  not  show  daily  activity  rhythms  under  the 


3000- 

2500 

2000- 

P  1500- 
o 

1000- 
500- 


6RM. 
I/    /46 


2M. 


6A.M.        I2N. 
I/I2/46 


6P.M.         I2M. 


6  AW. 
I/I3/46 


!2N. 


FIGURE  2.  Activity  pattern  of  35-gram  male  goldfish  between  1  P.M.  January  11  and  1 
P.M.  January  13,  1946.  Activity  is  expressed  as  number  of  impulses  recorded  each  hour.  Tem- 
perature 21.5°  C.  Fed  at  11 :05  A.M.  January  12. 

experimental  conditions  may  have  done  so  eventually  had  they  been  studied  for  lon- 
ger periods,  and  that  by  chance  the  observations  were  made  during  arhythmic 
periods. 

Activity  and  food 

The  fish  were  fed  rolled  oats,  commercial  fish  foods,  shredded  shrimp,  ground 
liver  or  chopped  earthworms  about  three  times  a  week,  usually  0.5  to  1  gram  at  each 
feeding.  The  effects  of  daily  feeding,  larger  amounts  of  food  and  starvation  were 
also  studied.  Under  the  conditions  of  the  experiments  the  type  of  food  given  had 
no  consistent  effects  upon  activity,  but  the  quantity  of  food  had  pronounced  effects, 
particularly  on  the  total  amount  of  activity.  A  well  fed  fish  was  usually  sufficiently 
active  that  the  number  of  impulses  recorded  in  the  course  of  a  24-hour  period  aver- 
aged between  500  and  1,500  an  hour,  and  averages  in  excess  of  2,500  impulses  an 


ACTIVITY  AND  OXYGEN  CONSUMPTION 


317 


hour  were  not  uncommon.  Starvation  caused  this  rate  to  decrease  markedly,  some- 
times to  fewer  than  100  impulses  an  hour,  although  as  a  rule  the  lowest  rates  did  not 
appear  until  the  fish  had  been  starved  for  a  week  or  so.  No  fish  was  observed  to 
become  completely  inactive  for  periods  of  more  than  an  hour  or  two,  however,  even 
when  starved  for  two  weeks.  The  effects  of  feeding  after  a  period  of  starvation 
were  striking,  activity  increasing  to  normal  "well  fed"  rates  within  a  few  minutes. 
Doubtless  the  swimming  movements  associated  with  feeding  accounted  for  some  of 
the  activity  recorded  following  the  introduction  of  food,  but  it  seems  that  the  nu- 
tritional state  also  affected  the  amount  of  activity.  Food  given  in  amounts  of  one 


3000- 


2500- 


^2000- 

H 

p I  500- 
o 

1000- 


500- 


TJ 


J" 


6RM.        I2M.       6A.M.        I2N. 
7/3/45  7/4/45 


6  P.M. 


I2M. 
I 


6A.M. 
7/5/45 


2N. 


FIGURE  3.     Activity  pattern  of  32-gram  male  goldfish  between  1  P.M.  July  3  and  1  P.M.  July  5, 

1945.     Units  as  in  Figure  2.     Temperature  23.5°  C. 

gram  or  less  was  usually  consumed  within  three  to  six  hours,  but  the  fish  remained 
active  (in  accordance  with  their  activity  patterns)  for  from  several  days  to  a  week 
after  they  had  been  fed.  Similarly,  Spencer  ( 1939)  found  the  goldfish  to  maintain 
a'high  rate  of  activity  for  several  hours  after  feeding,  although  in  his  experiments 
the  food  was  usually  consumed  within  15  minutes  or  less. 

The  effects  of  feeding  upon  activity  rhythms  were  not  studied  in  detail,  but  the 
available  data  bearing  on  this  question  indicate  that  although  the  rhythms  appearing 
under  natural  conditions  of  light  were  frequently  modified  by  the  quantity  of  food 
and  the  time  of  feeding,  they  were  not  causally  related  to  food.  Feeding  modified 
the  activity  patterns  of  some  fish  for  part  or  all  of  the  subsequent  24-hour  period, 
usually  by  prolonging  the  active  phase.  A  response  of  this  type  may  be  seen  in 
Figure  2.  The  fish  was  fed  0.5  gm.  of  rolled  oats  at  11 :05  A.M.  on  January  12  (the 
previous  feeding  being  on  January  9)  ;  it  will  be  noted  that  the  activity  level  re- 
mained relatively  high  for  a  much  longer  period  on  the  night  of  January  12  than  on 


318  W.  A.  SPOOR 

the  preceding  night.  On  the  other  hand  some  fish  showed  no  change  in  activity  in 
response  to  feeding,  provided  of  course  that  they  had  not  been  starved.  Variations 
in  the  quantity  of  food  and  in  the  time  and  frequency  of  feeding  did  not  seem  to  have 
•permanent  effects  on  the  activity  rhythms,  and  feeding  at  the  same  time  each  day 
did  not  cause  arhythmic  fish  to  become  rhythmic. 

Activity  and  light 

The  goldfish  did  not  seem  to  be  much  affected  by  changes  in  light  intensity  while 
they  were  not  following  daily  activity  rhythms,  but  they  were  usually  quite  responsive 
to  light  during  their  periods  of  rhythmic  behavior.  In  fact,  when  the  fish  were  well 
fed  and  undisturbed  the  activity  rhythms  seemed  to  be  closely  related  to  the  daily 
changes  in  natural  light,  as  Szymanski  (1914)  has  reported  previously.  This  view 
is  supported  by  several  observations  in  addition  to  the  fact  that  the  active  and  quies- 
cent phases  of  the  cycles  usually  coincided  with  day  and  night.  Periods  of  nocturnal 
activity  and  diurnal  quiescence  were  shown  by  the  32-gram  male  goldfish  mentioned 
above  in  July  and  December  of  1945.  Although  the  water  temperature  and  other 
factors  except  light  were  the  same  during  both  periods,  the  nocturnal  phase  of  ac- 
tivity usually  began  earlier  in  the  evening  (between  5  and  6  P.M.)  and  ended  later 
in  the  morning  (between  7  and  8  A.M.)  in  December  than  in  July,  when  it  usually 
began  between  7:30  and  8:30  P.M.  and  ended  between  5  and  6  A.M.  This  suggests 
of  course  that  the  nocturnal  phase  of  activity  was  limited  by  the  setting  and  rising 
of  the  sun.  This  fish  also  responded  readily  to  experimental  changes  in  light  in- 
tensity, particularly  during  the  day,  when  darkening  the  chamber  caused  its  ac- 
tivity to  increase  to  levels  usually  reached  only  at  night.  Records  were  also  ob- 
tained in  which  diurnally  active  and  nocturnally  quiescent  fish  remained  active  on 
nights  when  bright  moonlight  entered  the  room  in  which  they  were  kept.  Spencer 
(1939)  found  that  the  regular  diurnal  rhythm  of  the  goldfish  could  be  obliterated  by 
covering  the  tank  by  day  and  lighting  artificially  at  night.  This  procedure  was  ac- 
companied by  night  feeding,  however,  so  that  the  change  in  activity  may  not  be  at- 
tributed solely  to  the  reversed  lighting. 

On  the  basis  of  these  observations  attempts  were  made  to  maintain  goldfish  at 
definite  rates  of  activity  by  exposing  them  to  continuous  dim  light  and  to  continuous 
darkness  for  periods  lasting  as  long  as  three  weeks,  but  without  success.  The  fish 
did  not  maintain  constant  rates  of  activity  under  either  condition,  but  continued  to 
alternate  periods  of  increased  activity  with  periods  of  relative  quiescence.  In  order 
to  maintain  a  low  rate  of  activity  it  was  necessary  to  starve  the  fish  for  about  a  week, 
the  relationship  between  nutritional  state  and  amount  of  activity  being  similar  to 
that  described  in  the  preceding  section. 

Activity  and  disturbance 

The  goldfish  proved  to  be  extremely  sensitive  to  disturbances.  Noise,  slight, 
changes  in  the  water  level,  sudden  lights,  the  mere  presence  of  the  observer  in  the 
room,  or  such  minor  disturbances  as  the  quiet  opening  and  closing  of  the  door  to 
the  room  usually  caused  a  change  in  the  rate  of  activity.  Fish  that  had  been  active 
before  the  disturbance  almost  invariably  became  less  active,  sometimes  practically 
motionless,  while  quiescent  fish  frequently,  although  less  consistently,  became  ac- 
tive when  disturbed.  Whichever  the  response,  the  original  state  of  activity  was 


ACTIVITY  AND  OXYGEN  CONSUMPTION  319 

usually  resumed  within  a  few  minutes  after  the  disturbance  had  ceased.  The  degree 
of  response  seemed  to  be  related  to  the  amount  of  disturbance,  for  when  the  observer 
moved  slowly  and  quietly  the  change  in  activity  was  usually  less  pronounced,  and 
recovery  more  rapid,  than  after  ordinary  passage  through  the  room  or  adjustment 
of  the  apparatus.  The  effects  of  disturbances  upon  the  activity  of  an  otherwise 
quiescent  fish  may  be  seen  in  Figure  3.  The  room  was  entered  several  times  in  the 
course  of  the  afternoon  of  July  3  and  on  July  5,  although  the  experimental  chamber 
was  not  approached  and  the  fish  could  not  see  the  cause  of  the  disturbance.  It  is 
obvious  that  the  rates  of  activity  were  higher  than  at  corresponding  hours  on  July  4, 
when  the  room  was  not  entered.  Such  sensitivity  has  been  observed  in  other  species 
of  fish  by  Clausen  (1934),  who  found  that  a  shadow  passing  over  the  aquarium 
caused  increases  in  the  body  temperatures  of  perch  and  members  of  the  sunfish 
group. 

THE  RELATIONSHIP  BETWEEN  ACTIVITY  AND  OXYGEN  CONSUMPTION 
Method 

The  activity  and  corresponding  oxygen  consumption  of  individual  goldfish  were 
measured  in  observation  periods  ranging  in  length  from  11  to  210  minutes.  Ac- 
tivity was  measured  in  terms  of  the  number  of  impulses  recorded  in  a  given  period, 
and  the  "amount  of  oxygen  consumed  by  the  fish  in  that  period  was  determined  by 
means  of  a  continuous  flow  system.  A  control  chamber  similar  to  the  experimental 
and  housed  in  the  same  case  was  supplied  with  a  continuous  stream  of  water  from 
the  reservoir  supplying  the  fish.  The  water  in  each  chamber  was  covered  with  a 
layer  of  heavy  paraffin  oil  2.5  cm.  thick  to  retard  the  diffusion  of  oxygen  from  the 
air,  and  a  sample  of  the  effluent  from  each  chamber  was  analyzed  for  oxygen  by  the 
Winkler  method  at  the  beginning  and  end  of  each  period.  The  samples  were  col- 
lected in  narrow  necked  glass  stoppered  bottles  of  about  270  cc.  capacity  arranged 
to  serve  as  constant  level  drains  (Fig.  1).  Each  line  was  arranged  so  that  the 
water  passed  through  the  outlet  siphon  to  the  bottom  of  the  sampling  bottle  and 
overflowed  into  a  funnel  so  that  it  could  be  collected  for  flow  rate  determinations. 
Although  the  rates  of  flow  ranged  from  70  to  100  cc.  a  minute  in  the  course  of  the 
study,  the  rate  for  any  one  day's  series  of  samples  was  held  practically  constant. 
Due  care  was  taken  to  prevent  the  diffusion  of  oxygen  into  the  samples  and  to  ob- 
tain representative  samples  from  experimental  and  control  lines.  Samples  that 
were  contaminated  by  participate  matter  were  discarded.  The  permanganate  modi- 
fication was  used  in  most  of  the  analyses,  but  was  omitted  during  some  of  the 
shorter  periods.  The  results  obtained  with  and  without  the  modification  were 
quite  similar,  however,  which  was  not  unexpected  in  view  of  the  fact  that  from  four 
to  six  liters  of  water  passed  over  the  fish  each  hour. 

The  volume  of  water  flowing  through  the  system  in  the  course  of  an  observation 
period  being  known,  together  with  the  oxygen  content  of  the  water  leaving  the 
control  and  experimental  chambers  at  the  beginning  and  end  of  that  period,  the 
oxygen  consumed  by  the  fish  could  be  calculated.  The  calculations  took  into  ac- 
count the  change  in  the  amount  of  oxygen  in  the  constant  volume  of  water  in  the 
chamber.  The  volume  of  water  displaced  by  the  fish  was  too  small  to  affect  the 
calculations. 

The  samples  were  collected  wTith  the  foregoing  observations  on  activity  patterns 


320  W.  A.  SPOOR 

and  modifying  factors  in  mind,  the  periods  being  timed  to  yield  data  at  the  activity 
rates  desired,  and  the  method  of  sampling  being  modified  as  necessary  to  minimize 
disturbance  of  the  fish.  In  the  latter  connection  the  outlet  tubes  were  lengthened  so 
that  samples  were  collected  about  10  feet  away  from  the  chambers,  and  the  room  was 
not  entered  except  for  sampling  and  rate  of  flow  determinations.  Precautions  were 
taken  to  prevent  changes  in  the  water  level  in  the  experimental  chamber  as  there 
were  indications  that  small  changes  in  the  level  stimulated  the  fish.  These  pre- 
cautions were  necessary  also  because  the  volume  of  water  in  the  chamber,  as  well  as 
that  flowing  through  it,  entered  into  the  calculations  of  oxygen  consumption. 
Samples  were  discarded  if  subsequent  examinations  of  the  activity  records  showed 
that  they  had  been  collected  while  the  fish  was  undergoing  marked  changes  in  ac- 
tivity as  a  result  of  disturbance  or  in  accordance  with  an  activity  rhythm.  This  was 
necessary  because  although  the  activity  record  was  instantaneous  the  change  in  the 
oxygen  concentration  of  the  samples  tended  to  lag  somewhat  behind  that  in  the 
chamber,  the  sample  drawn  at  any  instant  representing  the  average  of  the  water 
flowing  into  the  bottle  in  the  few  minutes  preceding  its  removal.  The  temperature 
of  the  water  was  recorded  for  each  observation  period  in  order  to  avoid  discrepancies 
attributable  to  the  effect  of  temperature  on  metabolic  rate  (Ege  and  Krogh,  1914). 

Owing  to  its  viscosity  and  the  accumulation  of  emulsified  oil  and  water  at  the 
oil-water  interface,  the  layer  of  oil  interfered  with  the  movements  of  the  paddle 
shaft.  Its  thickness  was  therefore  reduced  to  1  cm.  within  the  central  glass  tube, 
thus  permitting  the  paddle  to  move  about  as  freely  as  with  a  water  surface.  This 
tube  extended  below  the  interface  far  enough  to  prevent  the  emulsion  from  accumu- 
lating around  the  paddle  shaft.  The  oil  within  the  tube  had  to  be  changed  now  and 
then,  however,  to  remove  the  small  amount  of  debris  that  entered  it  from  beneath. 
The  detector  contacts  and  bakelite  rod  were  cleaned  every  few  days  as  a  precaution 
against  their  becoming  coated  with  oil,  which  seemed  to  spread  slo\vly  up  the  paddle 
shaft. 

It  was  established  by  appropriate  tests  that  the  layer  of  paraffin  oil  was  effective 
in  preventing  the  diffusion  of  significant  amounts  of  oxygen  into  the  water  from  the 
atmosphere.  In  no  test  did  the  apparent  leakage  exceed  the  limits  of  error  of  the 
Winkler  method  itself  (Alice  and  Oesting,  1934),  and  it  was  usually  considerably 
less.  The  average  apparent  rate  of  change  for  the  contents  of  the  experimental 
chamber  was  0.0015  cc.  of  oxygen  a  minute,  which  was  so  much  smaller  than  the 
rate  at  wrhich  the  fish  consumed  oxygen  that  even  had  the  apparent  change  been 
real  it  would  have  had  but  little  effect  on  the  results.  It  should  be  mentioned  in  this 
connection  that  the  oil  layer  was  disturbed  relatively  little  by  the  movements  of  the 
fish,  inasmuch  as  the  wire  screen  kept  the  fish  out  of  the  oil  and  glass  plates  resting 
on  this  screen  lessened  the  churning  effects  of  the  water  beneath  it. 

Results 

Three  goldfish  were  studied  at  several  temperatures  in  a  total  of  104  observation 
periods.  As  the  results  on  all  three  were  much  alike,  data  on  but  one  of  the  fish, 
a  32-gram  male  on  which  over  two-thirds  of  the  measurements  were  made,  are 
presented  here. 

The  relationship  between  activity  and  oxygen  consumption  at  temperatures  be- 
tween 23  and  25°  C.  is  shown  in  Figure  4,  in  which  oxygen  consumption  in  cubic 


ACTIVITY  AND  OXYGEN  CONSUMPTION 


321 


centimeters  per  minute  is  plotted  against  activity  in  impulses  per  minute.  Fifty-nine 
observation  periods  are  represented,  each  point  corresponding  to  one  period.  The 
line  merely  indicates  the  trend,  and  has  not  been  fitted  to  the  data  mathematically. 
As  was  to  be  expected,  oxygen  consumption  and  activity  proved  to  be  closely  related, 
the  relationship  apparently  being  linear  above  the  basal  level  of  oxygen  consumption. 
Although  the  values  for  oxygen  consumption  at  any  one  rate  of  activity  are  seen  to 
vary  somewhat,  the  trend  is  clear  cut :  at  high  rates  of  activity  the  rate  of  oxygen 


.18 


.16- 


.15- 


.13- 


z 
o 

P   .12- 
o. 

01     .1    I    ' 


o 

0 


.10- 


.09- 


.08- 


.07- 


06- 


.05- 


.04 


.03 


10 


20 


30 


40         50         60 

ACTIVITY 


70 


80 


90         100 


FIGURE  4.     Activity  and  oxygen  consumption  of  32-gram  male  goldfish.     Activity  in  impulses/ 
minute;  oxygen  consumption  in  cubic  centimeters/minute.     Temperature  23  to  25°  C. 

consumption  is  correspondingly  high ;  at  low  activity  rates  less  oxygen  is  consumed. 
The  discrepancies  that  do  occur  may  well  have  been  due  to  errors  in  measurement, 
rather  than  to  a  lack  of  correspondence  between  activity  and  oxygen  consumption. 
In  this  connection  the  data  on  oxygen  consumption  follow  those  on  activity  quite 
closely  when  the  comparison  is  restricted  to  one  day's  series  of  measurements,  thus 
ruling  out  discrepancies  attributable  to  slight  differences  in  the  adjustment  of  the 
detector  contacts.  Such  a  series  is  shown  in  Figure  5,  which  is  based  on  data  ob- 
tained with  the  same  fish  in  a  series  of  thirteen  consecutive  15-  to  25-minute  pe- 
riods at  22°  C. 

According  to  the  slope  of  the  data  shown  in  Figure  4,  the  basal  oxygen  con- 


322 


W.  A.  SPOOR 


sumption  of  this  fish  was  in  the  vicinity  of  0.040  cc.  a  minute,  or  0.075  cc.  per  gram 
per  hour. 

DISCUSSION 

The  results  of  the  present  study  have  a  bearing  on  the  collection  and  interpreta- 
tion of  data  on  the  respiratory  metabolism  of  fishes,  and  in  the  light  of  these  results 
the  method  described  seems  to  offer  a  number  of  advantages  not  found  in  previous 
methods  which  have  been  employed  for  this  purpose. 

The  advantages  of  the  continuous  flow  method  for  measuring  respiratory  metab- 
olism in  fishes  have  been  discussed  by  Keys  ( 1930)  and  need  not  be  reviewed  here. 
In  view  of  the  relationship  between  oxygen  consumption  and  activity,  however,  the 


•100 


.03 


150 


215       230       245       300      315 


5OO 


30 


TIME 


FIGURE  5.  Activity  and  oxygen  consumption  of  32-gram  male  goldfish  in  each  of  thirteen 
consecutive  observation  periods  between  1  :35  P.M.  and  5  P.M.  November  13,  1945.  Units  as  in 
Figure  4.  Each  point  on  the  upper  line  represents  the  average  rate  of  oxygen  consumption  for 
the  15-  or  25-minute  period  preceding  it.  Each  point  on  the  lower  line  represents  the  average 
rate  of  activity  for  the  corresponding  period. 

observations  on  the  effects  of  disturbances  may  be  applied  to  the  use  of  this  method, 
inasmuch  as  the  process  of  sampling  may  disturb  the  fish.  Should  a  change  in  the 
rate  of  activity  (and  consequently  of  oxygen  consumption)  occur  at  the  time  of 
sampling,  the  sample  would  not  be  representative  of  the  volume  of  water  and  unit 
of  time  to  which  it  is  related  in  the  calculations.  The  resulting  error  could  be  of 
considerable  importance,  particularly  in  investigations  in  which  the  samples  con- 
sisted of  water  flowing  directly  from  the  experimental  chamber  and  overflowing 
through  a  sampling  bottle.  This  source  of  error  has  been  recognized  of  course,  and 
in  some  investigations  the  experimental  chamber  has  been  covered  in  attempts  to 
minimize  stimulation  of  the  fish.  It  seems  very  doubtful,  however,  whether  cover- 
ing a  goldfish  so  that  it  cannot  see  the  investigator  is  an  adequate  safeguard  against 
disturbance.  One  advantage  of  using  an  activity  detector  in  the  continuous  flow 
method  then  lies  in  the  fact  that  any  sudden  change  in  activity  occurring  at  the 
time  of  sampling  can  be  detected,  so  that  the  reliability  of  the  sample  may  be  judged. 
Furthermore,  the  activity  record  can  be  used  to  test  the  effectiveness  of  the  steps 
taken  to  avoid  disturbance. 


ACTIVITY  AND  OXYGEN  CONSUMPTION  323 

It  is  of  course  well  known  that  fluctuations  in  activity  during  the  test  periods 
constitute  a  major  obstacle  to  the  correct  interpretation  of  measurements  of  oxygen 
consumption,  and  numerous  attempts  to  overcome  this  difficulty  have  been  described 
(Ege  and  Krogh,  1914;  Hall,  1929;  Adkins,  1930;  Keys,  1930;  Wells,  1932,  1935; 
Clausen,  1933;  Smith  and  Matthews,  1942).  These  measures  include  the  use  of 
narcotics,  observing  that  the  fish  remains  quiet,  maintaining  constant  conditions  of 
light,  sampling  at  the  same  time  each  day,  restricting  the  movements  of  the  fish,  and 
maintaining  the  fish  in  an  experimental  chamber  until  it  appears  to  have  come  to 
rest  or  at  any  rate  to  have  reached  a  steady  state.  Although  such  measures  may 
permit  the  establishment  of  the  reality  of  a  change  in  oxygen  consumption  in  con- 
nection with  an  experimental  procedure,  they  do  not  appear  to  give  a  completely 
satisfactory  basis  for  the  interpretation  of  that  change.  The  interpretation  must  be 
based  on  knowledge  of  the  activity  of  the  fish,  inasmuch  as  oxygen  consumption  is 
affected  by  changes  in  the  basal  metabolic  rate  as  well  as  by  activity.  The  method 
employed  must  therefore  be  capable  of  supplying  information  on  activity  and  oxygen 
consumption  at  the  same  time,  so  that  the  fraction  of  the  respiratory  exchange  as- 
sociated with  basal  metabolism  may  be  distinguished  from  that  due  to  muscular  ac- 
tivity (Wells,  1935).  None  of  the  above  methods  seems  to  be  adequate  for  this 
purpose. 

Narcotics  are  of  doubtful  value  in  studies  of  this  type,  even  for  measuring  basal 
metabolic  rate  alone  (Adkins,  1930).  Among  other  objections  are  indications  that 
an  important  fraction  of  the  metabolic  functions  of  the  fish  may  be  suppressed  to 
such  an  extent  that  the  oxygen  consumption  falls  below  the  basal  level  as  it  is  gen- 
erally understood  (Keys  and  Wells,  1930).  In  fact,  Ege  and  Krogh  (1914)  con- 
sidered it  necessary  to  use  artificial  respiration  to  insure  the  survival  of  their  gold- 
fish, the  narcotic  having  interfered  with  normal  respiratory  movements.  The  other 
methods  are  open  to  criticism  because  they  are  based  on  the  assumption,  rather  than 
the  knowledge,  that  the  fish  is  quiescent  or  at  a  constant  level  of  activity  under  the 
conditions  of  the  experiment.  The  results  of  the  present  work  suggest  that  for  the 
goldfish  at  any  rate  this  assumption  may  be  unwarranted.  The  fact  that  a  goldfish 
is  quiescent  while  it  can  be  seen  should  not  be  taken  as  proof  that  it  remains  so 
while  unobserved,  and  it  does  not  seem  justifiable  to  assume  that  constant  environ- 
mental conditions  mean  constant  rates  of  activity.  So  far  as  the  goldfish  is  con- 
cerned, the  individual  variations  in  activity  open  to  question  the  reliability  of  methods 
based  on  sampling  at  the  same  time  each  day,  particularly  if  several  fish  are  being 
compared.  Confining  the  fish  to  a  small  respiration  chamber  to  restrict  its  move- 
ments gives  no  assurance  that  it  will  remain  quiescent  or  even  at  a  constant  rate  of 
activity,  and  the  fact  that  the  oxygen  consumption  varies  over  a  wide  range  in  such 
chambers  supports  this  objection.  This  method  would  seem  to  have  a  further  dis- 
advantage for  measuring  the  basal  metabolic  rate  in  that  a  fish  confined  to  a  small 
tube  must  swim  continuously,  however  slowly,  in  order  to  maintain  its  position  in 
the  current.  The  practice  of  leaving  the  fish  in  the  respiratory  chamber  until  its 
oxygen  consumption  has  reached  a  relatively  low  and  constant  rate  (Keys,  1930; 
Wells,  1935)  is  far  superior  to  the  earlier  techniques,  but  it  is  limited  in  its  applica- 
tion by  the  fact  that  it  gives  no  information  as  to  the  amount  of  activity  associated 
with  the  steady  state. 

The  requirements  of  a  satisfactory  method  appear  to  be  met  by  combining  an 
activity  detector  with  the  continuous  flow  system.  The  rate  of  activity  can  then  be 


324  W.  A.  SPOOR 

measured  at  the  same  time  that  oxygen  consumption  is  determined,  and  the  results 
interpreted  accordingly.  As  the  activity  record  is  continuous,  periods  of  quiescence 
can  be  selected  for  measuring  basal  oxygen  consumption,  so  that  it  is  not  necessary 
to  employ  special  techniques  designed  to  control  activity.  In  this  connection,  how- 
ever, starvation  may  be  used  as  a  means  of  prolonging  the  quiescent  state.  A  fur- 
ther advantage  of  the  present  method  lies  in  the  fact  that  the  fish  can  be  maintained 
in  good  health  in  the  experimental  chamber  for  months,  so  that  measurements  of 
its  respiratory  metabolism  need  not  be  obscured  by  the  excitement  and  other  effects 
of  handling. 

SUMMARY 

1.  Apparatus  for  making  continuous  records  of  the  activity  of  isolated  and  un- 
disturbed goldfish  is  described,  together  with  a  method  for  measuring  oxygen  con- 
sumption and  activity  simultaneously. 

2.  The  goldfish  were  quite  variable  in  their  patterns  and  rates  of  activity  under 
the   experimental   conditions.     Some   fish   were   diurnally   active   and   nocturnally 
quiescent,  others  followed  the  opposite  pattern  and   still  others  were  arhythmic 
throughout  the  periods  during  which  they  were  observed.     Moreover,  some  fish 
showed  both  rhythmic  and  arhythmic  states  of  activity  when  studied  for  periods 
extending  over  several  weeks  or  months. 

3.  Food,  light  and  minor  disturbances  had  pronounced  effects  on  the  activity  of 
the  goldfish. 

4.  Simultaneous  measurements  of  oxygen  consumption  and  activity  are  pre- 
sented which  indicate  that  the  two  are  closely  related  above  the  basal  level  of  oxygen 
consumption. 

5.  The  bearing  of  these  observations  on  the  collection  and  interpretation  of  data 
on  the  oxygen  consumption  of  the  goldfish  and  on  the  measurement  of  its  basal 
metabolic  rate  is  discussed,  and  certain  advantages  of  the  method  are  described. 

LITERATURE  CITED 

ADKINS,  M.,  1930.  A  method  for  determining  basal  metabolism  of  fishes.  Proc.  Soc.  Exp. 
Biol.  Med.,  28 :  259-263. 

ALLEE,  W.  C.  AND  R.  OESTING,  1934.  A  critical  examination  of  Winkler's  method  for  deter- 
mining dissolved  oxygen  in  respiration  studies  with  aquatic  animals.  Physiol.  Zool., 
7:  509-541. 

BOWEN,  E.  S.,  1932.  Further  studies  of  the  aggregating  behavior  of  Ameiurus  melas.  Biol. 
Bull.,  63 :  258-270. 

CLAUSEN,  R.  G.,  1933.  Fish  metabolism  under  increasing  temperature.  Trans.  Amcr.  Fish. 
Soc.,  63 :  215-219. 

CLAUSEN,  R.  G.,  1934.     Body  temperature  of  fresh  water  fishes.    Ecology,  15:  139-144. 

CLAUSEN,  R.  G.,  1936.     Oxygen  consumption  in  fresh  water  fishes.    Ecology,  17 :  216-226. 

CROZIER,  W.  J.  AND  T.  B.  STIER,  1925.  Critical  increment  for  opercular  breathing  rhythm  of 
the  goldfish.  Jour.  Gen.  Physiol..  1 :  699-704. 

EGE,  R.  AND  A.  KROGH,  1914.  On  the  relation  between  the  temperature  and  the  respiratory 
exchange  in  fishes.  Internal.  Revue  d.  ges.  Hydrobiol.  u.  Hydrog.,  7  :  48-55. 

HALL,  F.  G.,  1929.  The  influence  of  varying  oxygen  tensions  upon  the  rate  of  oxygen  con- 
sumption in  marine  fishes.  Amer.  Jour.  Physiol.,  88  :  212-218. 

KEYS,  A.  B.,  1930.  The  measurement  of  the  respiratory  exchange  of  aquatic  animals.  Biol. 
Bull,  59 :  187-198. 

KEYS,  A.  B.  AND  N.  A.  WELLS,  1930.  Amytal  anesthesia  in  fishes.  Jour.  Pharm.  Exp.  Thcrap., 
40:  115-128. 


ACTIVITY  AND  OXYGEN  CONSUMPTION  325 

KROGH,  A.,  1916.  The  respiratory  exchange  of  animals  and  man.  Monographs  on  Biochemistry. 
Longmans,  Green  and  Co.,  London. 

SCHLAIFER,  A.,  1938.  Studies  in  mass  physiology :  effect  of  numbers  upon  the  oxygen  consump- 
tion and  locomotor  activity  of  Carassius  auratus.  Physiol.  Zoo/.,  11:  408-424. 

SMITH,  D.  C.  AND  S.  A.  MATTHEWS,  1942.  The  effect  of  adrenalin  on  the  oxygen  consumption 
of  the  fish,  Girella  nigricans.  Aincr.  Jour.  Physiol.,  137  :  533-538. 

SPENCER,  W.  P.,  1939.  Diurnal  activity  rhythms  in  fresh-water  fishes.  Ohio  Jour.  Sci.,  39: 
119-132. 

SPOOR,  W.  A.,  1941.     A  method  for  measuring  the  activity  of  fishes.     Ecology,  22:  329-331. 

SZYMANSKI,  J.  S.,  1914.  Eine  Methode  zur  Untersuchung  der  Ruhe-  und  Aktivitatsperioden 
bei  Tieren.  Pfliigcr's  Arch.  gcs.  Physiol.,  158:  343-385. 

TORYU,  Y.,  1927.  The  respiratory  exchange  in  Carassius  auratus  and  the  gaseous  exchange  of 
the  air  bladder.  Sci.  Kept.  Tohoku  Imp.  Univ.,  4  Ser.  (Biology),  3:  87-96. 

WELLS,  N.  A.,  1932.  The  importance  of  the  time  element  in  the  determination  of  the  respira- 
tory metabolism  of  fishes.  Proc.  Nat.  Acad.  Sci.,  18:  580-585. 

WELLS,  N.  A.,  1935.  The  influence  of  temperature  upon  the  respiratory  metabolism  of  the 
Pacific  killifish,  Fundulus  parvipinnis.  Physiol.  Zoo/.,  8 :  196-227. 


INDEX 


A  BSTRACTS  of  scientific  papers  presented 
at  the  Marine  Biological  Laboratory,  sum- 
mer of  1946,  210. 

Activity  and  oxygen  consumption  of  the  gold- 
fish and  its  application  to  the  measurement 
of  respiratory  metabolism  in  fishes,  312. 

Amaroecium  constellatum,  II,  66. 

Annual  report  of  the  Marine  Biological  Labora- 
tory, 1. 

Arbacia  eggs,  the  effect  of  low  temperature  and 
of  hypotonicity  on  the  morphology  of  the 
cleavage  furrow,  272. 

Artemia  salina,  the  space-time  pattern  of  seg- 
ment formation  in,  119. 

gEERS,  C.  D.  Tillina  magna:  Micronuclear 
number,  encystment  and  vitality  in  di- 
verse clones;  capabilities  of  amicronucleate 
races,  256. 

BODENSTEIN,  DIETRICH.  Developmental  rela- 
tions between  genital  ducts  and  gonads  in 
Drosophila,  288. 

BROWN,  FRANK  A.,  JR.,  AND  LORRAINE  M. 
SAIGH.  The  comparative  distribution  of 
two  chromatophorotropic  hormones  (CDH 
and  CBLH)  in  Crustacean  nervous  sys- 
tems, 170. 

("^ARRIKER,  MELBOURNE  ROMAINE.  Ob- 
servations on  the  functioning  of  the  ali- 
mentary system  of  the  snail  Lymnaea 
stagnalis  appressa  Say,  88. 

CHEN,  TZE-TUAN.  Temporary  pair  formation 
in  Paramecium  bursaria,  112. 

Chromosome  movement,  hydrostatic  pressure 
effects  upon  the  spindle  figure  and,  145. 

Ciliates  of  the  family  Ancistrocomidae  Chatton 
and  Lwoff,  III,  189. 

Ciliates  of  the  family  Ancistrocomidae  Chatton 
and  Lwoff,  IV,  200. 

Cleavage  furrow  in  Arbacia  eggs,  272. 

T~)DT,  loci  of  action  in  cockroach  (Periplaneta 

americana),  247. 
Drosophila,   developmental   relations   between 

genital  ducts  and  gonads  in,  288. 

"pLECTRON  microscope  observations  of  the 
trichocysts  and  cilia  of  Paramecium,  141. 

("MESE,  ARTHUR  C.  Comparative  sensitivity  of 
sperm  and  eggs  to  ultraviolet  radiations,  81. 


JLJJABROBRACON,  a  strongly  intersexual 
female  in,  243. 

HALL,  C.  E.     See  M.  A.  JAKUS,  141. 

Histological  study  of  Syndisyrinx  franciscanus, 
~-an  endoparasitic  rhabdocoel  of  the  sea 
urchin,  295 

Hormones  (CDH  and  CBLH)  in  Crustacean 
nervous  systems,  the  comparative  distribu- 
tion of  two  chromatophorotropic,  170. 

INFLUENCE  of  texture  and  composition  of 
surface  on  the  attachment  of  sedentary 
marine  organisms,  57. 

JAKUS,  M.  A.,  AND  C.  E.  HALL.  Electron 
microscope  observations  of  trichocysts  and 
cilia  of  Paramecium,  141. 

L£  OLLROS,  J.  J.  See].  M.  TOBIAS,  247. 
KOZLOFF,  EUGENE  N.  Studies  on  ciliates 
of  the  family  Ancistrocomidae  Chatton  and 
Lwoff  (order  Holotricha,  suborder  Thig- 
motricha).  III.  Ancistrocoma  pelseneeri 
Chatton  and  Lwoff,  Ancistrocoma  dissimi- 
lis  sp.  nov.,  and  Hypocomagalma  phola- 
didis  sp.  nov.,  189. 

KOZLOFF,  EUGENE  N.  Studies  on  ciliates  of 
the  family  Ancistrocomidae  Chatton  and 
Lwoff  (order  Holotricha,  suborder  Thig- 
motricha).  IV.  Heterocineta  janickii  Ja- 
rocki,  Heterocineta  goniobasidis  sp.  nov., 
Heterocineta  fluminicolae  sp.  nov.,  and 
Enerthecoma  properans  Jarocki,  200. 

[EHMAN,  H.  E.  A  histological  study  of 
Syndisyrinx  franciscanus,  gen.  et  sp.  nov., 
an  endoparasitic  rhabdocoel  of  the  sea 
urchin,  Strongylocentrotus  franciscanus, 
295. 

Lymnaea  stagnalis  appressa  Say,  observations 
on  the  functioning  of  the  alimentary  sys- 
tem of  the  snail,  88. 

IV/f  ARINE  Biological  Laboratory,  annual  re- 
port, 1. 

Marine  Biological  Laboratory,  program  and 
abstracts  of  scientific  papers  presented, 
summer  of  1946,  210. 

MORRISON,  PETER  R.  Physiological  observa- 
tions on  water  loss  and  oxygen  consump- 
tion in  Peripatus,  181. 


327 


328 


INDEX 


/~\XYGEN  consumption  and  water  loss  in 
Peripatus,  181. 

Oxygen  consumption  and  activity  of  the  gold- 
fish, its  application  to  the  measurement  of 
respiratory  metabolism  in  fishes,  312. 

DARAMECIUM  bursaria,  temporary  pair 
formation  in,  112. 

Paramecium,  electron  microscope  observations 
of  the  trichocysts  and  cilia  of,  141. 

PEASE,  DANIEL  C.  Hydrostatic  pressure  ef- 
fects upon  the  spindle  figure  and  chromo- 
some movement.  II.  Experiments  on  the 
meiotic  divisions  of  Tradescantia  pollen 
mother  cells,  145. 

POMERAT,  C.  M.,  AND  C.  M.  WEISS.  The  in- 
fluence of  texture  and  composition  of  sur- 
face on  the  attachment  of  sedentary 
marine  organisms,  57. 

CAIGH,  LORRAINE  M.  .See  FRANK  A.  BROWN, 
JR.,  170. 

SCOTT,  ALLAN.  The  effect  of  low  temperature 
and  of  hypotonicity  on  the  morphology  of 
the  cleavage  furrow  in  Arbacia  eggs,  272. 

SCOTT,  SISTER  FLORENCE  MARIE.  The  de- 
velopmental history  of  Amaroecium  con- 
stellatum.  II.  Organogenesis  of  the  larval 
action  system,  66. 

Society  of  General  Physiologists,  papers  pre- 
sented at  the  meeting  of,  236. 

Spindle  figure  and  chromosome  movement, 
hydrostatic  pressure  effects  upon,  145. 


SPOOR,  W.  A.  A  quantitative  study  of  the 
relationship  between  the  activity  and 
oxygen  consumption  of  the  goldfish,  and 
its  application  to  the  measurement  of 
respiratory  metabolism  in  fishes,  312. 

Studies  on  ciliates  of  the  family  Ancistro- 
comidae  Chatton  and  Lwoff  (order  Holo- 
tricha,  suborder  Thigmotricha).  III.  An- 
cistrocoma  pelseneeri  Chatton  and  Lwoff, 
Ancistrocoma  dissimilis  sp.  nov.,  and  Hy- 
pocomagalma  pholadidis  sp.  nov.,  189. 

Studies  on  ciliates  of  the  family  Ancistro- 
comidae  Chatton  and  Lwoff  (order  Holo- 
tricha,  suborder  Thigmotricha).  IV. 
Heterocineta  janickii  Jarocki,  Heterocineta 
goniobasidis  sp.  nov.,  Heterocineta  flumini- 
colae  sp.  nov.,  and  Enerthecoma  properans 
Jarocki,  200. 

nPILLINA     magna:     Micronuclear     number, 

encystment  and  vitality  in  diverse  clones; 

capabilities  of  amicronucleate  races,  256. 
TOBIAS,  J.  M.,  AND  J.  J.  KOLLROS.     Loci  of 

action  of  DDT  in  cockroach  (Periplaneta 

americana),  247. 


u 


LTRAVIOLET     radiations,     comparative 
sensitivity  of  sperm  and  eggs  to,  81. 

EISS,  C.  M.     See  C.  M.  POMERAT,  57. 
WEISZ,  PAUL  B.     The  space- time  pattern 
of  segment  formation  in  Artemiasalina,  119. 
WHITING,  P.  W.     A  strongly  intersexual  female 
in  Habrobracon,  243. 


Volume  91 


<LS    (J 


Number  1 


THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE  MARINE   BIOLOGICAL  LABORATORY 

Editorial  Board 


E.  G.  CONKLIN,  Princeton  University 
E.  N.  HARVEY,  Princeton  University 
SELIG  HECHT,  Columbia  University 
LEIGH  HOADLEY,  Harvard  University 
L.  IRVING,  Swarthmore  College 
M.  H.  JACOBS,  University  of  Pennsylvania 
H.  S.  JENNINGS,  Johns  Hopkins  University 


FRANK  R.  LILLIE,  University  of  Chicago 
CARL  R.  MOORE,  University  of  Chicago 
GEORGE  T.  MOORE,  Missouri  Botanical  Garden 
G.  H.  PARKER,  Harvard  University 
A.  C.  REDFIELD,  Harvard  University 
F.  SCHRADER,  Columbia  University 
DOUGLAS  WmTAKER,  Stanford  University 


H.  B.  STEINBACH,  Washington  University 
Managing  Editor 


AUGUST,    1946 


Marine  Biological  lal>\>; 


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CONTENTS 


Page 
ANNUAL  REPORT  OF  THE  MARINE  BIOLOGICAL  LABORATORY.  ...      i 

POMERAT,  C.  M.  AND  C.   M.  WEISS 

The  influence  of  texture  and  composition  of  surface  on  the 
attachment  of  sedentary  marine  organisms 57 

SCOTT,  SISTER  FLORENCE  MARIE 

The  developmental  history  of  Amaroecium  constellatum.     II. 
Organogenesis  of  the  larval  action  system 66 

GIESE,  ARTHUR  C. 

Comparative  sensitivity  of  sperm  and  eggs  to  ultraviolet 
radiations 81 

CARRIKER,  MELBOURNE  ROMAINE 

Observations  on  the  functioning  of  the  alimentary  system  of 
the  snail  Lymnaea  stagnalis  appressa  Say 88 

CHEN,  TZE-TUAN 

Temporary  pair  formation  in  Paramecium  bursaria 112 


Volume  91 


Number  2 


THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE   MARINE   BIOLOGICAL  LABORATORY 


Editorial  Board 


E.  G.  CONKLIN,  Princeton  University 
E.  N.  HARVEY,  Princeton  University 
SELIG  HECHT,  Columbia  University 
LEIGH  HOADLEY,  Harvard  University 
L.  IRVING,  Swarthmore  College 
M.  H.  JACOBS,  University  of  Pennsylvania 
H.  S.  JENNINGS,  Johns  Hopkins  University 


FRANK  R.  LILLIE,  University  of  Chicago 
CARL  R.  MOORE,  University  of  Chicago 
GEORGE  T.  MOORE,  Missouri  Botanical  Garden 
G.  H.  PARKER,  Harvard  University 
A.  C.  REDFIELD,  Harvard  University 
F.  SCHRADER,  Columbia  University 
DOUGLAS  WHITAKER,  Stanford  University 


H.  B.  STEINBACH,  Washington  University 
Managing  Editor 


Marine  Biological  Labofototy 

L.I  Ell  A.  R  Y 

NOV  181946 

WOODS  HOLE,  MASS. 


OCTOBER,    1946 


Printed  and  Issued  by 

LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 

LANCASTER,  PA. 


BOOKS-WAR  VICTIMS 


'URING  THE  WAR,  the  libraries  of  half  the  world  were  destroyed  in  the 
fires  of  battle  and  in  the  fires  of  hate  and  fanaticism.  Where  they  were  spared 
physical  damage,  they  were  impoverished  by  isolation.  There  is  an  urgent  need 
—now — for  the  printed  materials  which  are  basic  to  the  reconstruction  of  dev- 
astated areas  and  which  can  help  to  remove  the  intellectual  blackout  of  Europe 
and  the  Orient. 

There  is  need  for  a  pooling  of  resources,  for  coordinated  action  in  order  that 
the  devastated  libraries  of  the  world  may  be  restocked  as  far  as  possible  with 
needed  American  publications.  The  American  Book  Center  for  War  Devastated 
Libraries,  Inc.,  has  come  into  being  to  meet  this  need.  It  is  a  program  that  is 
born  of  the  combined  interests  of  library  and  educational  organizations,  of  gov- 
ernment agencies,  and  of  many  other  official  and  non-official  bodies  in  the  United 
States. 

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physical,  economic,  social  and  industrial  rehabilitation  and  reconstruction  of 
Europe  and  the  Far  East. 

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from  individuals,  institutions,  and  organizations.  Each  state  will  be  organized 
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wanted. 

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CANNOT  ACCEPT  MATERIAL  WHICH  IS  SENT  COLLECT.  Reim- 
bursement cannot  be  made  for  packing  or  other  charges  beyond  actual  transpor- 
tation. When  possible,  periodicals  should  be  tied  together  by  volume.  It  will 
be  helpful  if  missing  issues  are  noted  on  incomplete  volumes. 


Your  Biological  News 

You  would  not  go  to  the  library  to  read  the  daily  newspaper — probably 
you  have  it  delivered  at  your  home  to  be  read  at  your  leisure.  Why,  then, 
depend  upon  your  library  for  your  biological  news  ? 

Biological  Abstracts  is  news  nowadays.  Abridgments  of  all  the  im- 
portant biological  literature  are  published  promptly — in  many  cases  before 
the  original  articles  are  available  in  this  country.  Only  by  having  your 
own  copy  of  Biological  Abstracts  to  read  regularly  can  you  be  sure  that 
you  are  missing  none  of  the  literature  of  particular  interest  to  you.  An 
abstract  of  one  article  alone,  which  otherwise  you  would  not  have  seen, 
might  far  more  than  compensate  you  for  the  subscription  price. 

Biological  Abstracts  is  now  published  in  seven  low  priced  sections,  as 
well  as  the  complete  edition,  so  that  the  biological  literature  may  be  avail- 
able to  all  individual  biologists.  Write  for  full  information  and  ask  for  a 
copy  of  the  section  covering  your  field. 

BIOLOGICAL  ABSTRACTS 

University  of  Pennsylvania 

Philadelphia,  Pa. 


MICROFILM  SERVICE 

* 

The  Library  of  The  Marine 
Biological  Laboratory  can 
supply  microfilms  of  ma- 
terial from  periodicals  in- 
cluded in  its  list.  Requests 
should  include  the  title  of 
the  paper,  the  author,  peri- 
odical, volume  and  date  of 
publication. 


Rates  are  as  follows:  $.30  for 
papers  up  to  25  pages,  and  $.10 
for  each  additional  10  pages  or 
fraction  thereof. 


LANCASTER  PRESS,  Inc. 

LANCASTER,  PA. 


THE  EXPERIENCE  we  have 
gained  from  printing  some 
sixty  educational  publica- 
tions has  fitted  us  to  meet 
the  standards  of  customers 
who  demand  the  best. 

We  shall  be  happy  to  have  workers  at 

the  MARINE  BIOLOGICAL  LABORATORY 

write  for  estimates  on  journals  or 
monographs.  Our  prices  are  moderate. 


INSTRUCTIONS  TO  AUTHORS 

The  Biological  Bulletin  accepts  papers  on  a  variety  of  subjects  of  biologi- 
cal interest.  In  general,  a  paper  will  appear  within  three  months  of  the  date  of 
its  acceptance.  The  Editorial  Board  requests  that  manuscripts  conform  to  the 
requirements  set  Below. 

Manuscripts.  Manuscripts  should  be  typed  in  double  or  triple  spacing  on 
one  side  of  paper,  SVz  by  11  inches. 

Tables  should  be  typewritten  on  separate  sheets  and  placed  in  correct 
sequence  in  the  text.  Explanations  of  figures  should  be  typed  on  a  separate 
sheet  and  placed  at  the  end  of  the  text.  Footnotes,  numbered  consecutively, 
may  be  placed  on  a  separate  sheet  at  the  end  of  the  paper. 

A  condensed  title  or  running  page  head  of  not  more  than  thirty-five  letters 
should  be  included. 

Figures.  The  dimensions  of  the  printed  page,  5  by  7%  inches,  should  be 
kept  in  mind  in  preparing  figures  for  publication.  Illustrations  should  be  large 
enough  so  that  all  details  will  be  clear  after  appropriate  reduction.  Explana- 
tory matter  should  be  included  in  legends  as  far  as  possible,  not  lettered  on  the 
illustrations.  Figures  should  be  prepared  for  reproduction  as  line  cuts  or  half- 
tones; other  methods  will  be  used  only  at  the  author's  expense.  Figures  to  be 
reproduced  as  line  cuts  should  be  drawn  in  black  ink  on  white  paper  or  blue- 
lined  co-ordinate  paper;  those  to  be  reproduced  as  halftones  should  be  mounted 
on  Bristol  board  and  any  designating  letters  or  numbers  should  be  made  di- 
rectly on  the  figures.  The  author's  name  should  appear  on  the  reverse  side  of 
all  figures.  The  desired  reduction  should  be  specified  on  each  figure. 

Literature  cited.  The  list  of  literature  cited  should  conform  to  the  style  set 
in  this  issue  of  The  Biological  Bulletin.  Papers  referred  to  in  the  manuscript 
should  be  listed  on  separate  pages  headed  "Literature  Cited." 

Mailing.  Manuscripts  should  be  packed  flat.  Large  illustrations  may  be 
rolled  in  a  mailing  tube,  but  all  illustrations  larger  than  9  by  12  inches  must 
be  accompanied  by  photographic  reproductions  or  tracings  that  may  be  folded 
to  page  size. 

Reprints.  Authors  will  be  furnished,  free  of  charge,  one  hundred  reprints 
without  covers.  Additional  copies  may  be  obtained  at  cost;  approximate 
figures  will  be  furnished  upon  request. 


THE  BIOLOGICAL  BULLETIN 

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

Subscriptions  and  similar  matter  should  be  addressed  to  The  Biologi- 
cal 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, 
$1.75.  Subscription  per  volume  (three  issues),  $4.50. 

Communications  relative  to  manuscripts  should  be  sent  to  the  Manag- 
ing Editor,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts, 
between  July  1  and  September  1,  and  to  the  Department  of  Zoology, 
Washington  University,  St.  Louis,  Missouri,  during  the  remainder  of 
the  year. 


Entered  as  second-class  matter  May  17,  1930,  at  the  post  office  at  Lancaster,  Pa., 

under  the  Act  of  August  24,  1912. 


BIOLOGY  MATERIALS 

The  Supply  Department  of  the  Marine  Biological  Labora- 
tory has  a  complete  stock  of  excellent  plain  preserved  and 
injected  materials,  and  would  be  pleased  to  quote  prices  on 
school  needs. 


PRESERVED  SPECIMENS 

for 
Zoology,  Botany,  Embryology, 

and  Comparative  Anatomy 

LIVING  SPECIMENS 

for 
Zoology  and  Botany 

including  Protozoan  and 
Drosophila  Cultures,  and 
Animals  for  Experimental  and 
Laboratory  Use. 

MICROSCOPE  SLIDES 

for 

Zoology,  Botany,  Embryology, 
Histology,  Bacteriology,  and 
Parasitology. 

CATALOGUES  SENT  ON  REQUEST 


Supply  Department 

MARINE 
BIOLOGICAL  LABORATORY 

Woods  Hole,  Massachusetts 


CONTENTS 


Page 
WEISZ,  P/vUL  B. 

The  space-time  pattern  of  segment  formation  in  Artemia 
salina 1 19 

JAKUS,  M.  A.,  AND  C.  E.  HALL 

Electron  microscope  observations  of  the  trichocysts  and  cilia 
of  Paramecium 141 

PEASE,  DANIEL  C. 

Hydrostatic  pressure  effects  upon  the  spindle  figure  and 
chromosome  movement.  II.  Experiments  on  the  meiotic 
divisions  of  Tradescantia  pollen  mother  cells 145 

BROWN,  FRANK  A.  JR.,  AND  LORRAINE  M.  SAIGH 

The  comparative  distribution  of  two  chromatophorotropic 
hormones  (CDH  and  CBLH)  in  Crustacean  nervous  systems  170 

MORRISON,  PETER  R. 

Physiological  observations  on  water  loss  and  oxygen  con- 
sumption in  Peripatus 181 

KOZLOFF,  EUGENE  N. 

Studies  on  ciliates  of  the  family  Ancistrocomidae  Chatton 
and  Lwoff  (order  Holotricha,  suborder  Thigmotricha).  III. 
Ancistrocoma  pelseneeri  Chatton  and  Lwoff,  Ancistrocoma 
dissimilis  sp.  nov.,  and  Hypocomagalma  pholadidis  sp.  nov. .  189 

KOZLOFF,  EUGENE  N. 

Studies  on  ciliates  of  the  family  Ancistrocomidae  Chatton 
and  Lwoff  (order  Holotricha,  suborder  Thigmotricha).  IV. 
Heterocineta  janickii  Jarocki,  Heterocineta  goniobasidis  sp. 
nov.,  Heterocineta  fluminicolae  sp.  nov.,  and  Enerthecoma 
properans  Jarocki 200 

ABSTRACTS  OF  SCIENTIFIC  PAPERS  PRESENTED  AT  THE  MARINE 

BIOLOGICAL  LABORATORY,  SUMMER  OF  1946 210 

PAPERS  PRESENTED  AT  THE  MEETING  OF  THE  SOCIETY  OF  GEN- 
ERAL PHYSIOLOGISTS.  236 


Volume  91 


Number  3 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 

THE   MARINE   BIOLOGICAL  LABORATORY 


Editorial  Board 


E.  G.  CONKLIN,  Princeton  University 

E.  N.  HARVEY,  Princeton  University 

SELIG  HECHT,  Columbia  University 

LEIGH  HOADLEY,  Harvard  University 

L.  IRVING,  Swarthmore  College 

M.  H.  JACOBS,  University  of  Pennsylvania 

H.  S.  JENNINGS,  Johns  Hopkins  University 


FRANK  R.  LILLIE,  University  of  Chicago 
CARL  R.  MOORE,  University  of  Chicago 
GEORGE  T.  MOORE,  Missouri  Botanical  Garden 
G.  H.  PARKER,  Harvard  University 
A.  C.  REDFffiLD,  Harvard  University 
F.  SCHRADER,  Columbia  University 
DOUGLAS  WHITAKER,  Stanford  University 


H.  B.  STEINBACH,  Washington  University 
Managing  Editor 


DECEMBER,   1946 


JAN -7 -19-47 

WOODS  HOLE, 


Printed  and  Issued  by 

LANCASTER  PRESS,  Inc. 

PRINCE  &  LEMON  STS. 

LANCASTER,  PA. 


BOOKS-WAR  VICTIMS 


D, 


'URING  THE  WAR,  the  libraries  of  half  the  world  were  destroyed  in  the 
fires  of  battle  and  in  the  fires  of  hate  and  fanaticism.  Where  they  were  spared 
physical  damage,  they  were  impoverished  by  isolation.  There  is  an  urgent  need 
— now — for  the  printed  materials  which  are  basic  to  the  reconstruction  of  dev- 
astated areas  and  which  can  help  to  remove  the  intellectual  blackout  of  Europe 
and  the  Orient. 

There  is  need  for  a  pooling  of  resources,  for  coordinated  action  in  order  that 
the  devastated  libraries  of  the  world  may  be  restocked  as  far  as  possible  with 
needed  American  publications.  The  American  Book  Center  for  War  Devastated 
Libraries,  Inc.,  has  come  into  being  to  meet  this  need.  It  is  a  program  that  is 
born  of  the  combined  interests  of  library  and  educational  organizations,  of  gov- 
ernment agencies,  and  of  many  other  official  and  non-official  bodies  in  the  United 
States. 

The  American  Book  Center  is  collecting  and  is  shipping  abroad  scholarly 
books  and  periodicals  which  will  be  useful  in  research  and  necessary  in  the 
physical,  economic,  social  and  industrial  rehabilitation  and  reconstruction  of 
Europe  and  the  Far  East. 

The  Center  cannot  purchase  books  and  periodicals ;  it  must  depend  upon  gifts 
from  individuals,  institutions,  and  organizations.  Each  state  will  be  organized 
to  participate  in  the  program  through  the  leadership  of  a  state  chairman.  Other 
chairmen  will  organize  interest  in  the  principal  subject  fields.  Cooperation  with 
these  leaders  or  direct  individual  contributions  are  welcomed. 

WHAT  IS  NEEDED:  Shipping  facilities  are  precious  and  demand  that 
all  materials  be  carefully  selected.  Emphasis  is  placed  upon  publications  issued 
during  the  past  decade,  upon  scholarly  books  which  are  important  contributions 
to  their  fields,  upon  periodicals  (even  incomplete  volumes)  of  significance,  upon 
fiction  and  non-fiction  of  distinction.  All  subjects — history,  the  social  sciences, 
music,  fine  arts,  literature,  and  especially  the  sciences  and  technologies — are 
wanted. 

WHAT  IS  NOT  NEEDED:  Textbooks,  out-dated  monographs,  recrea- 
tional reading,  books  for  children  and  young  people,  light  fiction,  materials  of 
purely  local  interest,  popular  magazines  such  as  Time,  Life,  National  Geographic, 
etc.,  popular  non-fiction  of  little  enduring  significance  such  as  Gunther's  Inside 
Europe,  Haliburton's  Royal  Road  to  Romance,  etc.  Only  carefully  selected 
federal  and  local  documents  are  needed,  and  donors  are  requested  to  write 
directly  to  the  Center  with  regard  to  specific  documents. 

HOW  TO  SHIP:  All  shipments  should  be  sent  PREPAID  via  the 
cheapest  means  of  transportation  to  THE  AMERICAN  BOOK  CENTER, 
C/O  THE  LIBRARY  OF  CONGRESS,  WASHINGTON  25,  D.  C.  Al- 
though the  Center  hopes  that  donors  will  assume  the  costs  of  transportation  of 
their  materials  to  Washington,  when  this  is  not  possible  reimbursement  will  be 
made  upon  notification  by  card  or  letter  of  the  amount  due.  THE  CENTER 
CANNOT  ACCEPT  MATERIAL  WHICH  IS  SENT  COLLECT.  Reim- 
bursement cannot  be  made  for  packing  or  other  charges  beyond  actual  transpor- 
tation. When  possible,  periodicals  should  be  tied  together  by  volume.  It  will 
be  helpful  if  missing  issues  are  noted  on  incomplete  volumes. 


Your  Biological  News 

You  would  not  go  to  the  library  to  read  the  daily  newspaper — probably 
you  have  it  delivered  at  your  home  to  be  read  at  your  leisure.  Why,  then, 
depend  upon  your  library  for  your  biological  news  ? 

Biological  Abstracts  is  news  nowadays.  Abridgments  of  all  the  im- 
portant biological  literature  are  published  promptly — in  many  cases  before 
the  original  articles  are  available  in  this  country.  Only  by  having  your 
own  copy  of  Biological  Abstracts  to  read  regularly  can  you  be  sure  that 
you  are  missing  none  of  the  literature  of  particular  interest  to  you.  An 
abstract  of  one  article  alone,  which  otherwise  you  would  not  have  seen, 
might  far  more  than  compensate  you  for  the  subscription  price. 

Biological  Abstracts  is  now  published  in  seven  low  priced  sections,  as 
well  as  the  complete  edition,  so  that  the  biological  literature  may  be  avail- 
able to  all  individual  biologists.  Write  for  full  information  and  ask  for  a 
copy  of  the  section  covering  your  field. 

BIOLOGICAL  ABSTRACTS 

University  of  Pennsylvania 

Philadelphia,  Pa. 


MICROFILM   SERVICE 


The  Library  of  The  Marine 
Biological  Laboratory  can 
supply  microfilms  of  ma- 
terial from  periodicals  in- 
cluded in  its  list.  Requests 
should  include  the  title  of 
the  paper,  the  author,  peri- 
odical, volume  and  date  of 
publication. 


Rates  are  as  follows:  $.30  for 
papers  up  to  25  pages,  and  $.10 
for  each  additional  10  pages  or 
fraction  thereof. 


LANCASTER  PRESS,  Inc. 

LANCASTER,  PA. 


THE  EXPERIENCE  we  have 
gained  from  printing  some 
sixty  educational  publica- 
tions has  fitted  us  to  meet 
the  standards  of  customers 
who  demand  the  best. 

We  shall  be  happy  to  have  workers  at 

the  MARINE  BIOLOGICAL  LABORATORY 

write  for  estimates  on  journals  or 
monographs.  Our  prices  are  moderate. 


INSTRUCTIONS  TO  AUTHORS 

The  Biological  Bulletin  accepts  papers  on  a  variety  of  subjects  of  biologi- 
cal interest.  In  general,  a  paper  will  appear  within  three  months  of  the  date  of 
its  acceptance.  The  Editorial  Board  requests  that  manuscripts  conform  to  the 
requirements  set  below. 

Manuscripts.  Manuscripts  should  be  typed  in  double  or  triple  spacing  on 
one  side  of  paper,  SVz  by  11  inches. 

Tables  should  be  typewritten  on  separate  sheets  and  placed  in  correct 
sequence  in  the  text.  Explanations  of  figures  should  be  typed  on  a  separate 
sheet  and  placed  at  the  end  of  the  text.  Footnotes,  numbered  consecutively, 
may  be  placed  on  a  separate  sheet  at  the  end  of  the  paper. 

A  condensed  title  or  running  page  head  of  not  more  than  thirty-five  letters 
should  be  included. 

Figures.  The  dimensions  of  the  printed  page,  5  by  7%  inches,  should  be 
kept  in  mind  in  preparing  figures  for  publication.  Illustrations  should  be  large 
enough  so  that  all  details  will  be  clear  after  appropriate  reduction.  Explana- 
tory matter  should  be  included  in  legends  as  far  as  possible,  not  lettered  on  the 
illustrations.  Figures  should  be  prepared  for  reproduction  as  line  cuts  or  half- 
tones; other  methods  will  be  used  only  at  the  author's  expense.  Figures  to  be 
reproduced  as  line  cuts  should  be  drawn  in  black  ink  on  white  paper  or  blue- 
lined  co-ordinate  paper;  those  to  be  reproduced  as  halftones  should  be  mounted 
on  Bristol  board  and  any  designating  letters  or  numbers  should  be  made  di- 
rectly on  the  figures.  The  author's  name  should  appear  on  the  reverse  side  of 
all  figures.  The  desired  reduction  should  be  specified  on  each  figure. 

Literature  cited.  The  list  of  literature  cited  should  conform  to  the  style  set 
in  this  issue  of  The  Biological  Bulletin.  Papers  referred  to  in  the  manuscript 
should  be  listed  on  separate  pages  headed  "Literature  Cited." 

Mailing.  Manuscripts  should  be  packed  flat.  Large  illustrations  may  be 
rolled  in  a  mailing  tube,  but  all  illustrations  larger  than  9  by  12  inches  must 
be  accompanied  by  photographic  reproductions  or  tracings  that  may  be  folded 
to  page  size. 

Reprints.  Authors  will  be  furnished,  free  of  charge,  one  hundred  reprints 
without  covers.  Additional  copies  may  be  obtained  at  cost:  approximate 
figures  will  be  furnished  upon  request. 


THE  BIOLOGICAL  BULLETIN 

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

Subscriptions  and  similar  matter  should  be  addressed  to  The  Biologi- 
cal 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, 
$1.75.  Subscription  per  volume  (three  issues),  $4.50. 

Communications  relative  to  manuscripts  should  be  sent  to  the  Manag- 
ing Editor,  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts, 
between  July  1  and  September  1,  and  to  the  Department  of  Zoology, 
Washington  University.  St.  Louis.  Missouri,  during  the  remainder  of 
the  year. 


Entered  as  second-class  matter  May  17,  1930,  at  the  post  office  at  Lancaster,  Pa., 

under  the  Act  of  August  24,  1912. 


BIOLOGY  MATERIALS 

The  Supply  Department  of  the  Marine  Biological  Labora- 
tory has  a  complete  stock  of  excellent  plain  preserved  and 
injected  materials,  and  would  be  pleased  to  quote  prices  on 
school  needs. 


PRESERVED  SPECIMENS 

for 

Zoology,  Botany,  Embryology, 
and  Comparative  Anatomy 

LIVING  SPECIMENS 

for 
Zoology  and  Botany 

including  Protozoan  and 
Drosophila  Cultures,  and 
Animals  for  Experimental  and 
Laboratory  Use. 

MICROSCOPE  SLIDES 

for 

Zoology,  Botany,  Embryology, 
Histology,  Bacteriology,  and 
Parasitology. 

CATALOGUES  SENT  ON  REQUEST 


Supply  Department 

MARINE 
BIOLOGICAL  LABORATORY 

Woods  Hole,  Massachusetts 


• 


CONTENTS 


'  •     ' 


Page 
WHITING,  P.  W. 

A  strongly  intersexual  female  in  Habrobracon  .............   243 

TOBIAS,  J.  M.,  AND  J.  J.  KOLLROS 

Loci  of  action  of  DDT  in  the  cockroach  (Periplaneta  ameri- 
cana)  .............................................  247 

BEERS,  C.  D. 

Tillina  magna:  Micronuclear  number,  encystment  and  vitality 

in  diverse  clones;  capabilities  of  amicronucleate  races  ......   256 

SCOTT,  ALLAN 

The  effect  of  low  temperature  and  of  hypotonicity  on  the 
morphology  of  the  cleavage  furrow  in  Arb'acia  eggs  ........  272 

BODENSTEIN,   DIETRICH 

Developmental  relations  between  genital  ducts  and  gonads 

in  Drosophila  ........  ^  .................................   288 

LEHMAN,  H.  E. 

A  histological  study  of  Syndisyrinx  franciscanus,  gen.  et  sp. 
nov.,  an  endoparasitic  rhabdocoel  of  the  sea  urchin,  Strongylo- 
centrotus  franciscanus  .................................  295 

SPOOR,  W.  A. 

A  quantitative  study  of  the  relationship  between  the  activity 
and  oxygen  consumption  of  the  goldfish,  and  its  application 
to  the  measurement  of  respiratory  metabolism  in  fishes.  ...  312 


J 

'  • 


.   VVHOI    LIBRARY 


UH    17JA    D