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Volume  189 


THE 


Number  I 


BIOLOGICAL 
BULLETIN 


AUGUST,  1995 


Published  by  the  Marine  Biological  Laboratory 


THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 
THE  MARINE  BIOLOGICAL  LABORATORY 


Associate  Editors 

PETER  A.  V.  ANDERSON,  The  Whitney  Laboratory,  University  of  Florida 

WILLIAM  D.  COHEN.  Hunter  College.  City  University  of  New  Yoft<arir 

Woods  Hole  OceanonraDhic  Institute 


DAVID  EPEL,  Hopkins  Marine  Station.  Stanford  University 
J.  MALCOLM  SHICK,  LJniversitv  of  Maine.  Orono 


Oceanographic  Institution 


AUG  1  7  1995 


Editorial  Board 


Woods  Ho(8,  MA  02543 


PETER  B.  ARMSTRONG,  University  of  California,  Davis 
THOMAS  H.  DIETZ,  Louisiana  State  University 
DAPHNE  GAIL  FALITIN,  University  of  Kansas 

WILLIAM  F.  GILLV.  Hopkins  Marine  Station.  Stanford 

University 

ROGER   T.    HANLON.    Marine   Biomedical    Institute. 
University  of  Texas  Medical  Branch 

MICHAEL  LABARBERA.  University  of  Chicago 
CHARLES  B.  METZ.  University  of  Miami 


K.  RANGA  RAO.  University  of  West  Florida 

BARUCH     RINKEVICH,     Israel    Oceanographic    & 
Limnological  Research  Ltd. 

RICHARD  STRATHMANN.  Friday  Harbor  Laboratories. 
University  of  Washington 

STEVEN  VOGEL.  Duke  University 

J.  HERBERT  WAITE,  University  of  Delaware 

SARAH  ANN  WOODIN,  University  of  South  Carolina 

RICHARD   K.   ZIMMER-FAUST,    University   of  South 

Carolina 


Editor:  MICHAEL  J.  GREENBERG.  The  Whitney  Laboratory,  University  of  Florida 
Managing  Editor:  PAMELA  L.  CLAPP.  Marine  Biological  Laboratory 


AUGUST,  1995 


Printed  and  Issued  bv 
LANCASTER  PRESS,  Inc. 

3575  HEMPLAND  ROAD 
LANCASTER,  PA 


THE  BIOLOGICAL  BULLETIN 


THE  BIOLOGICAL  BULLETIN  is  published  six  times  a  year  by  the  Marine  Biological  Laboratory.  MBL 
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ISSN  0006-3 185 


INSTRUCTIONS  TO  AUTHORS 


The  Biological  Bulletin  accepts  outstanding  original  research 
reports  of  general  interest  to  biologists  throughout  the  world. 
Papers  are  usually  of  intermediate  length  (10-40  manuscript 
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cepted after  formal  review.  A  paper  will  usually  appear  within 
four  months  after  its  acceptance. 

Very  short,  especially  topical  papers  (less  than  9  manuscript 
pages  including  tables,  figures,  and  bibliography)  will  be  pub- 
lished in  a  separate  section  entitled  "Research  Notes."  A  Re- 
search Note  in  The  Biological  Bulletin  follows  the  format  of 
similar  notes  in  Nature.  It  should  open  with  a  summary  para- 
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References  should  be  referred  to  in  the  text  by  number,  and 
listed  in  the  Literature  Cited  section  in  the  order  that  they  appear 
in  the  text.  Unlike  references  in  Nature,  references  in  the  Re- 
search Notes  section  should  conform  in  punctuation  and  ar- 
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Materials  and  Methods  should  be  incorporated  into  appropriate 
figure  legends.  See  the  article  by  Lohmann  ct  at  (October  1990, 
Vol.  179:  214-2 1 8)  for  sample  style.  A  Research  Note  will  usually 
appear  within  two  months  after  its  acceptance. 

The  Editorial  Board  requests  that  regular  manuscripts  con- 
form to  the  requirements  set  below;  those  manuscripts  that  do 
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1.  Manuscripts.  Manuscripts,  including  figures,  should 
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2.  Title  page.     The  title  page  consists  of  a  condensed  title 
or  running  head  of  no  more  than  35  letters  and  spaces,  the 
manuscript  title,  authors'  names  and  appropriate  addresses,  and 
footnotes  listing  present  addresses,  acknowledgments  or  contri- 
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3.  Figures.     The  dimensions  of  the  printed  page,  7  by  9 
inches,  should  be  kept  in  mind  in  preparing  figures  for  publi- 
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erally should  be  included  in  legends,  although  axes  should  always 
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4.  Tables,  footnotes,  figure  legends,  etc.     Authors  should 
follow  the  style  in  a  recent  issue  of  The  Biological  Bulletin  in 
preparing  table  headings,  figure  legends,  and  the  like.  Because 
of  the  high  cost  of  setting  tabular  material  in  type,  authors  are 
asked  to  limit  such  material  as  much  as  possible.  Tables,  with 
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numbered  with  consecutive  Roman  numerals,  and  placed  after 
the  Literature  Cited.  Figure  legends  should  contain  enough  in- 
formation to  make  the  figure  intelligible  separate  from  the  text. 
Legends  should  be  typed  double  spaced,  with  consecutive  Arabic 
numbers.  on  a  separate  sheet  at  the  end  of  the  paper.  Footnotes 
should  be  limited  to  authors'  current  addresses,  acknowledg- 
ments or  contribution  numbers,  and  explanation  of  unusual 
abbreviations.  All  such  footnotes  should  appear  on  the  title  page. 
Footnotes  are  not  normally  permitted  in  the  body  of  the  text. 

5.  Literature  cited.     In  the  text,  literature  should  be  cited 
by  the  Harvard  system,  with  papers  by  more  than  two  authors 
cited  as  Jones  el  al.,  1980.  Personal  communications  and  ma- 
terial in  preparation  or  in  press  should  be  cited  in  the  text  only, 
with  author's  initials  and  institutions,  unless  the  material  has 
been  formally  accepted  and  a  volume  number  can  be  supplied. 
The  list  of  references  following  the  text  should  be  headed  Lit- 
erature Cited,  and  must  be  typed  double  spaced  on  separate 
pages,  conforming  in  punctuation  and  arrangement  to  the  style 
of  recent  issues  of  The  Biological  Bulletin.  Citations  should  in- 
clude complete  titles  and  inclusive  pagination.  Journal  abbre- 
viations should  normally  follow  those  of  the  U.  S.  A.  Standards 
Institute  (USASI).  as  adopted  by  BIOLOGICAL  ABSTRACTS  and 
CHEMICAL  ABSTRACTS,  with  the  minor  differences  set  out  below. 
The  most  generally  useful  list  of  biological  journal  titles  is  that 
published  each  year  by  BIOLOGICAL  ABSTRACTS  (BIOSIS  List  of 
Serials:  the  most  recent  issue).  Foreign  authors,  and  others  who 
are  accustomed  to  using  THE  WORLD  LIST  OF  SCIENTIFIC  PE- 
RIODICALS, may  find  a  booklet  published  by  the  Biological 
Council  of  the  U.K.  (obtainable  from  the  Institute  of  Biology. 
41  Queen's  Gate.  London.  S.W.7.  England.  LI.K.)  useful,  since 
it  sets  out  the  WORLD  LIST  abbreviations  for  most  biological 


journals  with  notes  of  the  USASI  abbreviations  where  these  differ. 
CHEMICAL  ABSTRACTS  publishes  quarterly  supplements  of  ad- 
ditional abbreviations.  The  following  points  of  reference  style 
for  THE  BIOLOGICAL  BULLETIN  differ  from  USASI  (or  modified 
WORLD  LIST)  usage: 

A.  Journal  abbreviations,  and  book  titles,  all  underlined 
(for  italics) 

B.  All  components  of  abbreviations  with  initial  capitals 
(not  as  European  usage  in  WORLD  LIST  e.g.,  J  Cell.  Comp. 
Physiol.  NOT  J.  cell,  ami  p.  Phyxiol.) 

C.  All  abbreviated  components  must  be  followed  by  a  pe- 
riod, whole  word  components  must  not  (i.e..  J  Cancer  Res.) 

D.  Space  between  all  components  (e.g..  J.  Cell.  C<mip. 
Physiol..  not  J.Cell.Comp.Physiol.) 

E.  Unusual  words  in  journal  titles  should  be  spelled  out 
in  full,  rather  than  employing  new  abbreviations  invented  by 
the  author.  For  example,  use  Rit  I  'isindafjelags  hlcndinga  with- 
out abbreviation. 

F.  All  single  word  journal  titles  in  full  (e.g.,  I'eliger,  Ecol- 
ogy. Brain). 

G.  The  order  of  abbreviated  components  should  be  the 
same  as  the  word  order  of  the  complete  title  (i.e.,  Proc.  and 
Trans,  placed  where  they  appear,  not  transposed  as  in  some 
BIOLOGICAL  ABSTRACTS  listings). 

H.  A  few  well-known  international  journals  in  their  pre- 
ferred forms  rather  than  WORLD  LIST  or  USASI  usage  (e.g.. 
Nature,  Science,  Evolution  NOT  Nature,  Land..  Science,  N.Y.; 
Evolution.  Lancaster.  Pa.) 

6.  Reprints,  page  proofs,  and  charges.  Authors  receive  their 
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have  page  charges. 


Reference:  Biol.  Bull-  189:  1-5.  (August.  1995) 


A  Short  Story  of  Aequorin 

OSAMU  SHIMOMURA 

Marine  Biological  Laboratory.  Hoods  Hole.  Massachusetts  02543 


Discovery  of  Aequorin 

One  day  in  the  fall  of  1960,  shortly  after  my  arrival  at 
Princeton  from  Japan,  Dr.  Frank  Johnson  showed  me  a 
small  jar  containing  a  spoonful  of  white  powder.  He  ex- 
plained that  the  powder  was  a  freeze  dried  "squeezate" 
made  from  the  luminous  jellyfish  Aeqitorea.  and  that  it 
would  emit  light  when  mixed  with  water.  He  asked  me  if 
I  would  be  interested  in  studying  the  bioluminescence  of 
this  jellyfish.  The  powder  did  not  emit  any  light  when 
moistened.  But  I  was  quite  impressed  by  Dr.  Johnson's 
description  of  the  brilliant  luminescence  of  live  jellyfish 
and  the  great  abundance  of  specimens  around  Friday 
Harbor,  Washington.  So  my  response  was  a  definite  "yes." 
My  experience  in  bioluminescence  research  at  the  time 
was  meager  and  limited  to  only  the  luminescent  system 
of  the  ostracod  Cypridina.  I  imagined,  vaguely,  that  the 
jellyfish  would  probably  contain  a  kind  of  luciferin  and 
a  luciferase,  possibly  with  one  of  the  cofactors,  such  as 
ATP.  FMN.  or  NADH.  like  the  fireflies,  luminous  bac- 
teria, and  Cypridina  that  were  known  at  that  time. 

In  the  early  summer  of  1 96 1 ,  we  traveled  from  Prince- 
ton to  Friday  Harbor  in  Dr.  Johnson's  station  wagon, 
which  he  had  newly  purchased  for  the  excursion.  The  car 
was  fully  loaded  with  necessary  equipment  and  chemicals, 
including  a  MacNichol  integrating  photometer  of  gigantic 
size  (a  two-foot  cube),  and  four  travelers  (my  wife  and 
Yo  Saiga,  an  assistant,  came  along)  with  all  of  their  bag- 
gage on  the  roof.  It  took  us  seven  days  to  the  West  Coast, 
through  Chicago  and  Glacier  National  Park.  Dr.  Johnson 
was  the  only  driver  throughout  the  trip,  driving  12  hours 
a  day  with  an  admirable  toughness. 

Upon  arrival  at  the  Friday  Harbor  Laboratories,  we 
were  welcomed  by  Dr.  Robert  Fernald,  Director  of  the 
Lab.  We  set  up  our  work  space  in  Lab  1,  a  small  building 
consisting  of  two  rooms,  and  we  started  to  work.  There 
were  three  other  scientists  in  the  room,  and  one  of  them 

Received  23  March  1995;  accepted  5  June  1995. 


was  Dr.  Dixy  Lee  Ray.  future  governor  of  Washington 
State,  who  was  always  accompanied  by  a  dog,  her  well- 
known  trademark.  The  laboratory  area  was  a  sanctuary 
prohibited  to  common  dogs,  but  she  declared  that  the 
animal  was  her  assistant,  not  a  dog. 

The  jellyfish  were  abundant.  A  constant  stream  of 
floating  jellyfish  passed  along  the  side  of  the  lab  dock  every 
morning  and  evening,  riding  with  the  current  caused  by 
the  tide.  We  carefully  scooped  up  the  jellyfish  into  buckets, 
one  by  one,  using  a  shallow  dip-net.  The  specimens  of 
Aequorea  are  shaped  like  hemispherical  umbrellas  and 
are  nearly  transparent.  An  average  specimen  measures  3- 
4  inches  in  diameter  and  weighs  about  50  g.  The  light 
organs— about  100  granules— are  distributed  evenly  along 
the  edge  of  the  umbrella.  Thus,  the  margin  of  the  umbrella 
containing  light  organs  could  be  easily  cut  off  with  a  pair 
of  scissors,  yielding  a  thin  strip  called  a  "ring."  When  the 
rings  obtained  from  20-30  jellyfish  were  squeezed  through 
a  rayon  gauze,  a  liquid  called  "squeezate"  was  obtained. 
The  squeezate  was  only  dimly  luminescent,  but  when  it 
was  diluted  with  water,  the  luminescence  increased  sig- 
nificantly for  a  period  of  5-10  minutes,  as  the  granular 
light  organs  were  cytolyzed. 

We  tried  to  extract  luminescent  substance  from  the 
squeezate  by  every  thinkable  method,  but  all  failed;  and 
we  ran  out  of  ideas  after  only  a  few  days  of  work.  Con- 
vinced that  the  cause  of  our  failure  was  the  luciferin-Iu- 
ciferase  hypothesis  that  dominated  our  thinking,  I  sug- 
gested to  Dr.  Johnson  that  we  should  forget  the  idea  of 
extracting  luciferin  and  luciferase  and,  instead,  try  to  iso- 
late the  luminescent  substance  whatever  it  might  be.  I 
was,  however,  unable  to  convince  him.  He  did  not  agree 
with  my  idea,  which  had  neither  theoretical  backing  nor 
experimental  support.  Because  of  the  disagreement  on 
the  experimental  procedure,  I  started  to  work  alone  at 
one  side  of  a  table  while,  on  the  other  side.  Dr.  Johnson 
and  his  assistant  Yo  Saiga  continued  their  efforts  to  extract 
a  luciferin  by  grinding  luminous  tissues  with  sand.  It  was 
an  awkward  situation. 


O    SHIMOMURA 


The  basic  principle  of  isolating  a  bioluminescent  sub- 
stance is  to  extract  it  from  the  tissue  under  conditions 
that  reversibly  inhibit  luminescence,  or  that  cause  a  se- 
lective inactivation,  consumption,  or  removal  of  a  com- 
ponent necessary  for  light  emission.  In  the  case  of  a  lu- 
ciferin-luciferase  system,  for  example,  the  luciferin  is  usu- 
ally extracted  with  methanol,  which  stops  luminescence 
by  inactivating  the  luciferase.  And  the  luciferase  can  be 
obtained  from  an  aqueous  extract  after  the  luciferin  has 
been  exhausted  by  several  minutes  of  spontaneous  lu- 
minescence reactions.  If  a  cofactor  is  involved  in  light 
emission,  its  removal  or  exhaustion  can  cause  a  reversible 
inhibition  of  luminescence,  as  in  the  case  of  the  firefly 
bioluminescence  system  that  requires  ATP  as  the  cofactor. 

In  the  case  of  the  jellyfish  Aequorea,  however,  the  pre- 
sumed lack  of  luciferin  and  luciferase  severely  limited  the 
range  of  techniques  usable  for  the  extraction  of  the  light- 
emitting  principle.  I  did  believe  that  jellyfish  luminescence 
requires  molecular  oxygen  like  all  other  known  biolu- 
minescence systems  (later  proved  to  be  incorrect!),  but 
we  had  no  other  information  about  the  luminescence  sys- 
tem or  cofactor  requirements.  In  an  effort  to  find  a  way 
to  extract  the  luminescent  principle,  I  tried  to  inhibit  the 
luminescence  of  the  squeezate  by  using  the  anaerobic 
conditions  that  were  created  by  vacuum  or  by  the  addition 
of  reducing  agents,  but  nothing  worked.  The  results  forced 
me  to  assume  that  the  jellyfish  system,  like  that  of  Cy- 
pridhui.  requires  a  very  low  oxygen  tension — a  level  less 
than  that  attainable  in  my  evacuated  container.  Further- 
more, all  of  the  known  enzymatic  cofactors,  such  as  ATP, 
FMN,  and  DPNH,  showed  no  effect  on  the  luminescence 
when  added.  Finally,  the  only  recourse  was  to  try  various 
chemicals  available  in  the  stockroom  of  the  Lab,  with  the 
hope  that  one  would  reversibly  inhibit  the  luminescence. 
This  was  clearly  an  approach  that  relied  entirely  on  good 
luck,  and  I  was  not  surprised  when  all  of  my  efforts  failed. 
I  was  conceptually  exhausted,  and  could  not  come  up 
with  one  further  idea. 

I  spent  the  next  several  days  soul-searching,  trying  to 
imagine  the  reaction  that  occurs  in  luminescing  jellyfish 
and  searching  for  a  way  to  extract  the  luminescent  prin- 
ciple. I  often  meditated  on  a  drifting  rowboat  under  the 
clear  summer  sky.  Friday  Harbor  in  summer,  at  that  time, 
was  quiet  and  peaceful,  differing  from  the  present-day 
scene  that  is  almost  saturated  with  busy  pleasure  boats 
and  noisy  seaplanes.  A  rowboat  always  has  the  right  of 
way  over  one  with  a  motor,  so  nobody  disturbed  my  drift- 
ing vessel;  even  large  ferries  saved  me  a  wide  berth.  Thus, 
meditation  afloat  was  safe,  but  if  I  fell  asleep  and  the  boat 
was  carried  away  by  the  tidal  current,  then  I  had  to  row 
for  a  long  time  to  get  back  to  the  Lab. 

One  afternoon  on  the  boat,  a  thought  suddenly  struck 
iiie — a  thought  so  simple  that  I  should  have  had  it  much 
sooner:  "Even  if  a  luciferin-luciferase  svstem  is  not  in- 


volved in  the  jellyfish  luminescence,  another  enzyme  or 
protein  is  very  probably  involved  directly  in  the  light- 
emitting  reaction.  If  so,  the  activity  of  this  enzyme  or 
protein  can  probably  be  altered  by  a  pH  change,  at  least 
to  some  extent.  Indeed,  there  might  be  a  certain  level  of 
acidity  at  which  an  enzyme  or  protein  could  be  reversibly 
inactivated." 

I  immediately  went  back  to  the  lab  and  made  a  squeez- 
ate. Then.  1  mixed  a  small  portion  of  the  squeezate  with 
acidified  water  containing  various  amounts  of  acetic  acid. 
The  resultant  mixtures  at  pH  6.0  and  pH  5.0  were  clearly 
luminous,  but  at  pH  4.0. 1  saw  no  luminescence.  I  filtered 
off  the  liquid  from  the  rest  of  squeezate  and  mixed  the 
solid  part,  containing  the  granules  of  light  organs,  with 
water  of  pH  4.0.  After  the  mixture  was  filtered,  the  filtrate, 
now  free  of  cells  and  debris,  was  nearly  dark,  but  it  re- 
gained its  luminescence  upon  neutralization  with  a  small 
amount  of  sodium  bicarbonate.  Indeed,  the  experiment 
showed  that  the  luminescent  substance  of  the  jellyfish  can 
be  extracted. 

But  my  real  surprise  came  in  the  next  moment,  when 
I  added  a  small  amount  of  seawater  to  the  solution  and 
saw  that  its  light  became  explosively  strong.  The  experi- 
ment showed  that  some  component  of  seawater  activates 
the  luminescence.  Because  the  composition  of  seawater 
is  known.  I  quickly  discovered  that  the  activator  is  Ca2+. 
The  discovery  of  Ca2+  as  the  activator  in  turn  suggested 
that  EDTA  should  serve  as  a  better  inhibitor  of  lumines- 
cence than  acidification  during  the  extraction  of  the  light- 
emitting  principle.  On  the  basis  of  these  data,  we  devised 
a  method  of  extracting  the  light-emitting  principle. 

With  a  workable  procedure  in  hand  for  extracting  the 
luminescent  principle,  our  next  task  was  to  catch  and 
process  as  many  jellyfish  as  possible.  We  would  collect 
jellyfish  from  6  to  8  AM,  then  after  a  quick  breakfast,  we 
would  cut  rings  from  the  jellyfish  until  noon.  We  devoted 
all  afternoon  to  the  extraction.  After  dinner,  we  again 
collected  jellyfish  from  6:30  to  8:30  PM,  and  the  catch  was 
kept  in  an  aquarium  to  be  processed  next  day  together 
with  the  catch  of  the  next  morning.  We  soon  found  that 
the  bottleneck  of  the  operation  was  the  step  of  cutting 
rings  with  scissors,  which  is  a  delicate  and  very  slow  pro- 
cess. Even  after  considerable  practice,  it  took  more  than 
1  minute  to  cut  one  jellyfish  ring,  and  3  hours  of  work 
by  four  persons  could  not  produce  more  than  500  rings. 
To  increase  productivity,  we  hired  several  high  school  girls, 
trained  them,  and  paid  them  2  cents  for  each  ring  they 
cut.  We  also  decided  to  buy  jellyfish  from  the  kids  of 
scientists  living  on  the  campus,  paying  a  penny  for  each 
jellyfish.  This  job  provided  dual  benefits,  the  fun  of  catch- 
ing jellyfish  and  of  earning  money.  I  remember  a  6-  or  7- 
year-old  girl  who  grossed  more  than  10  dollars  in  2  days 
(probably  with  parental  help).  Unfortunately,  just  when 
our  operation  was  in  full  swing,  the  jellyfish  suddenly 


A  SHORT  STORY  OF  AEQUORIN 


vanished  from  the  area.  Thus,  we  extracted  and  processed 
only  about  10,000  specimens  of  Acc/itorca  that  summer. 
We  returned  to  Princeton  with  the  jellyfish  extract 
packed  in  dry  ice,  and  then  began  to  purify  the  light- 
emitting  principle  from  the  extract  by  repeated  chroma- 
tography  on  various  kinds  of  large  columns.  It  was  a  long 
process,  and  the  utmost  care  was  required  to  prevent  the 
luminescence  activity  from  being  lost,  which  could  be 
brought  about  by  many  causes.  We  completed  the  puri- 
fication in  early  1962,  obtaining  about  5  mg  of  the  light- 
emitting  principle.  The  substance  was  found  to  be  a  pro- 
tein with  a  molecular  weight  about  20,000,  and  it  emitted 
light  when  a  trace  of  Ca~+  was  added — whether  in  the 
presence  or  absence  of  oxygen,  to  our  astonishment.  We 
named  the  protein  "aequorin"  after  the  genus  name  of 
the  jellyfish.  Aequorin  is  an  extraordinary  protein  con- 
taining a  large  amount  of  energy  that  can  be  released  when 
calcium  is  added:  thus  it  resembles  a  charged  battery  that 
releases  the  energy  when  short-circuited.  The  system  was 
so  unusual  that  some  biochemistry  professors  expressed 
their  skepticism.  After  30  years  of  discovery,  however,  the 
importance  of  aequorin  and  its  use  as  a  calcium  probe 
are  firmly  established  in  biochemistry  and  physiology.  The 
word  "aequorin"  now  can  be  found  in  various  common 
dictionaries. 

The  Town  Dock 

In  1962,  using  the  methods  that  had  worked  in  the 
previous  year,  we  obtained  an  additional  amount  of  ae- 
quorin and  began  to  study  various  aspects  of  the  molecule, 
including  its  application  in  the  measurement  of  calcium 
ions.  We  also  wanted  to  know  the  mechanism  of  the  lu- 
minescence reaction  and  the  structure  of  the  light-emitting 
chromophore.  But  our  efforts  to  achieve  these  goals  were 
soon  blocked  by  an  insuperable  difficulty.  When  various 
methods  were  used  to  break  down  the  molecules  of  ae- 
quorin, the  first  step  of  the  reaction  was  always  an  intra- 
molecular chemical  change;  so  it  was  impossible  to  isolate 
intact  chromophores.  We  therefore  decided  to  postpone 
further  study  on  the  light-emitting  mechanism. 

In  1967,  Ridgway  and  Ashley  reported  their  observa- 
tion, with  the  aid  of  microinjected  aequorin,  of  transient 
Ca2+  signals  in  single  muscle  fibers  of  the  barnacle.  It  was 
the  first  report  on  the  use  of  aequorin  in  studying  intra- 
cellular  calcium,  and  it  was  soon  followed  by  hundreds 
of  papers.  Because  the  importance  of  aequorin  was  now 
evident,  we  wanted  to  study  the  chemistry  of  the  lumi- 
nescence reaction.  Although  the  structure  of  the  native 
light-emitting  chromophore  seemed  intractable,  I  thought 
that  the  structure  of  the  chromophore  after  the  lumines- 
cence reaction  could  be  determined.  For  a  structural  study 
of  the  chromophore,  I  estimated  that  100-200  mg  of  pure 
aequorin  would  be  needed  in  a  single  experiment.  About 


50,000  jellyfish  (2.5  tons)  would  be  needed  to  produce 
this  amount  of  aequorin.  But  to  process  50,000  jellyfish 
in  one  summer,  we  would  have  to  collect  and  cut  at  least 
3000  of  the  animals  each  day,  allowing  for  days  of  bad 
weather  and  poor  fishing.  This  was  a  workload  that  could 
not  be  accomplished  by  collecting  jellyfish  at  the  lab  dock 
and  cutting  ring  with  scissors  at  a  rate  of  one  ring  per 
minute. 

We  resumed  the  jellyfish  operation  at  Friday  Harbor 
in  the  summer  of  1967,  not  anticipating  that  it  would 
continue  for  the  next  20  years.  To  collect  more  jellyfish, 
we  expanded  our  fishing  ground  beyond  the  lab  dock, 
adding  the  Chevron  dock  (a  small  commercial  pier),  the 
town  dock  (public  pier),  and  the  shipyard  (a  covered  boat 
storage),  and  we  used  a  car  to  move  around  and  to  trans- 
port the  buckets  of  jellyfish.  When  the  current  carried  the 
stream  of  jellyfish  far  beyond  the  docks,  we  also  used  row- 
boats  to  collect  jellyfish,  a  tricky  activity  that  occasionally 
caused  a  collector  to  fall  into  very  cold  seawater.  The 
Chevron  dock  was  our  favorite  place  during  the  first  2-3 
years,  because  there  was  a  part  of  it  where  a  large  number 
of  jellyfish  would  stack  up  on  an  early  morning  tide.  We 
had  to  be  careful,  however,  not  to  make  noise  that  might 
awaken  sleeping  people  on  the  boats. 

The  town  dock  was  very  small — almost  nonexistent — 
in  the  late  '60s;  but  then  it  was  rapidly  expanded.  By  1975, 
the  dock  had  been  extended  far  enough  into  the  bay  to 
intersect  with  the  main  jellyfish  stream,  and  it  then  became 
a  highly  favorable  spot  for  fishing.  Indeed,  the  town  dock 
with  its  large  sign  saying  "Port  of  Friday  Harbor"  became 
our  main  fishing  ground,  and  the  collection  became  much 
easier  than  before.  We  harvested  jellyfish  every  morning 
and  evening.  The  collectors  were  usually  my  wife,  our  son 
and  daughter,  a  couple  of  assistants,  and  me.  Dr.  and 
Mrs.  Johnson  also  helped  for  the  first  several  years.  Be- 
cause the  jellyfish  are  nearly  transparent  in  seawater.  they 
cannot  easily  be  seen  with  untrained  eyes.  Our  children 
were  only  3-4  years  old  when  they  began  collecting  jel- 
lyfish with  specially  made  short  nets;  they  had  become  as 
efficient  as  an  average  adult  by  the  age  of  8;  and  through 
high  school  they  continued  to  be  great  helpers  in  my  pro- 
ject. 

Before  beginning  a  collection,  we  filled  buckets  about 
half-full  with  seawater  and  placed  them  strategically  along 
the  edge  of  pier,  then  gathered  jellyfish  until  the  buckets 
were  completely  full.  When  a  dense  stream  of  animals 
was  passing  the  dock,  we  could  collect  at  a  rate  of  5-10 
jellyfish  per  minute.  When  all  the  buckets  were  filled,  we 
poured  off  some  water  to  about  80%  capacity,  and  then 
covered  each  bucket  with  a  plastic  bag  to  prevent  seawater 
from  spilling  during  transportation.  The  buckets — each 
crammed  with  about  100  jellyfish  in  very  little  water- 
were  then  packed  into  the  trunk  of  a  car  (which  could 
accommodate  12  buckets)  and  rushed  to  the  lab.  More 


O.  SHIMOMURA 


buckets  were  usually  transported  to  the  lab  on  a  Boston 
Whaler  by  one  of" the  assistants.  Once  at  the  lab,  and  before 
any  rings  were  cut,  the  jellyfish  were  kept  in  aquaria  to 
revive.  In  this  manner,  we  were  able  to  collect  an  average 
of  3000-4oUO  jellyfish  each  day  at  the  town  dock. 

The  town  dock  was  very  good  for  jellyfish  fishing,  but 
there  were  some  problems.  Often  we  found  too  many 
boats  at  the  dockside;  this  decreased  the  open  space  where 
we  could  collect  jellyfish.  When  the  leading  area,  located 
halfway  along  the  main  dock,  was  fully  occupied,  we  had 
to  carry  the  heavy  buckets  of  jellyfish  all  the  way  to  our 
car,  which  would  be  parked  more  than  200  yards  away. 
The  biggest  problem,  however,  was  that  there  were  too 
many  boat  people  who  asked  us  questions.  "What  are  you 
doing?"  "What  are  you  collecting?"  "How  do  you  use 
them?"  Almost  every  passerby  felt  obliged  to  ask  us  a 
question  while  we  were  busily  collecting.  Most  people  were 
satisfied  by  our  simple  reply:  "These  are  for  scientific  re- 
search." Some  people  persisted  until  they  had  received  a 
complete  explanation  of  our  research. 

I  cannot  forget  a  funny  exchange  that  took  place  one 
early  morning.  An  old  lady  poked  her  head  out  from  the 
window  of  a  small  boat,  looked  at  the  jellyfish  on  my  net. 
and  asked  me,  "How  do  you  cook  them?" 

I  answered,  "We  don't  cook  those  jellyfish." 

She  gazed  at  me  distastefully,  "Do  you  eat  them  raw?" 
and  her  head  disappeared. 

"No!  We  don't  eat  them!"  But  my  reply  was  too  late. 

The  Jellyfish  Factory 

Cutting  the  jellyfish  rings  with  scissors  was  impossibly 
slow;  we  could  not  produce  the  amount  of  aequorin  that 
we  needed  using  this  technique.  This  problem  was  solved 
primarily  by  Dr.  Johnson.  He  constructed  the  first  model 
of  a  jellyfish-cutting  machine  in  the  summer  of  1967;  it 
was  essentially  a  strip  of  wire  screen  that  worked  like  a 
grater.  An  average  jellyfish  has  about  100  light  organs  the 
size  of  poppy  seeds  located  under  the  edge  of  its  umbrella. 
By  sliding  the  jellyfish  over  the  screen,  we  hoped  that  the 
light  organs  would  be  scraped  off  the  body  and  collected 
in  a  tray  under  the  screen.  We  found,  however,  that  the 
light  organs  were  not  scraped  off  by  the  wire  screen.  The 
next  version  of  the  cutting  machine  had  a  strip  of  coarse 
sandpaper  over  which  seawater  flowed  slowly;  the  sand- 
paper was  connected  to  one  end  of  the  first  version.  When 
jellyfish  were  slid  down — first  over  the  sandpaper,  then 
the  screen — most  of  the  light  organs  were  indeed  scraped 
off.  But  the  material  accumulated  in  the  tray  contained 
an  excessive  amount  of  slime,  and  the  quality  of  the  ma- 
terial was  much  poorer  than  that  of  the  hand-cut  rings. 
Thus,  the  manufacture  of  a  machine  based  on  the  prin- 
ciple of  a  grater  was  abandoned. 

Dr.  Johnson  next  purchased  two  circular  meat-slicing 
blades  (10"  diameter)  .it  a  local  hardware  store  and  began 


to  build  a  cutting  machine;  this  project  took  the  next  two 
summers  to  complete.  The  basic  plan  was  to  install  a  meat- 
slicing  blade  perpendicular  to  a  black  Lucite  board,  and 
with  the  blade  slowly  rotating,  cut  the  ring  offthe jellyfish. 
The  motor  from  a  small  laboratory  shaker  was  used  to 
rotate  the  blade.  The  jellyfish  were  rotated  with  a  hand 
tool  called  a  "peg."  a  small  disk  with  several  short  nails 
on  one  side  and  a  2-inch-long,  stick-shaped  handle  at- 
tached in  the  center  on  the  other  side.  A  jellyfish  on  the 
Lucite  board  was  grasped  by  the  nails  of  the  disk  and 
rotated  by  the  stick,  which  was  held  between  the  index 
finger  and  thumb.  The  setup  worked,  at  least  in  principle. 

A  number  of  improvements  were  made  over  the  next 
two  years.  A  razor  blade  was  installed  at  the  edge  of  the 
Lucite  board;  the  razor  blade  and  the  rotating  circular 
blade  were  in  contact  each  other  on  their  flat  sides  and 
the  jellyfish  was  cut  at  the  intersection  of  the  two  cutting 
edges.  It  made  cutting  so  sharp  and  smooth  that  the  jel- 
lyfish might  not  even  feel  that  their  rings  were  being  cut 
off.  To  make  the  rotation  of  jellyfish  easy,  a  seawater  outlet 
was  installed  near  the  center  of  the  board  to  lubricate  its 
surface.  An  ice  bath  was  installed  to  cool  the  ring  reservoir; 
this  prevented  a  loss  of  activity  from  the  rings  and  also 
served  as  a  preparation  for  the  extraction  process.  In  the 
summer  of  1969,  the  quality  of  the  machine-cut  rings 
finally  surpassed  that  of  the  hand-cut  ones.  We  therefore 
set  up  two  cutting  machines  and  used  them,  thereafter, 
to  cut  all  of  the  jellyfish. 

With  machines  that  could  cut  rings  at  10  times  the 
speed  of  a  hand-cutter,  and  with  a  sufficient  supply  of 
jellyfish,  our  mode  of  operation  had  to  be  changed.  We 
needed  a  large  working  space,  and  we  also  did  not  want 
to  disturb  other  researchers  with  our  messy,  smelly,  and 
noisy  experimental  processes.  Fortunately,  we  were  as- 
signed to  use  the  Gear  Locker,  a  small,  isolated  building 
that  had  been  used  for  storage  in  the  past.  Two  large  tanks 
installed  outside  the  building  were  used  for  temporary 
storage  of  collected  jellyfish. 

Ring  cutting  was  probably  the  most  important  step  in 
determining  the  quality  and  yield  of  purified  aequorin. 
Cutting  too  thick  would  increase  the  amount  of  impurities. 
Cutting  too  thin  would  decrease  the  yield  because  some 
of  the  light  organs  were  cut  through  and  destroyed. 
Therefore,  we  always  assigned  the  best  workers  to  do  this 
job.  Of  the  many  excellent  helpers  we  had  in  our  jellyfish 
operation,  I  remember  particularly  three  girls  who  worked 
for  many  summers  and  cut  rings  extremely  skillfully  and 
fast:  Debby  Nash,  Liz  Illg,  and  Laura  Norris;  the  first  was 
from  the  town  and  the  other  two  were  daughters  of  biology 
professors. 

Our  jellyfish  cutting  usually  began  at  1 1  AM.  A  counting 
person  would  put  80  jellyfish  into  each  bucket,  already 
half-full  of  seawater,  and  would  then  take  the  buckets  to 
the  cutters.  Two  cutters  cut  the  jellyfish  with  the  machines 


A  SHORT  STORY  OF  AEQUORIN 


that  were  installed  side-by-side:  the  cutter  would  place  a 
jellyfish  onto  the  cutting  board,  quickly  rotate  it  with  a 
peg  to  spread  out  the  edge  of  the  umbrella  where  the  light 
organs  are  located,  and  then — pushing  the  jellyfish  to  the 
cutting  blade  while  simultaneously  rotating  the  jellyfish 
quickly — cut  off  the  rings,  all  in  less  than  5  seconds.  The 
rings  would  fall  automatically  into  the  ice-cold  reservoir, 
and  the  ringless  jellyfish  body  was  slid  down  into  a  waste 
bucket.  These  buckets,  each  filled  with  about  200  spent 
jellyfish,  were  carried  to  the  nearest  seashore  about  50 
yard  away,  called  by  us  "jellyfish  cliff,"  and  dumped  onto 
the  rocks  below.  The  heaps  of  jellyfish  bodies,  several 
thousands  of  them,  were  carried  away  by  the  next  high 
tide. 

The  process  of  extracting  aequorin  from  rings  began  at 
2  PM;  it  was  carried  out  by  a  team  of  two  persons.  The 
extraction  was  done  in  batches  of  480  rings  (i.e..  six  buck- 
ets). The  first  person  would  drain  the  rings  on  a  nylon 
gauze,  then  mix  the  drained  rings  with  a  cold  EDTA  so- 
lution saturated  with  ammonium  sulfate.  The  rings  shrank 
quickly  and  were  also  desensitized  by  the  salt.  They  were 
cut  with  scissors  into  pieces  1-2  inches  long,  then  stirred 
with  a  cake  mixer  for  10  minutes  to  dislodge  the  granular 
light  organs  from  the  tissue.  The  mixture  was  squeezed 
through  a  nylon  gauze  to  remove  the  shrunken  ring  tissue, 
and  then  the  turbid  liquid  obtained  was  filtered  on  a 
Buchner  funnel  using  some  Celite.  The  filter  cake,  con- 
taining the  light  organs,  was  given  to  the  second  person, 
who  was  responsible  for  the  rest  of  the  extraction  process. 
The  second  person  put  the  filter  cake  into  a  2-liter  flask 
containing  cold  EDTA  solution  (1  liter),  then  shook  the 
flask  vigorously  to  extract  aequorin  from  the  light  organs 
into  the  EDTA  solution.  Finally,  the  mixture  was  filtered 
through  a  large  Buchner  funnel,  and  the  filtrate  containing 
aequorin  was  saturated  with  ammonium  sulfate  to  pre- 
cipitate the  protein.  The  first  person  in  the  team  would 
start  a  new  batch  of  rings  every  20  minutes,  and  the  second 
person's  work  would  also  take  20  minutes.  Thus,  3360 
jellyfish  rings  could  be  extracted  in  about  2  hours  and  40 
minutes. 

The  precipitates  of  crude  aequorin  were  purified  at  our 
laboratory  in  Princeton.  The  purification  was  done  in 
several  steps  of  column  chromatography.  mainly  by 
Sephadex  gel  filtration  and  DEAE-cellulose  chromatog- 
raphy. all  at  0°C.  It  was  indeed  a  lengthy,  time-consuming 
process,  notwithstanding  the  fact  that  aequorin  should  be 


purified  as  quickly  as  possible  because  it  is  constantly  de- 
composing through  spontaneous  weak  luminescence,  even 
in  the  presence  of  a  high  concentration  of  EDTA.  To 
purify  an  extract  of  50,000  jellyfish,  which  contains  a  large 
amount  of  total  protein,  chromatography  had  to  be  re- 
peated 30  times  for  only  the  first  gel  filtration  step,  and 
the  total  number  of  chromatography  runs  required  for 
complete  purification  was  more  than  60.  An  extract  of 
50.000  jellyfish  yielded  only  150-200  mg  of  purified  ae- 
quorin in  the  early  '70s,  but  as  the  techniques  improved, 
the  yield  gradually  increased,  exceeding  500  mg  by  1980. 
Since  1975,  all  of  the  steps  in  the  purification  have  been 
done  by  my  wife,  Akemi,  who  is  highly  knowledgeable  in 
handling  aequorin. 

The  purified  aequorin  was  used  in  various  studies  of 
luminescence  in  our  laboratory.  Thus,  the  chemical 
structure  of  the  light-emitter  was  determined  in  1973. 
Then  the  structure  of  the  aequorin  chromophore  "coe- 
lenterazine"  was  elucidated  and  the  regeneration  of  spent 
aequorin  into  active  aequorin  was  accomplished,  both  in 
1975.  The  molecular  characterization  of  various  aequorin 
isoforms  was  reported  in  1986.  The  improved  forms  of 
aequorin — "semisynthetic  aequorins"  with  widely  differ- 
ent calcium  sensitivities — were  produced  in  1988-1989. 
Purified  aequorin  has  also  been  supplied  to  hundreds  of 
cell  biologists  and  physiologists  who  study  intracellular 
calcium,  leading  to  many  important  findings  about  intra- 
cellular calcium.  Aequorin  was  cloned  in  1985  by  two 
groups  simultaneously,  one  in  Georgia  and  another  in 
Japan.  With  the  recent  progress  in  molecular  genetics, 
studies  involving  recombinant  aequorin  are  now  flou- 
rishing. 

Acknowledgments 

Our  work  on  aequorin  was  initiated  by  Dr.  Frank  H. 
Johnson,  and  developed  with  support  and  encouragement 
from  many  individuals.  I  thank  all  the  people  who  helped 
directly  or  indirectly  with  this  project.  The  work  was  made 
possible  by  the  excellent  facilities  of  the  Friday  Harbor 
Laboratories,  University  of  Washington,  and  of  Princeton 
University,  and  was  financially  supported  by  research 
grants  from  the  National  Science  Foundation  and  the  Na- 
tional Institutes  of  Health. 

Literature  Cited 

Ridgway.  E.  B.,  and  C.  C.  Ashley.  1967.     Calcium  transients  in  single 
muscle  fibers.  Biochem.  Biophyx.  Res  Commitn.  29:  229-234. 


Reference:  Binl  Bull  189:  6-12.  (August,  1995) 


Fine  Structure  of  Spermatozoa  of  the  Hagfish 
Eptatretus  burgeri  (Agnatha)1 


SACHIKO  MORISAWA 

Biological  Laboratory,  St.  Marianna  University,  School  of  Medicine, 
2-16-1  Si/gao.  Miyamae.  Kawasaki  216.  Japan 


Abstract.  Live  motile  spermatozoa  of  the  hagfish  £77- 
t  at  ret  us  burgeri  were  obtained  for  the  first  time,  and  their 
fine  structure  was  examined.  The  spermatozoon  is  char- 
acterized by  an  extremely  long  midpiece.  Two  of  the  four 
midpiece  mitochondria  are  extensively  elongated  and  ex- 
tend through  almost  the  entire  length  of  the  tail.  The  ac- 
rosome  contains  electron-dense  and  less  dense  materials 
in  two  different  compartments.  Amorphous  subacrosomal 
material  lies  between  the  acrosome  and  the  nucleus.  No 
distinct  perforatorium  rod  or  filamentous  structure  was 
observed  within  the  subacrosomal  material.  Two  cen- 
trioles  lie  almost  end  to  end  in  the  nuclear  fossa  near  the 
posterior  end  of  the  nucleus.  The  structure  of  the  acro- 
somal  complex  in  the  hagfish,  which  is  quite  different 
from  that  in  the  lamprey,  was  compared  to  that  of  other 
chordates  with  respect  to  its  function  in  sperm-egg  inter- 
action and  phylogeny. 

Introduction 

The  reproductive  life  of  the  lamprey  has  been  studied 
extensively  (Kille,  1960;  Nicander  et  a/..  1968),  but  little 
is  known  about  reproduction,  especially  fertilization,  in 
hagfish  (Dean,  1899;  Walvig,  l963;Gorbman,  1983).  The 
structures  of  the  testes  and  spermatozoa  of  hagfish  have 
been  described  in  Myxine glutinosa,  Bdellostorna  burgeri, 
and  B.  stouti  (Walvig,  1963);  M.  glutinosa  (Nicander, 
1970);  E.  burgeri  (Patzner,  1977,  1982);  Paramyxine 
atann  (Patzner,  1982);  and  E.  stouti  (Gorbman,  1990). 
Electron  micrographical  studies  on  the  formation  and 
structure  of  hagfish  spermatozoa  in  Myxine  circifrons,  M. 
sp.,  Eptatretus  stoutii,  E.  deat'i,  and  E.  sp.  were  performed 


1  This  article  is  dedicated  to  the  late  professor  Dr.  J.  C.  Dan  who 
introduced  the  author  to  biological  science. 

Received  22  February  1194;  accepted  5  June  -995. 


by  Jespersen  (1975).  Because  of  difficulties  in  catching 
mature  hagfish  males  alive  (Walvig,  1963;  Jespersen,  1975; 
Patzner,  1982).  there  had  been  few  studies  describing  live 
and  motile  spermatozoa  (Patzner,  1982). 

In  the  sea  near  the  Misaki  Marine  Biological  Station 
in  the  Kanagawa  Prefecture  of  Japan,  the  hagfish  species 
Eptatretus  burgeri  migrates  from  depths  of  50-100  m  to 
the  shore  (10-12  m  deep)  between  November  and  June 
(Kobayashi  el  ill..  1972);  in  contrast,  most  other  hagfish 
species  inhabit  the  deeper  sea  throughout  the  year  (Adam 
and  Strahan,  1963;  Jespersen,  \91 5).  Eptatretus  burgeri 
is  thought  to  breed  in  October,  while  living  in  deep  water 
(Kobayashi  et  al..  1972;  Patzner,  1977,  1978). 

By  catching  hagfish  from  the  shore  and  keeping  them 
in  an  aquarium  (Fernholm,  1975:  Ooka-Souda  et  al.. 
1985),  we  were  able  to  obtain  males  with  mature  sper- 
matozoa and  to  investigate  for  the  first  time  the  ultra- 
structural  details  of  these  motile  spermatozoa.  Because 
the  phylogeny,  as  well  as  the  fertilization,  of  living  ag- 
nathans  (hagfish  and  lampreys)  has  attracted  much  atten- 
tion from  biologists  (Brodal  and  Fa'nge,  1963;  Hardisty, 
1979),  we  compared  these  sperm  to  those  of  other  chor- 
dates. 

Materials  and  Methods 

Males  of  the  hagfish  Eptatretus  burgeri.  45-60  cm  in 
body  length,  were  caught  in  July  in  Sagami  Bay  near  the 
Misaki  Marine  Biological  Station  in  Kanagawa  Prefecture, 
Japan.  They  were  kept  in  a  seawater  tank  ( 1 5°C)  without 
food  under  controlled  light  (light  0700-1900)  until  they 
were  used  for  experiments  between  September  and  De- 
cember in  the  same  year.  After  an  animal  was  anesthesized 
with  0.5%  3-aminobenzoic  acid  ethyl  ester  (MS222, 
Sankyo  Pharmaceut.,  Tokyo),  its  abdomen  was  opened 
to  remove  the  testis.  Actively  motile  spermatozoa  were 


FINE  STRUCTURE  OF  HAGF1SH  SPERM 


obtained  when  pieces  of  the  posterior  portion  of  the  testis 
were  immersed  in  seawater. 

For  transmission  electron  microscopy  (TEM),  small 
pieces  of  testis  with  mature  spermatozoa  were  fixed  for  1 
h  with  2%  glutaraldehyde  in  0. 1  M  phosphate  buffer  (pH 
7.4),  and  postfixed  with  1%  osmium  tetroxide  in  the  same 
butter  for  1  h.  Dehydration  in  a  graded  alcohol  series, 
followed  by  propylene  oxide,  and  infiltration  with  Epon 
were  performed  using  an  automatic  electron  microscopy 
processor  (REM-20B.  Sakura,  Tokyo).  All  procedures 
were  done  at  room  temperature.  Thin  sections  were  ob- 
tained using  diamond  knives,  stained  with  uranyl  acetate 
and  lead  citrate,  and  then  examined  with  an  electron  mi- 
croscope (JEOL100  or  JEM-1200EX,  JEOL  Ltd..  Tokyo). 

For  scanning  electron  microscopy  (SEM).  a  testis  was 
minced  and  spermatozoa  were  released  onto  a  sheet  of 
filter  paper.  Sperm  on  the  paper  were  fixed  in  2%  glutar- 
aldehyde in  70%  seawater,  followed  by  postfixation  with 
2%  osmium  tetroxide  in  2.5%  NaHCO,.  Samples  were 
critical-point  dried  and  examined  using  a  scanning  elec- 
tron microscope  (US4,  JEOL  Ltd.,  Tokyo). 


Fine  structure  of  spermatozoa 

Figure  1  is  a  scanning  electron  micrograph  of  an  E. 
hurgeri  spermatozoon.  The  head  is  8-10  ^m  in  length 
and  about  0.5  and  1.2  Mm  wide  at  the  anterior  and  pos- 
terior regions,  respectively.  The  acrosome  is  at  the  anterior 
end  of  the  sperm  head  (Figs.  2  and  3a).  The  outer  acro- 
somal  membrane  is  in  close  approximation  with  the  over- 
lying plasma  membrane  at  the  posterior  1/2-2/3  of  the 
acrosome  (Fig.  3a).  The  inner  acrosomal  membrane  cov- 
ers a  conspicuous  subacrosomal  material  into  which  the 
apex  of  the  nucleus  projects,  and  the  double  structure  of 
the  nuclear  membranes  is  indistinct  in  this  area.  The  ac- 
rosomal contents  are  tightly  packed  in  the  anterior  region 
of  the  acrosome  (Fig.  3b),  but  somewhat  loosely  packed 


H 


Results 

Macroscopic  aspects  of  testis 

Four  male  hagfish  kept  in  the  laboratory  had  mature 
testes  between  late  September  and  early  October  in  1992. 
The  testis  appeared  as  a  single  nodular,  spiral  structure, 
longitudinally  located  along  the  right  side  of  the  mesentery 
as  described  in  E.  hurgeri  (Patzner.  1982)  and  in  other 
hagfish  (Walvig,  1963;Gorbman.  1990).  Toward  Decem- 
ber, after  the  breeding  season,  the  testis  became  thinner. 


Motilitv  and  light  microscopic  aspects  of  sperm 

When  a  small  piece  of  mature  testis  was  immersed  in 
seawater.  many  spermatozoa  were  released  from  the  testis 
and  exhibited  active  forward  motility  for  about  10  min. 
Motile  spermatozoa  could  be  obtained  during  late  October 
in  some  fish.  The  number  of  spermatozoa  with  normal 
ultrastructure  decreased  in  November.  Thus,  the  hagfish 
kept  in  the  laboratory  had  mature  spermatozoa  at  about 
the  same  time  as  their  natural  breeding  period. 

The  sperm  head  and  tail  could  be  identified  with  a  light 
microscope.  They  were  about  10  nm  and  35-40  ^m  in 
length,  respectively.  These  were  almost  the  same  as  those 
observed  by  Walvig  ( 1963).  After  active  swimming  in  sea- 
water  for  about  5  min.  some  spermatozoa  stuck  to  the 
glass  surface  with  the  tip  of  their  heads,  rotating  their  tails 
freely.  Some  spermatozoa  had  shortened  tails  with  a  knot 
near  the  end  of  the  tail.  Heads  of  some  spermatozoa  were 
bent  backward,  and  they  swam  with  their  heads  pointing 
backward. 


Figure  I.  A  scanning  electron  micrograph  of  an  Eptalrelus  burgeri 
spermatozo'  >n.  H.  head  (acrosomal  and  nuclear  regions);  T,  tail  (midpiece 
and  end  puce).  Bar  =  1  ^m. 


S.  MORISAWA 


tochondria  or  with  only  one  mitochondrion  are  observed 
only  in  sections  near  the  posterior  end  of  the  tail  (Fig.  4f). 
The  unique  feature  of  the  E.  burgeri  spermatozoon  is 
the  two  extremely  elongated  mitochondria  that  run  par- 
allel with  the  axoneme  throughout  nearly  the  entire  length 
of  the  tail.  Figure  5  diagrams  the  structure  of  various  re- 
gions of  the  E.  burgeri  spermatozoon. 

Discussion 

Spermatozoa  of  species  that  exhibit  external  fertiliza- 
tion, including  common  fishes,  usually  have  a  few  mi- 
tochondria in  the  short  midpiece  surrounding  the  cen- 
trioles  and  the  9  +  2  axoneme  without  accessory  structures 
in  the  flagellum.  The  sperm  midpiece  in  many  species 
with  internal  fertilization  [e.g..  mammals  (Phillips,  1977) 
and  viviparous  teleosts  (Grier,  1975)]  has  a  long  mito- 
chondrial  sheath  or  a  long  cytoplasmic  sleeve  that  contains 
many  separate  mitochondria.  Hagfish  sperm  have  four 
mitochondria,  but  two  of  them  extend  nearly  the  entire 
length  of  the  tail,  forming  a  long  midpiece,  as  in  sper- 
matozoa of  species  with  internal  fertilization. 

The  acrosomal  vesicle  of  protochordate  spermatozoa 
is  either  ovoid,  as  in  the  urochordate  Oikopk-nni  (Holland 
ct  a/.,  1988),  or  cap-shaped,  as  in  the  cephalochordate 
Bramhiostonui  (Baccetti  el  al,  1972),  and  a  distinct  ac- 


Figure  2.     An  electron  micrograph  of  the  longitudinal  section  ol  the 
sperm  head.  A,  acrosomal  region;  N.  nucleus.  Bar  =  I  pm. 


in  the  posterior  region  (Fig.  3c,d).  The  subacrosomal  ma- 
terial between  the  acrosome  and  the  nucleus  is  almost 
homogeneous  in  electron  density.  Neither  a  distinct  per- 
foratorium  rod  nor  filamentous  structures  are  detected 
within  the  subacrosomal  material. 

The  nucleus  increases  its  thickness  posteriorly  (Fig.  2). 
In  the  posterior  lateral  surface  of  the  sperm  head  is  a  small 
fossa  in  which  two  centrioles  are  located  almost  end  to 
end  (Fig.  4a).  The  axoneme  has  the  ordinary  9  +  2  ar- 
rangement of  doublets  (Fig.  4e,f). 

Four  mitochondria  encircle  the  axoneme  at  the  base 
of  the  flagellum  (Fig.  4c),  each  arranged  longitudinally 
(Fig.  4a).  Two  of  them  are  extensively  elongated  and  ex- 
tend almost  along  the  entire  length  f  the  axoneme  (Fig. 
4d,e).  Most  cross  sections  of  the  mic  liece  exhibit  a  9  + 
2  arrangement  of  axonemal  doublets  lanked  by  two  mi- 
tochondria ('Fig.  4e).  Axonemes  with  >ut  associated  mi- 


Figure  3.  Longitudinal  (a)  and  cross  sections  (h-d)  of  the  acrosomal 
region.  Labels  -h.  -c.  and  -d  in  figure  (a)  indicate  the  levels  of  sections 
shown  in  b.  c.  and  d,  respectively.  Acrosomal  vesicle  (AV)  and  the  un- 
derlying subacrosomal  material  (SM)  cover  the  anterior  end  of  the  nucleus 
(N).  IM.  inner  acrosomal  membrane:  NE,  nuclear  envelope;  OM,  outer 
acrosomal  membrane;  PM,  plasma  membrane.  Each  bar  =  200  nm. 


FINE  STRUCTURE  OF  HAGF1SH  SPhRM 


Figure  4.  Longitudinal  and  cross  sections  of  various  regions  of  the  tail,  (a)  The  base  of  a  flagellum.  The 
proximal  centriole  (PC)  and  distal  centriole  (DC)  lie  almost  longitudinally.  Elongated  mitochondria  (M)  are 
arranged  along  the  axoneme  (AX),  (b)  A  cross  section  through  a  centriole.  (c)  An  oblique  section  through 
the  base  of  a  flagellum.  Four  mitochondria  encircle  the  axoneme.  (d)  A  longitudinal  section  of  the  winding 
and  twisting  flagellum.  Two  long  mitochondria  flank  the  axoneme.  (e)  Cross  sections  of  three  flagellae.  Two 
mitochondria  flank  the  axoneme  in  the  plane  of  two  central  singlets,  (f)  Cross  sections  of  flagellae  through 
near  endpiece  (left)  and  endpiece  (right).  Note  that  the  axoneme  without  mitochondria  has  incomplete 
doublets.  Bar  =  250  nm  (in  a,  b,  e,  f)  and  500  nm  (in  c,  d). 


rosomal  process  is  produced  dc  novo  following  the  acro- 
somal  exocytosis  at  the  anterior  end  of  the  sperm  (Holland 
ft  a/.,  1988).  Spermatozoa  of  vertebrates  such  as  amphib- 
ians ( Yoshizaki  and  Katagiri,  1982;  Fig.  6c),  reptiles  (Fu- 
rieri,  1970;  Fig.  6d),  birds  (Okamura  and  Nishiyama, 
1978;  Fig.  6e),  and  mammals  (Yanagimachi  and  Noda, 
1970;  Fig.  60  have  a  cap-shaped  acrosomal  vesicle  and 
underlying  subacrosomal  material,  which  cover  the  an- 


terior portion  of  the  nucleus.  The  exocytosis  of  the  ac- 
rosomal vesicle  occurs  at  several  points,  and  a  new  ac- 
rosomal process  does  not  protrude  (Yanagimachi  and 
Usui,  1974;  Okamura  and  Nishiyama,  1978;  Yoshizaki 
and  Katagiri.  1982). 

A  variety  of  acrosomal  structures  are  found  in  rish 
spermatozoa.  The  lamprey,  which  is  a  cyclostome,  has 
spermatozoa  that  carry  a  spherical  acrosomal  vesicle  at 


10 


S.   MORISAWA 


H 
10|jm 


MP 


EP 


AX 


Figure  5.  Schematic  drawings  of  the  Eplalre/ns  btirgeri  spermatozoon,  (a)  Whole  view  at  low  magnifi- 
cation; a,  surface  view;  a',  side  view  of  the  head,  (b)  Head  and  regions,  (c)  Acrosfjmal  region  indicated  by  a 
rectangle  in  the  figure  b.  (d-g)  A  longitudinal  and  cross  section  of  the  flagellum.  AV,  acrosomal  vesicle;  AX, 
axoneme;  DC,  distal  centriole;  EP,  endpiece;  H,  head;  M.  mitochondria;  MP,  midpiece;  N,  nucleus;  NE. 
nuclear  envelope;  PC,  proximal  centriole;  PM,  plasma  membrane;  SM,  subacrosomal  material. 


the  anterior  end  of  their  heads,  subacrosomal  material 
between  the  acrosome  and  the  nucleus,  and  a  long  per- 
foratorium  rod  through  the  nucleus  (Follenius,  1 965; 
Stanley,  1967;  Nicander  and  Sjoden,  1971;  Jaana  and 
Yamamoto,  1981)  (see  Fig.  6b).  When  the  sperm  reach 
the  outer  chorion  of  the  egg  during  fertilization  (Nicander 
and  Sjoden,  1971),  or  when  they  are  exposed  to  fixatives 
(Jaana  and  Yamamoto,  1981 ),  a  long  acrosomal  process 
is  formed.  Spermatozoa  of  the  elasmobranch  Squalus 
sitckleyi.  a  species  that  has  internal  fertilization,  have  a 
cap-shaped  acrosomal  vesicle  and  a  subacrosomal  rod 
(Stanley,  1971).  Spermatozoa  of  the  sturgeon,  Acipenser 
tninMiiontainis.  which  have  a  scalloped  and  cap-shaped 
acrosomal  vesicle  and  filamentous  structure  in  the  sub- 
acrosomal material  and  in  the  canals  through  the  nucleus, 
form  an  acrosomal  process  upon  the  acrosome  reaction 
(Cherr  and  Clark,  1984).  In  Holostei  (Afzelius,  1978)  and 
Teleostei  (Mattei,  1970),  spermatozoa  lack  an  acrosome. 
In  hagfish,  the  act  :>somal  vesicle  of  thi  spermatozoa  covers 
the  protrusion  ol  the  nucleus  with  i  -iderlying  subacro- 
somal material  (Figs.  3,  6).  In  our  ^  reliminary  experi- 
ments, the  acrosomal  exocytosis  of  the  ^ptairems  hurgeri 


sperm  occurred  not  only  at  the  apical  point  of  the  sperm 
head  but  at  several  points,  and  was  not  followed  by  con- 
spicuous formation  of  a  long  process.  Such  features  are 
common  in  the  spermatozoa  of  higher  animals.  The  role 
of  the  acrosomal  complex  of  the  hagfish  spermatozoa  re- 
mains to  be  studied. 

The  acrosome  reaction  occurs  inside  or  on  the  surface 
of  the  egg  envelope  to  allow  sperm  penetration.  In  the 
case  of  external  fertilization  in  teleosts,  spermatozoa  reach 
the  egg  plasma  membrane  through  a  narrow  micropyle 
that  has  been  perforated  in  the  chorion;  the  sperm  lack 
an  acrosome.  Lamprey  eggs  have  a  two-layered  chorion 
( Afzelius  eta/.,  1968)  that  has  no  micropyle  (Kille,  1960); 
the  sperm  penetrate  the  chorion  with  the  acrosome  re- 
action (Nicander  and  Sjoden,  1971 ).  Sturgeon  eggs  have 
numerous  micropyles,  and  the  sperm  form  an  acrosomal 
process  (Cherr  and  Clark,  1984).  In  hagfish  eggs,  one  mi- 
cropyle with  an  outer  opening  diameter  of  4.2  ^m,  4.7 
/urn,  or  4  /urn  in  Myxine glutinosa  (Kosmath  el  al..  198 1 ). 
Eptatretus  bnrgeri  (Kosmath  el  a/..  1981),  or  E.  stout  i 
(Koch  et  al.,  1993),  respectively,  is  perforated  at  the  animal 
pole  through  the  thick  and  hard  chorion.  Inasmuch  as 


FINE  STRUCTURE  OF  HAGFISH  SPERM 


I  I 


Figure  6.  Comparison  of  the  acrosomal  region  of  the  spermatozoa  of  various  vertebrates,  (a)  Agnathans 
(Eplalretus  hurgeri);  (b)  agnathans  (Liinipclru  planm):  (c)  amphibians  (Bulb  hnln  nipon/ciis);  (d)  reptiles 
(Chelonia  sp.):  (e)  birds  (Gallus  gallits):  (l'|  mammals  (.\fesocnci'tu\  uiirtiiux).  b-f  were  redrawn  from  Stanley 
(1967).  Voshizaki  and  Katagiri  (1982).  Furieri  (1970),  Okamura  and  Nishiyama  (1978).  Yanagimachi  and 
Noda  (1970),  respectively.  Acrosomal  vesicle,  spotted;  perforatorium  (subzonal  material),  shaded:  nucleus. 
*;  inner  acrosomal  cap  with  granular  substance  (Furieri,  1970),  triangles. 


the  head  of  the  hagfish  spermatozoon  is  2.5-3  iim  wide 
in  M  g/utinosa  (Walvig.  1963),  about  1.2  ^m  wide  (see 
Results)  and  1.5  urn  wide  (Walvig,  1963)  in  /:.  hnrgeri, 
and  about  1.5  nm  wide  (Jespersen.  1975)  and  2.5-3  ,um 
wide  (Koch  ct  al..  1993)  in  E.  stout i,  the  spermatozoa 
could  access  the  egg  surface  directly.  Judging  by  the  fea- 
tures of  the  anterior  portion  of  the  spermatozoa  and  the 
micropyle.  the  relationship  between  sperm  and  egg  in 
hagfish  may  differ  from  those  in  lampreys  and  teleosts. 
The  exocytosis  of  the  acrosomal  vesicle  at  several  points 
in  hagfish  (our  preliminary  observation)  as  seen  in  higher 
vertebrates  may  be  different  from  the  acrosome  reaction 
in  sturgeon.  The  relationship  between  the  existence  of  egg 
micropyle  and  no  acrosome  in  spermatozoa  has  been 
considered  in  teleosts  having  external  fertilization  (Bac- 
cetti  and  Afzelius.  1976).  In  hagfish  and  sturgeon,  how- 
ever, gametes  have  both  structures,  although  the  mode  of 
fertilization  in  hagfish  is  still  unknown.  The  structure  of 
the  micropyle  in  hagfish  and  sturgeon  should  be  studied 
in  detail  to  understand  the  role  of  these  structures  in  fer- 
tilization. 

The  hagfish  has  a  phylogenetically  interesting  position 
in  the  Chordata.  Analysis  of  sperm  function — for  exam- 
ple, the  interaction  between  sperm  and  egg  during  fertil- 
ization and  the  acrosome  reaction — would  contribute  to 


an  understanding  of  both  the  mode  and  the  phylogenical 
aspects  of  fertilization  in  hagfish. 

Acknowledgments 

The  author  is  indebted  to  Prof.  R.  Yanagimachi,  Uni- 
versity of  Hawaii,  for  revising  the  original  manuscript. 
She  thanks  Prof.  M.  Morisawa,  Misaki  Marine  Biological 
Station.  University  of  Tokyo,  Dr.  S.  Ooka-Souda,  Atomi 
College,  and  Dr.  N.  Usui.  Teikyo  University,  for  their 
valuable  advice.  She  also  thanks  Dr.  H.  Kabasawa.  Keikyu 
Aburatubo  Marine  Park,  and  the  staff  of  the  MMBS  for 
providing  the  material,  and  the  staff  of  the  Central  Facility 
of  Electron  Microscopy.  St.  Marianna  University,  for 
technical  assistance.  This  work  was  supported  in  part  by 
a  Grant-in-Aid  for  Scientific  Research  from  the  Ministry 
of  Education.  Science  and  Culture.  No.  06839024. 

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Reference:  Bio/  Bull  189:  13-21.  (August.  1995) 


Oxidase  Activity  Associated  with  the  Elevation  of  the 
Penaeoid  Shrimp  Hatching  Envelope 

PATRICIA  S.  GLAS1.  JEFFREY  D.  GREEN2,  AND  JOHN  W.  LYNN1  * 

1  Louisiana  State  University,  Department  of  Zoology  and  Physiology,  Baton  Rouge,  Louisiana  70803: 

and 2  Louisiana  Stale  University — Medical  School.  Department  of  Anatomy. 

New  Orleans,  Louisiana  70112 


Abstract.  When  penaeoid  shrimp  spawn  into  seawater. 
the  ova  elevate  a  hatching  envelope  (HE)  within  30- 
50  min.  By  60  min  after  spawning,  the  bilayered  HE  is 
completely  formed.  In  other  animal  systems,  peroxidatic 
enzymes  are  responsible  for  the  hardening  of  the  extra- 
embryonic  coat.  In  this  study,  observations  are  made 
consistent  with  the  involvement  of  an  oxidase  in  the  as- 
sembly of  the  shrimp  HE.  As  observed  by  electron  mi- 
croscopy, eggs  of  Sicyoniti  ingentis  and  Trachypenaeus 
simtlis  spawned  in  seawaters  containing  peroxidase  in- 
hibitors had  abnormally  assembled  HEs  compared  to 
control  eggs  in  seawater.  Dihydrotetramethylrosamine,  an 
oxidase-sensitive  fluorescent  dye,  supravitally  stained  the 
cortex  of  S.  ingentis  eggs  at  the  time  of  initial  HE  for- 
mation. The  HE  fluoresced  from  elevation  (40-50  min 
postspawn  in  5.  ingentis)  until  60-70  min  postspawn.  By 
first  cleavage  (90-120  min  postspawn).  HE  staining  was 
no  longer  visible,  although  staining  persisted  in  the  egg 
cortex.  In  eggs  treated  with  the  peroxidase  inhibitors  3- 
amino- 1 ,2.4-triazole  or  sodium  sulfite,  the  egg  cortex  flu- 
oresced, but  no  fluorescence  appeared  in  the  HE  before, 
during,  or  after  its  elevation. 

Introduction 

Formation  of  extracellular  egg  coats  has  been  the  subject 
of  studies  especially  in  frogs  (for  review.  Schmell  et  ai. 
1983).  sea  urchins  (for  review.  Schuel.  1978.  1985;  Kay 
and  Shapiro,  1985).  and  crustaceans.  The  crab  and  lobster 


Received  16  September  1994;  accepted  24  May  1995. 

*  Author  to  whom  correspondence  should  be  addressed. 

Abbreviations:  3-amino-1.2,4-triazole  (ATA);  3.3-diaminobenzidine 
(DAB):  artificial  seawater  (ASW);  hatching  envelope  (HE):  scanning 
electron  microscopy  (SEM):  transmission  electron  microscopy  (TEM); 
fertilization  envelope  (FE):  dihydrotetramethylrosamine  (DHTMR); 
penvitelline  space  (PVS). 


egg  coats  were  described  by  Goudeau  and  her  colleagues 
(Goudeau  and  Becker,  1982;  Goudeau  and  Lachaise, 
1980,  1983;  Talbot  and  Goudeau,  1988),  and  Pillai  and 
Clark  (1987,1 988,  1 990)  described  the  elevation  and  for- 
mation of  the  hatching  envelope  of  the  penaeid  shrimp 
Sicyonia  ingentis. 

The  presence  of  an  ovoperoxidase  enzyme  has  been 
demonstrated  as  crucial  in  the  assembly  of  the  sea  urchin 
fertilization  envelope  (FE)  (Kay  and  Shapiro,  1985,  for 
review).  Peroxidases  catalyze  reactions  in  which  hydrogen 
peroxide  serves  as  a  substrate  that,  when  bound  to  per- 
oxidase, oxidizes  other  substances  much  more  rapidly  than 
alone  (Kiernan,  1990).  Ovoperoxidase  in  sea  urchins 
forms  a  complex  with  the  protein  proteoliaisin  and  is  in- 
corporated into  the  FE  as  it  catalyzes  di-  and  tri-tyrosine 
linkages.  This  hardens  the  sea  urchin  FE  mechanically  so 
that  it  is  resistant  to  sperm  proteases  and  environmental 
stress.  Extra-embryonic  coat  assembly  involving  di-  and 
tri-tyrosine  linkages  mediated  by  an  ovoperoxidase-pro- 
teoliaisin  complex  has  been  described  in  detail  (for  review, 
Kay  and  Shapiro.  1985.  1987;  Weidman  et  ai,  1985,  1987; 
Somers  et  ai,  1989).  In  fish.  Kudo  et  ai  (1988)  demon- 
strated a  peroxidatic  reaction  in  the  fertilized  fish  egg  cho- 
rion.  This  is  believed  to  be  part  of  the  hardening  reaction 
in  the  chorion  necessary  to  provide  a  microenvironment 
for  the  developing  embryo  (Kudo  and  Inoue.  1986,  1989; 
Kudo  and  Teshima,  1991;  Kudo.  1992). 

These  peroxidatic  reactions  were  visualized  by  substrate 
localization  using  3.3-diaminobenzidine  (DAB)  (Daems 
et  ai.  1964;  Katsura  and  Tominaga.  1974;  KJebanofft'/ 
ai.  1979;  Kudo  et  ai,  1988;  Green  et  ai.  1990).  Ovoper- 
oxidase inhibitors  such  as  3-amino-l,2,4-triazole  (ATA) 
or  sodium  sulfite  have  been  used  to  prevent  normal  as- 
sembly of  sea  urchin  FE  (Veron  el  ai.  1977;  Showman 
and  Foerder.  1979).  In  the  presence  of  inhibitors,  lack  of 
incorporation  of  the  ovoperoxidase  enzyme  is  implicated 


13 


14 


P.  S.  GLAS  ET  AL 


by  an  absence  of  DAB  precipitation  staining  (Katsura  and 
Tominaga,  1974;  Klebanoff  ct  al.,  1979;  Green  el  at, 
1990).  Although  ovoperoxidase  has  been  demonstrated 
in  the  cortical  granules  in  mammals,  other  enzymes  are 
believed  to  account  for  the  change  in  zona  pellucida  per- 
meability during  the  zona  reaction  (Wasserman,  1987; 
Bleil  and  Wasserman.  1980). 

In  eggs  of  the  penaeoid  shrimp  Sicyimia  ingentix  and 
Trachypenaeus  similis.  the  hatching  envelope  (HE)  is  el- 
evated 30-50  min  after  spawning  in  seawater.  This  ele- 
vation is  the  result  of  exocytosis  of  at  least  two  distinct 
types  of  cortical  vesicles  in  5.  ingentis  (Pillai  and  Clark 
1988;  1990)  and  T.  ximilix(L\nn  ct  at.  1991;Glas,  1994). 
Assembly  results  in  an  HE  with  a  thin,  electron-dense 
outer  layer  and  a  thick,  more  electron-translucent  inner 
core.  When  peroxidase  inhibitors  were  added  to  the 
spawning  media,  abnormal  hatching  envelopes  elevated 
(Lynn  el  al..  1993)  indicating  that  a  peroxidatic  reaction 
may  be  necessary  for  normal  HE  assembly.  However,  at- 
tempts to  localize  a  peroxidase  with  DAB  did  not  show 
significant  staining.  This  apparently  conflicting  evidence 
suggests  the  presence  of  an  oxidatic  enzyme,  but  not  nec- 
essarily ovoperoxidase. 

Materials  and  Methods 

Gamete  collection 

Animals  were  collected  as  previously  described  by  Pillai 
ct  at  (1988)  and  Lynn  ct  al.  (1991,  1992.  1993).  Eggs 
were  collected  in  70  X  50  mm  crystallizing  dishes  con- 
taining artificial  seawater  ( ASW)  (Cavanaugh,  1956).  The 
dishes  were  gently  swirled  for  10  min  postspawning  to 
prevent  clumping  of  the  eggs. 

Inhibitor  and  localisation  substrates 

The  peroxidase  inhibitors  ATA  or  sodium  sulfite  were 
added  to  dishes  of  eggs  in  ASW  10  min  after  spawning  to 
give  a  final  concentration  of  100  nM  ATA  (Lynn  et  at. 
1 993)  or  20  mM  sodium  sulfite  in  seawater.  Hatching  en- 
velope elevation  was  monitored  with  light  microscopy. 

Samples  for  light  microscopy  were  removed  from  the 
spawning  dishes  at  intervals  to  correct  for  temperature 
changes  from  the  microscope  light.  Transmission  electron 
microscopy  (TEM)  samples  were  removed  and  processed 
by  fixation,  osmication,  dehydration  (Lynn  ct  at.  1992), 
infiltration,  and  embedding  in  modified  Spurr's  resin 
(Spurr,  1969).  Sections  were  double  stained  with  uranyl 
acetate  and  lead  citrate  (Venable  and  Coggeshall,  1965). 
Scanning  electron  microscopy  (SEM)  samples  were  pro- 
cessed through  dehydration,  then  critical-point  dried, 
sputter-coated,  and  viewed. 

For  fluorescent  localization  of  enz  me  activity,  a  per- 
oxidase-sensitive,  rhodamine  analog  lye,  dihydrotetra- 
methylrosamine  (DMTMR)  (Molecuk  r  Probes,  D-638), 


was  added  directly  to  spawning  dishes  containing  5.  in- 
gentix eggs.  The  dye  was  in  an  aqueous  stock  solution  of 
2  mg/ml  of  which  100  ^'  per  100  ml  egg  suspension  was 
used.  An  FITC  filter  cube  with  excitation  at  485  nm  and 
transmission  at  510  nm  was  used  to  observe  eggs  for  flu- 
orescence. 

Localization  of  peroxidase  activity  with  DAB  as  a  sub- 
strate was  performed  as  described  by  Klebanoff  et  at 
(1979).  Samples  were  removed  from  the  spawning  dish 
and  washed  in  a  solution  of  0.1  M  TRIS-0.45  M  Nad, 
pH  8.0,  to  remove  salts  that  might  interfere  with  the  re- 
action. The  wash  solution  was  replaced  with  a  reaction 
solution  (2  mg/ml  of  3,3-diaminobenzidine,  0.01%  H:O: 
in  0.1  M  Tris-0.45  M  NaCl.  pH  8.0).  and  the  reaction 
was  allowed  to  proceed  for  10  min.  The  reaction  was 
stopped  by  the  addition  of  two  times  the  volume  of  5.0% 
glutaraldehyde/1.6%.  formaldehyde  fixative  in  0.1  A/Tris- 
burler,  pH  8.0.  Samples  were  then  processed  for  TEM. 
Both  stained  and  unstained  sections  were  observed. 

Permeability  of  the  hatching  envelope 

To  ascertain  permeability  of  the  HE,  fluorescently 
tagged  dextrans  (500  ng/ml  egg  suspension)  (Sigma)  were 
added  to  the  ASW  and  ATA  seawater  media  after  the 
hatching  envelope  was  visible.  Eggs  were  observed  with 
epifluoresence  until  90  min  postspawn.  A  flow-through 
chamber  was  used  on  an  inverted  microscope  to  allow 
changing  the  solution  without  agitating  the  eggs.  Dex- 
trans of  4400,  10,000,  40.000,  76,000,  and  155,000  mo- 
lecular weight,  conjugated  to  fluorescein  isothiocyanate 
(FITC-dextran)  or  tetramethylrhodamine  isothiocyanate 
(TRITC-dextran)  probes  were  used.  Eggs  were  incubated 
in  the  presence  of  the  dextran  at  room  temperature.  Eggs 
were  transferred  to  a  flow-through  chamber,  which  was 
purged  two  times  with  seawater.  The  eggs  were  observed 
for  the  absence  of  fluorescence  within  the  perivitelline 
space  (PVS)  by  using  FITC  or  TRITC  excitation  and  bar- 
rier filters  on  a  Nikon  diaphot  inverted  microscope.  Dye 
exclusion  was  indicated  by  decreased  fluorescence  com- 
pared to  the  medium  outside  the  hatching  envelope  in 
the  PVS  when  eggs  were  observed  at  an  equatorial  focus. 

Enzyme  assays 

Enzyme  assays  were  performed  on  supernatants  from 
the  spawned  eggs  to  detect  ovoperoxidase  secretion  from 
the  egg.  The  guaiacol  assay  was  used  as  described  by  Deits 
et  at  (1984)  with  28  mAI  guaiacol,  1.0  mM  H:O:,  and 
50-500  n\  supernatant  from  settled  eggs  in  the  spawning 
dishes.  Supernatant  from  a  dish  with  spawned  eggs  was 
assayed  at  10.  30.  45.  and  60  min  postspawn.  Protein  as- 
says were  as  described  by  Lowry  el  at  ( 195 1 ). 


OX1DASH  ACTIVITY  IN  SHRIMP  EGGS 


15 


Results 


Hist i  meal  descriptions 


For  clarity,  a  brief  description  of  hatching  envelope 
(HE)  formation  in  5.  ingentis  and  T.  similis  is  reiterated 
here.  Eggs  spawned  from  penaeoid  shrimp  release  a  jelly 
coat  upon  contact  with  seawater  (Pillai  and  Clark,  1987; 
Lynn  el  a/.,  1991).  The  first  cortical  vesicle  exocytosis 
occurs  about  30-35  min  postspawn  in  5.  ingentis.  and  the 
products  interact  with  a  surface  coat  to  form  the  outer, 
electron-dense  layer  of  the  HE  (Pillai  and  Clark,  1988). 
The  HE  is  elevated  by  40-50  min  postspawn.  HE  eleva- 
tion is  reported  to  be  independent  of  fertilization  (Clark 
et  ui.  1980;  Pillai  and  Clark,  1987).  In  T.  similis.  the  first 
exocytosis  also  occurs  about  30  min  postspawn.  However, 
the  second  exocytosis  follows  more  rapidly  so  that  the  HE 
is  elevated  by  40  min  postspawn  (Glas.  1994).  Formation 
of  the  first  polar  body  occurs  5  to  10  min  before  the  HE 
is  apparent.  The  second  polar  body  appears  at  30-45  min 
postspawn  beneath  the  elevating  HE.  and  both  polar  bod- 
ies remain  throughout  HE  elevation.  During  this  period, 
granular  material  is  observed  accumulating  in  the  PVS. 
At  90  min  postspawn,  the  HE  is  retractile  and  an  expan- 
sive PVS  is  visible.  Transmission  electron  micrographs 
reveal  an  HE  with  a  distinct  bilayered  appearance. 

The  criteria  for  successful  assembly  and  elevation  of 
the  HE  were  based  on  the  morphology  of  the  envelope. 
These  included  the  continued  elevation  of  the  HE  without 
collapse  and  a  bilayered  appearance.  The  extra-embryonic 
envelopes  remain  around  the  zygote  until  the  time  of 
hatching,  about  24  h  later. 

Assessment  ofperoxidase  activity 

In  S.  ingentis  eggs  spawned  into  ASW,  a  distinct  PVS 
was  visible  at  75  min  postspawn  (Fig.  1).  separating  the 
oolemma  from  the  elevated  HE.  With  TEM,  the  HE  ap- 
peared as  a  well-formed  structure  with  flocculent  material 
juxtaposed  on  the  interior  of  an  electron-dense  outer  layer. 
Remnants  of  the  jelly  layer  remained  outside  the  HE  (Fig. 
2).  Materials  within  the  PVS  were  seen  in  close  association 
with  the  oolemma  or  the  thickening  HE.  With  higher 
magnification,  the  bilayered  configuration  of  the  HE  was 
evident  as  a  distinct  electron-dense  outer  layer  and  a  floc- 
culent, more  electron-translucent  inner  layer  (Fig.  3).  The 
exterior  of  the  egg  envelope  had  no  noticeable  ridges  or 
marks. 

S.  ingentis  eggs  treated  with  ATA  elevated  HEs  by 
45  min  postspawn.  Envelope  elevation  in  these  samples 
often  preceded  that  in  the  control  samples  by  5  min,  and 
initial  elevation  was  frequently  greater  than  in  control  egg 
HEs.  At  75  min  postspawn,  inhibitor-treated  eggs  had  en- 
velopes that  were  more  refractile  and  less  birefringent  than 
the  control  eggs,  and  the  envelopes  often  collapsed  to  the 
oolemma  (Fig.  4).  The  envelopes  were  very  fragile,  were 


often  wrinkled  and  folded,  and  were  easily  removed  (Lynn 
el  al..  1993;  Glas,  1994).  Transmission  electron  micro- 
graphs of  envelopes  and  eggs  of  ATA-treated  samples  re- 
vealed no  significant  differences  between  control  and 
treated  eggs  in  the  appearance  of  the  PVS  or  the  release 
of  cortical  vesicles  (compare  Figs.  2  and  5).  HEs  of  eggs 
in  ATA.  however,  showed  a  dense  outer  layer,  but  the 
more  electron-translucent  layer  was  absent  or  poorly  de- 
veloped (Fig.  6).  This  resulted  in  a  thinner  envelope  than 
seen  in  the  controls  (Figs.  3  and  6). 

In  5".  ingentis.  even  at  75  min  postspawn,  definitive 
DAB  localization  was  absent  in  the  hatching  envelope  in 
ASW  (data  not  shown).  The  intense  black  precipitant  in- 
dicative of  peroxidase  activity  in  other  systems  such  as 
the  sea  urchin  FE  was  not  present  (see  Klebanoff  et  al.. 
1979).  Similarly,  DAB  staining  was  also  not  apparent  in 
the  elevated  HE  of  T.  similis  eggs. 

In  T.  similis  eggs,  the  HE  appeared  as  a  distinct  re- 
fractile coat  around  the  egg  by  40  min  postspawn  and 
remained  so  at  90  min  postspawn  (Fig.  7).  The  intact  HE 
prevented  observation  of  the  oolemma  in  scanning  elec- 
tron micrographs  (Fig.  8).  Handling  and  dehydration 
techniques  caused  the  envelope  to  collapse,  so  that  large 
folds  in  the  HE  were  observed  with  SEM.  At  higher  mag- 
nifications, small  ridge-like  contours  on  the  exterior  of 
the  envelope  were  visible.  These  ridges  were  arranged  in 
distinctive  polygonal  patterns  encompassing  areas  of 
smooth  envelope  (Fig.  9). 

In  transmission  electron  micrographs  of  T.  similis  eggs 
at  90  min  postspawn,  the  HE  appeared  as  a  bilayered 
structure  (Fig.  10).  The  ridge-like  contours  seen  with  SEM 
correlated  with  cone-shaped  protrusions  of  electron-dense 
material  (Fig.  10).  This  material  appeared  continuous  with 
the  electron-dense  outer  layer  of  the  hatching  envelope. 
A  flocculent,  less  electron-dense  layer  formed  interiorly 
to  the  outer  layer,  and  was  three  to  four  times  the  thickness 
of  the  outer  dense  layer  (Fig.  10). 

T.  similis  eggs  treated  with  100/um  ATA  at  10  min 
postspawn  elevated  HEs  about  30-35  min  postspawn.  By 
90  min  postspawn,  eggs  incubated  in  ATA  had  envelopes 
that  appeared  less  refractile  (Fig.  11).  Frequently,  the 
granular  material  observed  in  the  PVS  was  coarser  and 
more  abundant  than  in  control  eggs.  When  90-min-post- 
spawn  eggs  were  observed  with  SEM,  a  ridge-like  pattern 
was  distinguishable  (Fig.  12),  but  was  much  less  prominent 
(Fig.  13)  than  in  the  control  eggs  (Fig.  9). 

At  90  min  postspawn,  eggs  treated  with  ATA  appeared 
to  have  a  thinner  HE  that  consisted  of  the  electron-dense 
layer  (Fig.  14)  with  the  flocculent,  more  electron-trans- 
lucent layer  absent  or  greatly  reduced  when  examined 
with  TEM.  The  inhibitor-treated  HEs  often  folded  on 
themselves,  so  that,  in  section,  the  ridges  sometimes  ap- 
peared to  be  on  the  "inside"  of  the  envelope  (Fig.  14). 

When  S.  ingentis  eggs  were  treated  with  the  oxidase- 
sensitive  fluorescent  dye  dihydrotetramethylrosamine 


16 


P.  S.  GLAS  ET  AL. 


Figures  1-6.  Sicvimia  ingenlis  eggs.  HE,  hatching  envelope:  PVS.  perivitelline  space;  ATA,  3-amino- 
1,2,4-tnazole. 

Figure  1.  With  phase  microscopy,  eggs  in  artificial  seawater  show  a  distinctive  retractile  HE  surrounding 
the  PVS  (*).  The  second  polar  body  (pb)  is  visible  within  the  PVS.  Bar  equals  100  jim. 

Figure  2.  With  transmission  electron  microscopy,  eggs  in  artificial  seawater  show  the  jelly  layer  (JL) 
outside  of  the  HE  (HE).  The  PVS  (*).  separating  the  HE  and  oolemma  (O).  contains  materials  that  may  be 
added  to  the  HE.  Bar  equals  1  jim. 

Figure  3.  At  higher  magnification,  the  bilayered  structure  of  the  HE  is  apparent.  The  outer  dense  layer 
(DL)  forms  a  smooth  exterior  while  the  inner  flocculent  layer  (IF)  appears  to  be  incorporating  more  material 
from  the  PVS  (*).  Bar  equals  0.25  /im. 

Figure  -4.  Eggs  in  3-amino-l,2,4-triazole  (ATA)  seawater  have  HEs  (HE)  that  often  collapse.  The  first 
polar  body  (pb)  is  visible  outside  of  the  collapsed  HE.  Bar  equals  100  ^m. 

Figure  5.  With  transmission  electron  microscopy,  eggs  in  ATA  seawater  show  HEs  (HE)  that  do  not 
have  the  structural  bilayered  appearance  of  those  in  control  eggs.  The  inner  electron  translucent  layer  is 
missing.  The  PVS  (*)  separates  the  oolemma  (O)  from  the  HE.  Bar  equals  1  pm. 

Figure  6.  Higher  magnification  shows  that  the  ATA-treated  HEs  are  thinner  and  do  not  appear  to  have 
the  flocculent  inner  layer  attached  to  the  dense  layer  (DL).  Bar  equals  0.25  ^m. 


(DHTMR)  (Whitaker  et  a/.,  1991),  no  fluorescence  was 
visible  in  the  egg  before  HE  elevation.  Sperm  attached  to 
the  egg  surface  were  visibly  fluorescent  (Fig.  15a,  b).  A 
fluorescent  band  appeared  in  the  cortex  of  the  egg  as  the 
HE  became  visible  (Fig.  16a,  b).  A  thin,  fluorescent  outline 
of  the  HE  appeared  after  HE  elevation.  The  HE  fluores- 
cence intensified  briefly  and  remained  in  the  region  of 
the  newly  elevated  HE  until  about  6  i  min  postspawn  (Fig. 
17a,  b).  By  the  two-cell  stage,  the  fK  irescence  in  the  HE 
was  no  longer  detectable  (Fig.  18a,  I  i. 

The  cortex  of  eggs  incubated  in  A  TA  was  fluorescent 
immediately  before  HE  elevation,  as  in  controls.  However, 


unlike  the  controls  (Figs.  15a,  b;  16a,  b:  17a.  b),  eggs  in 
ATA  showed  no  fluorescence  associated  with  the  HE  dur- 
ing and  after  elevation  (Fig.  19a,  b). 

Eggs  incubated  in  another  peroxidase  inhibitor,  sodium 
sulfite,  showed  no  cortical  fluorescence  immediately  be- 
fore HE  elevation.  Continued  monitoring  showed  no  flu- 
orescence by  60  min  postspawn  in  the  elevated  HE  (Fig. 
20a.  b). 

Halching  envelope  permeability 

S.  ingentis  eggs  were  incubated  with  fluorescently  la- 
beled dextrans  of  a  range  of  sizes  to  examine  the  perme- 


()\|])\SI      \(    IIVI1  V    IN   SIIRIMI'   I  (i(iS 


17 


Figures  7-14.  Trachypenaeus  s/m///v  eggs.  HE,  hatching  envelope;  PVS.  perhitelline  space;  ATA,  3- 
amino-1.2.4-triazole. 

Figure  7.  Eggs  in  artificial  seawater  show  a  jelly  layer  (JL)  outside  of  the  retractile  HE  (HE)  that  surrounds 
the  PVS  (*).  Bar  equals  100  ^m. 

Figure  8.  Scanning  electron  microscopy  shows  the  HE  covering  the  developing  zygote  to  act  as  an 
environmental  barrier.  The  HE  collapses  during  processing,  resulting  in  some  wrinkling  of  the  envelope. 
Bar  equals  100  ^m. 

Figure  9.  Higher  magnification  of  the  HE  shows  an  exterior  series  of  ridges  (arrowhead)  not  seen 
in  Sicyonia  ingenlis  egg  HEs.  The  ridges  form  octagonal  patterns  on  the  surface  of  the  HE.  Bar  equals 
1  nm. 

Figure  10.  Transmission  electron  microscopy  shows  the  distinct  bilayered  HE  with  the  ridges  ap- 
pearing to  be  constructed  of  the  electron-dense  material  (DL).  The  arrowhead  indicates  a  ridge  now 
seen  in  cross  section.  Note  the  ring  material  in  the  PVS  associated  with  the  flocculent  inner  layer  (large 
arrow).  The  flocculent  layer  is  three  to  four  times  the  thickness  of  the  outer  dense  layer.  Bar  equals 
0.5  Mm. 

Figure  11.  Phase  microscopy  of  an  egg  in  ATA  shows  a  less  refractile  HE  (HE)  surrounding  the  PVS 
(*).  Bar  equals  100  nm. 

Figure  12.  Scanning  electron  microscopy  of  an  egg  in  ATA  seawater  shows  an  egg  with  more  folds  than 
in  ASW.  The  collapse  of  the  HE  causes  the  envelope  to  be  folded  on  itself.  Bar  equals  100  urn. 

Figure  13.  At  higher  magnification,  the  ridges  (arrowhead)  are  not  as  prominent  as  in  the  control  eggs. 
The  height  of  the  ridges  appears  lower  than  in  normal  eggs.  Bar  equals  1  ^m. 

Figure  14.  Transmission  electron  microscopy  shows  that  the  envelope  is  folded  so  that  ridges  (arrowheads) 
appear  to  be  on  the  interior  and  exterior  of  the  HE.  The  electron-dense  layer  is  present,  but  the  electron- 
translucent  layer  is  greatly  reduced.  Bar  equals  0.5  nm. 


18 


P.  S.  GLAS  ET  AL. 


Figures  15-18.  A  time  series  of  Sicyonia  inguntis  eggs  in  artificial 
seawater  shows  the  elevation  of  the  hatching  envelope  (HE)  under  light 
microscopy  (a)  and  labeling  of  the  HE  with  an  oxidase  sensitive  fluorescent 
dye,  dihydrotetramethylrosamine  (b). 

Figure  15.  (a)  Normal  egg  before  elevation  at  35  min  postspawn. 
Two  sperm  are  visible  on  the  exterior  of  the  egg  (arrowhead).  Arrow 
indicates  first  polar  body,  (b)  No  fluorescence  is  visible  in  the  cortex; 
however,  the  sperm  are  fluorescent  (arrowhead).  The  first  polar  body 
(arrow)  can  also  be  seen. 

Figure  16.  At  47  min  postspawn.  the  HE  has  begun  to  lift  from  the 
oolemma.  (a)  The  HE  (HE)  is  separating  from  the  oolemma  and  has 
extranumerary  sperm  on  its  exterior,  (b)  The  cortex  can  be  seen  to  react 
with  DHTMR. 

Figure  17.  By  60  min  postspawn.  the  HE  is  fully  formed,  (a)  A  normal 
HE  (HE)  with  the  second  polar  body  (pb)  visible  in  the  perivitelline 
space,  (b)  The  HE  fluoresces  brightly,  as  does  the  cortex  of  the  egg.  The 
polar  body  does  not  fluoresce. 

Figure  18.  At  100  min  postspawn.  the  two-cell  stage  can  be  seen,  (a) 
The  cells  are  visible  within  the  ele\ated  HE  (HE),  (b)  The  cortex  still 
fluoresces,  but  the  HE  does  not.  Bar  equals  100  urn. 


ability  of  the  HE  (Table  I).  Based  on  the  relative  fluores- 
cent intensity  inside  vs.  outside  the  PVS  at  an  equato- 
rial focus,  both  the  control  and  ATA-treated  eggs  had 


fluorescent  dextrans  within  the  PVS  when  incubated 
with  the  4400  KDa  sugar.  When  incubated  with  the 
10,000  KDa  dextran,  the  ATA-treated  eggs  contained  flu- 
orescent dextrans  within  the  PVS,  but  eggs  in  ASW  did 
not.  The  entry  of  the  dextrans  was  variable  in  the  ATA- 
treated  eggs  with  40,000  and  76.000  Da  dextrans,  although 
the  control  eggs  revealed  no  fluorescent  dextrans  within 
the  PVS  (Table  I).  Even  though  extreme  care  was  used, 
the  presence  of  high  molecular  weight  fluorescent  dextrans 
in  the  PVS  of  ATA-treated  eggs  may  have  been  due  to 
damage  of  the  envelope  allowing  the  dextrans  to  enter 
the  PVS. 

Enzyme  assays 

A  spectrophotometric  assay  using  the  colorometric 
substrate  guaiacol  (Foerder  and  Shapiro,  1977)  failed  to 
indicate  peroxidase  activity  in  the  supernatant  within 
3  min  at  10,  30,  45,  or  60  min  postspawn.  These  time 
points  encompass  the  period  before,  during,  and  after  HE 
elevation. 

Discussion 

Histochemical  localization  and  identification  of  per- 
oxidase using  DAB  staining  is  well  documented  (Kay  and 


Figure  19.  Sicytmia  inxciiim  eggs  were  treated  with  the  peroxidase 
inhibitor  3-amino-l,2.4-triazole  and  then  incubated  with  DHTMR.  (a) 
In  the  inhibitor,  the  eggs  elevate  the  hatching  envelope  (HE),  which  then 
frequently  collapses  back  to  the  egg  surface,  (b)  With  DHTMR.  the  cortex 
can  be  seen  to  fluoresce  weakly  on  one  side  of  the  egg  and  is  not  consistent 
with  fluorescence  in  the  HE  (compare  with  same  time  point  of  controls 
in  Fig.  17a.  h). 

Figure  20.  Sicyoniu  i/ww/.v  eggs  were  also  treated  with  sodium  sulfite. 
a  peroxidase  inhibitor,  and  then  incubated  with  DHTMR.  (a)  The 
hatching  envelope  (HE)  is  elevated,  but  collapses  around  the  egg  as  seen 
with  light  microscopy.  The  second  polar  body  (pb)  is  visible,  (b)  Flu- 
orescent microscopy  shows  no  labeling  of  the  HE  or  cortex  although  the 
sperm  shows  fluorescence  (arrowhead).  Bar  equals  100  pm. 


OXIDASE  ACTIVITY  IN  SHRIMP  EGGS 


19 


Table  I 

Presence  of  fluorescent  dextrans  in  the  perivitelline  space 
iilicr  lialcliinn  envelope  e/evanoii 

Molecular  Control  Treated 

weight         (artificial  seawater)      (3-amino-1.2.4-triazole  +  seawatcr) 


4.400 

10.000 

40.000 

76.000 

155.000 


-,  '/:+  Vi- 


Each  score  represents  a  separate  trial  of  10  or  more  visibly  undamaged 
eggs. 

+  not  excluded  from  the  perivitelline  space;  -  excluded  from  the 
perivitelline  space. 

'/2+  50%  had  fluorescence  in  the  perivitelline  space:  'A—  50%  excluded 
fluorescence. 


Shapiro,  1985,  for  review;  Kiernan,  1990;  Green  et  ai, 
1990).  These  studies  report  intense  black  staining  as  in- 
dicative of  the  peroxidase  reaction  in  sea  urchin  eggs  and 
mammalian  tissue.  DAB  staining  due  to  peroxidatic  ac- 
tivity has  been  reported  in  the  FEs  of  the  sea  urchins 
Hemicentrotus pulcherrimiis,  Temnopleunis  torewnaticus, 
Strongylocentrotus  purpuratiis,  and  Lytechinus  pictus 
(Katsura  and  Tominaga,  1974;  Klebanoff  et  a/..  1979; 
Green  et  ai.  1990).  Using  DAB,  Kudo  et  ai  (1988)  also 
demonstrated  peroxidase  activity  in  the  vitelline  envelope 
of  unfertilized  fish  eggs  and  in  the  chorion  and  micropylar 
region  of  fertilized  fish  eggs.  In  addition.  DAB  reactivity 
remains  detectable  over  an  extended  time  (Klebanoff  et 
ai,  1979;  Kay  and  Shapiro,  1985.  for  review;  Green  et 
ai.  1990).  Heavy  precipitation  indicative  of  peroxidase 
activity  was  not  visualized  in  the  HE  of  eggs  from  either 
S.  ingentis  or  T.  similis  by  using  the  DAB  methodology. 
Staining  was  not  observed  at  any  of  the  time  points  sam- 
pled (30,  45,  75,  or  90  minutes  postspawn). 

The  peroxidase  inhibitors  ATA  and  sodium  sulfite  are 
believed  to  interfere  with  tyrosine  cross-linking  by  oxi- 
dative  inhibition  of  the  enzyme  or  enzymes  responsible 
for  FE  elevation  and  transformation  (Kay  and  Shapiro, 
1985,  for  review;  Kay  and  Shapiro.  1987;  Lynn  et  ai, 
1988;  Green  et  ai.  1990).  Inhibitor-treated  sea  urchin  eggs 
have  FEs  that  are  thinner  and  less  refractile  than  the  nor- 
mal envelopes.  Treatment  of  penaeid  shrimp  eggs  with 
the  peroxidase  inhibitors  ATA  and  sodium  sulfite  results 
in  "soft"  HEs  that  are  less  refractile  and  tend  to  collapse. 
The  morphology  of  the  envelope  shows  a  thinner  envelope 
that  lacks  the  bilayered  appearance.  These  inhibitors  are 
not  detrimental  to  the  development  of  the  shrimp  egg 
(Lynn  el  ai.  1993). 

In  contrast,  interference  by  "peroxidase"  inhibitors  with 
normal  HE  assembly  is  also  implicated  by  changes  in  HE 
permeability  as  exhibited  by  fluorescently  labeled  dex- 
trans. In  these  experiments,  the  HE  is  more  permeable  to 


substances  of  10,000  and  40,000  kDa  in  peroxidase-in- 
hibited  medium  than  in  artificial  seawater.  The  variability 
of  exclusion  in  the  higher  ranges  may  be  due  to  other 
influences  including  time  of  dextran  addition,  damage  to 
the  envelopes,  or  possibly,  whether  fertilization  had  taken 
place.  Variation  due  to  damage  of  the  envelopes  was  min- 
imized by  developing  a  system  with  minimum  handling 
of  the  eggs.  Further  implications  of  differences  in  HE  per- 
meability due  to  sperm  interaction  were  not  considered 
during  this  study  since  envelope  elevation  and  formation 
were  previously  reported  to  be  independent  of  fertilization 
(Pillai  and  Clark,  1987). 

The  paradox  of  these  findings  resulted  in  the  use 
of  a  fluorescent  probe,  dihydrotetramethylrosamine 
(DHTMR),  a  rhodamine  analog.  DHTMR  reacts  with 
oxidizable  substrates  and  is  pH  independent  in  its  ab- 
sorption and  emission  (Whitaker  et  ai.  1991).  Whitaker 
et  ai  (1991)  describe  this  compound  as  a  neutral,  lipo- 
philic  substrate  for  horseradish  peroxidase  and  hydrogen 
peroxide.  An  advantage  of  the  dye  is  that  it  can  be  used 
with  in  vitro  preparations,  thereby  eliminating  the  use  of 
fixatives  and  the  accompanying  interference  by  aldehyde 
groups.  The  cytoplasm  of  spawned  eggs  placed  in  this  dye 
fluoresces,  indicating  the  presence  of  an  oxidative  capacity. 
Similar  fluorescence  is  reported  in  mammalian  and  pro- 
tozoan systems  in  relation  to  phagocytosis  and  the  im- 
mune response  (Whitaker  et  ai.  1991).  The  dye  reaction 
in  shrimp  eggs  may  be  indicative  of  several  reactions. 
These  reactions  could  include  a  peroxidase,  a  hydroxide, 
or  superoxide-based  change  that  may  correspond  to  the 
"respiratory  burst"  in  sea  urchin  eggs  (Turner  et  ai.  1985; 
Heinecke  and  Shapiro,  1989;  Epel,  1990.  for  review)  or 
respiratory  activity  associated  with  mitochondria.  Thus, 
it  appears  that  DHTMR  may  react  with  one  or  more  sub- 
strates in  the  shrimp  egg,  but  the  substrate  cannot  be  de- 
fined as  ovoperoxidase.  In  shrimp,  bound  sperm  also  flu- 
oresced,  a  result  in  keeping  with  observations  by  Lindsay 
and  Clark  (1992)  of  a  pH  (hydroxide)  change  involved  in 
sperm  acrosomal  filament  formation.  It  is  unclear  why 
all  sperm  that  were  attached  to  eggs  did  not  fluoresce,  but 
there  did  not  appear  to  be  any  correlation  to  the  fertilizing 
sperm.  Since  this  was  not  the  main  point  of  this  study, 
further  investigations  will  be  required  to  resolve  this  in- 
teresting phenomenon. 

Comparison  of  the  amino  acid  composition  of  the  sea 
urchin  FE  (Foerder  and  Shapiro,  1977)  with  the  amino 
acid  composition  of  the  shrimp  HE  (Pillai  and  Clark. 
1990)  reveals  that  the  relative  abundance  of  tyrosine  res- 
idues is  considerably  lower  in  the  shrimp  HE  than  in  the 
sea  urchin  FE.  Because  tyrosine  is  a  major  substrate  for 
ovoperoxidase  activity,  lack  of  an  abundant  supply  may 
suggest  there  is  not  a  major  role  for  an  ovoperoxidase  in 
shrimp  eggs.  Assays  for  rie  di-  and  tri-tyrosine  compo- 
nents in  the  shrimp  HE  have  not  been  performed. 


20 


P.  s.  GLAS  ET  .-in- 


sufficient ovoperoxidase  is  released  by  sea  urchin  eggs 
to  allow  quantitative  analysis  of  the  supernatant  (Deits  ct 
til..  1984;  Green  el  ai.  1990).  In  peneaoid  shrimp,  how- 
ever, if  an  ovoperoxidase  is  released  following  spawning, 
the  quantities  are  insufficient  to  detect  between  spawning 
and  90  minutes  postspawn.  Although  HE  assembly  could 
be  acting  as  a  barrier  to  release  into  the  supernatant,  we 
do  not  believe  this  is  the  case  for  two  reasons.  First,  mul- 
tiple time  points  were  used  for  the  assay  and  included 
times  before,  during,  and  after  the  HE  assembly  was 
"complete."  Second,  the  relative  permeability  of  the  as- 
sembled envelope  allows  molecules  of  at  least  10,000  Da 
through,  as  demonstrated  by  the  dextran  experiments.  It 
is  very  likely  that  even  larger  molecules  would  pass  the 
envelope  prior  to  the  final  "tanning"  process.  An  alter- 
native explanation  might  be  that  the  enzyme  is  bound  so 
tightly  to  the  assembling  envelope  that  none  escapes.  Al- 
though this  is  possible,  it  is  inconsistent  with  the  reports 
in  other  systems  and  contradictory  to  the  results  of  the 
DAB  assays,  which  failed  to  demonstrate  localization  in 
the  HE. 

Ovoperoxidase  from  sea  urchins  is  reported  to  have 
oxidoreductase  activity  requiring  Mn+:  ions  and  certain 
phenols  related  to  the  respiratory  burst  (Turner  et  ai. 
1985;  1986;  1988;  Heinecke  and  Shapiro.  1989).  Phenol 
oxidase  in  crustaceans  oxidizes  diphenols  to  the  corre- 
sponding quinones  that  react  in  the  tanning  of  crustacean 
exoskeletons  to  cross-link  proteins  within  the  chitin  matrix 
(Stevenson,  1985).  Perhaps  the  oxidase  of  the  shrimp  em- 
bryo is  a  closer  analog  of  phenol  oxidase  than  of  ovoper- 
oxidase. Pillai  and  Clark  (1990)  report  that  carbohydrates 
similar  to  those  found  in  chitin  are  abundant  in  the  pe- 
neaoid hatching  envelope.  Lectin-binding  assays  dem- 
onstrate the  presence  of  oligosaccharides,  especially  man- 
nose  and  TV-acetylglucosamine,  in  the  HE  (Pillai  and 
Clark,  1990;  Lin,  1992;  Glas.  1994).  These  sugars  are  the 
major  components  of  chitin.  A  phenol-oxidase-driven 
cross-linkage  of  carbohydrates  within  the  HE  would  be 
consistent  with  the  presence  of  an  oxidase.  The  action  of 
such  an  enzyme  is  also  consistent  with  the  thinner  HE  in 
the  presence  of  the  oxidase  inhibitors  ATA  and  sodium 
sulfite. 

Thus,  an  oxidase  is  probably  involved,  as  indicated  by 
the  morphology  of  the  HE  after  exposure  to  the  peroxidase 
inhibitors  ATA  and  sodium  sulfite  and  the  staining  with 
DHTMR.  The  active  enzyme  is  not  similar  to  the  ovo- 
peroxidase identified  in  the  sea  urchin,  as  shown  by  the 
lack  of  localization  with  DAB  or  quantitation  by  the 
guaiacol  assay;  neither  does  the  enzyme  appear  to  be 
analogous  to  the  enzyme  that  stimulates  the  peroxidatic 
reaction  in  fish  fertilization.  Nevei  •  'ieless,  a  peroxidase- 
inhibitor-sensitive  enzyme  is  involv  J  with  the  assembly 
of  the  penaeoid  HE.  Further  identifu  ition  of  such  an  ac- 
tive enzyme  is  required  for  a  full  ui  derstanding  of  the 


mechanisms  responsible  for  HE  assembly,  elevation,  and 
function. 

Acknowledgments 

The  authors  would  like  to  express  their  appreciation  to 
Dr.  Wallis  Clark,  Jr.,  and  his  students  at  University  of 
California,  Bodega  Marine  Laboratory,  for  procurement 
of  the  Sicyonia  ingentis  and  use  of  laboratory  space.  We 
are  also  grateful  to  Jim  Hanifen  and  his  colleagues  at 
Louisiana  Department  of  Wildlife  and  Fisheries  for  pro- 
viding the  Trachypenaeus  similis.  We  thank  Becky  Dem- 
ler,  Ron  Bouchard.  Cindy  Henk,  and  Dr.  Sharon  Mat- 
thews of  the  Louisiana  State  University  Life  Sciences  Mi- 
croscopy Facility  for  their  assistance.  Research  supported 
by  Louisiana  Sea  Grant  #NA89-AA-D-SG226  project  #R/ 
SA-1  to  JWL  and  JDG  and  Sigma  Xi  Grant-in-Aid  of 
Research  to  PSG. 

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Reference:  Biol.  Bui/.  189:  22-28.  (August,  1995) 


Sensitivity  of  Metabolic  Rate,  Growth,  and  Fecundity 

of  Tadpole  Shrimp  Triops  longicaudatus  to 

Environmental  Variation 

DAVID  A.  SCHOLNICK 

University  of  Colorado,  Department  of  E. P.O.  Biology,  Boulder,  Colorado  80309 


Abstract.  The  influence  of  fluctuations  of  ambient  oxy- 
gen tensions  and  temperature  on  the  rate  of  oxygen  con- 
sumption (Vo2)  was  determined  for  the  tadpole  shrimp. 
Triops  longicaudatus.  1'O2  was  oxygen  dependent  up  to 
185  torr  Po2,  and  (?10  for  oxygen  consumption  between 
20°  and  30°C  was  1 .9.  From  these  results  it  was  estimated 
that  oxygen  consumption  increases  more  than  1100 
//I  •  g~ '  •  h"  '  in  T.  longicaudatus  for  typical  diurnal  changes 
in  temperature  and  oxygen  in  desert  ephemeral  pools. 
Elevated  I  'o2  may  be  coupled  with  increased  growth  rate 
and  fecundity,  because  these  characteristics  were  highly 
sensitive  to  changes  in  ambient  temperature  and  oxygen 
tension.  Depressing  mean  daily  temperatures  by  2.3°C 
significantly  decreased  body  mass,  whereas  hyperoxia  (200 
torr)  significantly  increased  growth  compared  to  that  of 
animals  raised  under  hypoxic  conditions  (70  torr).  Fe- 
cundity was  dependent  on  animal  mass  and  ambient  oxy- 
gen tension.  Thus,  for  a  22-day  season,  one  T.  longicau- 
datus female  could  produce  30  more  eggs  per  10  torr  in- 
crease in  oxygen  tension  and  43  more  eggs  per  1  °C  change 
in  mean  daily  temperature.  These  results  indicate  that 
there  are  selective  pressures  for  metabolic  sensitivity  to 
the  high  temperature-high  oxygen  conditions  of  the 
ephemeral  environments  inhabited  by  T.  longicaudatus. 

Introduction 

Tadpole  shrimp  (Triops  longicaudatus  LeConte)  are 
primitive  branchiopod  crustaceans  that  face  extreme  en- 
vironmental conditions  in  the  ephemeral  desert  pools  that 
they  inhabit.  These  pools  are  characterized  by  large  diurnal 
oscillations  (greater  than  200  torr  O,  per  day)  in  the  dis- 
solved gasses  produced  by  the  photosynthesis  and 

Received  29  September  1994:  accepted  26  May  1995. 


metabolism  of  their  biota,  and  by  large  and  rapid  fluc- 
tuations in  water  temperature  (greater  than  1 5°C  per  day; 
Scholnick,  1994).  In  addition,  pool  water  may  persist  for 
as  little  as  10  days  during  the  summer  when  evaporation 
rates  are  high.  As  a  result,  species  such  as  T.  longicaudatus, 
which  survive  dry  periods  as  dormant  eggs,  must  complete 
their  life  cycles  rapidly  while  experiencing  extreme  envi- 
ronmental fluctuations. 

Metabolic  control  in  tadpole  shrimp  presents  a  unique 
problem  because  of  the  need  for  rapid  development  in  a 
variable  environment.  Only  a  few  physiological  studies 
have  been  conducted  on  tadpole  shrimp.  Home  (1971) 
and  Scott  and  Grigarick  (1979)  reported  that  Triops  eggs 
do  not  hatch  until  temperatures  are  greater  than  14°C. 
Hillyard  and  Vinegar  (1972)  reported  that  oxygen  con- 
sumption in  immature  T.  longicaudatus  was  three  times 
more  sensitive  to  temperature  than  in  adults.  In  another 
species  of  tadpole  shrimp  (Lepidurus  lemmoni),  metabo- 
lism was  sensitive  to  changes  in  ambient  oxygen  tensions 
(Eriksen  and  Brown,  1980).  Therefore,  it  is  unclear  what 
effects  simultaneous  fluctuations  of  both  oxygen  and 
temperature  have  on  the  metabolism,  growth,  and  repro- 
ductive success  of  tadpole  shrimp  inhabiting  desert 
ephemeral  pools. 

This  investigation  examines  the  influence  of  simulta- 
neous fluctuations  in  oxygen  and  temperature,  which  oc- 
cur naturally  in  desert  ephemeral  habitats,  on  rates  of 
oxygen  uptake  in  Triops  longicaudatus.  The  effects  of 
these  fluctuations  on  growth  and  fecundity  were  also  ex- 
amined. 

Materials  and  Methods 

Specimens  of  Triops  longicaudatus  LeConte  were 
hatched  from  and  raised  with  rehydrated  soils  collected 
from  four  previously  studied  ephemeral  pools  near  Moab, 


22 


METABOLIC  SENSITIVITY  IN  TADPOLE  SHRIMP 


23 


Utah  (Scholnick,  1994).  Soils  containing  eggs  from  the 
four  pools  were  mixed  and  randomly  subsampled  for  dif- 
ferent treatments.  Animals  were  raised  in  plastic  tubs  (70 
X  50  X  13  cm)  or  10-gallon  glass  aquaria  under  cycles  of 
temperature  ( 1 8°-32°C)  and  light  ( 1 2L:  1 2D)  to  simulate 
summer  conditions.  Diets  consisted  of  naturally  occurring 
protozoa  and  algae  of  rehydrated  soils,  supplemented  with 
commercially  purchased  live  Tuhifex. 

Metabolic  measurements 

Respiration  rates  ( I  'o2,  microliters  of  oxygen  consumed 
per  gram  wet  weight  per  hour,  standardized  to  STP)  were 
determined  for  oxygen  tensions  of  77,  127,  and  185  ton- 
al 20°,  25°,  and  30°C.  I'o2  values  were  determined  in  a 
temperature-equilibrated  flow-through  system.  The  sys- 
tem consisted  of  an  elevated  4.5-1  water  reservoir  that  was 
equilibrated  with  different  partial  pressures  of  O:  and  used 
as  a  pressure  head  to  maintain  constant  flow.  Flow  was 
measured  volumetrically  and  regulated  by  the  height  of 
the  reservoir  and  the  diameter  of  the  tubes.  Flow  rates 
were  set  between  6.5  and  8  ml  •  min~',  depending  on  an- 
imal  size  and   temperature,   and   were   held   constant 
throughout  each  experiment  (less  than  0. 1  ml  change  over 
5  h).  The  oxygen  content  of  the  water  was  measured  with 
an  Orion  oxygen  meter  and  probe  (#840  Orion  Research. 
Boston)  fitted  with  a  0.5-ml  flow-through  cell  and  a  stir 
bar.  Voltage  output  was  connected  to  a  personal  computer 
through  an  analog-to-digital  converter  and  sampled  every 
20  s.  A  133-ml  animal  chamber  was  suspended  in  a  water 
bath  equipped  with  a  thermostat  and  was  continually 
stirred  with  an  enclosed  stir  bar  to  ensure  mixing.  Water 
was  collected  downstream  and  pumped  back  to  the  res- 
ervoir with  a  water  pump.  A  series  of  valves  made  it  pos- 
sible to  measure  the  oxygen  content  of  the  incurrent  water 
(water  coming  directly  from  the  reservoir)  or  the  excurrent 
water  (water  coming  directly  from  animal  chamber)  with- 
out disrupting  flow. 

Animals  of  similar  masses  (mean  of  364  ±  10.5  mg 
ranging  from  290  to  440  mg)  were  given  1  h  to  adjust  to 
the  temperature  and  oxygen  tensions  of  the  chamber  be- 
fore measurements  began.  Animals  were  selected  from 
simulated  pools  at  predetermined  times  when  oxygen  and 
temperature  levels  were  similar  to  experimental  condi- 
tions. I  'O2  was  measured  for  individual  animals,  and  each 
animal  was  measured  only  once.  Measurement  periods 
ranged  from  3  to  4  h  for  each  animal  at  a  given  temper- 
ature and  Po2. 

The  design  of  the  animal  chamber  and  oxygen  sampling 
system  conformed  to  the  principles  of  a  single-chamber 
system  as  denned  in  Frappell  et  al.  ( 1 989).  In  this  system, 
the  time  constant  for  the  washout  of  oxygen  for  the  animal 
chamber  and  from  the  electrode  chamber  (T,  +  r2)  was 
24  min.  while  the  time  constant  for  the  electrode  circuit 


alone  (r:)  was  less  than  2  min.  Because  r2  was  reasonably 
small  with  respect  to  (r,  +  r2)  the  system  could  be  con- 
sidered a  single  chamber  in  which  r2  =  0. 

Measurements  of  growth 

Growth  rates  of  T.  longkaudatm  under  natural  con- 
ditions were  determined  in  four  study  pools  near  Moab, 
Utah,  during  the  summer  of  1 993.  Animals  were  individ- 
ually caught  in  a  small  sieve,  blotted  dry  through  the  sieve, 
and  weighed.  Animals  were  held  in  a  beaker  containing 
pool  water  until  all  animals  were  weighed.  Wet  weight 
was  determined  daily  by  weighing  10  to  20  animals  from 
each  pool  on  an  Ohaus  portable  balance  (#CT  10-3,  Flor- 
ham.  Park,  NJ). 

The  influence  of  temperature  on  growth  rate  was  de- 
termined, as  described  above,  in  laboratory-simulated 
ephemeral  pools  where  the  average  temperature  was  either 
25.7°  or  23.4°C.  For  the  high-temperature  condition 
(mean  =  25.7°C),  temperatures  were  cycled  between  19° 
and  31°C  by  using  heat  lamps;  the  average  temperature 
change  was  0.85°C-h~'.  For  the  low-temperature  con- 
dition (mean  =  23.4°C),  temperature  fluctuated  from  19° 
to  29°C;  average  temperature  change  was  0.65°C-h~'. 
Animals  were  hatched  at  treatment  temperature  cycles, 
and  growth  rates  were  determined  from  daily  measure- 
ments of  wet  weight.  Average  oxygen  partial  pressure  of 
125  ±  2  torr  was  maintained  in  each  treatment  by  vig- 
orously bubbling  air  into  each  tank.  Temperature  treat- 
ments were  replicated  five  times  with  4-5  animals  in  each 
tank  at  the  beginning  of  the  experiment. 

Sensitivity  of  growth  to  oxygen  was  determined  from 
animals  raised  in  laboratory-simulated  pools  as  described 
above  at  200  ±  5  and  70  ±  7  torr  Po2  by  either  continually 
bubbling  100%  oxygen  or  13%  oxygen,  remainder  nitro- 
gen. Temperature  was  cycled  in  a  manner  identical  to  the 
low-temperature  condition  described  above,  and  growth 
rates  were  determined  by  changes  in  wet  weight  per  day. 
Oxygen  treatments  were  replicated  five  times  with  3-5 
animals  in  each  tank  at  the  beginning  of  the  experiment. 
Animals  were  hatched  at  treatment  temperature  cycles 
and  growth  rates  were  determined  from  daily  measure- 
ments of  wet  weight. 

Fecundity 

Animals  were  raised  in  simulated  ephemeral  pools 
where  temperature  and  light  were  cycled  at  125  or  200 
torr  O:  as  described  above.  Fecundity,  or  egg  production, 
was  determined  by  chilling  gravid  animals  (eggs  were  vis- 
ible in  brood  pouches)  to  8°C.  When  animals  became 
hypothermic,  the  brood  pouches  opened  and  the  eggs  were 
released.  All  eggs  released  per  female  were  collected  and 
counted.  Brood  pouches  were  checked  to  ensure  that  all 
eggs  had  been  liberated.  This  procedure  resulted  in  zero 


24 


D.  A.  SCHOLNICK 


1000 
^   900- 
0>  800- 
3-   700- 

g      600' 

"-   500- 


R  300  ^ 


en 


200- 
100 


O 


y=2.6x+355 
,  y=2.3x+202 
I  y=2.4x+62 


30'C 


25'C 


20'C 


"50  75          100         125         150         175 

Oxygen  tension  (torr) 


200 


Figure  1.  Mean  oxygen  consumption  (microliters  of  oxygen  per  gram 
wet  weight  per  hour)  at  different  oxygen  tensions  for  Triops  longicaudatus. 
Values  presented  are  means  ±  SEM.  Lines  represent  linear  regressions 
with  r2  greater  than  0.7  in  all  cases. 


adult  mortality.  However,  egg  viability  appeared  to  be 
severely  compromised  as  a  result  of  the  "forced"  release. 
Attempts  to  dry  and  rehydrate  eggs  were  unsuccessful. 

Statistics  and  calculations 

An  analysis  of  covariance  was  applied  to  the  mean 
weights  for  each  treatment  group  to  determine  statistical 
differences  between  growth  rates.  ANCOVA  was  weighted 
by  the  number  of  individuals  in  each  tank  to  account  for 
uncontrollable  differences  in  animal  number.  Higher 
sample  size  was  assumed  to  be  a  more  accurate  predictor 
of  mean  population  mass  and  was  therefore  given  more 
weight  in  the  analysis.  A  probability  of  <0.05  was  con- 
sidered statistically  significant. 

Sensitivity  of  oxygen  consumption  to  change  in  tem- 
perature, Qw,  was  calculated  by  using  the  general  formula: 


where  M2  and  Mt  are  metabolic  rates  at  the  higher  (7^) 
and  lower  (  7%)  temperatures,  respectively. 

In  order  to  reduce  mass-specific  effects,  animals  of  sim- 
ilar masses  were  chosen  for  metabolic  measurements.  Be- 
cause mass  did  not  vary  significantly  between  treatment 
groups  (p  <  0.05  ANOV  A),  Vo2  (n\  •  g^  '  •  h~  '  )  was  deter- 
mined by  dividing  oxygen  consumption  by  wet  mass. 

Results 

Rates  of  oxygen  consumption  (I'O2)  in  Triops  longi- 
caudatus are  sensitive  to  changes  in  ambient  temperature 
and  oxygen  (Fig.  1).  Temperature  sensitivity,  or  Q{0,  at 
125  torr  was  1.85  between  20°  and  ^0°C,  1.82  between 
20°  and  25°C,  and  1.92  between  2?  J  and  30°C.  There 


was  a  positive  linear  relationship  between  I'o2  and  oxygen 
tension  throughout  the  physiological  range.  Temperature 
had  very  little  effect  on  oxygen  sensitivity.  The  average 
change  in  I  'O2  per  torr  oxygen  tension  was  2.5  ^1  •  g"1  •  h~'. 

Changes  in  I  'o2  for  oxygen  and  temperature  fluctua- 
tions typical  of  diurnal  cycles  in  desert  pools  (Scholnick, 
1994)  were  predicted  from  the  metabolic  responses  pre- 
sented in  Figure  1.  Metabolic  rates  were  estimated  to  in- 
crease more  than  sixfold  during  a  typical  diurnal  cycle 
(Fig.  2)  when  temperature  and  oxygen  vary  between  18° 
and  32.5°C  and  60  and  226  torr,  respectively.  Therefore, 
animals  could  experience  a  change  in  }'o2  as  great  as  1 100 
^l-g~''h~'  during  a  single  6-h  diurnal  period.  Growth 
rates  in  the  field  ranged  from  97.7  to  44.7  mg-  day"'  be- 
tween pools  (Fig.  3).  The  average  growth  rate  for  field 
animals  was  53  mg-day"1,  similar  to  the  rate  of  about  57 
mg-day~'  for  animals  raised  in  the  laboratory  (Fig.  4). 
Depressing  the  mean  daily  temperatures  by  2.3°C  resulted 
in  a  significant  decrease  in  body  mass  (p  <  0.02  ANCOVA; 
Fig.  4).  On  average,  animals  raised  at  the  higher  temper- 
ature cycle  weighed  over  80  mg  more  than  those  raised 
at  the  low  temperature  cycle  after  5  days  of  age.  At  day 
9,  about  the  time  when  egg  laying  is  initiated,  body  mass 
was  27%  greater  for  animals  raised  at  the  higher  average 
daily  temperature. 

Animals  raised  under  hyperoxic  conditions  (200  torr) 
grew  significantly  faster  (an  increase  of  more  than  twofold 
until  day  20)  and  were  significantly  larger  (p  <  0.01  AN- 
COVA) at  every  age  over  5  days  than  animals  raised  under 
hypoxic  conditions  (70  torr;  Fig.  5).  The  results  for  the 


^   1400 


c 
o 

"o. 

E 


c 
o 
O 


- 

"o 

T3 
0) 


1200- 


1000- 


800- 


600- 


400- 


200- 


0 


6:30  9:30  14  16  20:30        6:30  9:30  14  16  20:30 
Time  of  Day 

Figure  2.  Predicted  change  in  oxygen  consumption  rates  (microliters 
per  gram  per  hour)  for  Triops  longicaudatus  for  typical  diurnal  fluctu- 
ations in  oxygen  and  temperature  measured  in  the  field  (Scholnick,  1 994). 
Changes  in  oxygen  consumption  rates  are  based  on  temperature  and 
oxygen  sensitivity  presented  in  Figure  I. 


METABOLIC  SENSITIVITY  IN  TADPOLE  SHRIMP 


25 


1200 


1000 


O) 

CO 
C/) 

CD 


800- 


600- 


400- 


200- 


10  15 

Time  (days) 


20 


Figure  3.  Growth  rates  for  Triops  longicaudatus  from  four  desert 
pools  near  Moab.  Utah,  during  the  summer  months  of  1 993.  Each  point 
represents  an  average  wet  mass  of  10-20  animals  in  each  pool. 


oxygen  and  temperature  treatments  were  statistically 
identical  whether  the  entire  growth  curve  (linearized  using 
log  time)  or  only  the  linear  portion  of  the  curve  was  used. 

There  was  a  positive  linear  relationship  between  num- 
ber of  eggs  and  wet  mass  (Fig.  6).  At  a  Po2  of  200  torr. 
the  brood  pouches  of  an  800-mg  animal  contained  an 
average  of  50  more  eggs  than  those  of  a  300-mg  animal. 
Rearing  temperature  (mean  cyclic  temperature  of  either 
25.7°  or  23.4°C)  had  no  effect  on  the  relationship  between 
body  mass  and  egg  number.  The  slope  of  the  regression 
line  for  animals  raised  at  elevated  ambient  oxygen  tensions 
(200  torr)  was  significantly  greater  than  that  of  the  regres- 
sion line  for  animals  raised  at  oxygen  saturation  ( 1 25  torr 
O::/><0.01  ANCOVA). 

Small  changes  in  temperature  or  oxygen  dramatically 
change  the  estimate  of  total  number  of  eggs  laid  in  one 
season  (Table  I).  Based  on  the  independent  influence  of 
temperature  or  oxygen  on  body  mass  (Figs.  4  and  5)  and 
the  relationship  between  body  mass  and  fecundity  (Fig. 
6),  an  increase  of  2°-3°C  in  average  diurnal  temperature 
was  calculated  to  increase  fecundity  by  about  99  eggs  over 
a  22-day  period.  This  estimate  assumes  that  animals  pro- 
duce one  brood  every  3  days  (estimated  from  Ahl.  1983) 
and  the  first  brood  is  produced  at  a  wet  weight  of  200  mg 


(Fig.  6;  Table  I).  Animals  experiencing  hyperoxic  condi- 
tions could  produce  378  more  eggs  over  a  22-day  season 
than  animals  raised  under  hypoxic  conditions  at  the  lower 
average  daily  temperature  (Table  I). 

Discussion 

The  results  presented  in  this  study  indicate  that  Triops 
longicaudatus  maintains  a  high  degree  of  metabolic  sen- 
sitivity despite  the  wide  range  of  environmental  conditions 
in  its  natural  habitat.  The  net  effect  of  temperature  and 
oxygen  sensitivity  is  to  increase  oxygen  consumption  dur- 
ing the  day  when  temperature  and  oxygen  tensions  are 
high.  Development  and  fecundity  are  related  to  Po2  and 
temperature,  and  hence  to  elevated  1'O2.  Shortened  de- 
velopment time  and  increased  fecundity  are  critical  for 
successful  completion  of  life  cycles  in  ephemeral  envi- 
ronments. 

Branchiopods  are  primitive  crustaceans  that  are  often 
prominent  in  ephemeral  systems  (Hessler  el  at..  1982). 


1200 


1000- 


800- 


o> 

CO 


600  ] 


400 


200- 


•  -High  temp  cycle 
x-Low  temp  cycle 


X 

X    X 
X 

x  x 


10         15         20 
Time  (days) 


25       30 


Figure  4.  Effect  of  cyclic  temperature  regimes  on  growth  rate  of  Triops 
limgicaiidalu.s.  Each  point  represents  the  mean  wet  mass  of  all  animals 
raised  in  a  single  pool  (see  Materials  and  Methods  for  details).  Squares 
represent  mean  wet  mass  for  animals  raised  at  a  mean  temperature  of 
25.7°C.  cycled  between  19°  and  31°C  (r  =  57.7.v  -  190.7,  r2  =  0.91). 
Crosses  represent  mean  wet  mass  for  animals  raised  at  a  mean  temperature 
of  23.4°C.  cycled  between  19"  and  29°C  (y  =  56.8.V  -  271.3,  r2  =  0.89). 
Positions  ot  regression  lines  were  significantly  different  (p  <  0.02). 


26 


D.  A.  SCHOLNICK 


1000 


CT 

10 
CO 

ro 


800- 


600 


400- 


200- 


•  -Hyperoxic  (200  torr) 
x-Hypoxic  (70  torr) 


10          15 
Time  (days) 


20 


25 


Figure  5.  Effect  of  oxygen  tension  on  growth  rate  of  7V/iy.v  longi- 
caudatus.  Each  point  represents  the  mean  mass  of  all  individuals  in  a 
single  pool  (see  Materials  and  Methods  for  details).  Squares  represent 
mean  wet  mass  for  animals  raised  at  constant  hyperoxia  (200  torr).  Crosses 
represent  mean  wet  mass  for  animals  raised  at  constant  hypoxia  (70 
torr).  Slopes  of  lines  are  significantly  different  (ANCOVA  p  <  0.01). 


The  two  extant  genera  of  Notostraca  ( Triops  and  Lcpi- 
diirus)  exhibit  an  extremely  high  degree  of  morphological 
stasis  (Fryer,  1985).  The  specific  adaptations  these  species 
use  may  have  evolved  quite  early  and  remained  relatively 
unchanged  throughout  geologic  time.  Of  the  two  genera, 
Triops  prefers  warm  habitats,  whereas  Lepit/iinis  prefers 
cooler  regions  and  more  permanent  pools  (Fryer,  1988). 
Eriksen  and  Brown  (1980)  determined  that  l'o:  in  Lcpi- 
chinis  Icmmoni  is  dependent  upon  both  oxygen  (35%  de- 
crease in  I'o2  from  saturation  to  critical  CK)  and  temper- 
ature (£>,„  ranging  from  2.1  to  2.7  from  18°-28°C).  In 
addition,  Shtcherbakov  and  Muragina  (1953)  reported  a 
Qio  of  2.2  at  15°C  for  Triops  cancriformis,  and  Hillyard 
and  Vinegar  (1972)  reported  a  lower  Qw  of  1 .55  between 
26°  and  30°C  for  T.  longicaudatus,  although  it  is  unclear 
what  oxygen  tensions  were  used  to  measure  metabolic 
rate.  The  relatively  high  temperature  sensitivity  of  the 
Notostraca  suggests  that  diurnal  fluctuations  in  temper- 
ature, even  in  vernal  pools  where  oscillations  may  be  less 
extreme  (Wiggins  el  al..  1980),  may  be  essential  to  elevate 
metabolism  and  thus  increase  rates  of  growth  and  repro- 
duction. 

Many  crustaceans  from  environments  that  regularly 
experience  large  fluctuations  of  oxygen  and  temperature 


160 


"     120 


A  =  Hyperoxic  (200  torr) 

D  =  Low  Temperature  (125  torr) 

•  =  High  Temperature  (125  torr) 


£ 


100 
o 
o 

CD     80 


100   200   300   400   500   600   700   800   900   1000 

Mass  (mg) 

Figure  6.  Relationship  between  mass  and  fecundity  for  Triops  ton- 
giciniilalii*  Triangles  represent  animals  raised  at  constant  hyperoxic 
conditions  (200  torr)  and  a  mean  temperature  of  25. 7°C,  cycled  between 
19°  and  3I°C.  Open  squares  represent  animals  raised  at  a  mean  tem- 
perature of  23.4°C  (cycled  between  19°  and  29°C),  and  closed  squares 
represent  animals  raised  at  mean  temperature  of  25.7°C  (cycled  between 
19°  and  31°C)  and  normoxia.  Regression  lines  are  for  all  data  at  125 
torr  and  animals  raised  at  hyperoxia.  Slope  of  hyperoxic  line  is  signifi- 
cantly different  than  temperature  line  (ANCOVA  p  <  0.05). 


are  known  to  regulate  oxygen  consumption  (see  Taylor, 
1988.  for  a  review).  Numerous  mechanisms,  including 
increasing  ventilation  volume  and  changing  heart  rate, 
pH,  or  the  oxygen-transporting  properties  of  the  blood, 
have  been  shown  to  aid  in  the  regulation  of  oxygen  uptake. 
Thus,  metabolic  independence  appears  to  be  adaptive  for 
many  crustaceans.  The  results  presented  here  suggest  the 
opposite — that  there  are  strong  selection  pressures  on  T. 
longicaudatus  to  maintain  metabolic  sensitivity  in  pre- 
dictably high  temperature-high  oxygen  environments. 

Lowering  the  average  ambient  temperature  by  2.3°C 
significantly  decreased  the  body  mass  of  the  tadpole 
shrimp  but  did  not  change  rate  of  growth  except  early  in 
life  (<5  days).  This  result  suggests  that  temperature  sen- 
sitivity before  adulthood  may  be  critical.  Hillyard  and 


Table  I 

Estimated  fecundity  <>/  a  \ingli'  female  T.  longicaudatus 

n\ci  11  .V-i/tir  MUVIW 


Dav  first  clutch 

Cumulative  no. 

oviposited 

of  eggs  laid 

High  temp,  cycle  (25.7°C) 

7 

287 

Low  temp,  cycle  (23.4°C) 

8 

188 

Hyperoxic  (200  torr) 

7 

452 

Hypoxic  (70  torr) 

1  1 

74 

Assuming  one  clutch  oviposited  every  3  days  (estimated  from  Ahl, 
1983)  and  first  clutch  oviposited  at  200  mg  wet  mass  (Fig.  5). 


METABOLIC  SENSITIVITY  IN  TADPOLE  SHRIMP 


27 


Vinegar  (1972)  reported  that,  between  26°  and  30°C,  T. 
longicaiulatiis  oxygen  consumption  rates  were  almost 
three  times  more  temperature-sensitive  in  immature  an- 
imals than  in  adult  animals.  Therefore,  temperature  sen- 
sitivity in  Trio/is  may  he  elevated  immediately  after 
hatching,  when  pools  have  recently  filled  and  temperature 
fluctuations  are  reduced  (Scholnick.  1994). 

Unlike  changes  in  temperature,  changes  in  ambient 
oxygen  tensions  affect  growth  rate  throughout  the  life  of 
the  animal.  This  result  is  not  surprising  considering  the 
large  metabolic  oxygen  sensitivity  exhibited  by  T.  longi- 
caudatus.  The  dependence  of  metabolism  on  oxygen  ten- 
sion, even  above  saturation,  suggests  that  oxygen  diffusion 
is  limiting  I'o2  throughout  the  physiological  range  of  the 
animal.  Although  an  extracellular  hemoglobin  with  a  rel- 
atively high  affinity  for  oxygen  has  been  described  for  T. 
longicaudatus  (P^  =  6.8  at  pH  =  7  and  22°-23°C;  Home 
and  Beyenbach,  1971),  the  unspecialized  respiratory 
structures  (bulbous  epipodite.  Fryer,  1988;  and  entire  ab- 
dominal phyllopod)  in  this  primitive  crustacean — unlike 
the  specialized  respiratory  structures  in  more  advanced 
Crustacea — appear  to  be  inadequate  for  adjusting  oxygen 
delivery  at  variable  oxygen  tensions.  In  view  of  the  high 
degree  of  morphological  stasis  exhibited  by  tadpole  shrimp 
(Fryer,  1985)  and  the  possible  reproductive  advantages  of 
maintaining  oxygen  sensitivity,  the  selection  pressure  for 
more  specialized  respiratory  structures  is  probably  low. 

T.  longicaudatus  does  not  appear  to  fit  the  typical  pat- 
tern of  an  active  oxygen  conformer  (for  reviews  see  Her- 
reid,  1980;  Prosser,  1991).  ]'o2  in  T.  longicaudatus  is 
highly  dependent  on  ambient  oxygen  tensions  well  above 
oxygen  saturation.  Therefore,  J'o2  is  seemingly  influenced 
by  ambient  oxygen  tension  at  all  oxygen  tensions  within 
this  species'  physiological  range.  Few  studies  have  ex- 
amined the  physiological  effects  of  hyperoxia;  however, 
ventilation  was  found  to  decrease  in  both  crayfish  (Mas- 
sabuau  ci  ai.  1984)  and  lugworm  (Toulmond  and  Tcher- 
nigovtzeff,  1984)  in  response  to  an  increase  in  oxygen.  In 
the  few  decapod  crustaceans  studied,  \'O2  remains  inde- 
pendent of  oxygen  tensions  above  saturation  (Dejours  and 
Beekenkamp,  1977;  Jouve-Duhamel  and  Truchot,  1983). 
T.  longicaudatus  apparently  does  not  stabilize  Vo2  against 
hyperoxia.  Furthermore,  predawn  measurements  of  oxy- 
gen in  ephemeral  desert  pools  (Scholnick,  1994)  indicate 
that  partial  pressures  of  oxygen  typically  do  not  drop  below 
40  torr  and  should  therefore  not  have  a  large  negative 
effect  on  I  'o2 . 

Growth  in  T.  longicaudatus  is  dependent  on  high  tem- 
perature. In  addition,  growth  and  survivorship  are  strongly 
influenced  by  the  large  fluctuations  of  temperature  and 
oxygen  that  are  characteristic  of  these  habitats  (Home, 
1971;  Scholnick,  1 994).  Home  ( 1 97 1 )  and  Scott  and  Gri- 
garick  (1979)  found  that  T.  longicaudatus  eggs  did  not 
hatch  until  temperatures  were  greater  than  14°C  and  that 


the  rates  of  hatching  were  highest  above  22°C  (Takahashi, 
1977).  Additionally,  Scott  and  Grigarick  (1978)  reported 
high  mortality  rates  and  slow  growth  rates  (about  9  times 
lower  growth  rates  than  those  reported  in  this  study)  for 
animals  raised  at  25°  or  30°C  compared  to  those  raised 
under  diurnal  oscillations,  as  in  this  study.  Therefore, 
hatching,  growth  rate,  and  survivorship  in  T.  longicau- 
datus all  appear  to  be  extremely  dependent  on  the  high 
temperature  conditions  characteristic  of  ephemeral  desert 
pools. 

A  small  change  in  average  daily  temperature  or  oxygen 
tension  has  a  large  effect  on  the  total  number  of  eggs  that 
can  be  laid  in  a  season.  Calculations  based  on  differences 
in  mass  indicate  that  over  a  22-day  season  an  individual 
can  produce  about  30  more  eggs  in  response  to  an  increase 
of  1°C  in  mean  temperature.  In  addition,  a  10-torr  in- 
crease in  ambient  oxygen  results  in  production  of  about 
43  more  eggs  during  a  22-day  season.  Therefore,  small 
changes  in  temperature,  oxygen,  or  both  between  seasons 
can  dramatically  affect  fecundity  and  reproductive  success. 
The  dependence  of  initial  oviposition  times  on  temper- 
ature and  oxygen  would  exaggerate  this  effect  when  rainfall 
is  limited  and  seasons  are  compressed.  Ahl  (1991)  and 
Seaman  el  al.  (1991)  reported  a  similar  relationship  be- 
tween carapace  length  and  fecundity  for  the  tadpole 
shrimp  Lepidunis  packardi  and  Triops granarius,  respec- 
tively. Although  it  was  not  possible  to  measure  egg  via- 
bility, a  change  in  viability  would  be  unlikely  to  offset  the 
large  effect  of  oxygen  and  temperature  on  fecundity.  Food 
for  tadpole  shrimp  is  abundant  in  ephemeral  pools  (Dod- 
son.  1987).  suggesting  that  the  limiting  factor  in  these 
environments  is  not  food  availability  but  the  time  nec- 
essary to  complete  the  life  cycle. 

The  results  from  this  investigation  suggest  that  meta- 
bolic sensitivity  in  high  temperature-high  oxygen  envi- 
ronments enhances  the  reproductive  success  of  Triops 
longicaudatus.  Shortened  development  times  and  in- 
creased fecundity  are  critical  for  success  in  ephemeral  en- 
vironments. Because  Vo2  is  associated  with  aerobic  energy 
metabolism  and  appears  to  be  diffusion-limited  in  Triops 
longicaudatus,  the  combination  of  hyperoxia  and  high 
temperature  may  engender  a  higher  rate  of  energy  me- 
tabolism and  in  turn  a  higher  growth  and  reproductive 
output.  Thus,  there  appear  to  be  reproductive  advantages 
for  metabolic  sensitivity  to  the  hot,  hyperoxic  conditions 
in  the  ephemeral  pools  inhabited  by  Triops  longicaudatus. 

Acknowledgments 

The  author  is  grateful  to  Dr.  G.  K.  Snyder  for  valuable 
advice  and  comments  on  the  manuscript.  This  work  was 
supported  in  part  by  National  Science  Foundation  Grant 
DCB88 18647  to  G.  K.  Snyder. 


28 


D.  A.  SCHOLNICK 


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Reference:  fl/o/  Bull  189:  29-35.  (August. 


Microfilament  Contraction  Promotes  Rounding  of 

Tunic  Slices:  An  Integumentary  Defense  System  in  the 

Colonial  Ascidian  Aplidium  yamazii 


EUICHI  HIROSE1  AND  TERUHISA  ISHII2 

*  Biological  Laboratory,  College  of  Agriculture  and  Veterinary  Medicine,  Nilion  University,  Fujisawa. 
Kanagawa  252,  Japan;  and  -Shimoda  Marine  Research  Center.  University  of'Tsukiiha.  Shimoda. 

Shizuoka  415,  Japan 


Abstract.  In  Aplidium  yamazii.  when  a  slice  of  a  live 
colony  (approximately  0.5  mm  thick)  was  incubated  in 
seawater  for  1 2  h.  the  slice  became  a  round  tunic  fragment. 
This  tunic  rounding  was  inhibited  by  freezing  of  the  slices, 
incubation  with  Ca2+-Mg2+-free  seawater.  or  addition  of 
cytochalasin  B.  Staining  of  microfilaments  in  the  slices 
with  phalloidin-FITC  showed  the  existence  of  a  cellular 
network  in  the  tunic.  Contraction  of  this  cellular  network 
probably  promotes  rounding  of  the  tunic  slice.  In  electron 
microscopic  observations,  a  new  tunic  cuticle  regenerated 
at  the  surface  of  the  round  tunic  fragments;  the  tunic  cu- 
ticle did  not  regenerate  in  newly  sliced  specimens  nor  in 
specimens  in  which  rounding  was  experimentally  inhib- 
ited. 

Based  on  these  results,  an  integumentary  defense  system 
is  proposed  in  this  species  as  follows.  ( 1 )  When  the  colony 
is  wounded  externally,  contraction  of  the  cellular  network 
promotes  tunic  contraction  around  the  wound.  (2)  The 
wound  is  almost  closed  by  tunic  contraction.  (3)  Tunic 
contraction  increases  the  density  of  the  filamentous  com- 
ponents of  the  tunic  at  the  wound,  and  it  may  accelerate 
the  regeneration  of  tunic  cuticle  there. 

Introduction 

The  integumentary  tissues  of  metazoans  commonly 
function  to  protect  the  body  from  a  hostile  environment 
and  as  a  transporting  surface,  and  they  display  various 
structures.  Some  of  these  tissues  have  a  keratinous,  col- 
lagenous.  or  chitinous  cuticle,  and  some  have  a  ciliated 
surface  or  a  mucous  layer.  The  body  of  urochordates 

Received  18  November  1994;  accepted  23  May  1995. 


(tunicates)  is  usually  covered  with  a  leathery  or  gelati- 
nous matrix  called  the  tunic.  The  tunic  is  a  peculiar 
integumentary  tissue  in  metazoans  for  the  following  two 
reasons.  First,  the  tunic  contains  cellulosic  fibers  that 
link  proteins  (De  Leo  el  til..  1977;  Van  Daele  et  ai. 
1992).  Second,  live  free  cells,  called  tunic  cells,  are  dis- 
tributed within  the  tunic,  which  is  thus  a  mesenchyme- 
like  tissue.  In  ascidians,  the  tunic  cells  are  involved  in 
various  biological  functions,  such  as  phagocytic  activity 
(De  Leo  et  ai.  1981:  Hirose  et  ai.  1994a),  conduction 
of  impulses  (Mackie  and  Singla.  1987),  and  biolumi- 
nescence  (Aoki  et  ai.  1989).  Because  it  is  unique,  in- 
vestigations on  tunic  morphology  and  functions  may 
lend  perspective  to  our  understanding  of  integumentary 
and  mesenchymal  tissues. 

Aplidium  yamazii  is  a  colonial  ascidian  (Polyclinidae. 
Aplousobranchia)  with  a  gelatinous,  transparent  tunic  in 
which  elongated  forms  of  zooids  are  embedded  separately 
from  each  other.  The  tunic  is  overlaid  by  a  thin  cuticle, 
and  the  cuticular  surface  has  numerous  minute  protru- 
sions, each  about  60  nm  in  height  (Hirose  et  ai.  1990). 
The  tunic  cuticle  has  a  dense  structure  that  is  probably 
an  effective  barrier  to  the  invasion  of  microorganisms  into 
the  tunic.  Various  kinds  of  tunic  cells  are  distributed  in 
the  tunic  of  this  species  (Hirose  et  ai.  1994b).  and  no 
colonial  vascular  network  (tunic  vessels)  connects  the 
zooids.  When  tunic  slices  are  incubated  in  seawater,  they 
spontaneously  round  up  to  form  tunic  balls.  This  phe- 
nomenon is  presumed  to  represent  the  mechanism  by 
which  external  injuries  of  the  tunic  are  healed.  In  this 
study,  we  examine  the  mechanism  of  rounding  of  the  slices 
and  discuss  its  functions  as  an  integumentary  defense  sys- 
tem of  this  tissue. 


29 


30 


E.  HIROSE  AND  T.  ISHII 


Materials  and  Methods 

Animals 

The  colonies  of  Apliilhtin  yainaiii  were  collected  in 
Nabeta  Bay,  Shimoda  (Shizuoka  Prefecture,  Japan).  They 
were  temporarily  kept  in  running  seawater  in  the  labo- 
ratory or  reared  in  a  culture  box  immersed  in  Nabeta  Bay. 
A.  vamaiii  forms  a  relatively  flat  colony,  about  2  to  3  mm 
thick,  spreading  on  a  flat  substratum;  its  rod-shaped  zooids 
are  embedded  separately  in  a  common  tunic  (Fig.  1 ). 

Tunic  rounding  assay 

Colonies  were  transversely  sliced  with  a  razor  blade 
into  pieces  that  were  0.5  mm  thick  or  less  (e.g..  7  X  2  X 
0.5  mm).  The  tunic  slices  (Hiroset'/a/.,  1994a)  were  com- 
posed of  tunic,  tunic  cells,  and  small  fragments  of  zooids; 
many  of  the  zooid  fragments  were  washed  out.  The  fresh 
tunic  slices  were  placed  in  a  plastic  petri  dish  filled  with 
filtered  seawater  (FSW)  or  artificial  seawater  (ASW),  and 
were  incubated  overnight  at  17°  to  20°C.  During  the  in- 
cubation, each  specimen  shrank  and  became  a  single  tunic 
ball. 

Video  recording  of  tunic  rounding 

The  tunic  slices  were  put  in  a  100-ml  beaker  filled  with 
FSW  (16°-18°C),  and  the  process  of  tunic  rounding  was 
recorded  with  a  time-lapse  videocassette  recorder  (AG- 
6010;  National,  Osaka,  Japan)  and  a  video  camera  ( WV- 
1800;  National)  equipped  with  a  55-mm  macro  lens  (Mi- 
cro-Nikkor;  Nikon,  Tokyo).  Recording  was  performed  at 
about  1/60  of  the  actual  speed.  The  time  course  of  trans- 
formation was  analyzed  by  hourly  measurements  of  the 
length  of  the  longest  diagonal  line  that  could  be  drawn 
within  the  profile  of  the  rounding  specimens. 

Tunic  rounding  assay  under  experimental  conditions 

Some  of  the  slices  were  frozen  at  -20°C.  thawed  at 
room  temperature,  and  then  incubated  in  ASW. 

Live  slices  were  incubated  in  three  kinds  of  experi- 
mental media:  Ca2+-Mg24-free  artificial  seawater  (CMF- 
ASW),  various  concentrations  of  colchicine-ASW.  and 
various  concentrations  of  cytochalasin  B-ASW.  Because 
cytochalasin  B  was  dissolved  in  dimethylsulfoxide 
(DMSO)  before  dilution  in  ASW,  the  assay  was  also  car- 
ried out  in  1%  DMSO-ASW  as  a  control.  All  media 
also  contained  penicillin  (100  ILI/ml)  and  streptomycin 
(1  Mg/ml). 

Staining  with  phalloidin-fluorescein  isothiocyanate 
(FITC) 

Microfilaments  were  visualized  in  colony  slices  by  la- 
beling with  phalloidin-FITC.  The  specimens  were  fixed 


tm 


tc 


Figure  I .  Schematic  drawing  showing  the  frontal  section  of  a  colony. 
A  zooid  (z)  is  embedded  in  the  tunic  matrix  (tm).  Tunic  cells  (tc)  are 
distributed  throughout  the  tunic.  There  are  no  blood  vessels  in  the  tunic. 


with  3.5%  formaldehyde  in  Ca2+-free  artificial  seawater 
(CF-ASW)  for  10  min.  made  permeable  with  0. 1%  Triton 
X-100  in  CF-ASW  for  5  min,  and  washed  with  phosphate- 
buffered  saline  (PBS).  They  were  incubated  with  1  //g/ml 
phalloidin-FITC  in  PBS  for  30  min  and  then  were  rinsed 
extensively  with  PBS.  Some  fixed  specimens  were  embed- 
ded in  O.C.T.  compound  so  that  cryostat  sections  could 
be  made.  Sections  that  were  20  ^m  thick  were  stained 
with  2  /ig/ml  phalloidin-FITC  in  PBS  for  30  min  and 
rinsed  with  PBS.  These  specimens  were  observed  under 
a  microscope  equipped  with  epifluorescence  and  Nomar- 
ski  differential  interference  contrast  optics. 

Chemicals 

ASW.  CF-ASW,  and  CMF-ASW  were  obtained  from 
Jamarine  Lab.,  Osaka,  Japan.  Colchicine,  cytochalasin  B, 
and  phalloidin-FITC  were  from  Sigma  Chemical  Co.,  St. 
Louis.  Missouri.  O.C.T.  compound  was  from  Miles  Inc., 
Naperville,  Illinois. 

Electron  microscopy 

The  specimens  were  fixed  in  2.5%  glutaraldehyde-ASW 
or  2.5%^  glutaraldehyde-0.1  Mcacodylate-0.45  At  sucrose 
(pH  7.4).  They  were  rinsed  with  the  same  buffer,  postfixed 
in  1%  osmium  tetroxide-0. 1  A/cacodylate  (pH  7.4).  and 
dehydrated  through  graded  ethanol.  For  scanning  electron 
microscopy  (SEM).  the  specimens  were  dried  in  a  critical- 
point  dryer,  coated  with  Au-Pd,  and  examined  in  a  Hitachi 
S-570  scanning  electron  microscope  at  20  kV.  For  trans- 
mission electron  microscopy  (TEM).  the  dehydrated 
specimens  were  cleared  with  n-butyl  glycidyl  ether  and 
embedded  in  low-viscosity  epoxy  resins.  Thin  sections 
were  stained  with  uranyl  acetate  and  lead  citrate  and  were 
examined  in  a  Hitachi  HS-9  transmission  electron  mi- 
croscope. 


ROUNDING  OF  ASCIDIAN  TUNIC  SLICES 


31 


Figure  2.  A  freshly  cut  tunic  slice  (A)  and  a  rounding  tunic  specimen 
after  incubation  in  FSW  for  24  h  (B).  Arrowhead  indicates  a  zooid  frag- 
ment extruded  from  the  tunic  ball.  Magnifications  of  these  two  figure 
parts  are  the  same.  Scale  bar  =  1  mm. 


Results 

Freshly  cut  tunic  slices  were  basically  thin  rectangular 
pieces  of  tunic  (Fig.  2A),  and  each  of  them  rounded  up 
into  an  elastic  tunic  ball  after  incubation  in  ASW  (Fig. 
2B).  The  tunic  ball  was  completely  filled  with  tunic  matrix, 
and  no  hollows  remained.  During  the  incubation,  some 
of  the  zooid  fragments  were  pushed  out  from  the  rounding 
tunic  specimens,  and  the  others  were  packed  inside  the 
tunic  ball,  but  rounding  occurred  even  if  every  zooid  frag- 
ment in  a  tunic  slice  was  lost.  Tunic  rounding,  therefore, 
did  not  depend  on  the  presence  of  zooids  or  zooid  frag- 
ments. The  size  of  a  tunic  ball  depended  on  the  initial 
size  of  the  slice  and  the  quantity  of  zooid  fragments  that 
were  lost  during  rounding.  For  instance,  tunic  slices  of 
about  2.5  X  5  X  0.5  mm  transformed  to  tunic  balls  of  2 
to  2.7  mm  in  diameter.  In  a  few  cases,  one  tunic  slice 
would  round  up  into  two  or  three  balls  connected  to  each 
other  by  thin  strands  of  tunic  material,  or  a  tunic  slice 
deformed  into  a  rodlike  or  irregularly  shaped  mass  of 
tunic.  Within  a  tunic  ball  or  deformed  tunic  mass,  the 
tunic  cells  were  alive  and  some  were  motile.  Noticeable 
tunic  rounding  began  4  to  5  h  after  a  slice  was  prepared, 
and  proceeded  gradually  for  about  20  h;  typical  time 
courses  are  shown  in  Figure  3. 


10         15        20        25 
Incubation  time  (hr) 


30        35 


Figure  3.  Time  course  of  rounding  of  the  tunic  slices.  The  length  of 
the  longest  diagonal  line  was  measured  in  three  specimens  every  hour 
during  the  incubation  in  FSW. 


Table  I  shows  the  results  of  the  tunic  rounding  assay 
under  experimental  conditions.  In  ASW  (control),  most 
of  the  tunic  slices  became  tunic  balls;  those  that  did  not 
form  balls  stuck  on  the  surface  of  the  petri  dishes  and 
rounding  or  deformation  of  the  tunic  partially  occurred 
in  the  periphery  or  in  some  small  areas  in  these  slices. 
Tunic  slices  that  were  frozen  and  thawed  did  not  round 
up,  and  the  hardness  and  shapes  of  these  tunic  slices  were 
almost  unchanged  (Fig.  4A).  Tunic  rounding  was  com- 
pletely inhibited  in  CMF-ASW,  and  the  tunic  slices  were 
transformed  into  disorganized  soft  gel  (Fig.  4B).  These 
specimens  were  so  soft  that  they  were  easily  taken  to  bits 
by  handling  with  forceps.  Cytochalasin  B  also  inhibited 
the  rounding,  with  1  ng/m\  of  cytochalasin  B  being  enough 
for  complete  inhibition.  These  tunic  slices  became  softer 
than  either  new  or  frozen  slices,  and  they  were  swollen  to 
some  extent  (Fig.  4C).  Because  tunic  rounding  normally 
occurred  in  1%  DMSO-ASW.  the  small  amount  of  DMSO 


Table  I 

Rounding  of  colony  slices  under  experimental  conditions 

No.  of         No.  of  rounding 
Medium3  Concentration     specimens        specimens  (%) 


ASW  (control) 

224 

213(95) 

Freeze  treatment 

67 

0(0) 

CMF-ASW 

49 

0(0) 

Cytochalasin  B-ASW 

5  /ig/ml 

95 

0(0) 

1  Mg/ml 

67 

0(0) 

0.5  Mg/ml 

18 

13(72) 

0.3  ^g/ml 

18 

10(56) 

(1.1  ,ig/ml 

37 

32  (86) 

DMSO-ASW 

1% 

56 

51  (91) 

CoIchicine-ASW 

10  jig/ml 

30 

28(93) 

lOO^g/ml 

47 

42(89) 

3  ASW  =  artificial  seawater;  CMF-ASW  =  Ca:+-Mg2+-free  artificial 
seawater;  DMSO  =  dimethylsulfoxide. 


32 


E.  HIROSE  AND  T.  ISH1I 


Figure  4.  Tunic  slices  assayed  under  experimental  conditions  after 
a  24-h  incubation:  frozen  and  thawed  once  (A),  incubated  in  Ca2+-Mg2+- 
free-ASW  (B),  incubated  in  cytochalasm  B-ASW  (5  Mg/ml)  (C),  incubated 
in  colchicine-ASW  ( 10  Mg/ml)  (D):  Tunic  rounding  occurred  only  in  D. 
The  slice  was  transformed  into  disorganized  soft  gel  in  B.  Magnifications 
are  the  same  for  all  parts  of  this  figure.  Scale  bar  =  1  mm. 


contained  in  cytochalasin  B-ASW  did  not  have  an  inhib- 
itory effect  (see  Table  I).  Colchicine  did  not  inhibit  tunic 
rounding  even  in  high  concentrations  (Fig.  4D). 

The  inhibitory  effects  on  rounding  by  cytochalasin  B 
suggest  that  microfilaments  promote  tunic  rounding.  Mi- 
crofilaments  in  the  tunic  slices  were  visualized  by  labeling 
with  phalloidin-FITC.  which  stained  cellular  microfila- 
ments of  tunic  cells,  particularly  their  filopodia.  In  A.  ya- 
nnnii.  a  type  of  tunic  cell,  called  an  elongated  tunic  cell, 
extended  long  cellular  processes  that  were  stained  exten- 
sively with  phalloidin-FITC.  The  elongated  tunic  cells  ap- 
peared to  form  a  cellular  network  by  contacting  each  other 
with  their  cellular  processes  (Hirose  et  ai.  1994b)  (Fig. 
5A).  But,  this  cellular  network  disappeared  in  tunic  slices 
that  had  been  incubated  in  CMF-ASW  (Fig.  5B)  or  cy- 
tochalasin B-ASW  (Fig.  5C).  In  these  specimens,  most  of 
the  tunic  cells  were  almost  spherical,  with  shortened  fi- 
lopodia or  none  at  all.  Cryostat  sections  were  prepared 


from  pieces  of  the  colony  (Fig.  5D)  and  rounding  tunic 
balls  (Fig.  5E).  The  cells  were  distributed  rather  uniformly 
in  the  tunics  of  colony  pieces.  After  rounding,  the  number 
of  tunic  cells  and  filamentous  materials  increased  signif- 
icantly in  cortical  area  of  the  specimens. 

SEM  observation  of  a  fresh  slice  reveals  the  tunic  matrix 
consisting  of  fine  filamentous  materials  that  densely  in- 
tertwine: the  surface  of  the  slice  has  a  sponge-like  structure 
(Fig.  6 A).  After  some  rounding,  a  dense,  sheetlike  material 
covered  the  surface  of  the  tunic  ball,  so  the  filamentous 
materials  were  not  exposed  (Fig.  6B);  and  TEM  obser- 
vation disclosed  an  electron-dense,  thin  layer  covering  the 
tunic  matrix  (Fig.  6C).  This  thin  layer  is  a  regenerating 
tunic  cuticle.  The  ascidian  tunic  is  always  overlaid  by  a 
cuticle  that  entirely  covers  the  matrix.  In  A.  yamazii,  the 
intact  tunic  cuticle  has  protrusions  of  about  60  nm  (Hirose 
et  a/..  1990)  (Fig.  6D),  and  tiny  protrusions  were  also 
found  in  the  regenerating  cuticle  of  the  rounding  tunic 
(Fig.  6C,  arrows).  Under  experimental  conditions  in  which 
the  tunic  did  not  round,  the  cuticle  did  not  regenerate, 
and  the  filamentous  materials  remained  exposed.  In  tunic 
slices  that  were  frozen  and  thawed  once,  the  surface  struc- 
tures were  almost  the  same  as  those  of  freshly  sliced  spec- 
imens (Fig.  6E).  In  the  tunic  slices  incubated  in  CMF- 
ASW  or  cytochalasin  B-ASW  (Fig.  6,  F  and  G),  the  fila- 
mentous materials  of  the  tunic  were  loosely  packed  in 
comparison  with  newly  sliced  specimens. 

Discussion 

Tunic  slices  of  Aplidium  yamazii  gradually  round  up 
in  seawater,  usually  becoming  round  tunic  masses  within 
24  h.  In  other  words,  the  superficial  area  of  the  specimen 
is  minimized  by  rounding.  Because  tunic  slices  that  had 
been  frozen  did  not  round,  live  tunic  cells  are  probably 
necessary  for  rounding.  Tunic  rounding  was  also  inhibited 
by  cytochalasin  B,  which  suggests  that  microfilaments  are 
involved  in  the  process.  Phalloidin-FITC  staining  allowed 
visualization  of  the  distribution  of  microfilaments  in  the 
tunic  slices,  and  it  revealed  a  network  of  tunic  cells  in- 
terconnected by  their  long  filopodia.  We  deduce  that  tunic 
rounding  is  promoted  by  contraction  of  this  cellular  net- 
work in  the  tunic;  that  is,  the  network  contracts,  carrying 
with  it  the  surrounding  gelatinous  tunic  matrix.  As  shown 
in  Figure  5E,  the  number  of  tunic  cells  and  filamentous 
materials  increase  in  the  cortical  area  of  the  tunic  ball. 
This  suggests  that  shrinkage  of  the  tunic  occurs  in  the 
cortical  area,  and  that  the  contraction  of  the  cellular  net- 
work probably  promotes  this  tunic  shrinkage.  The  com- 
plete inhibition  of  rounding  in  CMF-ASW  may  be  caused 
by  the  disappearance  of  the  cellular  network  (Fig.  5B).  In 
contrast  to  microfilaments,  microtubules  are  probably  not 
essential  for  tunic  rounding,  because  high  concentrations 
of  colchicine  were  not  inhibitory. 


ROUNDING  OF  ASCIDIAN  TUNIC  SLICES 


33 


IDJ 


IB 


Figure  5.  Microfilaments  in  the  tunic  specimens  stained  with  phalloidin-FITC.  In  a  newly  sliced  tunic, 
elongated  tunic  cells  extending  tilopodia  form  a  cellular  network  (A).  The  cellular  network  has  disappeared 
in  the  tunic  slices  incubated  in  Ca2*-Mg2*-free-ASW  (B),  or  incubated  in  cytochalasin  B-ASW  (10  Mg/ml) 
(C).  Cryostat  sections  of  colony  pieces  (D)  and  rounding  tunic  balls  (E).  showing  cortical  area  ot  the  specimens. 
Arrow  indicates  tunic  cuticle.  Magnifications  are  the  same  for  all  parts  of  this  figure.  Scale  bar  =  50  jim. 


The  tunic  cells  forming  the  network  have  been  de- 
scribed as  "elongated  tunic  cells"  (Hirose  et  al..  1994b) 
and  probably  correspond  to  the  "myocytes"  described 
in  Diploxoma  species  (Mackie  and  Singla.  1987).  The 
myocytes  also  form  a  network  in  the  tunic,  and  the  net 
of  myocytes  itself  is  supposed  to  conduct  impulses  that 
trigger  its  contraction,  according  to  electrophysiological 
studies  (Mackie  and  Singla,  1987).  Elongated  forms  of 
tunic  cells  were  also  reported  in  Leptodinides  echinatits 
(Hirose.  1992),  although  it  is  uncertain  whether  they 
form  a  network.  On  the  other  hand,  similar  types  of 
tunic  cells  or  tunic  cell  network  have  not  been  described 
in  other  colonial  ascidians  that  have  colonial  vascular 
networks  in  the  tunic,  such  as  Clavelina  miniata  (Aoki 
et  al.,  1989),  Perophora  viridis  (Deck  et  ill..  1966),  and 
Botryllus  and  Botrylloides  species  (Zaniolo,  1981;  Hi- 
rose et  ul.,  1991).  Although  the  epidermal  cells  of  the 
vascular  network  show  contractility  (Mukai  et  ill..  1978) 
and  impulse  conductivity  (Mackie  and  Singla,  1983), 
the  tunic  cell  network  may  be  uniquely  developed  in 


some  colonial  species  that  lack  a  colonial  vascular  net- 
work. 

The  fine-structure  study  revealed  that  the  tunic  cuticle, 
a  thin,  electron-dense  layer,  had  regenerated  in  the 
rounding  tunic  ball  and  covered  the  entire  surface.  In 
contrast,  the  tunic  cuticle  was  lacking  and  filamentous 
tunic  materials  were  exposed  at  the  surface  of  newly  sliced 
tunic  and  in  the  specimens  in  which  tunic  rounding  was 
inhibited.  Rounding  (or  tunic  shrinkage  at  the  cortical 
area)  may  be  necessary  for  cuticle  regeneration  at  the  ex- 
posed surface  of  the  tunic. 

Like  newly  sliced  tunics,  frozen  specimens  were  gelat- 
inous, and  the  filamentous  tunic  materials  were  inter- 
twined densely  at  the  surface.  When  the  slices  were  in- 
cubated in  CMF-ASW  or  cytochalasin  B-SW,  the  speci- 
mens became  much  softer  than  newly  sliced  tunic  or 
frozen  specimens;  moreover,  the  filamentous  materials  of 
the  tunic  were  loosely  packed,  and  the  cellular  network 
in  the  tunic  was  not  present.  The  cellular  network  of  elon- 
gated tunic  cells  may  also  be  important  for  maintaining 


34 


E.  HIROSE  AND  T.  ISHII 


Figure  6.  SEM  (A,  B,  E,  F,  and  G)  and  TEM  (C  and  D)  observation  of  tunic  slices  assayed  under 
experimental  conditions:  newly  sliced  surface  of  the  tunic  (A),  surface  of  rounding  tunic  (B  and  C).  tunic 
cuticle  of  intact  tunic  (D),  surface  of  tunic  slice  frozen  and  thawed  once  (E),  tunic  slice  incubated  in  Ca2+- 
Mg2+-free-ASW  (F),  tunic  slices  incubated  in  cytochalasin  B-ASW  (lO^g/mll  (G).  All  SEM  micrographs 
are  at  the  same  magnification.  Arrows  indicate  some  minute  protrusions  of  the  tunic  cuticle.  Scale  bars  = 
1  ^m  in  A,  0.2  ^m  in  C  and  D. 


tension  in  the  tunic  and  the  organization  of  the  tunic 
filaments. 

Tunic  rounding  is  presumed  to  represent  the  healing 
mechanism  that  is  initiated  when  the  exterior  of  the  tunic 
is  injured.  We  propose  the  following  integumentary  de- 
fense system  in  the  A.  yamaiii  tunic.  When  the  colony  is 
externally  wounded,  contraction  of  the  network  of  elon- 
gated tunic  cells  promotes  contraction  of  the  tunic  around 
the  wound.  Tunic  contraction  almost  closes  the  wound, 
and  it  minimizes  the  exposed  area  that  lacks  tunic  cuticle. 
At  the  same  time,  tunic  contraction  increases  the  density 
of  the  filamentous  components  of  the  tunic  at  the  wound, 
and  it  may  accelerate  the  regeneration  of  tunic  cuticle  so 
as  to  cover  the  exposed  surface  of  the  wound.  The  tunic 
cuticle  has  a  dense  structure  that  is  effective  in  preventing 
the  invasion  of  microorganisms.  Tunic  rounding  is,  how- 
ever, a  slow  process  in  which  noticeable  rounding  begins 
4-5  h  after  production  of  a  slice,  so  this  phenomenon 
may  not  be  an  effective  defensive  mechanism  in  the  early 


stages  after  an  injury.  If,  in  A.  yamaiii.  microorganisms 
invade  through  the  wounded  part  before  the  completion 
of  cuticle  regeneration,  phagocytic  tunic  cells  might  be 
expected  to  phagocytize  those  invaders  (Hirose  el  al, 
1994a).  This  integumentary  defense  system  is  unique 
among  metazoans,  and  it  appears  to  be  especially  suited 
to  the  organization  of  this  species,  which  has  a  large 
amount  of  tunic  outside  the  epidermis  and  no  vascular 
network  in  the  tunic.  In  this  system,  the  contractile  cellular 
network  in  the  tunic  may  work  like  the  dermal  or  epi- 
dermal muscle  does  in  other  metazoans. 

Acknowledgments 

This  study  was  supported  in  part  by  grants  from  Nihon 
University  and  from  the  Ministry  of  Education,  Science 
and  Culture  of  Japan  (#07456092).  Most  of  this  study 
was  performed  at  Shimoda  Marine  Research  Center 
(SMRC),  University  of  Tsukuba,  and  we  are  grateful  to 


ROUNDING  OF  ASCIDIAN  TUNIC  SLICES 


35 


the  staff  of  SMRC,  particularly  Dr.  Y.  Saito,  for  providing 
facilities.  We  also  thank  anonymous  referees  for  their 
valuable  comments.  The  present  study  includes  contri- 
bution No.  583  from  SMRC. 

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Reference:  Binl  Bull  189:  36-41.  (August,  1995) 


Life  Histories  and  Senescence  of  Botryllus  schlosseri 
(Chordata,  Ascidiacea)  in  Monterey  Bay 


NANETTE  E.  CHADWICK-FURMAN1  *   AND  IRVING  L.  WEISSMAN: 

*  Hopkins  Marine  Station  of  Stanford  University,  Pacific  Grove,  California  93950:  and  ^Department  of 
Pathology,  Stanford  University  Medical  School.  Stanford.  California  94305 


Abstract.  The  colonial  ascidian  Botryllus  schlosseri  is  a 
model  organism  for  research  on  invertebrate  histocom- 
patibility,  development,  and  evolutionary  biology.  None- 
theless, the  basic  life  history  of  Pacific  Ocean  populations 
of  the  species  remains  unknown.  We  determined  field  rates 
of  growth,  reproduction,  and  senescence  in  four  cohorts 
of  B.  schlosseri  colonies  in  Monterey  Bay,  California. 
Colonies  grew  exponentially  as  juveniles  and  reached  sizes 
of  up  to  1400  zooids  within  69  days.  After  a  juvenile  phase 
lasting  at  least  49  days,  the  colonies  began  to  reproduce 
sexually.  Each  zooid  produced  up  to  10  clutches,  each 
with  a  maximum  of  5  eggs,  resulting  in  very  high  fecundity 
of  up  to  8000  eggs  per  colony.  Following  a  short  period 
(maximum  70  days)  of  continuous  sexual  reproduction, 
colonies  abruptly  senesced  and  died  while  still  bearing  a 
full  clutch  of  eggs.  Senescence  progressed  through  four 
distinct  stages  over  1-2  weeks,  and  inevitably  led  to  the 
simultaneous  death  of  all  zooids  in  the  colony.  Although 
senescence  was  the  main  cause  of  mortality,  some  colonies 
died  as  a  result  of  predation  or  undetermined  causes.  Cer- 
tain life  history  traits  varied  significantly  between  cohorts 
that  settled  at  different  times  of  year.  For  example,  life- 
spans  in  the  field  varied  from  about  3  months  for  spring 
to  8  months  for  fall-born  colonies,  but  the  lifetime  fecun- 
dity of  colonies  did  not  vary  between  cohorts.  The  mor- 
phologies and  life  histories  of  colonies  monitored  in  the 
field  and  reported  here  differed  from  those  of  colonies 
cultured  previously  in  the  laboratory. 

Introduction 

The  colonial  ascidian   Botryllus  schlosseri  Pallas  is 
widely  employed  in  studies  on  invertebrate  alloimmunity 


Received  14  September  1493;  accepted  5  June 
*Present  address:  Interuniversity  Institute  for  Marine  Sciences.  P.O. 
Box  469,  Eilat.  Israel. 


(reviewed  by  Weissman  et  at..  1990;  Rinkevich,  1992; 
Sabbadin  et  al.  1992),  development  (Milkman,  1967; 
Mukai  and  Watanabe,  1976;  Rinkevich  et  al..  1992;  Lau- 
zon  et  al..  1993).  and  evolutionary  ecology  (Grosberg. 
1988;  Harvell  and  Grosberg,  1988;  Buss,  1990).  Most  of 
this  research  has  been  conducted  under  laboratory  con- 
ditions. Life  histories  of  B.  schlosseri  colonies  in  the  field 
are  known  for  some  populations  in  the  Atlantic  Ocean 
(Grave.  1933;  Grosberg,  1988)  and  Mediterranean  Sea 
(Brunetti,  1974).  The  life  histories  of  Pacific  Ocean  pop- 
ulations are,  however,  little  known,  despite  their  extensive 
use  in  laboratory  investigations  (Scofield  et  al.,  1982; 
Rinkevich  and  Weissman,  1987;  Lauzon  et  al..  1933,  and 
references  therein)  (Carwile,  1989).  B.  schlosseri  was 
probably  introduced  to  the  Pacific  Ocean  sometime  during 
the  last  century  as  one  of  the  fouling  organisms  on 
wooden-hulled  vessels  or  concomitant  with  the  culture  of 
Atlantic  oysters,  which  transferred  whole  organisms  and 
their  encrusting  communities  from  the  Atlantic  to  the 
Pacific  Ocean  (Carlton,  1987;  Hewitt,  1993).  Recent 
morphological  and  genetic  studies  indicate  that  B.  schlos- 
seri at  Woods  Hole  (Atlantic  Ocean)  and  Monterey  (Pa- 
cific Ocean)  are  the  same  species  (Boyd  et  al..  1990).  Re- 
sults of  laboratory  cultures  from  both  of  the  latter  pop- 
ulations have  been  used  to  infer  evolutionary  processes 
in  nature  (Harvell  and  Grosberg,  1988;  Weissman  et  al., 
1990;  Rinkevich.  1992,  and  references  therein).  Thus,  it 
becomes  important  to  understand  the  life  history  patterns 
of  B.  schlosseri  from  different  populations  and  under  dif- 
ferent culture  conditions. 

We  present  here  the  life  histories  of  B.  schlosseri  col- 
onies growing  under  field  conditions  in  Monterey  Bay, 
California.  We  describe  patterns  of  growth,  sexual  repro- 
duction, and  senescence  in  cohorts  that  settled  at  four 
times  of  year.  We  then  compare  these  field  life  histories 


36 


ASCIDIAN  LIFE  HISTORIES  AND  SENESCENCE 


37 


with  those  of  laboratory-cultured  colonies  from  the  same 
population. 

Materials  and  Methods 

We  conducted  the  present  study  during  1990-1991  in 
the  Monterey  Municipal  Marina,  Monterey  County,  Cal- 
ifornia (36°  37.41^,  121°  54'W).  Surface  seawater  tem- 
peratures varied  from  11.4°C  in  January  to  16.5°C  in 
August  (E.  C.  Haderlie,  pers.  comm.).  This  site  is  described 
in  detail  by  Haderlie  and  Donat  (1978)  and  Carwile 
(1989).  Colonies  of  Botryllm  schlosseri  grow  on  docks, 
floats,  and  pilings  throughout  the  marina  and  seasonally 
dominate  the  fouling  community  (pers.  obs.,  N.  E.  Chad- 
wick-Furman).  Colonies  of  B.  schlosseri  sexually  repro- 
duce throughout  the  year  at  Monterey;  sexual  generations 
overlap  and  cohorts  are  not  discrete  (Carwile.  1989). 

To  determine  life  history  patterns,  we  monitored  co- 
horts of  B  schlosseri  that  settled  at  four  arbitrarily  chosen 
dates:  19  May  1990,  3  July  1990,  15  October  1990,  and 
25  January  1991.  To  obtain  each  cohort,  we  collected  10 
large  colonies  from  wooden  pilings  at  0-1  m  depth  in  the 
marina,  transported  them  to  Hopkins  Marine  Station  of 
Stanford  University,  and  maintained  them  in  flowing  sea- 
water  at  ambient  temperature.  We  secured  the  colonies 
with  string  to  glass  plates  and  placed  them  vertically  in 
aquaria,  with  an  empty  plate  facing  each  colony.  Within 
a  few  days,  they  released  swimming  larvae  that  rapidly 
settled  and  metamorphosed  into  sessile  zooids  on  the  fac- 
ing plates.  We  then  isolated  each  newly  settled  zooid  on 
a  separate  5.0  X  7.5  cm  glass  plate,  and  allowed  it  to  firmly 
attach  during  1  week  in  the  laboratory.  For  each  cohort, 
we  transplanted  at  least  25  newly  settled,  isolated  zooids 
to  the  marina  field  site. 

In  the  marina,  we  placed  the  zooids  in  wooden  racks 
and  hung  them  face  down  from  floating  docks  at  0.5- 
1.0m  depth  (after  Brunetti,  1974;  Boyd  el  a/.,  1986; 
Grosberg,  1988).  Sessile  organisms  colonized  the  racks 
and  formed  a  fouling  community  around  the  experimental 
plates  (see  Carwile,  1989.  for  community  description). 
No  epibionts  were  observed  to  settle  on  individuals  of  B 
schlosseri. 

At  each  sample  interval,  every  4-7  days,  we  observed 
the  growing  colonies  in  the  laboratory  and  then  returned 
them  to  the  field  within  a  few  hours  (for  details  of  methods, 
see  Brunetti,  1974;  Grosberg,  1988).  They  showed  no  ad- 
verse effects  of  handling  (see  also  Milkman.  1967).  To 
avoid  effects  of  crowding  on  colony  growth,  we  removed 
all  other  organisms  from  the  plates  during  each  sample 
interval  (after  Brunetti  and  Copello,  1978;  Grosberg. 
1988).  Colonies  grew  over  both  sides  of  the  plates,  but  did 
not  fill  all  of  the  space  provided.  About  every  7  days,  de- 
pending on  the  time  of  year,  all  the  zooids  in  each  colony 
passed  through  an  asexual  growth  cycle  (hereafter  termed 


"cycle").  During  each  cycle,  the  zooids  produced  buds, 
then  shrank  and  were  replaced  by  their  buds,  which 
formed  a  new  asexual  generation  of  zooids  in  each  colony. 
The  replacement  of  zooids  during  each  cycle  in  Botryllm 
is  described  in  detail  by  Mukai  and  Watanabe  (1976)  and 
Grosberg  (1988).  Here  we  report  colony  age  in  terms  of 
both  the  number  of  cycles  and  the  days  since  settlement 
(after  Brunetti  and  Copello,  1978;  Grosberg,  1988). 

During  each  sample  interval,  we  determined  the  num- 
ber of  zooids,  number  of  eggs,  cycle  stage  (see  Mukai  and 
Watanabe,  1976),  and  the  general  condition  of  each  col- 
ony. In  colonies  of  less  than  800  zooids,  we  counted  zooid 
number  directly.  For  larger  colonies,  zooid  number  was 
estimated  by  placing  a  grid  over  the  colony  surface, 
counting  all  zooids  under  a  single  grid-square  (1.5  X 
1.8  cm),  and  multiplying  by  the  number  of  squares  oc- 
cupied by  the  entire  colony  (maximum  =  12).  To  deter- 
mine the  number  of  eggs  per  colony,  we  visually  estimated 
the  number  of  eggs  per  zooid,  then  multiplied  by  the  total 
number  of  zooids.  Eggs  were  observed  from  the  exterior 
of  whole,  undissected  colonies. 

The  causes  of  mortality  were  determined  by  analyzing 
colony  morphology.  In  senescing  colonies,  the  entire  or- 
ganism deteriorated  in  distinct  stages  during  the  1  -2  weeks 
preceding  death  (Brunetti,  1974;  Rinkevich  el  al.,  1992). 
Colonies  that  senesced  left  behind  a  residue  of  decaying 
tissue  that  distinguished  them  from  colonies  killed  by 
other  agents.  In  cases  of  predation.  colonies  showed  lo- 
calized lesions  and  then  sections  of  dead  tissue  that  in- 
creased in  area  for  several  weeks  before  the  complete  con- 
sumption of  the  colony  by  predators. 

Results 

Morphology  and  growth 

In  the  Monterey  Marina,  members  of  all  cohorts  of 
Botryllus  schlosseri  exhibited  the  same  general  morphol- 
ogy. Colonies  were  flat  and  roughly  circular  to  oval  in 
outline.  Their  zooids  were  closely  packed,  with  almost  no 
space  between  adjacent  zooids  or  systems  (circular  groups 
of  zooids).  Each  colony  formed  a  compact  disk  that  did 
not  fragment. 

Juvenile  colonies  in  all  cohorts  grew  exponentially  (Fig. 
1 ).  Colonies  that  settled  in  May  grew  significantly  faster 
than  those  in  all  other  cohorts  (Fig.  2A,  Table  I)  and 
reached  a  size  of  up  to  1400  zooids  in  69  days.  Members 
of  the  July  and  October  cohorts  grew  the  slowest,  at  rates 
that  did  not  differ  significantly  (Fig.  2a,  Table  I).  Colonies 
that  settled  during  October  and  January  delayed  their  ex- 
ponential growth  until  the  spring  months  (Fig.  1 ).  Some 
colonies  reduced  their  growth  rates  after  commencing 
sexual  reproduction  (Fig.  1 ).  In  addition,  15.6%  of  all  col- 
onies (N  =  122)  shrank  slighth  (by  15.2%  +  11.1%  in 
zooid  number.  A  +  SD)  during  the  2  weeks  preceding 


38 


N.  E.  CHADWICK-FURMAN  AND  I.  L.  WEISSMAN 
10.000-,    January  25  10.000-5     May    19 


5  10          15          20          25          30 

64        107       153      198      233      261 

Age  (cycles) 
Age  (days) 

Figure  1 .  Growth,  reproduction  and  lifespan  in  four  cohorts  of  the  colonial  ascidian  Bolrvllus  schlosseri 
in  Monterey  Bay.  For  clanty,  growth  curves  are  presented  for  only  four  colonies  per  settlement  date  (out  of 
>25  original);  they  represent  the  extremes  of  those  that  survived  to  reproduce  in  each  cohort:  the  smallest 
in  final  size  (triangle),  largest  in  final  size  (circle),  shortest-lived  (asterisk),  and  longest-lived  (square).  Horizontal 
bars  indicate  the  period  of  sexual  reproduction  for  the  entire  cohort.  Note  that  colony  size  is  plotted  on  a 
logarithmic  scale. 


death.  The  only  other  lapse  in  exponential  growth  oc- 
curred when  members  of  the  October  cohort  were  attacked 
by  an  unknown  predator  during  cycles  5-15  (Fig.  1 ). 

Sexual  reproduction 

After  a  period  of  exponential  somatic  growth,  the  col- 
onies entered  sexual  reproduction.  The  May  and  July  co- 
horts reached  sexual  maturity  the  earliest,  at  ages  that  did 
not  differ  significantly  (Fig.  2b,  Table  I).  The  minimum 
age  at  sexual  maturity  was  49  days  (7  cycles).  Members 
of  the  October  cohort  began  to  reproduce  when  signifi- 
cantly older  than  other  cohorts  (Table  I,  Fig.  2b).  They 
overwintered  as  small  juveniles  and  postponed  reproduc- 
tion until  spring,  at  a  minimum  age  of  1 53  days  ( 1 5  cycles) 
(Fig.  1). 

The  size  at  which  colonies  began  to  reproduce  varied 
widely  (range  =  38  to  1297  zooids)  but  did  not  differ 
significantly  between  most  cohorts  (Table  I).  Colonies  that 
settled  in  July  reproduced  at  the  smallest  sizes;  those  in 
January  and  May  at  the  largest  (Fig.  2c).  Due  to  differences 
in  growth  rate,  some  cohorts  matured  at  similar  sizes  but 
widely  different  ages  (compare  May  and  July  cohorts.  Fig. 
2b  and  c). 


Most  zooids  in  each  colony  produced  eggs  continuously 
throughout  the  period  of  sexual  reproduction.  The  du- 
ration of  reproduction  extended  for  7-70  days  (I- 
10  cycles),  with  one  clutch  of  eggs  produced  during  each 
cycle.  Some  cohorts  produced  significantly  more  clutches 
than  did  others  (Table  I).  Each  zooid  contained  up  to  five 
eggs  per  cycle,  although  most  zooids  produced  only  one 
to  two  eggs  per  cycle  (Fig.  2d).  Colonies  did  not  interrupt 
clutch  production  or  reduce  the  number  of  eggs  per  zooid 
as  they  aged;  mature  colonies  still  contained  a  full  clutch 
of  eggs  when  they  died. 

Lifetime  fecundity  was  very  high  (maximum  =  about 
8000  eggs).  Such  high  fecundity  was  possible  because  of 
the  large  number  of  zooids  in  adult  colonies  and  their 
ability  to  produce  multiple  clutches.  Lifetime  fecundity 
did  not  differ  significantly  between  colonies  that  settled 
at  different  times  of  year  (Table  I,  Fig.  20. 

Longevity  and  survivorship 

Colonies  grown  in  the  field  at  Monterey  had  short,  sub- 
annual  lifespans.  Maximum  lifespan  ranged  from  almost 
3  months  (82  days)  in  the  May  cohort,  to  just  over 
8  months  (247  days)  in  the  October  cohort  (Fig.  3). 


ASCIDIAN   LIFE  HISTORIES  AND  SENESCENCE 


39 


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Cohort 

Figure  2.  Variation  in  six  life-history  traits  among  four  cohorts  of 
the  colonial  ascidian  Botrvllus  schlosseri  in  Monterey  Bay.  Error  bars: 
positive  standard  de\iations  of  the  means. 


The  percentage  of  colonies  that  survived  to  first  repro- 
duction was  high  in  most  cohorts  (Fig.  3).  Colonies  that 
settled  in  October  had  lower  survivorship  to  maturity  than 
did  the  other  three  cohorts.  An  undetermined  predator 
began  to  attack  members  of  the  October  cohort  during 
cycle  5.  Mortality  increased  gradually,  and  by  the  15th 
cycle,  only  21%  of  the  cohort  remained  (Fig.  3).  Nev- 
ertheless, the  remaining  October  colonies  all  eventually 
reproduced  sexually  and  lived  longer  than  those  of  any 
other  cohort  (Figs.  1  and  3).  In  all  cohorts,  survivorship 
decreased  rapidly  after  commencement  of  sexual  repro- 
duction, and  all  colonies  then  died  within  10  cycles 
(Fig.  3). 

Senescence  caused  most  of  the  mortality  in  field-raised 
colonies  (54.9%  of  colonies.  A'  =  122).  After  a  period  of 
continuous  sexual  reproduction,  colonies  passed  through 
four  stages  of  degeneration,  as  previously  described  for  B. 


schlosseri (Brunetti.  1974;  Rinkevich  cl  a/.,  1992).  First, 
blood  vessels  narrowed  and  blood  flow  slowed.  Then,  the 
zooids  shrank  and  became  densely  pigmented.  In  the  third 
stage,  circular  systems  (groups)  of  zooids  were  discon- 
nected and  became  disorganized.  In  the  fourth  and  final 
stage,  the  protective  tunic  softened  and  disintegrated,  and 
all  of  the  tissue  died.  A  film  of  tunic  material  persisted 
for  at  least  1  week  after  death,  and  marked  the  former 
extent  of  the  colony.  Senescence  was  not  reversible.  In  all 
cases,  the  initial  stages  of  senescence  led  to  the  death  of 
the  entire  colony  within  1-2  weeks.  Some  colonies  se- 
nesced  while  still  in  the  juvenile  stage,  at  an  age  of  at  least 
70  days  (=10  cycles),  and  died  without  reproducing  sex- 
ually. The  occurrence  and  timing  of  senescence  did  not 
appear  to  be  related  to  the  position  of  the  colonies  in  the 
racks  or  to  other  extrinsic  factors. 

Other  agents  of  mortality  included  predation  (12.3%  of 
colonies,  A^  =  122.  described  above),  and  undetermined 
causes  of  death  early  in  life  (32.8%,  N  =  122).  In  the  latter 
case,  small  juvenile  colonies  suddenly  disappeared  from 
the  field  site  without  showing  any  previous  signs  of 
damage. 

Discussion 

We  demonstrate  here  that  Botryllus  schlosseri  colonies 
raised  in  the  field  at  Monterey  have  characteristic  mor- 
phologies, which  are  readily  distinguishable  from  those 
of  colonies  grown  under  laboratory  conditions.  In  the  field, 
isolated  colonies  are  rounded  and  compact  (Brunetti, 
1974;Grosberg,  1988;  Carwile.  1989;  this  paper).  In  con- 


Table  I 

Tukey-Kramer  multiple  comparisons  lest  for  differences  in  life  history 
traits  benveen  cohorts  of  the  ascidian  Botryllus  schlosseri  grown 
in  Monterey  Bay,  California,  during  1990-1991 


Life-history  trait 


Cohort* 


Growth  rate 

(#buds/zooid/cycle) 

Age  at  1st  reprod. 
(#  cycles) 

Size  at  1st  reprod. 
(#  zooids) 

Number  of 
eggs/zooid/clutch 

Clutch  number 


Fecundity 

(total  #  eggs/colony) 


Jan  May          Jul  Oct 


May          Jan  Oct          Jul 


May          Jan  Oct          Jul 


Jan  May          Oct          Jul 


Jan 


May          Jul  Oct 


*  Cohorts  that  did  not  differ  significantly  (p  >  0.05)  are  conjointly 
underlined.  See  text  for  details 


40 


N.  E.  CHADWICK-FURMAN  AND  I.  L.  WEISSMAN 


January  25 
(N   =  28) 


May    19 

(N   =  35) 


5 
32 


10 
69 


15 
103 


1) 
CL, 


July  3 
(N   =   26) 


5 
33 


10 
64 


15 
99 


20 
141 


Age  (cycles) 
Age  (days) 


October   15 
(N   =  33) 


in  Monterey  Bay.  Presented 


Figure  3.     Survivorship  curves  of  the  colonial  ascidian  Botryllus  vcW 
are  four  cohorts,  each  settled  at  the  date  shown.  Arrows  indicate  the  beginning  of  sexual  reproduction  in 
each  cohort.  Note  that  survivorship  is  plotted  on  a  logarithmic  scale. 


trast,  colonies  grown  in  the  laboratory  tend  to  branch  and 
produce  extensions  along  the  substratum;  these  extensions 
may  fragment  into  subcolonies  (Rinkevich  and  Weissman, 
1987;  N.  E.  Chadwick-Furman,  pers.  obs.). 

In  addition,  the  life  history  patterns  of  colonies  in  the 
field  seem  to  differ  from  those  in  the  laboratory.  Members 
of  all  field  cohorts  at  Monterey  exhibited  the  same  general 
features:  rapid  growth,  short  and  intense  reproduction, 
short  lifespan,  and  senescence  soon  after  reaching  maturity 
(Figs.  1-3).  In  contrast,  Monterey  colonies  raised  in  the 
laboratory  have  been  observed  to  grow  slowly  or  shrink 
over  many  months,  to  cease  reproduction  long  before 
death,  and  to  live  for  more  than  2  years  (Boyd  et  a/..  1986; 
Rinkevich  and  Weissman,  1987;  Rinkevich  et  ai.  1992). 
Also  in  Mediterranean  populations,  the  same  life  history 
differences  are  exhibited  between  field-  and  laboratory- 
raised  colonies  (Brunetti,  1974;  Brunetti  and  Copello. 
1978). 

Several  factors  may  be  responsible  for  these  differences. 
Under  laboratory  conditions,  water  motion  is  slower,  and 
paniculate  food  is  less  varied  and  abundant  than  for  B. 
schlosseri  populations  in  the  field  (Milkman,  1967;  Bru- 
netti and  Copello,  1978;  Carwile,  1989).  In  addition,  the 
absence  of  natural  grazers  in  the  laboratory  may  lead  to 
the  formation  of  a  fouling  film  that  inhibits  the  attachment 
and  growth  of  colonies  (Boyd  et  a/..  1986;  N.  E.  Chadwick- 
Furman,  pers.  obs.).  Laboratory  culture  is  important  for 


the  maintenance  of  genetically  defined  stocks  that  are 
employed  in  histocompatibility  studies  and  other  inves- 
tigations (reviewed  in  Sabbadin  et  ai.  1992;  Rinkevich  et 
ai,  1992).  Laboratory  culture  at  summer  temperatures 
also  allows  continued  production  of  experimental  tissues 
during  the  winter  when  field  colonies  in  Monterey  slow 
their  growth  (Boyd  et  ai,  1986).  Unfortunately,  however, 
the  life  history  traits  exhibited  by  laboratory  cultures,  in- 
cluding patterns  of  growth,  reproduction,  and  longevity, 
may  not  reflect  the  evolutionary  or  ecological  processes 
that  act  upon  B.  schlosseri  in  nature. 

In  the  field  at  Monterey,  life  history  variation  between 
cohorts  is  probably  related  to  seasonal  environmental  cy- 
cles. Factors  known  to  correlate  with  such  variation  in 
other  field  populations  ofB.  schlosseri  include  water  tem- 
perature and  paniculate  food  concentration  (Millar.  1971; 
Brunetti,  1974).  In  the  present  study,  we  observed  slowed 
growth  and  delayed  reproduction  of  young  colonies  during 
the  winter  months  when  temperature  and  planktonic  food 
levels  are  at  their  annual  minima  in  Monterey  Bay  (Had- 
erlie  and  Donat,  1978;  Boyd  et  ai,  1986;  Carwile,  1989, 
and  references  therein).  The  colonies  began  to  grow  ex- 
ponentially and  reproduce  sexually  in  the  spring  to  sum- 
mer when  the  above  two  factors  reach  their  annual  max- 
ima. In  some  localities,  B.  schlosseri  colonies  completely 
cease  sexual  reproduction  during  the  winter  when  tem- 
peratures fall  below  11°C  (Millar,  1971).  At  Monterey, 


ASCIDIAN  LIFE  HISTORIES  AND  SENESCENCE 


41 


however,  sea  temperature  remains  above  1 1  °C  all  year, 
so  mature  colonies  continue  to  produce  eggs  even  during 
January  (see  Methods). 

The  present  study  had  several  weaknesses.  Although 
colonies  were  cultured  in  the  sea.  they  did  not  grow  under 
completely  natural  conditions.  The  periodic  removal  of 
competitors  may  have  led  to  inflated  rates  of  growth  and 
reproduction.  Thus,  values  reported  here  are  probably 
maximal  in  the  absence  of  space  competition.  Also,  we 
did  not  monitor  colonies  immediately  following  natural 
settlement  in  the  field.  Thus,  survival  rates  are  probably 
inflated  because  we  did  not  determine  natural  mortality 
rates  during  the  first  1-2  weeks  of  life.  Finally,  we  con- 
ducted our  study  in  an  artificial  habitat,  on  a  non-native 
population  of  B.  schlosseri.  As  such,  the  life  histories  pre- 
sented here  are  not  those  of  a  natural  field  population. 
Members  of  this  species  probably  have  been  introduced 
over  much  of  their  current  range,  possibly  from  native 
populations  in  the  Mediterranean  Sea  (Carlton,  1987; 
Hewitt.  1993).  Indeed,  a  weakness  of  most  life  history 
studies  on  this  species  is  that  they  have  been  conducted 
on  introduced  populations  or  in  manmade  fouling  envi- 
ronments, or  both  (Millar,  1971;  Brunetti,  1 974;  Grosberg, 
1988;Carwile,  1989). 

In  spite  of  the  above  drawbacks,  the  data  presented 
here  give  the  most  complete  picture  to  date  of  life  histories 
and  morphologies  of  Pacific  Ocean  populations  of  B. 
schlosseri.  The  life  histories  of  Monterey  Bay  colonies  are 
quite  similar  to  those  of  iteroparous  colonies  at  Woods 
Hole  in  the  western  Atlantic  (Grave,  1933;  Grosberg, 
1988),  and  at  the  Venice  Lagoon  in  the  Mediterranean 
(Brunetti.  1974). 

We  present  here  the  first  description  of  senescence  in 
the  field  for  Monterey  B.  schlosseri.  Our  observations 
confirm  that  senescence  progresses  under  field  conditions 
through  essentially  the  same  stages  as  in  the  laboratory, 
and  takes  about  1-2  weeks  (Brunetti  and  Copello,  1978; 
Rinkevich  el  ill..  1992).  Senescence  appears  to  be  con- 
trolled intrinsically,  as  inferred  from  the  synchronized 
death  of  segregated  laboratory  clones  (Rinkevich  el  al., 
1992).  The  specific  factors  that  regulate  the  timing  and 
initiation  of  senescence  in  B.  schlosseri.  and  in  other  as- 
cidians,  remain  unknown. 

Acknowledgments 

We  thank  the  staff  of  Hopkins  Marine  Station  and  the 
Pathology  Department  of  Stanford  University  Medical 
School,  especially  Kathi  Ishizuka,  Karla  Palmeri,  and 
Margaret  Finney.  The  manuscript  benefitted  from  exten- 
sive comments  by  Richard  Grosberg,  Robert  Lauzon, 
Baruch  Rinkevich,  and  anonymous  reviewers.  This  pro- 
ject was  supported  by  a  Frederick  B.  Bang  Grant  from 
the  American  Association  of  Immunologists  and  a  post- 


doctoral fellowship  from  the  National  Cancer  Institute 
(PHS  Grant  Number  CA09302,  DHHS)  to  N.  E.  C.-F., 
and  by  USPHS  grants  to  I.  L.  W. 

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Reference:  Biol.  Bull  189:  42-4X.  (August,  1995) 


The  Interaction  of  Photoperiod  and  Temperature  in 
Diapause  Timing:  A  Copepod  Example 

NELSON  G.  HAIRSTON,  JR..  AND  COLLEEN  M.  KEARNS 

Sec/ion  of  Ecology  and  Systematics,  Cornell  University.  Ithaca.  New  York  14853 


Abstract.  In  many  organisms,  photoperiod  and  tem- 
perature are  thought  to  be  the  most  significant  token  cues 
for  seasonally  timed  life  history  events,  including  diapause 
in  arthropods.  A  common  pattern  in  many  species  of  ter- 
restrial insects  and  several  copepod  species  is  the  existence 
of  a  critical  daylength  on  one  side  of  which  the  animals 
do  not  enter  diapause  and  on  the  other  side  of  which  they 
do.  Temperature  plays  a  secondary  role  as  modifier  of  the 
critical  daylength.  In  some  species,  however,  including 
the  freshwater  copepod  Diaptonuis  sanguineus,  the  frac- 
tion of  females  making  subitaneous  eggs  (eggs  that  hatch 
immediately)  undergoes  a  very  gradual  transition  as  day- 
length  changes  over  the  natural  range  of  photoperiods 
experienced  in  nature.  Here  we  show  that  temperature  is 
as  important  as  photoperiod  in  cuing  diapause  timing  in 
a  population  of  D.  sanguiueus  living  in  Bullhead  Pond. 
Rhode  Island.  When  ecologically  relevant  photoperiod 
and  temperature  cues  are  provided  in  the  laboratory,  the 
copepods  rapidly  switch  from  producing  subitaneous  eggs 
to  producing  diapausing  eggs  in  a  way  that  is  typical  of 
the  seasonal  switch  seen  in  the  pond.  We  provide  a  graph- 
ical model  that  illustrates  how  copepod  sensitivities  to 
photoperiod  and  temperature  interact  to  produce  an 
abrupt  transition,  and  we  discuss  how  natural  selection 
should  act  on  D.  sanguinem  diapause  response  to  produce 
the  variation  in  diapause  timing  seen  within  and  between 
natural  populations. 

Introduction 

An  organism  that  times  its  life  history  to  seasonal  vari- 
ations in  its  habitat  must  be  able  to  detect  some  com- 
ponent of  the  environment  that  indicates  time  of  year. 
To  this  end.  many  plants  and  animals  perceive  and  re- 
spond to  daylength  (or  change  in  daylength)  as  a  cue  for 

Received  6  January  1995:  accepted  4  May  1995. 


such  phenological  events  as  leafing-out.  flowering,  and 
seed  set  in  plants  (Harper,  1977;  Begon  et  ai.  1990),  dia- 
pause and  dispersal  in  insects  (Tauber  et  ai.  1986).  and 
hibernation,  molting,  or  migration  in  various  vertebrates 
(Hairston,  1994).  Other  features  of  the  environment  (e.g., 
temperature,  resource  availability:  Danilevskii,  1965; 
Tauber  el  ai.  1986)  can  also  play  a  role,  but  are  typically 
thought  to  be  less  important  because  they  are  less  reliable 
seasonal  indicators.  For  example,  photoperiod  is  the  most 
common  environmental  factor  cuing  the  onset  of  diapause 
in  temperate-zone  insect  populations,  and  temperature  is 
typically  seen  as  one  of  several  possible  modifiers  of  the 
photoperiod  response  (Lees.  1955;  Danilevskii.  1965; 
Beck,  1980;  Tauber  et  ai.  1986).  A  common  pattern  for 
insects  that  overwinter  in  diapause  is  response  to  a  critical 
photoperiod  below  which  essentially  all  individuals  in  a 
population  enter  diapause  and  above  which  no  individuals 
enter  diapause  (e.g..  Kogure,  1933;  Tauber  and  Tauber, 
1981,  Tauber  et  ai.  1986).  Temperature  affects  the  timing 
of  diapause  by  altering  the  critical  photoperiod:  as  tem- 
perature declines,  the  photoperiod  that  induces  diapause 
increases  (Danilevskii,  1965;  Tauber  et  ai.  1986).  Other 
effects  of  temperature  on  diapause  induction  in  insects 
include  determination  of  whether  any  photoperiod  re- 
sponse exists  and  alteration  of  photoperiod  responses  due 
to  changes  in  temperature  or  due  to  diel  thermoperiod 
(Tauber  el  ai.  1986:  Eizaguirre  et  ai.  1994). 

The  reason  typically  given  for  photoperiod  primacy  is 
that  daylength  is  the  most  reliable  predictor  of  seasonal 
changes,  and  so  most  insects  living  in  seasonal  environ- 
ments have  evolved  a  means  of  sensing  this  cue  (Lees, 
1955:  Tauber  et  ai.  1986).  This  argument  holds  especially- 
true  for  terrestrial  habitats  in  which  brief  periods  of  un- 
seasonable warming  or  cooling  could  send  a  false  signal 
about  time  in  the  season.  In  contrast,  animals  living  in 
large-volume  aquatic  habitats  (lakes  and  the  oceans)  are 
buffered  from  short-term  temperature  fluctuations  by  the 


42 


DAYLENGTH.  DEGREES.  AND  DIAPAUSE 


43 


100r 


M 
Q> 


O 
w 

D 
O 
9) 

C 
CO 

15 

3 
CO 


20   • 


10 


11 


12  13 

Daylength  (h) 


14 


15 


16 


Figure  1.  The  timing  of  the  switch  from  production  of  subitaneous  (immediately  hatching)  eggs  to 
diapausing  eggs  by  Diaplomus  sanguineus  from  Bullhead  Pond.  Rhode  Island.  The  continuous  line  connecting 
error  bars  shows  the  mean  trajectory-  over  9  years  (±  95%  CI)  for  copepods  in  the  lake,  expressed  as  a 
function  of  photoperiod  on  the  date  that  the  animals  were  collected.  The  line  connecting  individual  open 
data  points  shows  the  photoperiod  response  of  females  reared  in  the  laboratory  at  9°C  (from  Hairston  and 
Olds.  1986).  The  line  connecting  closed  data  points  shows  the  photopenod  response  of  females  reared  at 
the  temperatures  prevailing  at  the  daylengths  indicated  (this  study). 


thermal  inertia  of  water.  The  temperature  of  the  medium 
in  which  they  live  is  a  more  reliable  indicator  of  the  passing 
of  the  seasons  than  is  the  case  on  land.  We  might  expect, 
then,  that  temperature  plays  a  more  significant  role  in 
regulating  timing  of  diapause  in  aquatic  animals  than  in 
their  terrestrial  counterparts.  Among  pelagic  copepods. 
significant  temperature  modification  of  photoperiod  re- 
sponse has  been  found  in  every  instance  investigated,  both 
for  cyclopoid  copepods  with  a  late-instar  diapause  (Wat- 
son and  Smallman.  1971:  Alekseev,  1990)  and  for  cal- 
anoid  copepods  with  egg  diapause  (Marcus,  1982:  Walton. 
1985;  Hairston  el  «/.,  1990:  Ban.  1992).  Furthermore, 
Alekseev  (1990)  has  pointed  out  that  the  effect  of  tem- 
perature in  delaying  critical  photoperiod  is  about  twice 
as  great  in  pelagic  copepods  (0.65  hours  per  C°)  as  it  is 
in  terrestrial  insects  (0.3  hours  per  C°). 

An  enhanced  effect  on  critical  photoperiod  is  only  one 
way  in  which  temperature  might  be  expected  to  influence 
the  timing  of  diapause.  In  some  species,  diapause  response 
to  changing  photoperiod  can  be  gradual  rather  than  a  dis- 
tinct threshold  (Tauber  el  til.,  1986).  In  this  instance  the 
temperature-photoperiod  interaction  must  be  somewhat 
different.  An  opportunity  to  explore  such  an  interaction 
was  presented  by  the  data  available  on  the  timing  and 
control  of  diapause  in  a  population  of  Diaptoinm  san- 
gnim>its.  a  small  (ca.  1mm).  herbivorous,  obligately  sex- 
ual, freshwater  copepod.  This  population  is  found  in 
Bullhead  Pond,  a  small  Rhode  Island  lake  that  has  a  sur- 
face area  of  2.4  ha  and  a  maximum  depth  of  4  m.  While 
active  during  winter  these  copepods  make  subitaneous 


(immediately  hatching)  eggs  (Hairston  and  Munns.  1984): 
in  early  spring  they  switch  to  making  diapausing  eggs, 
and  they  continue  to  do  so  until  they  are  eliminated  in 
late  spring  or  early  summer  by  sunfish  predation.  Nine 
years  of  data  on  the  timing  of  the  switch  to  diapause  show 
that  most  females  make  the  transition  during  a  relatively 
brief  3-week  period  between  10  March  and  10  April  (Fig. 
1 ).  The  timing  of  the  switch  is  apparently  an  adaptation 
to  avoid  an  annual  springtime  increase  in  fish  predation 
(Hairston  and  Munns,  1984:  Hairston  and  Walton.  1986: 
Hairston  and  Dillon.  1990).  The  phenological  pattern 
shown  in  Figure  1  looks  superficially  like  a  typical  critical 
photoperiod  response.  However,  when  the  copepods  are 
reared  in  the  laboratory  at  a  range  of  photoperiods,  and 
at  a  single  water  temperature  (9°C)  approximating  that 
in  the  lake  at  the  time  of  the  switch  to  diapause,  the  tran- 
sition from  production  of  subitaneous  eggs  to  diapausing 
eggs  is  very  gradual  (Fig.  1;  Hairston  and  Olds,  1986). 
This  experiment  suggests  that  the  copepods  in  the  lake 
require  a  more  complex  signal  than  photoperiod  alone  to 
achieve  not  only  the  appropriate  mean  timing,  but  also 
the  observed  rate,  of  transition  from  subitaneous  to  dia- 
pausing eggs. 

In  laboratory  and  field  studies  of  D.  sanguineus.  Hair- 
ston et  a/.  (1990)  showed  that  temperature  has  a  striking 
effect  on  the  timing  of  diapause,  but  their  data  are  inad- 
equate to  reveal  the  nature  of  the  temperature-photoperiod 
interaction  in  cuing  diapause  timing.  Here  we  provide 
evidence  that  temperature  and  photoperiod  acting  to- 
gether are  sufficient  to  effect  the  rapid  transition,  as  ob- 


44 


N.  G.  HAIRSTON  AND  C.  M.  KEARNS 


served  in  Bullhead  Pond,  from  subitaneous  to  diapausing 
eggs.  We  suggest  a  graphical  depiction  of  the  D.  sanguineus 
diapause  response  that  illustrates  how  its  graded  reaction 
to  photoperiod  alone  can  result  in  a  distinct  seasonal  phe- 
nology when  combined  with  temperature.  The  resulting 
model  is  intented  as  a  description  of  the  interaction  of 
the  two  cues  in  effecting  diapause  timing,  not  as  a  deeper 
mechanistic  explanation  of  its  physiological  basis. 

Materials  and  Methods 

Our  objective  was  to  test  the  hypothesis  that  photo- 
period  and  temperature  are  together  sufficient  to  cue  D. 
sanguineus  to  make  subitaneous  or  diapausing  eggs  in  a 
pattern  consistent  with  the  seasonal  phenology  observed 
in  Bullhead  Pond.  For  testing,  we  chose  four  photoperiod- 
temperature  combinations  that  span  the  range  of  dates  at 
which  copepods  in  the  lake  switched  between  the  two  egg 
types.  Our  choice  of  conditions  was  based  on  the  average 
distribution  of  switch  dates  in  Bullhead  Pond  for  9  years 
(Fig.  1;  Hairston.  1987;  Hairston  and  De  Stasio,  1988: 
Hairston,  unpub.  data),  and  lie  ±  1.5  and  ±  2.5  standard 
deviations  from  the  mean  switch  date  (=  Julian  day  85, 
26  March).  The  standard  deviation  of  switch  date  is  about 
7  days  (Hairston  and  Dillon,  1990),  so  temperature  and 
photoperiod  conditions  mimicked  Julian  days  67.5  (8 
March),  74.5  (15  March),  95.5  (5  April),  and  102.5  (12 
April).  Temperatures  at  these  times  were  established  using 
averages  of  1 1  years  of  in  .tint  pond  measurements  (using 
linear  extrapolation  between  measurement  dates),  and 
these  values  were  paired  with  the  photoperiods  obtained 
from  standard  tables.  Photoperiod-temperature  combi- 
nations (L:D  X  °C)  were  set  up  in  four  controlled  en- 
vironment chambers  as  follows:  11.75:12.25  X  4.3. 
12.00:12.00  x  6.0,  12.75:11.25  X  8.0,  and  13.25:10.75 
X  10.5.  Photoperiods  were  established  at  the  15-min 
increment  nearest  to  that  for  the  chosen  date,  with 
illumination  provided  by  daylight  fluorescent  lamps 
( 17-38  nmo\  •  s~'  •  irT:  depending  upon  location  within 
the  growth  chamber):  temperatures  were  maintained 
±0.5C°. 

Live  fifth-instar  copepodids  of  D.  sanguineus  were  col- 
lected from  Bullhead  Pond  on  2  March  1993.  We  mated 
144  females  in  125-ml  glass  jars  (one  female  and  two  males 
per  jar)  at  8:16  L:D  photoperiod  and  4.8°C  (i.e..  short- 
day  and  cold  conditions  to  ensure  that  all  clutches  pro- 
duced were  subitaneous;  see  Hairston  and  Olds.  1986. 
1987).  The  copepods  were  fed  laboratory-cultured 
Chlamydomonas  sp.  every  few  days.  From  these  matings 
we  obtained  121  ovigerous  females  over  a  period  of  17 
days.  An  additional  82  ovigerous  females  were  obtained 
over  the  same  time  period  from  the  stock  20-1  carboy  of 
copepods  in  which  the  plankton  were  transported  from 
Bullhead  Pond.  As  egg-carrying  females  were  found,  they 


were  distributed  evenly  among  the  four  environmental 
treatments.  When  egg  clutches  hatched,  the  nauplii  were 
placed  in  250-ml  glass  jars,  one  family  per  jar,  and  again 
fed  Chlamydomonas.  As  these  animals  reached  copepodid 
stage,  their  diet  was  supplemented  with  Euglena  gradlis. 
Mature  males  and  females  were  taken  from  separate  fam- 
ilies within  each  environmental  treatment  and  mated  as 
previously  described.  Individual  ovigerous  females  from 
these  laboratory-reared  cultures  were  isolated  in  7-ml  wells 
of  12-well  plastic  tissue  culture  plates  and  monitored  daily 
for  hatching.  Based  on  established  procedure  (Hairston 
and  Munns.  1984;  Hairston  and  Olds,  1984,  1986).  eggs 
hatching  within  2  weeks  (warm  temperatures)  or  4  weeks 
(cold  temperatures)  of  laying  were  scored  as  subitaneous, 
and  those  that  had  not  hatched  by  this  time  were  scored 
as  diapausing  eggs. 

Results 

In  each  of  the  four  environmental  conditions,  between 
207  and  356  female  copepods  were  reared  from  nauplius 
to  ovigerous  adult  (Table  I),  although  in  the  two  lower 
temperature  treatments  a  substantial  fraction  of  the  egg 
sacs  produced  were  nonviable  (i.e..  eggs  turned  grey  and 
decomposed).  The  fractions  of  egg  sacs  that  were  either 
subitaneous  or  diapausing  were  calculated  relative  to  the 
total  number  of  viable  sacs  (Table  I).  Because  the  nature 
of  the  nonviable  egg  sacs  could  not  be  determined,  we 
make  the  null  assumption  that  viability  was  independent 
of  egg  type. 

A  plot  of  percent  subitaneous  clutches  produced  at  each 
of  the  treatment  conditions  as  a  function  of  daylength 
(Fig.  1 )  shows  that  photoperiod  and  temperature  together 
produce  a  diapause  phenology  remarkably  similar  to  that 
observed  in  the  wild.  The  fit  is  much  better — both  in  mean 
timing  and  in  the  rapidity  of  the  population  switch  to 
diapause — than  that  found  previously  for  photoperiod 
alone  (Hairston  and  Olds.  1986). 


Table  I 

Pholopenod  and  temperature  combinations  under  which  diapause 
response  inn  determined  lor  Diaptomus  sanguineus  (reasons  for 
condition  chosen  given  in  the  le\ll.  the  total  number  of  egg  clutches 
produced  (one  clutch  per  female),  the  number  oj  viable  clutches,  anil 
the  percentages  oj  viable  dutches  that  were  either  subitaneous 
or  diapausing 

Treatment 

Clutches 

Daylength       Temp.  Subitaneous       Diapausing 

(h)  (°C)         Total       Viable          clutches  clutches 


11.75 

4.3 

356 

265 

91.3 

8.7 

12.00 

6.0 

293 

121 

74.7 

25.6 

12.75 

8.0 

245 

243 

12.8 

87.2 

13.25 

10.5 

207 

203 

2.0 

98.0 

DAYLENGTH.  DEGREES.  AND  DIAPAUSE 


45 


100 


v> 


10 


11  12  13 

Daylength  (h) 


14 


15 


Figure  2.  The  fraction  of  subitaneous  eggs  (versus  diapausing  eggs)  produced  by  niaptoniiix  sanguineus 
when  reared  in  the  laboratory  at  different  combinations  of  photopenod  and  temperature.  The  data,  taken 
from  three  previous  studies  (Hairston  and  Olds,  1 986,  1 987;  Hairston  el  at..  1990)  and  the  current  investigation, 
show  distinct  diapause  responses  to  daylength  at  different  temperatures.  Solid  lines  connect  values  from 
experiments  at  equal  temperatures  (4°C  and  9°C).  The  dotted  6°C  line  is  a  hypothetical  relationship  based 
on  the  single  6°C  point  and  the  4°C  and  9°C  lines.  The  dashed  line  connects  the  four  photopenod-temperature 
treatments  reported  here  and  thus  shows  the  trajectory  of  diapause  phenology  of/),  sanguineus  as  photoperiod 
and  temperature  increase  during  spring  in  Bullhead  Pond,  Rhode  Island. 


Discussion 

For  Diapttimus  sanguineus  in  Bullhead  Pond,  the  grad- 
ual effect  of  photoperiod  at  a  constant  temperature  (Hair- 
ston and  Olds,  1 986,  see  Fig.  I )  is  converted  into  a  discrete 
seasonal  timing  of  diapause  by  the  addition  of  a  thermal 
cue.  Similar  to  our  result,  those  of  Marcus  ( 1 982)  show  a 
gradual  shift  from  subitaneous  to  diapausing  egg  produc- 
tion as  a  function  of  photoperiod  for  the  calanoid  copepod 
Labidocera  aesliva.  despite  a  seasonal  transition  to  dia- 
pause in  nature  that  is  quite  discrete  (Marcus,  1979).  In 
her  study,  however,  temperature  had  a  weaker  effect  on 
the  diapause  response  than  we  have  observed  for  D.  .san- 
guineus. Our  result  contrasts  with  the  photoperiod-dom- 
inated  pattern  seen  in  many  insect  species  (Danilevskii, 
.1965:  Tauber  et  ai,  1986)  and  in  several  previous  studies 
of  diapause  in  pelagic  copepods.  Photoperiod  plays  a  pri- 
mary role  in  the  switch  to  diapausing  eggs  in  the  calanoid 
copepod  Ewytemom  affinis  (Ban,  1992)  and  in  the  late- 
instar  diapause  of  the  cyclopoid  copepods  Diacydops  na- 
vus  (Watson  and  Smallman.  1971)  and  Metacyclops  mi- 
nut  us  (Alekseev,  1990).  Copepod  density  can  also  act  as 
a  significant  modifier  of  the  photoperiod  cue  (E.  affinis. 
Ban,  1992;  M.  minutus.  Alekseev.  1990);  and  in  Diapto- 
mus  birgei.  photoperiod  is  only  a  secondary  factor  in  cuing 
diapause  (Walton.  1985). 

How  does  temperature  sensitivity  convert  a  gradual 
photoperiod  response  into  a  relatively  discrete  seasonal 


switch  to  diapause  for  the  D  sanguineus  population?  An 
answer  emerges  when  we  plot,  in  a  single  figure  (Fig.  2), 
all  of  the  available  data  for  D.  sanguineus  diapause  re- 
sponse at  a  range  of  photoperiods  and  temperatures 
(Hairston  and  Olds.  1986,  1987;  Hairston  et  ai,  1990; 
this  study).  In  this  plot,  the  slope  of  the  line  relating  the 
percentage  of  subitaneous  clutches  to  the  daylength  is 
much  shallower  at  4°C  than  at  9°C.  The  single  data  point 
at  6°C  is  intermediate  between  these  two  lines,  and  we 
conjecture  that  a  6°C  line  would  have  an  intermediate 
slope.  Data  points  at  higher  temperatures  (i.e..  8-1 1°C) 
are  variable,  but  uniformly  give  low  values  for  the  per- 
centage of  subitaneous  clutches.  The  dashed  line  in  Figure 
2  shows  what  happens  as  both  temperature  and  photo- 
period  change  early  in  the  season  in  Bullhead  Pond.  At 
short  photoperiods,  when  water  temperatures  are  4°C  or 
below,  the  copepods  make  subitaneous  eggs.  As  spring 
comes  on,  not  only  does  photoperiod  increase,  but  so  does 
temperature:  thus  the  appropriate  daylength-response 
shifts  from  the  4°C  line  to  the  6°C  line  to  the  9°C  line, 
and  so  on.  The  result  is  the  relatively  discrete  seasonal 
switch  to  production  of  diapausing  eggs  seen  in  Bullhead 
Pond.  Note  also  that  the  data  in  Figure  2  could  be  ex- 
pressed equally  well  by  plotting  the  percentage  of  subi- 
taneous clutches  against  temperature,  with  lines  of  equal 
photoperiod  radiating  from  the  upper  left-hand  corner. 
The  photopenod-temperature  interaction  would  be  still 
apparent. 


46 


N.  G.  HAIRSTON  AND  C.  M.  KEARNS 


Our  depiction  of  the  response  of  D.  sangiiineus  to  com- 
bined photoperiod  and  thermal  cues  (Fig.  2)  illustrates 
only  the  phenotypes  expressed  by  the  copepods  when  ex- 
posed to  environments  of  differing  photoperiod  and  tem- 
perature combinations.  Behind  this  response  surface  lies 
the  physiological  mechanisms  by  which  the  copepods  de- 
tect daylength  (or  nightlength)  and  temperature  and  then 
react  by  producing  the  appropriate  egg  type. 

Two  alternative  photoperiodic  clock  mechanisms  have 
received  substantial  attention  in  the  entomological  liter- 
ature. The  circadian  oscillator  model  posits  an  internal 
pacemaker  of  intrinsic  period,  which  is  then  either  com- 
pared (physiologically)  to  the  environment  (i.e.,  photo- 
period)  or  drives  other,  "slave,"  oscillators  whose  phases 
are  differentially  altered  by  the  environment  (Pittendrigh, 
1981;  Saunders.  1982;  Gillanders  and  Saunders,  1992). 
The  hourglass  model  hypothesizes  the  production  of  some 
chemical  substance  that  accumulates  incrementally  during 
the  dark  (or  the  light)  phase  of  a  light-dark  cycle,  up  to 
some  critical  threshold  (Lees,  1973;  Skopik  and  Bowen. 
1976;  Veerman  ft  a/.,  1988).  Some  recent  studies  have 
emphasized  models  that  have  an  underlying  oscillator  but 
can  exhibit  hourglass-like  behavior  (e.g.,  Vas  Nunes  et  at., 
1991).  In  addition  to  providing  a  mechanism  for  critical 
photoperiod  response,  each  model  provides  a  mechanism 
for  the  influence  of  temperature  in  shifting  critical  pho- 
toperiod earlier  or  later  in  the  season  (Pittendrigh  el  ai. 
1991;  Vas  Nunes  eta/.,  1991;  Kimura  and  Masaki,  1993). 
The  conversion,  in  D.  sangiiinens.  of  a  graded  photoperiod 
response  to  a  much  sharper  seasonal  phenology  by  the 
addition  of  a  thermal  cue  results  simply  from  a  rightward 
shift  (towards  longer  photoperiods)  of  the  diapause  re- 
sponse curve  under  low  temperatures  and  a  leftward  shift 
(towards  shorter  photoperiods)  under  high  temperatures 
(Fig.  3;  explained  in  detail  below).  Because  both  clock 
models  could,  in  principle,  accommodate  this  behavior, 
the  data  presented  here  do  not  permit  us  to  distinguish 
between  the  two  alternatives.  Nevertheless,  experiments 
designed  to  determine  the  photoperiodic  clock  mechanism 
in  this  copepod  should  provide  essential  clues  for  under- 
standing both  the  range  of  expression  of  diapause  response 
by  D.  sangiiineus  to  different  environments  and  the  pos- 
sible constraints  on  the  genetic  covariance  structure  of 
sensitivity  to  photoperiod  and  thermal  cues  (i.e.,  how  se- 
lection on  copepod  sensitivity  of  diapause  expression  to 
photoperiod  might  influence  trait  sensitivity  to  temper- 
ature). 

The  pattern  of  photoperiod-temperature  response  pro- 
posed here  does  not  in  fact  differ  radically  from  typical 
thermal  alteration  of  critical  photoperiod.  Figure  3A  shows 
a  family  of  hypothetical  critical  daylength  responses 
modified  by  temperature  that  would  produce  a  spring 
switch  from  production  of  subitaneous  to  diapausing  eggs 
by  a  pelagic  copepod  species.  If  the  lake  temperature  in- 


creased with  lengthening  photoperiod  according  to  the 
patterns  listed  in  Figure  3E,  the  copepods  would  make 
subitaneous  eggs  until  the  threshold  photoperiod  and 
temperature  combination  that  induced  a  complete  switch 
to  diapausing  eggs  was  reached.  Figure  3B  shows  critical 
response  curves  more  typical  of  many  insect  species  in 
which  the  transition  to  diapause  is  not  rectilinear  but 
somewhat  graded,  with  a  thermal  effect  on  critical  pho- 
toperiod of  0.3  h  per  C°  (c.f..  Danilevskii.  1965,  pp.  1 14- 
115).  Tracking  the  environment  of  Figure  3E  produces  a 
seasonal  switch  to  diapause  more  rapid  than  that  seen 
along  any  constant  temperature  line,  though  not  as  rapid 
as  that  seen  in  Figure  3A.  In  contrast  to  these  two  patterns, 
the  photoperiod  response  lines  for  different  temperatures 
in  Figure  2  radiate  from  100%  subitaneous  clutches  at 
short  photoperiod  (i.e.,  there  is  no  temperature  effect  at 
short  daylength).  Suppose  now  that  these  lines  of  constant 
temperature  reconverge  at  long  photoperiods  on  0%  sub- 
itaneous clutches  (100%  diapause),  as  illustrated  in  Figure 
3C.  Tracking  the  Figure  3E  environment  again  produces 
a  seasonal  switch  to  diapause,  just  as  seen  in  Figure  2. 
The  differences  between  each  of  these  three  patterns  lie 
in  the  steepness  of  the  slopes  of  the  photoperiod  responses. 
It  is  not  difficult,  however,  to  envision  any  one  pattern 
being  derived  from  one  of  the  others  through  a  simple 
change  in  how  rapidly  diapause  responds  to  photoperiod 
at  each  temperature. 

A  gradual  transition  from  production  of  subitaneous 
eggs  to  diapausing  eggs  in  a  single  population  implies  that 
individual  females  must  respond  differently  to  any  given 
photoperiod-temperature  combination  (i.e.,  some  fraction 
make  subitaneous  eggs  while  the  others  make  diapausing 
eggs).  For  D.  sangiiineus.  we  know  that  there  is  significant 
heritable  variation  for  diapause  response  at  13  h  of  light 
and  9°C  in  the  laboratory  (Hairston  and  Dillon,  1990). 
If  the  model  in  Figures  2  and  3C  is  an  accurate  represen- 
tation of  the  sensitivity  of  the  copepods'  diapause  response 
to  temperature  and  photoperiod,  it  provides  a  means  of 
envisioning  how  natural  selection  might  act  on  this  genetic 
variation  to  alter  the  timing  of  diapause  in  natural  pop- 
ulations. For  example,  500  m  from  Bullhead  Pond  lies 
Little  Bullhead  Pond.  In  1979,  the  D.  sangiiineus  popu- 
lation in  this  pond  switched  to  diapause  in  late  March, 
the  same  time  as  the  population  in  Bullhead  Pond.  In 
1981.  a  drought  dried  Little  Bullhead  Pond  and  killed  all 
of  the  fish,  thus  removing  the  principal  selection  force 
maintaining  the  March  diapause  date.  The  pond  refilled 
in  1982.  and  by  1983  the  mean  timing  of  diapause  had 
moved  to  late  April,  apparently  as  a  response  to  the  altered 
selection  regime  (Hairston  and  Walton,  1986;  Hairston 
and  De  Stasio.  1988).  Bullhead  Pond,  which  is  deeper 
than  Little  Bullhead  Pond,  did  not  dry,  no  fish  were  killed, 
and  the  timing  of  diapause  in  that  population  changed 
little  between  1979  and  1983  (Hairston  and  Walton.  1986; 


DAYLENGTH,  DEGREES.  AND  DIAPAUSE 


47 


I 

u 


O 

3 
o 

0) 
CO 


CO 


IUU 

1 

80 

_ 

1 

1 

60 

- 

ii 

10 

9 

_    7 

6 

5 

4 

3°C 

40 

_ 

1 

1 

20 

- 

1 

o 

| 

B 


8     9     10    11    12    13    14    15    16    17   18 


3  "C 


8  9  10  11  12  13  14  15  16  17  18 
Daylength  (h) 


8  9  10  11  12  13  14  15  16  17  18 


3°C 


8°C 


8     9    10   11    12   13   14   15   16   17    18 
Daylength  (h) 


Date                Feb 

1      10      21       Mar 

6     18      29          Apr 

10      20 

May       5     20 

Daylight  (h) 
Temp  (°C) 

10     10.5  11 
333 

11.5  12      12.5 
468 

13      13.5 
10      11 

14      14.5 
13      18 

Figure  3.  Three  patterns  of  springtime  diapause  responses  under  varying  combinations  of  photoperiod 
and  temperature.  (A)  Strict  critical-photoperiod  response  with  thermal  effect.  (B)  Critical-photoperiod  response 
with  a  graded  transition  from  100%  non-diapause  to  100%  diapause  covering  a  photoperiod  range  of  about 
2  h  and  a  temperature  delay  of  1.5  h  per  1°C.  as  is  typical  of  many  insects.  (C)  Gradual  transition  from 
100%-  non-diapause  to  100%-  diapause  with  lengthening  daylength  as  seen  for  D  sungiiiiu-iis  (Fig.  2),  but 
with  the  added  assumption  that  all  animals  produce  diapausing  eggs  at  long  photoperiods  independent  of 
temperature.  (D)  Same  graph  as  in  C,  but  with  response  line  relabeled  to  show  the  effect  of  selection  for 
later  diapause.  (E)  Springtime  pattern  of  increasing  photoperiod  and  temperature  typical  of  Bullhead  Pond, 
Rhode  Island  (see  Table  I).  These  values  are  used  in  A.  B.  and  C  to  illustrate  projected  diapause  phenologies. 
In  each  case  the  mean  switch  to  diapause  occurs  at  12.3  h  daylength  and  7°C. 


Hairston  and  Dillon.  1990).  What  features  of  the  photo- 
period  and  temperature  responses  of  the  Little  Bullhead 
Pond  populations  were  altered  by  selection  to  produce 
the  change  in  diapause  phenology?  For  diapause  timing 
to  be  heritable,  the  hypothetical  temperature-specific 
photoperiod-response  lines  in  Figures  2  and  3C  must  vary 
depending  upon  genotype.  That  is,  for  some  genotypes 
the  lines  lie  above  the  population  mean,  and  for  others 
the  lines  lie  below  the  mean.  Directional  selection  for  later 
diapause  would  simply  move  the  population  mean  re- 


sponse lines  upward  (as  in  Figure  3D).  The  effect  is  a  joint 
one  on  both  thermal  and  photoperiodic  responses  of  the 
copepods:  at  a  given  temperature,  the  slope  of  the  response 
to  change  in  daylength  is  shallower,  but  also,  at  a  given 
photoperiod.  the  position  of  each  of  the  isothermal  lines 
is  raised.  With  the  new  temperature-specific  photoperiod- 
response  lines,  the  timing  of  diapause  shifts  to  later  in  the 
season  (Figure  3D). 

Populations  of  D.  su/iguini'its  living  in  different  lakes 
and  ponds  exhibit  both  distinct  photoperiod  responses 


48 


N.  G.  HAIRSTON  AND  C.  M.  K.EARNS 


under  controlled  laboratory  conditions  (Hairston  and 
Olds,  1986,  1987)  and  distinct  seasonal  diapause  phe- 
nologies in  the  field  (Hairston  and  Olds,  1984;  Hairston 
ei  al..  1985).  Both  the  mean  timing  of  diapause  and  the 
temporal  pattern  of  the  switch  from  subitaneous  to  dia- 
pausing  eggs  vary  between  populations.  In  ephemeral 
pools,  water  depths  and  temperatures  fluctuate  depending 
upon  local  rainfall,  leading  to  a  decoupling  of  temperature 
and  photoperiod  as  predictors  of  time  in  the  season.  Here, 
D.  sanguineits  can  show  late-season  reversals  to  subita- 
neous egg  production  (Hairston  and  Olds,  1987).  The 
graphical  model  developed  here  provides  a  framework  for 
investigating  the  genetic  covariance  structure  underlying 
such  interpopulation  variation  in  diapause  responses  to 
photoperiod  and  to  temperature,  and  how  this  structure 
differs  to  produce  the  variety  of  diapause  phenologies  ob- 
served in  nature. 

Acknowledgments 

We  thank  C.  Tauber,  M.  Tauber,  S.  Ellner,  L.  Polish- 
chuck,  and  N.  Hairston,  Sr.,  for  helpful  discussions,  and 
K.  Batson  for  technical  assistance.  This  research  was  sup- 
ported by  National  Science  Foundation  grant  BSR- 
91 18894  to  NGH  and  S.  Ellner. 

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Saunders,  D.  S.  1982.     Insect  Clocks.  2nd  ed.  Pergamon  Press.  New 

York.  409  pp. 

Skopik,  S.  D.,  and  M.  F.  Bowen.  1976.     Insect  photopenodism:  an 
hourglass  measures  photoperiodic  time  in  Oxlrinia  nubilahs.  J  Coinp. 
Physio/.  Ill:  249-259. 
Tauber,  C.  A.,  and  M.  J.  Tauber.  1981.     Insect  seasonal  cycles:  genetics 

and  evolution.  Ann.  Rev.  Ecol.  Syst.  12:  281-308. 
Tauber,  M.  J.,  C.  A.  Tauber,  and  S.  Masaki.  1986.     Scasnnal  Adap- 
tations c/  Insects.  Oxford  Univ.  Press,  New  York,  411  pp. 
Vas  Nunes,  M.,  R.  D.  Lewis,  and  D.  S.  Saunders.  1991.     A  coupled 
oscillator  feedback  system  as  a  model  for  the  photoperiodic  clock  in 
insects  and  mites.  II.  Simulations  of  photoperiodic  responses.  /  Theor. 
Biol  152:  294-317. 

Veerman,  A.,  M.  Beekman,  and  R.  L.  Veenendaal.  1988.  Photoperiodic 
induction  of  diapause  in  the  large  white  butterfly,  Pieris  brassicae: 
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1069. 

Walton,  W.  E.  1985.  Factors  regulating  the  reproductive  phenology  of 
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Watson,  N.  H.,  and  B.  N.  Smallman.  1971.  The  role  of  photoperiod 
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Reference:  Bml.  Hull  189:  49-5X.  (August.  1995) 


Process-Specific  Recruitment  Cues 
in  Marine  Sedimentary  Systems 

SARAH  A.  WOODIN,  SARA  M.  LINDSAY,  AND  DAVID  S.  WETHEY 

Department  of  Biological  Sciences,  University  of  South  Carolina,  Columbia,  South  Carolina  29208 


Abstract.  In  marine  sediments,  many  of  the  processes 
associated  with  high  post-settlement  mortality  of  infauna 
have  similar  effects  on  the  sediment  surface.  In  most  cases 
the  original  sediment  surface  is  either  removed,  buried, 
or  mixed  with  subsurface  sediment.  The  experiments  re- 
ported here  tested  the  ability  of  new  juvenile  infauna  to 
discriminate  between  undisturbed  and  recently  disturbed 
sediment  surfaces  (i.e..  subsurface  sediment  exposed). 

Recently  settled  juveniles  of  two  polychaete  species 
(Nereis  vexillosa  and  Arenicola  cristata)  and  one  bivalve 
species  (Alercenaria  mercenaria)  were  exposed  to  simu- 
lated erosional  and  mixing  events  as  well  as  to  fresh  feces. 
burrow  tailings,  and  feeding  tracks.  Where  the  disturbance 
buried  or  removed  several  millimeters  of  the  sediment 
surface,  the  time  to  initiate  burrowing  or  the  percentage 
of  individuals  failing  to  burrow  increased  significantly  over 
times  and  percentages  for  juveniles  on  undisturbed  sur- 
faces. In  all  cases  the  results  were  consistent  with  the  hy- 
pothesis that  new  juveniles  reject  (or  are  significantly 
slower  to  burrow  into)  disturbed  sediment  surfaces,  if  the 
disturbance  is  less  than  several  hours  old.  For  example. 
51%  of  nereid  juveniles  did  not  burrow  when  placed  on 
subsurface  sediments,  whereas  100%  burrowed  into  sur- 
face sediments;  their  average  burrowing  time  on  surface 
sediments  was  29.3s  compared  with  109.7s  on  fecal 
mounds  of  arenicolid  polychaetes  or  106.1  s  on  burrow 
tailings  ofthalassinid  crustaceans.  Individuals  that  did  not 
indicate  acceptance  of  a  sediment  surface  by  burrowing 
were  all  rapidly  eroded  from  the  surface  in  the  presence 
of  flow.  Erosion  of  nonburrowing  individuals  occurred 
within  90  s  of  initiation  of  flow.  Burrowing  individuals 
were  not  eroded.  The  decision  as  to  the  acceptability  of 
a  sediment  was  made  within  30  s.  These  data  imply  that 


Received  12  December  1994;  accepted  2  May  1995. 
Contribution  Number  1051  of  the  Belle  W.  Baruch  Institute  for  Marine 
and  Coastal  Research. 


the  new  juveniles  are  utilizing  cues  associated  with  a  pro- 
cess, the  disturbance  of  surface  sediments,  in  addition  to 
the  species-specific  cues  described  elsewhere. 

Introduction 

Recruitment  is  of  fundamental  importance  to  com- 
munity structure  because  it  is  the  foundation  upon  which 
all  subsequent  interactions  within  the  community  take 
place.  When  recruitment  fails,  organisms  do  not  have  the 
opportunity  to  interact  as  adults  (Underwood  and  Denley, 
1984).  Therefore,  the  often  substantial  variation  in  success 
of  recruitment  (Loosanoff,  1964;  Wethey,  1985;  Feller  el 
at.,  1992)  can  profoundly  influence  the  dynamics  of  adult 
populations.  Two  major  classes  of  processes  combine  to 
determine  the  eventual  recruitment  success  of  species  with 
planktonic  larvae,  assuming  that  competent  larvae  are  in 
the  plankton.  First,  larval  transport  on  geographic  and 
local  scales  can  determine  how  many  larvae  actually  reach 
a  site  and  are  retained  (Boicourt,  1982;  Cameron  and 
Rumrill,  1982;  Kendall  el  al..  1982;  Levin.  1984:  Jonsson 
el  u/.,  1991 ).  Only  larvae  that  arrive  on  the  bottom  have 
an  opportunity  to  exercise  a  behavioral  choice  of  substra- 
tum (Highsmith.  1982;  Butman  el  al.,  1988).  Second, 
mortality  and  emigration  occurring  during  or  after  settle- 
ment and  metamorphosis  can  determine  the  success  of 
the  larvae  that  settle  (Sellmer,  1967;  Muus,  1973;  Sigurd- 
son  et  at.,  1976;  Bell  and  Coull.  1980;  Brenchley,  1981; 
Levin,  1981;  Wilson.  1981;  Watzin,  1983;  Luckenbach, 
1984;Connell,  1985;  Wethey,  1985;  Elmgren  et  al..  1986: 
Woodin,  1986;EysterandPechenik,  1987;  Walters,  1992). 
Thus,  both  presettlement  and  postsettlement  events  are 
involved  in  recruitment. 

The  literature  on  recruitment  is  dominated  by  two  types 
of  papers,  those  on  mortality  and  those  on  larval  choice. 
This  dichotomy  is  interesting  because  the  mortality  lit- 
erature is  primarily  directed  toward  elucidating  how  var- 


49 


50 


S.  A.  WOODIN  ET  AL. 


ious  postsettlement  factors  cause  greater  mortality,  i.e., 
on  negative  influences.  The  literature  on  choice,  if  it  were 
parallel,  would  be  on  cues  by  which  new  recruits  avoid 
sites  that  have  characteristics  associated  with  high  mor- 
tality. Instead,  most  of  the  choice  literature  is  directed 
toward  identification  of  positive,  species-specific  cues  that 
are  associated  with  the  presence  of  either  prey  (Hadfield, 
1984;  Morse  and  Morse,  1984)  or  mates  (Highsmith,  1982: 
Crisp,  1984).  We  would  argue  that  given  the  cues  asso- 
ciated with  high  mortality  and  the  characteristics  of  re- 
cruits that  allow  them  to  recognize  such  cues,  there  would 
be  strong  selection  for  retention  of  individuals  with  such 
traits  (for  examples  of  larvae  with  such  traits  see  Johnson 
and  Strathmann,  1989,  and  Grosberg.  1981). 

A  characteristic  common  to  the  majority  of  the  mor- 
tality sources  for  new  juveniles  in  sediments,  such  as  pre- 
dation,  erosion,  and  deposition,  is  that  these  mortality 
sources  alter  the  sediment  surface  either  by  removing  it 
or  by  covering  it  with  a  layer  of  subsurface  or  a  mixed 
surface-subsurface.  A  testable  deduction  consistent  with 
our  proposed  selection  regime,  then,  is  that  new  recruits 
distinguish  between  recently  disturbed  and  undisturbed 
sediment  surfaces.  The  experiments  described  here  were 
designed  to  test  this  hypothesis.  We  confined  ourselves  to 
sediments  that  are  stable  and  rarely  move  as  bedload  (for 
events  characteristic  of  more  mobile  habitats,  see  Emerson 
and  Grant,  1991).  We  defined  the  disruptions  of  interest 
to  be  those  that  either  covered  the  original  surface  of  the 
sediment  or  removed  at  least  3-5  mm  vertically.  Biotic 
erosional  forces,  such  as  tellinid  bivalve  feeding,  and 
physical  erosion  can  easily  remove  at  least  this  much  sed- 
iment. We  also  asked  whether  the  behavior  of  new  ju- 
veniles on  disturbed  surfaces  is  likely  to  result  in  rates  of 
recruitment  that  are  reduced  compared  with  those  on  un- 
disturbed surfaces. 

Materials  and  Methods 

Background  information  on  infauna 

New  juveniles  of  infaunal  bivalves  and  polychaetes  were 
used  to  contrast  the  responses  of  taxa  that  can  burrow  as 
new  juveniles  (the  polychaetes)  and  those  that  cannot  (the 
bivalves).  The  use  of  new  juveniles  rather  than  settling 
larvae  also  avoids  the  potentially  serious  problem  of  dif- 
ferentiating between  competent  larvae  and  incompetent 
larvae  of  similar  size  and  appearance  (Bachelet  et  ai. 
1992).  Bivalves  and  polychaetes  are  quite  different  at  the 
initiation  of  metamorphosis.  For  example,  bivalves  have 
a  shell  at  settlement  but  usually  cannot  burrow  for  a  week 
or  more  after  metamorphosis  begins  (Belding.  1930:  Car- 
riker,  1 96 1 :  Gustafson  and  Reid.  1986).  Polychaetes  lack 
a  shell  but  can  burrow  at  this  developmental  stage  (Wilson. 
1952;  Roe,  1975).  Bivalves  generally  cannot  emigrate  from 
a  habitat  by  swimming  oft"  the  bottom  once  their  velum 


has  been  lost:  they  can,  however,  both  crawl  and  use  water 
currents  to  waft  away  from  a  site  after  erosion  off  the 
bottom  sediment  (Sigurdsson  et  ai.  1976;  Sastry,  1979). 
Many  polychaete  juveniles  can  crawl,  waft,  and  actively 
swim  away  from  a  site,  even  after  they  metamorphose 
(e.g..  Roe.  1975).  The  important  point  of  similarity  is  that 
sediment-dwelling  juveniles  of  both  taxa  can  crawl  away 
as  well  as  waft  away.  Thus  rejection  of  a  site  may  occur 
at  the  juvenile  as  well  as  the  larval  stage. 

In  all  experiments,  we  used  juveniles  that  had  initiated 
metamorphosis  less  than  one  week  before  they  were  tested. 
The  polychaetes  were  Nereis  vexillosa,  a  nereid,  and  Ar- 
cnicola  cristata,  an  arenicolid.  These  larvae  are  lecitho- 
trophic  and  were  not  fed.  The  juveniles  of  both  species 
were  fed  Isochrysis  galbana  T-ISO  strain  as  well  as  a  mix- 
ture of  diatom  species.  Nereis  is  common  near  the  Uni- 
versity of  Washington  Friday  Harbor  Laboratories  on  San 
Juan  Island,  Washington.  Females  lay  egg  masses  con- 
taining hundreds  of  eggs  (Johnson,  1943).  Arenicola  also 
lays  egg  masses  that  are  initially  attached  to  the  bottom. 
Egg  masses  of  both  species  were  collected  in  the  field  and 
transported  to  the  laboratory,  where  they  were  placed  in 
individual  micro-airlifts  (Strathmann.  1987)  until  the  lar- 
vae hatched  at  four  to  five  setigers  (Nereis)  or  three  to 
four  setigers  (Arenicola).  Swimming  larvae  were  cultured 
in  1 1-cm-i.d.  glass  culture  dishes.  When  the  larvae  develop 
six  setigers,  at  a  length  of  about  500  ^m  for  Nereis  and 
about  600  /urn  for  Arenicola.  the  larvae  will  burrow  and 
build  tubes  in  acceptable  sediments.  Both  polychaete  spe- 
cies actively  reject  some  sediments  by  either  initiating  a 
crawling  sequence  or  standing  perpendicular  to  the  sed- 
iment surface,  attached  only  by  a  mucus  thread.  In  flowing 
water  such  individuals  are  easily  eroded  oft"  their  attach- 
ment point  (see  below).  Sediments  into  which  the  recruits 
did  not  burrow  within  the  observation  period  were  clas- 
sified as  unacceptable. 

The  bivalve  species  used  in  these  experiments  was  Mer- 
cenaria  mercenaria.  Late  pediveligers  of  Mercenaria  were 
cultured  in  the  laboratory  with  stirring  and  fed  Isochrysis 
galhana  T-ISO  strain  until  settlement  and  initiation  of 
metamorphosis.  The  individuals  of  Mercenaria  averaged 
0.307  mm  (SD  =  0.035)  in  length.  New  spat  of  Mercenaria 
do  not  penetrate  the  sediments  and  burrow;  rather  they 
nestle  into  the  substratum  to  about  half  the  length  of  the 
shell,  position  themselves  with  the  hinge  down,  and  gape 
slightly  (Carriker.  1961).  Initiation  time  for  spat  was  de- 
fined as  the  time  at  which  the  foot  of  the  individual  pen- 
etrated the  sediment  surface  and  the  shell  was  pulled 
down.  In  some  types  of  sediments  the  spat  fail  to  show 
this  nestling  behavior;  instead  they  remain  on  the  surface, 
typically  resting  on  one  valve  or  sometimes  actively 
crawling  across  the  sediment  without  any  evidence  of 
movement  of  the  hinge  and  shell  below  the  sediment  sur- 


PROCESS-SPECIFIC  CUES 


51 


face.  Sediments  eliciting  such  behaviors  were  classified  as 
unacceptable. 

Behavioral  observations 

Glass  dishes  with  a  1.5-  to  2-cm  layer  of  test  sediment 
on  the  bottom,  covered  by  a  layer  of  seawater  2  cm  deep, 
were  used  for  observations.  Individual  juveniles  were 
gently  pipetted  into  the  water  column  and  allowed  to  drift 
onto  the  sediment  surface.  Individuals  that  contacted  the 
sediment  surface  forcefully,  landed  on  a  sand  grain  larger 
than  their  length,  or  (for  the  worms)  landed  in  any  position 
other  than  their  ventral  surface  were  not  used  in  the 
analyses.  Timing  of  responses  to  the  sediments  began 
when  the  individual  made  contact  with  the  sediment  sur- 
face. A  dissecting  microscope  was  used  to  monitor  indi- 
viduals continuously  from  their  arrival  on  the  sediment 
surface  until  they  disappeared  below  the  surface  (poly- 
chaetes)  or  nestled  in  among  the  surface  sediments  and 
began  to  feed  (bivalves),  or  until  the  designated  obser- 
vation period  expired.  The  length  of  the  observation  pe- 
riod depended  upon  the  species  (Nereis,  5  min;  Mercen- 
aria,  6  min;  Arenicola,  1  min)  and  was  set  to  a  minimum 
of  10X  the  mean  time  individuals  took  to  initiate  bur- 
rowing in  preliminary  observations.  In  the  experiments 
with  Nereis  and  Mercenaria,  4  to  10  individuals  were  sep- 
arately added  to  each  dish  or  core.  To  avoid  the  possibility 
of  interactions,  individuals  that  landed  within  2  cm  of 
another  animal  were  not  used. 

Experimental  sediments 

Experimental  sediments  were  collected  in  the  field  and 
immediately  transported  to  the  laboratory.  Sediments 
were  collected  as  two  types:  surface  sediments  (the  top  1- 
to  2-mm-thick  layer)  and  subsurface  sediments  (the  1-  to 
2-mm-thick  layer  exposed  after  removal  of  the  top  4  to 
5  mm).  In  the  field,  the  surface  sediments  were  collected 
with  a  paint  scraper.  Areas  of  disturbance  such  as  fecal 
mounds,  burrow  scrapings,  tubes,  and  feeding  traces  were 
avoided.  Subsurface  sediments  were  collected  by  removing 
the  top  4  to  5  mm  of  sediment  and  then  collecting  the 
next  1  to  2  mm  with  a  paint  scraper.  In  field-processed 
sediments,  each  type  of  sediment  was  collected  and  placed 
in  a  1 1-cm-i.d.  glass  dish,  creating  a  layer  1.5  to  2.0  cm 
deep.  Once  in  the  laboratory,  each  dish  was  filled  with 
seawater  to  a  depth  of  at  least  2  cm.  Dishes  were  stored 
floating  in  a  seawater  table  and  were  used  within  2  h.  In 
addition,  we  used  several  other  types  of  sediments:  fresh 
feces  of  arenicolid  polychaetes  (Abarenicola  pacified),  fresh 
burrow  scrapings  of  thalassinid  crustaceans  (Upogebia 
pugeltensis),  1:1  mixtures  by  volume  of  surface  and  sub- 
surface sediments,  and  sediments  that  had  been  reworked 
physically  by  storm  events.  All  were  used  within  2  h  of 


collection,  including  the  1 : 1  mixtures  that  were  made  from 
field-collected  sediments. 

Juvenile  Nereis  were  also  tested  on  sediments  contain- 
ing active  macrofauna.  Sediments  from  False  Bay  were 
defaunated  by  freezing,  thawing,  and  refreezing,  then 
rinsed  thoroughly  with  seawater  and  sieved  on  a  0.5-mm 
mesh.  Cores  (7  cm  X  6  cm  i.d.)  of  defaunated  sediment 
were  either  innoculated  with  known  numbers  and  species 
of  macrofauna  or  left  as  controls.  After  two  weeks,  juvenile 
Nereis  were  exposed  to  the  surfaces  of  each  type.  Within 
the  macrofauna-addition  cores,  the  juveniles  were  added 
to  the  feeding  areas  of  the  macrofauna  (half  of  the  tested 
cores)  as  well  as  to  sites  outside  of  the  feeding  areas  (re- 
mainder of  the  macrofauna-addition  cores).  The  macro- 
fauna  used  were  three  species  of  spionid  polychaete  (Spio 
sp.,  Rhynchospio  gliitaeus,  and  Pygospio  elegans)  and  a 
tellinid  bivalve  (Macoma  nasuta). 

Several  sites  were  used  for  sediment  collection.  In  all 
cases  adults  of  the  species  to  be  tested  were  common  at 
the  collection  site.  For  Nereis  vexillosa  the  sites  were  a 
semiprotected  fine  sandflat,  False  Bay,  Washington 
(48°29;  N:  123°04'  W,  median  grain  size  0.18  mm,  silt- 
clay  10%)  and  a  more  enclosed  bay  with  a  mud-gravel 
sediment  mixture.  Snug  Harbor.  Washington  (48°34'  N, 
123°  10'  W,  median  grain  size  1.66  mm,  silt-clay  6%).  For 
Arenicola  cristata  and  Mercenaria  mercenaria  the  sites 
were  medium-  to  fine-grained  sandflats  on  the  landward 
side  of  Pawleys  Island,  South  Carolina  (33°24'  N,  79°8' 
W,  median  grain  size  0.39  mm,  silt-clay  0.05%)  and  at 
Oyster  Landing,  North  Inlet,  South  Carolina  (33°20'  N, 
79°  12'  W,  median  grain  size  0.38  mm,  silt-clay  0%).  The 
numbers  of  replicates  per  treatment  differed  among  ex- 
periments and  are  given  in  the  Results  section. 

All  experiments  on  Nereis  vexillosa  were  run  in  June 
1 990  at  the  Friday  Harbor  Laboratories.  The  controls  were 
run  on  every  day  of  the  experiment,  and  each  experi- 
mental treatment  was  run  on  most  days.  The  experiments 
on  Arenicola  cristata  were  run  in  July  1992.  Those  on 
Mercenaria  mercenaria  were  run  in  May  1991.  Controls 
were  always  alternated  with  experimental  sediments  to 
ensure  that  the  juveniles  were  still  responsive. 

Hume  obsen'aiions  and  design 

A  rectangular  pipe  flume,  1.3  X  10  cm  in  cross  section 
and  1.26  m  in  length,  was  fitted  with  a  2.5  X  2.5  cm  sed- 
iment box  0.8  m  from  the  inlet.  Honeycomb  material  (5- 
mm  cells,  1 1  cm  long)  was  used  as  a  flow  straightener  in 
the  inlet.  A  hot  film  flow  sensor  (TSI  1231W)  was  flush- 
mounted  0.5  cm  upstream  from  the  test  section.  The  sen- 
sor was  calibrated  in  the  wall  of  a  cylindrical  pipe.  The 
flume  geometry  is  scaled  up  from  the  larval  flume  used 
by  Eckman  et  al,  (1990)  in  their  studies  of  barnacle  cy- 
prids.  Rectangular  pipe  flow  was  turbulent,  with  boundary 


52 


S.  A.  WOODIN  ET  AL 


layer  growth  from  the  top,  bottom,  and  sides  of  the  flume. 
The  test  section  was  small  enough  that  flow  characteristics 
varied  by  no  more  than  10%  over  its  area.  The  flow  near 
the  sediment  surface  can  be  summarized  by  the  boundary 
shear  velocity  u*,  which  is  a  measure  of  the  turbulent 
momentum  transfer  from  the  water  to  the  sediment,  and 
is  calculated  from  the  covariance  between  the  vertical  (v ) 
and  horizontal  (u)  velocity  fluctuations: 


»*  =  Vcov  (u.v) 

In  our  flow  treatments  the  boundary  shear  velocity  was 
approximately  1.0  cms"1,  which  is  close  to  the  critical 
erosion  condition  for  sediments  in  False  Bay  (critical  ero- 
sion u*  =  1.2  cm  s~':  Miller  and  Sternberg,  1988).  In  our 
flow  treatments,  which  are  typical  of  tidal  flows,  surface 
sand  grains  wiggled  and  surface  floe  was  eroded.  In  storms 
with  15-cm  waves,  »*  can  be  3.5  cm  s~'  or  higher,  and 
large  amounts  of  sediment  are  resuspended  (Miller  and 
Sternberg.  1988). 

Individual  juveniles  were  introduced  to  the  flume 
through  an  opening  about  1  cm  upstream  of  the  test  sec- 
tion. As  in  the  still-water  dish  experiments,  individuals 
were  timed  from  the  moment  of  initial  contact  with  the 
sediment  surface.  A  dissecting  microscope  with  a  video 
camera  was  used  to  observe  the  behavior  of  six-setiger 
Nereis  juveniles  on  sediments  of  three  types:  surface  sed- 
iments of  False  Bay  origin;  clean,  not  previously  seawater- 
aged,  foundry  sand;  and  surface  sediments  of  False  Bay 
origin  contaminated  with  the  dibromobenzyl  alcohol 
produced  by  the  terebellid  polychaete  Thelepus  crispus. 
Contaminated  sediments  resulted  from  introducing  T. 
crispus  into  the  sediments  for  a  minimum  of  48  h  (for 
concentration  data  as  well  as  extraction  details,  see 
Woodin  el  a/.,  1993).  Such  sediments  are  known  to  be 
rejected  by  juveniles  of  Nereis  in  both  the  field  and  the 
laboratory  (Woodin  el  at..  1993).  In  most  cases,  juveniles 
were  observed  in  still  and  flowing  water. 

Statistical  analysis 

All  experiments  were  analyzed  using  PC  SAS  version 
6.04  (SAS  Institute.  Cary.  NC).  The  data  were  of  two  types: 
(a)  times  to  initiation  of  burrowing  or  nestling  and  (b) 
percentages  of  juveniles  per  dish  accepting  or  rejecting 
the  sediment.  In  the  first  case  only  individuals  that  ini- 
tiated burrowing  or  nestling  were  used.  The  burrowing 
times  were  analyzed  by  analysis  of  variance  (Arenicola) 
or  nested  analysis  of  variance  with  dishes  nested  under 
treatment  (Nereis  and  Mercenaria).  If  a  large  number  of 
the  individuals  failed  to  initiate  burrowing,  making  the 
number  of  observations  per  dish  per  treatment  highly  un- 
balanced, then  the  analysis  was  done  on  the  average  time 
to  initiation  of  burrowing  for  each  dish.  In  these  cases, 
the  analysis  was  not  nested.  Simultaneous  comparisons 


among  treatments  were  made  with  the  Tukey's  studen- 
tized  range  test.  All  data  were  examined  for  normality 
and  homogeneity  of  variance  and  transformed  if  neces- 
sary. The  analyses  and  transformations  used  are  indicated 
for  each  experiment  below.  The  percentage  burrowing 
data  for  the  nereid  juveniles  were  normalized  using  an 
arcsine  square  root  transformation  and  analyzed  by 
analysis  of  variance  followed  by  an  a  posteriori  Tukey's 
studentized  range  test,  except  for  the  data  from  Snug  Har- 
bor. Those  data  were  non-normal  with  unequal  variances 
per  treatment,  so  the  data  were  analyzed  by  separate  Fish- 
er's exact  tests  using  a  conservative  probability  of  0.01. 
For  the  data  on  Mercenaria  and  Arenicola  juveniles  where 
only  two  treatments  were  involved,  a  Fisher's  exact  test 
was  used. 

Results 

Juveniles  on  field-collected  sediment 

When  sediment  from  a  semiprotected  fine  sand  site 
(False  Bay,  WA)  was  used,  juveniles  of  Nereis  vexillosa 
clearly  differentiated  between  surface  and  subsurface  lay- 
ers; 100%  burrowed  into  surface  sediments  and  51%  bur- 
rowed into  subsurface  sediments  (Table  I:  False  Bay — 
Calm).  The  same  was  true  for  1:1  mixtures  by  volume  of 
surface  and  subsurface  sediments;  25%'  failed  to  burrow 
within  the  5-min  observation  period.  In  contrast,  the  re- 
jection rates  were  only  12%  and  14%  for  freshly  collected 
burrow  cleanout  sediments  of  thalassinid  crustaceans 
(burrow  tailings)  and  arenicolid  polychaete  feces,  respec- 
tively. The  percentages  of  juveniles  rejecting  the  sediment 
were  significantly  greater  for  the  subsurface  and  1 : 1  mix- 
ture treatments  than  for  the  surface,  feces.  and  burrow 
tailings  (ANOVA.  arcsine  square  root  transformation:  df 
=  4.  19;  MSE  =  0.0599:  F  =  6.39;  p  <  0.005;  multiple 
comparison  tests:  Table  I).  For  individuals  that  did  bur- 
row, times  to  initiate  burrowing  were  significantly  shorter 
on  undisturbed  surface  sediments  than  on  all  treatments 
including  naturally  disturbed  sediments  (ANOVA,  recip- 
rocal transformation  of  average  times  per  dish:  df  =  4, 
19;  MSE  =  0.00017;  F=  6.10,  p<  0.005)  (Table  I:  False 
Bay— Calm). 

The  surface  versus  subsurface  trials  were  repeated  with 
juveniles  of  Nereis  in  a  mud-gravel  mixed  sediment  (Snug 
Harbor.  WA).  The  results  were  similar  to  those  for  the 
fine  sand  habitat  (False  Bay,  WA).  One  hundred  percent 
of  the  nereid  juveniles  burrowed  into  the  surface  sedi- 
ments, but  only  50%  burrowed  into  the  subsurface  sedi- 
ments. For  individuals  that  did  burrow,  times  to  initiate 
burrowing  were  significantly  shorter  in  all  treatments  other 
than  the  subsurface  sediments  (Table  I:  Snug  Harbor) 
(ANOVA,  average  times  per  dish:  df  =  3,  6;  MSE  =  79.53; 
F  =  40.29,  />  <  0.0005).  Because  of  resuspension  problems. 


PROCESS-SPECIFIC  CUES 


53 


Table  I 

Responses  of  nereid juveniles  to  selected  sediment  types:  Pan  A, 
reieelion  percentages  by  dish:  Par!  B.  initiation  tunes  in  seconds  of 
individuals  l/uil  burrowed 


Part  A.  Percentage  of  [ndmduals  That  Did  Not  Burrow 
False  Bav  —                 False  Ba\  — 

Snug  Harbor  — 

Calm 

Storm 

Calm 

Sediment 

A 

Reject 

Ar 

Reject 

A 

Reject 

Surface 

45 

0  (0)  A 

10 

0  (0)  A 

40 

0(0)  A 

Subsurface 

31 

51  (I2IB 

8 

0(0)  A 

30 

50  (26.5)  B 

1:1  mix 

39 

25  (7)  B 

NA 

NA 

NA 

NA 

Feces 

50 

14(8)  A 

NA 

NA 

21 

0(0)  A 

Bur.  tailings 

50 

12  (6)  A 

NA 

NA 

20 

0(0)  A 

Part  B.  Initiation  Times  (s)  of  Indiv 

iduals  That  Burrowed 

False  Bay- 

False  Bay— 

Snug  Harbor  — 

Calm 

Storm 

Calm 

Sediment 

A' 

Inn.  (s) 

N 

Imt.  (s) 

N 

Imt.  (s) 

Surface 

45 

29.3  (3.8)  A 

10 

58.4(21.6)  A 

40 

15.3(3.8)  A 

Subsurface 

15 

126.6(11.0)8 

8 

41.8(6.8)  A 

15 

97.8(5.9)8 

1:1  mix 

30 

117.6(25.7)8 

NA 

NA 

NA 

NA 

Feces 

43 

109.7(19.5)8 

NA 

NA 

21 

25.2(10.3)  A 

Bur.  tailings 

44 

106.1  (24.3)  B 

NA 

NA 

20 

31.6(3.5)  A 

'1:1  mix'  are  volumetric  mixtures  of  surface  and  subsurface  sediments: 
'feces'  are  fresh  feces  of  the  polychaete  Abaremcola  pacifica:  'bur.  tailings' 
are  fresh  burrow  cleanouts  of  the  thalassinid  crustacean  Vpogehia  pug- 
etleii.si.s.  'Reject'  is  the  mean  percentage  of  individuals  per  dish  which 
did  not  burrow.  'Init.  (s)'  is  the  time  in  seconds  to  initiation  of  burrowing 
into  the  sediment.  These  are  means  and  standard  errors  of  individuals 
by  dish.  'NA'  means  not  available.  Letters  after  each  number  indicate 
the  results  of  a  posteriori  Tukey's  studentized  range  tests  (/>  <  0.05)  for 
that  column  category,  with  the  exception  of  the  percent  rejection  data 
for  Snug  Harbor,  which  are  Fisher's  exact  tests.  Numbers  with  the  same 
letter  within  a  column  are  not  significantly  different  from  one  another. 
False  Bay  is  a  semiprotected.  muddy  sand  site,  while  the  sediment  at 
Snug  Harbor  is  a  protected  mud-gravel  mixture.  'False  Bay — Storm'  are 
results  from  False  Bay  sediments  following  a  windstorm  that  caused 
whitecaps  within  the  bay.  "A"  is  total  number  of  juveniles  used  in  that 
treatment. 


we  were  unable  to  make  satisfactory  1 : 1  mixtures  of  the 
sediments  at  this  site. 

The  results  for  time  to  initiate  burrowing  for  the  ju- 
veniles ofArenicola  cristala  were  similar  to  those  for  Ner- 
eis vexillosa.  The  time  to  initiate  burrowing  was  signifi- 
cantly longer  for  juveniles  on  subsurface  sediments  than 
on  surface  sediments  (ANOVA,  log  transformed  times: 
df  =  1.21:  MSE  =  0.0497:  F  =  26.97;  p  <  0.000 1 )  (Table 
IIB).  The  percentage  burrowing  into  each  treatment  was 
equivalent:  93%  burrowed  into  surface  sediments;  86% 
burrowed  into  subsurface  sediments. 

New  juveniles  (spat)  of  Mercenaria  mercenaria  clearly 
differentiated  between  surface  and  subsurface  sediments, 
both  in  mud  and  sand  (Table  IIA).  The  initiation  times 
were  significantly  shorter  on  surface  sediments  than  on 


subsurface  sediments  (nested  ANOVA,  log  base  10  trans- 
formed data:  df  =  1,4;  MSE  =  0.064;  F  =  39.4;  p  <  0.005) 
(Table  IIA).  Correspondingly,  for  both  mud  and  sand 
sediment  combined,  100%  nestled  into  surface  sediments, 
but  only  40%  to  50%  nestled  into  subsurface  sediments 
(Table  IIA).  The  initiation  times  were  not  significantly 
different  between  mud  and  sand  sediments  ( nested 
ANOVA,  log  base  10  transformed  data:  df  =  1,  24;  MSE 
=  3.13:  F  =  0.08;  p  =  0.78).  Particle  size  appears  to  be 
much  less  important  than  whether  the  sediment  is  from 
the  surface  or  the  subsurface. 

Storm-mixed  sediments 

At  one  of  our  sites.  False  Bay,  storms  have  been  ob- 
served to  resuspend  and  transport  surface  sediment  layers 
at  a  rate  of  16  mgcm~:  s"1  (average  sediment  flux  rate 
without  regard  to  direction)  and  5.4  mg  cm"2  s  '  (depo- 
sition or  vertical  sediment  flux  rate)  (Miller  and  Steinberg, 
1988).  When  a  storm  occurred  during  these  experiments, 
we  collected  surface  and  subsurface  sediments  and  ex- 
posed the  nereid  juveniles  to  them.  As  expected  under 
these  conditions — surface  and  subsurface  sediments 
thoroughly  mixed  to  a  depth  of  several  centimeters — there 
was  no  significant  difference  in  time  to  initiate  burrowing 
on  surface  compared  with  subsurface  sediments  (Table  I: 
False  Bay— Storm:  nested  ANOVA:  df  ==  1.  6;  MSE 
=  1578.48;  F  =  0.65;  />  =  0.45).  One  hundred  percent  of 
the  nereid  juveniles  burrowed  into  the  surface  and  the 
subsurface  sediments.  This  result  is  quite  different  from 
that  obtained  with  1 : 1  mixtures  of  sediment  in  which  sub- 
surface sediment  was  mixed  with  surface  sediment  without 
the  agitation  and  aeration  typical  of  storm-induced  sed- 
iment mixing. 

Feeding  traces 

Juvenile  Nereis  were  also  tested  on  sediments  contain- 
ing active  macrofauna:  the  spionid  polychaetes  Spio  sp., 
Rhynchospio  g/iitaeu.s;  and  Pygospio  elegans  and  the  tel- 
linid  bivalve  Macoma  nasuta.  Individuals  of  each  species 
were  introduced  into  separate  cores  of  defaunated  sedi- 
ment and  maintained  in  the  laboratory  in  running  sea- 
water  for  two  weeks  before  use.  Only  cores  with  obvious 
feces  after  the  two-week  incubation  were  used.  Controls 
were  cores  without  added  fauna:  these  control  cores  were 
also  maintained  in  running  seawater  in  the  laboratory  for 
two  weeks  before  use.  Although  100%.  of  the  nereid  ju- 
veniles burrowed  in  all  treatments,  times  to  initiate  bur- 
rowing were  significantly  different  among  the  treatments 
(nested  ANOVA  on  log  transformed  data:  df  =7,  16; 
MSE  =  0.0883:  F  =  1 8.76:  p  <  0.000 1 ).  The  feeding  traces 
of  Macoma  caused  a  significant  increase  in  time  to  ini- 
tiation of  burrowing  (Table  IIIA).  Of  the  species  tested, 
Macoma  is  the  infaunal  organism  with  the  deepest  feeding 


54  S.  A.  WOODIN  ET  AL 

Table  II 

Times  in  seconds  to  initiate  nestling  or  harrowing  into  the  sediment  ('Init.  Time')  by  juveniles  of  Mercenaria  mercenana  and  Arenicola  cristata 


Sediment 


Type 


Init.  Time 


Perc.  Reject 


Mercenaria  mercenaria 

Surface 

10 

35.  7  (7.  7)  A 

10 

0(0)  A 

Mud 

Subsurface 

5 

156.6(35.5)  B 

10 

50(50)B 

Muddv  sand 

Surface 

10 

21.7(6.7)  A 

10 

0(0)  A 

Subsurface 

6 

128.4(7.1)  B 

10 

40  (20)  B 

Arenicola  cristata 

Surface 

13 

41.4  (6.4)  A 

14 

7  A 

Muddv  sand 

Subsurface 

12 

133.9(23.2)8 

14 

14  A 

Percentage  of  the  individuals  that  did  not  nestle  or  burrow  within  the  observation  period  ('Perc.  Reject').  Letters  after  each  number  indicate  the 
results  of  a  posteriori  Tukey's  studentized  range  tests  (p  <  0.05)  for  initiation  times  in  seconds  Clnit.  Time')  or  a  Fisher's  exact  test  for  percentage 
rejecting  the  sediment  ('Perc.  Reject').  Numbers  with  the  same  letter  are  not  significantly  different  from  one  another.  W  is  number  of  juveniles  in 
that  treatment.  Means  and  standard  errors  are  for  individuals  by  dish  for  Mercenaria  mercenaria  and  by  treatment  for  Arenicola  cristata. 


traces:  2  to  5  mm  compared  with  1  mm  or  less  for  the 
spionid  polychaetes. 

Times  to  burrow  were  significantly  greater  on  recent 
feeding  traces  than  in  adjacent  areas  (nested  ANOVA  on 
log  transformed  data:  df  =  3,  32;  MSE  =  0. 1 1 32;  F  =  5.33; 
p  <  0.01)  (Table  IIIB).  The  distances  between  sites  of 
different  types  on  a  single  core  surface  were  3  cm  or  less, 
showing  that  differentiation  can  occur  on  small  spatial 
scales. 


Table  III 

Times  in  seconds  ('Init.  Time')  for  juveniles  of  'Nereis  vexillosa  to 
initiate  burrowing 


Macrofauna 


Type  of  Location 


Init.  Time 


Part  A. 

Spw(P) 

Control 

7.3  (0.7)  A 

15 

Feeding 

8.3  (1.0)  A 

15 

Pygospio  (P) 

Control 

15.1  (3.4)  A 

15 

Feeding 

7.3(1.6)  A 

16 

Rhynchospio  (P) 

Control 

10.4(2.7)  A 

15 

Feeding 

10.7(2.0)  A 

17 

Macoma  (B) 

Control 

14.6  (1.9)  A 

20 

Feeding 

59.5  (19.6)B 

22 

Part  B. 

Macoma  (B) 

Outside  recent  disturb. 

24.5  (6.8)  A 

17 

New  feeding  areas 

59.5  (19.6)B 

22 

Means  and  standard  errors  of  initiation  times  by  core.  Part  A.  Results 
from  core  surfaces  with  known  macrofauna  present,  feeding  areas  only 
('feeding')  or  from  surfaces  of  cores  held  in  the  same  tank  but  without 
macrofaunal  additions  ('control').  Part  B.  Results  for  cores  with  Macoma. 
different  locations  within  the  same  core.  All  cores  with  Macoma  added 
but  sites  designated  as  recently  disturbed  or  undisturbed.  Recently  dis- 
turbed sites  were  areas  of  recent  feeding  activity  by  Macoma.  The  surface 
floe  layer  was  missing  and  the  feeding  traces  were  clearly  defined.  Letters 
in  parentheses  after  the  species  indicate  taxon:  'P'  polychaete  and  'B' 
bivalve.  Letters  after  each  number  indicate  the  results  of  a  posteriori 
Tukey's  studem'ized  range  tests  (p  <  0.05)  for  that  column  category 
within  that  section  of  the  table.  '/V  is  total  number  of  juveniles  used  in 
that  treatment. 


Flow  versus  no-flow  conditions 

In  the  flume  with  surface  sediments  as  the  test  substrate, 
times  to  initiate  burrowing  by  Nereis  juveniles  were  similar 
for  still  water  and  flow  treatments  (Table  IV:  Burrowed: 
Init.),  and  burrowing  individuals  were  not  eroded  from 
such  surfaces.  All  but  3  of  1 7  juveniles  successfully  bur- 
rowed in  the  flow  treatment;  all  burrowed  without  flow 
(Table  IV).  In  the  flow  treatment,  flow  was  initiated  as 
soon  as  the  individual  appeared  to  reach  the  sediment 
surface.  The  three  that  were  eroded  blew  off  at  2,  3,  and 
135  s.  The  first  two  probably  had  not  made  contact  with 
the  sediment  surface  before  flow  was  initiated. 

Observations  were  also  made  on  individuals  placed  onto 
sediments  likely  to  be  unacceptable  to  the  nereid  juveniles: 
clean  foundry  sands  and  sediment  contaminated  with  the 
bromobenzyl  alcohol  of  Thelepus  crispus  (Woodin  et  al., 
1993).  The  nereidsdid  not  burrow  immediately  on  either 
of  these  sediments;  in  contrast,  on  uncontaminated  surface 
sediments,  the  nereid  juveniles  burrowed  on  average 
within  1 1  s  both  with  and  without  flow  (Table  IV).  To 
ensure  that  individuals  had  made  contact  with  the  sedi- 
ment surface  in  the  treatments  with  foundry  sand  and 
with  contaminated  sediments,  flow  was  not  initiated  until 
at  least  20  s  after  the  juveniles  made  contact  with  the  sed- 
iments. Individuals  remaining  on  the  surface  for  more 
than  40  s  were  eroded  within  2  min  in  all  but  3  of  17 
cases.  One  of  the  three  eventually  burrowed  (at  210s), 
while  the  other  two  eroded  off.  Times  to  erosion  once 
flow  was  initiated  are  given  in  Table  IV.  On  average,  in- 
dividuals were  eroded  in  less  than  80  s  with  flow  (Table 
IV:  Erosion  Time  in  Flow). 

Discussion 

Surfaces  of  sedimentary  habitats  are  dynamic  land- 
scapes, changing  in  response  to  both  biotic  and  physical 
forces.  Currents,  winds,  and  waves  can  mix,  resuspend, 
transport,  move  as  bedload,  and  deposit  sediments,  al- 


PROCESS-SPECIFIC  CUES 

Table  IV 

Responses  of  nereid juveniles  to  selected  \edimcnt  nyo  in  the  flume 


55 


Burrowed 

Eroded 

A' 

Init.  (s) 

N 

Seconds 

Erosion  time  in  flow 

Perc.  reject 

Surface  Sed. 

No  flow 

7 

9.8(14.7)  A 

0 

NA 

NA 

0  A 

Row 

14 

10.4  (5.8)  A 

3 

46.7(76.5) 

46.7  (76.5) 

17.6  A 

All  with  Flow 

Surface  sed. 

14 

10.4(5.8) 

3 

46.7  (76.5)  A 

46.7  (76.5)  A 

17.6  A 

Foundry  sand 

0 

8 

170.1  (135.3)  B 

80.7  (96.8)  A 

100  B 

Theleptis  sed. 

1 

210 

9 

51.3(17.5)  AB 

15.9(15.2)  A 

90.0  B 

'Burrowed'  means  those  individuals  that  burrowed  into  the  sediment.  'Init.  (s)'  is  the  time  in  seconds  to  initiation  of  burrowing  into  the  sediment. 
'Eroded'  is  the  time  in  seconds  from  contact  with  the  sediment  surface  to  erosion  and  includes  both  time  with  and  without  flow.  'Erosion  Time  in 
Row'  is  the  time  in  seconds  from  initiation  of  flow  to  erosion  off  the  surface.  'Perc.  Reject'  is  the  percentage  of  the  individuals  that  did  not  burrow. 
'A"  is  the  number  of  juveniles  per  category  of  a  treatment.  'NA'  means  not  available.  For  example,  in  the  no-flow  treatment  for  surface  sediments. 
7  juveniles  burrowed,  none  were  eroded,  and  0%  rejected  the  sediment.  Letters  after  each  number  indicate  the  results  of  a  posteriori  Tukey's 
studentized  range  tests  (/>  <  0.05)  for  that  column  category  with  the  exception  of 'Perc.  Reject',  which  are  results  of  Fisher's  exact  tests.  Numbers 
with  the  same  letter  within  a  column  are  not  significantly  different  from  one  another  at  the  0.05  level.  Means  and  standard  deviations  are  given. 


tering  the  landscape  (Miller  and  Sternberg,  1988).  Biotic 
events  such  as  predator  excavations,  surface  deposit  feed- 
ing, pit  feeding,  burrowing,  and  defecation  also  shape  the 
sediment  surface  (Hughes,  1969;  Rhoads  and  Young. 
1970;  Brenchley,  1981;  Grant,  1983;  Smith  el  al.,  1986; 
Nowell  ct  al..  1984;  Posey.  1986;  Krager  and  Woodin, 
1993).  Many  of  these  events  require  a  response  from  the 
infauna.  Sediment  deposition,  whether  biotic  or  physical 
in  origin,  can  result  in  a  period  of  burrowing  and  rees- 
tablishment  or  even  in  the  death  of  the  infauna  (Gallucci 
and  Kawaratani.  1975;  Nichols  el  al.  1978;  Turk  and 
Risk.  1981;  Wilson.  1981). 

Recently  settled  juveniles  of  infauna  are  particularly 
susceptible  to  a  number  of  these  surface-associated  pro- 
cesses. Feeding  by  surface  deposit-feeders,  such  as  spionid 
polychaetes  and  tellinid  bivalves,  can  result  in  spatial  par- 
titioning of  large  individuals  (Holme,  1950;  Levin,  1981) 
or  mortality  of  small  ones  (Wilson,  1980;  Levin,  1981; 
Elmgren  el  al..  1986;  Hines  et  al..  1989;  Olafsson.  1989). 
Sediment  deposition  can  also  increase  rates  of  mortality 
for  juveniles  (Wilson,  1981;  Brenchley.  1982:  Posey, 
1986).  These  biotic  and  physical  events  have  similar  effects 
on  the  original  sediment  surface,  causing  either  burial  or 
removal  of  the  original  surface.  Given  this  commonality 
of  effect  on  surfaces  and  on  mortality  rates,  selection 
should  favor  those  recruits  capable  of  (a)  distinguishing 
between  recently  disturbed  (exposed  subsurface  sedi- 
ments) and  undisturbed  surfaces  and  (b)  emigrating  from 
sites  with  disturbance.  The  data  presented  here  show 
clearly  that  new  juveniles  of  two  polychaete  species  and 
a  bivalve  species  can  distinguish  between  recently  dis- 
turbed and  undisturbed  sediment  surfaces  and  modify 
their  behavior  accordingly.  In  all  cases,  burrowing  times 


are  significantly  greater  on  recently  disturbed  surfaces  than 
on  undisturbed  surfaces  (Tables  I  to  III);  in  some  cases, 
rates  of  complete  rejection,  where  the  juvenile  did  not 
burrow,  are  higher  on  disturbed  surfaces  (Tables  I  and 
II).  This  was  true  for  simulated  erosional  events  (surface 
compared  with  subsurface:  Tables  I  and  II),  as  well  as  for 
simulated  mixing  (surface  compared  with  1:1  mix:  Table 
I).  Time  to  acceptance  was  also  significantly  longer  for 
new  juveniles  offered  fresh  feces  or  burrow  tailings  than 
for  those  given  undisturbed  surface  sediments  (Table  I). 
Finally,  exposure  to  feeding  traces  made  by  large  infauna 
such  as  the  tellinid  bivalve  Macoma  nasuta  caused  sig- 
nificant increases  in  burrowing  times  (Table  III).  All  of 
these  results  are  consistent  with  the  hypothesis  that  ju- 
veniles distinguish  between  undisturbed  and  recently  dis- 
turbed sediment  surfaces,  whether  the  disturbance  is  de- 
positional  or  erosional,  physical  or  biotic.  A  corollary  of 
this  hypothesis  is  that  there  is  a  selective  advantage  to 
individuals  able  to  distinguish  between  recently  disturbed 
and  undisturbed  sediment  surfaces.  This  does  not,  of 
course,  mean  that  the  currently  undisturbed  site  will  re- 
main undisturbed,  either  by  biotic  or  physical  forces 
(Wilson,  1981:  Krager  and  Woodin,  1993). 

When  disturbed  sediments  were  accepted  by  the  ju- 
veniles, times  to  initiation  of  burrowing  or  nestling  were 
significantly  (4-  to  5-fold)  longer  than  in  undisturbed  sed- 
iments (Tables  I  and  II).  Under  these  conditions,  juveniles 
remained  on  the  sediment  surface  for  1.5  to  2  min  or 
more.  Such  behaviors  can  increase  the  probability  of  ero- 
sion. Our  flume  data  clearly  indicate  that  individuals  that 
did  not  burrow  were  eroded  (Table  IV).  None  of  the  in- 
dividuals that  burrowed  was  eroded.  All  but  3  of  the  17 
individuals  that  did  not  burrow  within  40  s  of  contacting 


56 


S.  A.  WOODIN  ET  AL 


the  bottom  were  eroded  within  2  min  of  initiation  of  flow. 
On  average,  erosion  of  individuals  from  the  surface  oc- 
curred in  less  than  80  s  after  initiation  of  flow.  Flow  in 
these  experiments  had  an  average  boundary  shear  velocity 
of  1.0  cm  s"1,  which  is  much  less  than  storm  conditions 
(False  Bay.  WA:  it*  =  3.5  cm  s  '  or  more:  Miller  and 
Sternberg,  1988)  and  is  comparable  to  conditions  for 
maximum  flood  tidal  flows  [Oyster  Landing.  SC:  i<*:  0.7 
to  1.0  cm  s  '  (Palmer  and  Gust,  1985;  Wethey  el  a/.,  un- 
pub.  data)]  in  these  habitats  or  for  small-amplitude  waves 
(Denny  and  Shibata,  1989).  Thus  our  flume  results  are  a 
highly  conservative  estimate  of  the  probability  of  erosion. 
Interestingly,  the  temporal  spacing  between  small-ampli- 
tude waves  typical  of  these  habitats  and  likely  to  cause 
erosion  is  of  the  same  order  of  magnitude  as  the  time 
necessary  to  initiate  burrowing  (Pond  and  Pickard,  1983: 
Denny,  1988;  Miller  and  Sternberg,  1988). 

Travel  by  erosion  or  wafting  is  a  well-known  phenom- 
enon (Sigurdsson  el  ai.  1976;  Sastry,  1979)  and  may  also 
help  explain  some  of  the  results  of  Butman  and  her  col- 
laborators on  the  effect  of  still  water  and  flow  conditions 
on  larval  settlement  selectivity  (e.g.,  Snelgrove  el  ai, 
1993).  Some  species  show  more  settlement  selectivity  with 
flow  than  without  (Mitliniu  lateralis:  Grassle  ct  ai.  1 992b), 
whereas  other  species  are  relatively  insensitive  to  the  pres- 
ence or  absence  of  water  motion  (Capitella  sp.  1:  Butman 
and  Grassle,  1992;  Grassle  el  ai.  1992a).  For  species  that 
rely  on  erosion  from  the  surface  to  transport  them  short 
distances,  such  as  the  nereid  juveniles  used  in  these  ex- 
periments, the  no-flow  treatment  would  result  in  lowered 
rates  of  emigration  and  thus  less  apparent  selectivity. 

Larvae  and  new  juveniles  are  known  to  reject  other 
habitats  on  the  basis  of  chemical  cues  (see  review  by  Paw- 
lik.  1992),  although  the  literature  on  negative  settlement 
cues  is  not  extensive.  In  all  of  these  cases,  the  compound 
that  causes  rejection  is  intimately  associated  with  the  or- 
ganism and  is  produced  by  that  organism  or  a  symbiont. 
For  example,  the  terebellid  polychaete  Thelepm  crispns 
releases  a  brominated  aromatic  compound  into  the  sed- 
iments surrounding  its  tube,  and  this  inhibits  the  recruit- 
ment of  Nereis  vexillosa  (Woodin  el  ai,  1993).  To  date, 
all  the  reported  negative  responses  are  to  organism-specific 
cues  (Pawlik,  1992).  In  the  data  reported  here,  the  negative 
response  is  to  a  process-specific  event,  disruption  of  the 
surface  sediments  either  by  removal  or  burial  (Tables  I 
to  III).  A  process-specific  cue  is  distinctly  different  from 
an  organism-specific  cue.  For  example,  although  a  number 
of  organisms  have  bioactive  compounds  that  deter  settle- 
ment, these  compounds  vary  greatly  in  their  effectiveness 
on  different  taxa,  and  the  effect  of  the  compound  is  typ- 
ically limited  to  the  surface  of  the  organism  itself  or  the 
immediately. surrounding  area  (Pawlik,  1992;  Woodin  el 
a!..  1993).  With  process-specific  cues,  the  effect  is  confined 
to  the  area  affected  by  the  process,  which  can  be  physical 


or  biotic.  and  is  thus  not  limited  to  the  distribution  or 
abundance  or  even  the  habitat  of  a  given  taxon.  Given 
the  mortality  associated  with  sediments  that  are  disturbed, 
the  selective  advantage  accrued  to  individuals  able  to  dif- 
ferentiate between  disturbed  and  undisturbed  habitats  and 
vary  emigration  rates  in  response  is  potentially  enormous. 

Acknowledgments 

Dr.  Mark  Luckenbach  kindly  provided  late  pediveliger 
Mercenuria  mercenaria  larvae.  The  Director  of  the  Friday 
Harbor  Laboratories  gave  us  research  space  and  access  to 
research  sites.  The  Snug  Harbor  Resort  allowed  us  to  use 
their  property  as  a  research  site.  The  Director  of  the  Belle 
W.  Baruch  Institute  for  Marine  and  Coastal  Research  al- 
lowed us  access  to  research  sites.  E.  R.  W.  Wethey  col- 
lected egg  masses  and  provided  field  assistance.  C.  Rich- 
mond, M.  Grove.  J.  Hilbish,  L.  Levin.  W.  H.  Wilson.  R. 
Zimmer-Faust,  and  two  anonymous  reviewers  made 
valuable  comments  on  the  manuscript.  S.  M.  Lindsay 
was  supported  by  an  NSF  graduate  research  fellowship. 
This  research  was  supported  by  NSF  grant  OCE-89002 1 2 
to  S.  A.  Woodin  and  ONR  grant  N00014-82-K-0645  and 
NSF  grant  OCE-86-00531  to  D.  S.  Wethey. 

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survival  in  the  littoral  barnacle  Semibalanus  balanoides  (L.):  patterns 

and  consequences.  Pp.  194-202  in  The  Ecology  of  Rocky  Coasts. 

P.  G.  Moore  and  R.  Seed,  eds.  Hodder  and  Stoughton.  Sevenoaks, 

Kent,  UK. 
Wilson,  D.  P.  1952.     The  influence  of  the  nature  of  the  substratum  on 

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larvae  of  Ophelia  bicornis  Savigny.  Ann.  Inst.  Oceanogr.  27:  49-156. 
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terebellid  polychaete  on  the  survivorship  of  nereid  polychaete  larvae. 

./  Exp  Mar  Biol.  Ecol  46:  73-80. 
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Reference:  Biol  Bull  189:  59-68.  (August.  1995) 


Functional  Significance  of  Varices  in  the  Muricid 
Gastropod  Ceratostoma  foliatum 

THOMAS  H.  CAREFOOT  AND  DEBORAH  A.  DONOVAN 
Department  of  Zoology,  University  of  British  Columbia,  Vancouver.  Canada  V6T  1Z4 


Abstract.  Functional  significance  of  varices  in  the  mur- 
icid  gastropod  Ceratostoma  foliatum  was  investigated 
from  the  standpoints  of  (1)  frequency  of  landing  in  the 
two  upside-down  orientations  after  short  vertical  falls  of 
less  than  five  body  lengths  through  seawater  and  energy 
costs  of  righting  from  these  upside-down  positions,  and 
(2)  scaling  relationships  of  varix  areas  with  other  body 
dimensions.  Field  manipulations  showed  that  C.  foliatum 
occupied  habitats  that  mostly  permit  short  falls  of  less 
than  five  body  lengths  upon  dislodgment,  as  might  occur 
during  predation  by  fish.  After  short  vertical  falls  in  the 
laboratory,  animals  landed  48%  of  the  time  on  their  ap- 
erture sides  (upright),  15%  on  their  right  sides  (on  right 
and  middle  varices),  and  37%  on  their  left  sides  (on  left 
and  middle  varices).  These  frequencies  differed  signifi- 
cantly from  the  expected  frequencies  calculated  on  the 
basis  of  the  percentage  circumference  delineated  by  each 
varix  pair  (50%,  31%.,  and  19%,  respectively).  Righting 
from  the  right-side  orientation  was  slower  and  four  times 
more  energetically  costly  than  from  the  left-side  orien- 
tation, underscoring  the  advantage  conferred  by  animals, 
if  not  landing  in  the  upright  position  after  short  falls,  pref- 
erentially landing  on  their  left  sides.  Removal  of  individual 
varices  showed  that  the  large,  right  varix  is  most  influential 
in  producing  this  "destabilization."  Landings  are  biased 
to  the  side  from  which  rightings  are  easiest  due  to  a  com- 
bination of  the  location  of  center  of  mass  within  the  left 
side  of  the  main  body  whorl  and  the  broad  right  varix 
possibly  acting  as  an  upward-trailing  vane. 

Morphometric  relationships  of  shell  length,  live  weight, 
varix  areas,  aperture  dimensions,  and  labial  spine  (tooth) 
length  were  investigated  over  a  wide  range  of  body  sizes 
in  an  attempt  to  infer  varix  function.  Aperture  area  scaled 
allometrically  with  length.  Right-,  middle-,  and  left-varix 


Received  17  October  1994;  accepted  1  May  1995. 


areas  also  grew  relatively  larger  as  the  animals  increased 
in  length.  In  contrast,  combined  varix  areas  around  the 
aperture  increased  in  direct  proportion  with  aperture  area, 
forming  a  broad  shelf  surrounding  the  aperture.  We  infer 
from  this  that,  in  addition  to  their  effects  on  landing  ori- 
entation from  both  long  and  short  vertical  falls,  the  varices 
of  C.  foliatum  may  function  to  protect  the  aperture,  and 
thus  protect  the  soft  body  parts  that  protrude  from  it  dur- 
ing feeding  and  locomotion. 

Introduction 

The  function  of  shell  ornamentation  in  prosobranch 
gastropods  has  intrigued  scientists  for  decades.  Shell  or- 
namentation is  most  well-developed  in  the  family  Muri- 
cidae,  where  it  consists  of  ridgings,  or  varices,  elaborated 
from  thickenings  of  the  outermost  shell  edge  during 
growth.  In  the  adult  leafy  hornmouth  snail,  Ceratostoma 
foliatum.  three  such  varices  predominate  (Fig.  1).  The 
newest  varix  is  the  right  one.  New  varices  are  added  in 
growth  spurts,  which  causes  the  varices  to  shift  position, 
with  the  present  right  one  coming  to  occupy  a  new  middle 
position,  and  the  present  middle  one  becoming  a  new  left 
varix. 

The  precise  function  of  shell  sculpturing  in  C.  foliatum 
and  other  muricid  gastropods  is  not  known,  although 
suggestions  include  ( 1 )  stabilizing  the  shell  in  shifting  sub- 
stratum; (2)  aiding  in  feeding;  (3)  supporting  or  protecting 
sensory  structures;  (4)  perceiving  vibration;  (5)  protecting 
the  snail  from  predators;  (6)  strengthening  the  shell  (as 
seen  for  thicker-shelled  Nucella  lapillus,  which  withstand 
predatory  attacks  by  crabs  better  than  thinner-shelled  va- 
rieties); and  (7)  in  C.  foliatum.  destabilizing  the  falling 
orientation  such  that  landing  is  more  often  in  the  aperture- 
down  position  (Ebling  et  a/..  1964;  Kitching  el  ai,  1966; 
Carter,  1967;  Fotheringham.  1971;  Spight  and  Lyons, 
1974;  Palmer,  1977;  Vermeij,  1974,  1979,  pers.  com.; 


59 


60 


T    H    CAREFOOT  AND  D.  A.  DONOVAN 


7^-  Siphon 


Right 
varix 


Left-remnant 
varix 

OBLIQUE  TOP  VIEW 


Middle 
varix 


RIGHT -SIDE  VIEW 


Left-remnant 
varix 


Right 

varix  Position  of 
labial  spine 
(not  shown) 


BOTTOM  VIEW 


Aperture 
area 


Figure  1.  Top:  Oblique  plan  view  of  Ceralostoma  foliation  to  show 
varix  arrangement.  Middle  and  bottom  Views  from  the  right  side  and 
bottom,  respectively,  to  show  areas  of  right-,  left-,  middle-,  and  left- 
remnant  varices,  and  aperture,  as  measured  for  allometry.  The  dots  in- 
dicate the  location  of  center  of  mass  in  a  live  but  withdrawn  animal. 


Morris  el  al.,  1980).  Other  possible  paradigms  are  (8) 
camouflaging  the  snail  (reminiscent  of  the  shell  debris 
employed  by  the  carrier  shell,  Xenophora  conchyliophord); 
(9)  reducing  or  preventing  rolling  in  currents;  and  (10) 
increasing  apparent  size  to  deter  predators. 

The  problem  for  molluscan  functional  morphologists 
is  that  few  of  these  hypotheses  are  testable.  Even  the  ele- 
gant demonstration  by  Palmer  (1977)  that  the  middle  va- 
rix of  C.  foliatwn  (see  Fig.  1 )  acts  to  destabilize  the  ani- 
mal's orientation  during  falling,  causing  it  to  land  aperture 
down,  is  open  to  question. 

First,  the  requirement  for  a  falling  distance  of  at  least 
10  body-lengths  to  ensure  35%-70'r  landing  success  (ap- 
erture down)  would  rarely  be  satisfied  in  C.  foliatwn 's 
rock-  and  seaweed-strewn  natural  habitat.  Our  field  ob- 


servations (shown  later)  suggest  that  few  individuals  of 
this  species  occupy  positions  that  permit  even  five  body- 
lengths  of  free-fall  to  the  bottom.  Most  would  bump,  roll, 
or  slide,  even  from  near-vertical  slopes. 

Second,  Palmer  suggests  that  kelp  greenling  prey  upon 
C.  foliatwn  by  dislodging  the  snails  and  consuming  their 
feet  when  they  are  exposed  during  righting.  However,  we 
have  been  unable,  using  the  precipitin  test  of  Pickavance 
(1970).  to  identify  the  presence  of  C.  foliatwn  antigens  in 
the  stomach  contents  of  42  kelp  greenling  fish  coexisting 
with  C.  foliatwn.  This  is  despite  positive  responses  from 
stomach  contents  of  kelp  greenling  experimentally  fed  on 
C.  foliatwn,  and  a  robust  response  of  C.  foliatwn  antisera 
(induced  in  rabbits)  to  C.  foliatwn  antigen  preparations 
in  control  tests  (unpub.  data).  C.  foliatwn  lives  most  com- 
monly in  the  low  intertidal  and  subtidal  regions  to  a  depth 
of  30  m  and  feeds  predominantly  on  barnacles  and  bi- 
valves (Spight  et  al..  1974;  Spight  and  Lyons,  1974;  Kent. 
1981 ).  Although  little  is  known  of  its  natural  biology,  its 
principal  predators  might  be  seastars,  such  as  the  large, 
fast-moving  sunflower  star.  Pycnopodia  helianthoides. 
rather  than  fish.  If  this  is  the  case,  dislodgment  due  to 
predation  is  less  likely. 

Third,  although  this  by  no  means  discounts  a  desta- 
bilizing function  for  the  middle  varix  of  C.  foliation,  the 
fact  that  many  other  muricids  have  tri-radiate  varices  of 
spines  rather  than  blades  suggests  that  other  selective  fac- 
tors are  operating. 

Finally,  if  the  primary  function  of  varices  in  C.  foliatwn 
is  to  provide  a  large  right  varix  for  protection  or  stability 
during  locomotion  and  feeding,  then  the  destabilizing 
function  of  the  middle  varix  might  be  only  secondarily 
important. 

Two  features  of  C.  foliatwn 's  growth  are  relevant  to 
this  introduction  of  varix  function.  First,  the  varices  are 
produced  relatively  rapidly.  Spight  and  Lyons  (1974)  re- 
corded varix  production  about  once  per  year  in  older  an- 
imals, each  one  taking  1-2  months  for  completion.  Our 
in  situ  scuba  observations  indicate  that  varix  growth  must 
be  as  rapid,  or  more  so,  in  the  field.  We  have  only  once 
observed  a  partially  formed  varix,  indicating  that  varices 
are  produced  in  growth  spurts  of  extremely  short  duration 
or  that  they  are  produced  secretively.  In  either  case,  it  is 
clear  that  a  fully  formed  varix  is  vital  to  the  well-being  of 
the  animal.  Second,  the  three-varix  morphology  of  the 
adult  snail  is  not  typical  of  its  entire  life.  While  young 
(<25  mm),  the  snail  produces  multiple  axial  ribs,  up  to 
7-10  per  whorl.  This  changes  to  a  three-varix  pattern  in 
the  adult,  suggesting  that  the  three-varix  morphology, 
whatever  its  function,  is  more  important  in  older  stages. 

Palmer  (1977)  constructed  his  argument  for  varix 
function  in  C.  foliatwn  around  the  premise  that,  on  dis- 
lodgment and  subsequent  free-fall,  it  would  be  advanta- 
geous for  the  snail  to  land  upright.  He  was  not  concerned 


FUNCTION  OF  CERATOSTOMA  VARICES 


61 


with  landing  orientations  other  than  upright,  yet  the  other 
two  landing  orientations  occurred  with  43%- 100%  fre- 
quency, depending  on  height  of  fall,  in  his  study  (Palmer. 
1977).  Lacking  any  other  information,  similar  values 
could  he  predicted  from  falls  of  the  bump-and-roll  type. 
Based  on  shell  shape  and  angle  of  the  varices  (Fig.  2a), 
and  discounting  for  the  present  any  varix  or  center  of 
mass  influence  on  falling,  an  animal  that  landed  randomly 
might  be  expected  to  land  50%  of  the  time  on  its  right 
and  left  varices  (i.e..  upright),  31%  on  its  right  and  middle 
varices  (right-side  posture),  and  19%  on  its  middle  and 
left  varices  (left-side  posture). 

Two  implications  derive  from  these  considerations, 
both  of  which  focus  on  the  advantages  of  landing  in  a 
left-side  orientation  over  a  right-side  orientation.  First,  an 
animal  should  be  able  to  right  itself  more  readily,  that  is 
more  quickly  and  with  less  energy  expenditure,  from  the 
smaller-angled  left-side  orientation  (Fig.  2c)  than  from 
the  larger-angled  right-side  one  (Fig.  2b).  This  is  because 
the  foot  has  a  shorter  reach  to  gain  purchase  on  the  sub- 
stratum. In  contrast,  from  the  proposed  less-favorable 
landing  position  (Fig.  2b),  the  foot  must  reach  further  and 
the  shell  be  levered  through  a  greater  angle  to  right  it. 
Note  that  righting  from  the  "easy"  orientation  exposes 
the  foot  to  lesser  risk  of  predation  than  from  the  "hard" 
orientation.  The  second  implication  is  that  relative  varix 
height  affects  the  ability  of  the  animal  to  right  itself  due 
to  the  change  in  the  angles  the  animal  experiences  in  re- 
lation to  the  substratum  (Fig.  2b,  c).  Thus,  a  larger  middle 
varix  relative  to  the  other  varices  favors  the  righting  pro- 
cess from  both  easy  and  hard  orientations  by  decreasing 
the  distance  that  the  foot  must  traverse.  By  the  same  token, 
a  larger  right  varix  impedes  righting  from  the  hard  ori- 
entation by  increasing  the  distance  of  foot  extension. 
However,  because  a  large  right  varix  is  required  to  produce 
eventually  a  large  middle  varix,  any  such  argument  of 
effect  of  relative  varix  sizes  from  the  hard  orientation  is 
self-defeating. 

Palmer's  (1977)  interest  was  in  the  destabilizing  effect 
of  the  middle  varix  during  falls  mainly  in  excess  of  10 
body  lengths  in  height.  We  are  interested  here  in  dislodg- 
ment  effects  from  heights  less  than  this,  and  especially  in 
the  energetic  consequences  of  the  animal  landing  and 
having  to  right  from  the  left-  and  right-side  orientations. 
We  are  also  interested  in  the  relationship  of  varix  areas 
to  other  body  dimensions,  in  particular  to  aperture  area, 
since  three  varices  combine  to  form  a  broad  shelf  sur- 
rounding the  aperture,  which  may  provide  either  protec- 
tion or  stabilization  (Fig.  1,  bottom).  Hypotheses  to  be 
tested  are  ( 1 )  that  landing  orientations,  other  than  ones 
ending  with  the  animal  upright,  will  favor  the  "easy"  side, 
despite  the  smaller  circumference  occupied  by  this  side; 
and  (2)  that  righting  times  from  the  "easy"  posture  will 
be  shorter  than  from  the  "hard"  and.  correlativelv.  that 


Middle 

a)  varix 


h  =  130° 


Figure  2.  Schematic  representation  of  varices  and  shell-righting  pos- 
tures in  Ceralostomu  folialum.  from  head-on  views,  (a)  Extent  of  cir- 
cumference occupied  by  each  varix-pair  (1 10°  is  equivalent  to  31%  of 
the  circumference;  70°  to  19%).  (h)  Righting  from  the  "hard"  right-side 
landing  orientation  (resting  on  the  middle  and  right  varices).  The  angle 
"h"  denotes  the  extent  of  traverse  of  the  foot  during  righting  from  the 
"hard"  orientation  (130°).  (c)  Righting  from  the  "easy"  left-side  landing 
orientation  (resting  on  the  middle  and  left  varices).  The  angle  "e"  denotes 
the  extent  of  traverse  of  the  foot  during  righting  from  the  "easy"  ori- 
entation (110°). 


energy  expenditure  will  be  less.  Additionally,  morpho- 
metric  relationships  of  various  shell  structures  are  inves- 
tigated and  interpreted  in  an  attempt  to  infer  varix  func- 
tion. 

Materials  and  Methods 

Collection  of  animals 

Animals  were  collected  from  subtidal  locations  in 
Barkley  Sound,  on  the  west  coast  of  Vancouver  Island, 
and  in  Telegraph  Cove,  West  Vancouver,  British  Colum- 
bia. They  were  brought  to  the  University  of  British  Co- 
lumbia and  held  in  tanks  supplied  with  recirculated  sea- 
water.  Barnacle-encrusted  rocks  and  mussels  were  pro- 
vided as  sources  of  food. 

Field  tests  of  falling  distances 

One  hundred  snails  were  selected  at  random  during 
scuba  dives  at  depths  of  3-10  m  along  a  200-m  section 
of  Barkley  Sound  shoreline  characterized  by  large  boulders 
and  rock  walls.  Each  snail  was  manually  dislodged  and 
its  unimpeded  vertical  falling  distance  recorded.  The  div- 
ers moved  from  habitat  to  habitat,  selecting  and  testing 
the  first  individual  of  Ceratostoma foliation  seen  in  each. 
This  procedure  allowed  a  variety  of  habitats  to  be  sampled, 
such  as  sloping  and  vertical  rock  walls  and  overhangs. 
Length  of  each  snail  was  recorded  to  determine  falling 


62 


T.  H.  CAREFOOT  AND  D.  A.  DONOVAN 


distance  relative  to  body  length  (apex  to  tip  of  siphonal 
canal). 

I  'O:  ami  righting  times 

VO2  (oxygen  consumption  in  microliters  of  oxygen  per 
hour)  of  individual  animals  was  measured  in  a  closed  res- 
pirometry  system  by  means  of  a  polarographic  oxygen 
electrode  coupled  with  a  DATACAN  data  acquisition  and 
analysis  program  (SABLE  Systems  Ltd.,  Salt  Lake  City). 
A  stir-bar  in  the  chamber  ensured  continuous  and  thor- 
ough mixing.  Temperature  was  maintained  at  12°C,  the 
same  as  in  the  holding  tanks.  Respirometers  were  made 
of  clear  plastic  and  varied  in  size  (85,  120,  and  700  cm3) 
for  use  with  different-sized  animals.  The  snails  were  placed 
in  an  aperture-down  position  in  the  chamber  and  allowed 
to  rest  for  20  min  while  the  system  equilibrated.  Oxygen 
consumption  was  then  measured  for  20  min  to  establish 
a  resting  ]'O2.  A  narrow-gauge  wire  hook  inserted  into 
the  chamber  through  a  small  hole  in  the  top  was  used  to 
flip  each  snail  randomly  to  either  its  right  ("hard")  side 
or  its  left  ("easy")  side.  It  took  less  than  5-10  s  to  flip  a 
snail  onto  its  side.  The  snails  were  observed  as  they  righted 
themselves  and  times  of  the  following  specific  events  were 
recorded:  ( 1 )  appearance  of  the  snail's  foot  from  under 
the  operculum,  (2)  attachment  of  the  snail's  foot  to  the 
bottom  of  the  chamber,  and  (3)  completion  of  righting, 
as  defined  by  the  settling  of  the  shell  onto  the  back  of  the 
snail.  Righting  time  was  measured  as  the  time  from  first 
appearance  of  the  snail's  foot  to  when  the  shell  settled 
over  the  back  of  the  snail.  Oxygen  consumption  was  re- 
corded during  the  righting  episode.  After  the  20-min  rest 
period,  the  snail  was  flipped  onto  its  other  side  and  the 
procedure  repeated.  Three  I  'O:  values  were  generated  for 
each  animal  from  this  procedure:  ( 1 )  normal  upright  rest- 
ing r<9:  (snails  that  crawled  in  the  chamber  were  not 
tested),  (2)  J'02  when  righting  from  the  easy  orientation, 
and  (3)  \'O2  when  righting  from  the  hard  orientation. 

I  '0:'s  were  multiplied  by  righting  times  from  each  up- 
side-down orientation  to  give  absolute  cost  in  microliters 
of  oxygen  for  each  righting  episode.  Costs  in  microliters 
of  oxygen  were  converted  to  joules  by  multiplying  by  an 
oxycalorific  coefficient  (Q^  of  21.10  mJ-^l  O:  ',  rep- 
resenting an  accepted  value  for  catabolism  of  carbohydrate 
(Elliot  and  Davison,  1975).  This  value  was  chosen  because 
most  gastropods  rely  on  glycogen  stores  for  energy  during 
activity  (Carefoot,  1987). 

Landing  frequencies 

To  determine  the  frequency  of  landing  in  each  of  the 
three  orientations,  upright,  left  side  (easy),  and  right  side 
(hard),  1 37  snails  of  varying  size  were  dropped  in  aquaria 
with  seawater  depths  about  5  times  the  length  of  the  snail. 
We  chose  this  depth  because  ( 1 )  we  did  not  wish  to  invoke 


the  destabilizing  effect  of  the  middle  varix,  shown  by  Pal- 
mer (1977)  to  occur  at  depths  greater  than  10  body- 
lengths,  and  (2)  we  believed  from  our  field  observations 
that  short  falls,  or  bump-and-roll  falls,  would  be  most 
commonly  experienced  by  snails  in  the  field.  Each  snail 
was  held  just  below  the  water  surface  and  released.  Release 
positions  were  random.  Each  snail  was  tested  10  times 
and  the  average  landing  frequency  calculated.  After  this, 
the  length  of  each  snail  was  measured. 

In  another  series  of  experiments  to  test  the  effect  of 
varix  removal  on  landing  frequency,  snails  were  dropped 
20  times  from  random  release  positions  and  their  landing 
orientations  noted,  then  dropped  20  more  times  after  re- 
moval of  a  single  varix.  Varices  were  removed  by  grinding 
them  down  flush  with  the  shell  surface  with  a  rotary 
grinder.  Fifty  snails  were  used  for  each  of  the  three  treat- 
ments, or  150  snails  total.  A  given  landing  orientation 
was  calculated  as  the  mean  percentage  of  50  animals  each 
dropped  20  times.  Before-and-after  comparisons  were 
done  on  arcsine-transformed  percentage  values  using  ei- 
ther paired  Student's  /  tests  or  Wilcoxon  signed-rank  tests, 
depending  on  whether  the  data  were  normally  distributed. 

I  'arix  angles  and  righting  angles 

A  protractor  was  used  to  measure  the  angles  between 
varices  shown  in  Figure  2a.  To  standardize  the  measure- 
ments, the  middle  of  the  protractor  was  aligned  with  the 
siphon  and  upward-facing  middle  varix.  Angles  through 
which  a  snail's  foot  had  to  traverse  during  righting  from 
the  two  upside-down  landing  positions  shown  in  Figure 
2b  and  2c  were  also  measured  with  a  protractor.  In  this 
case,  the  middle  of  the  protractor  was  aligned  with  the 
upward-facing  varix  in  each  position.  The  angles  were 
determined  for  88  shells  ranging  in  length  from  25- 
82  mm. 

Center  of  mass 

Center  of  mass  with  the  snail  in  the  withdrawn  position 
was  ascertained  by  suspending  five  live  snails  of  varying 
sizes  (16-37  g)  from  threads  attached  at  about  midpoint 
in  their  right  and  middle  varix  edges,  and  from  a  point 
near  the  siphon.  Each  snail  was  suspended  in  seawater 
successively  from  each  of  the  three  positions.  Centers  of 
mass  were  estimated  from  the  point  of  intersection  of  the 
three  lines  generated  by  following  the  visual  extensions 
of  the  threads  from  which  the  animals  were  hung  down 
into  the  body  of  the  snail.  Penciled  reference  marks  on 
the  shells  were  used  to  help  assess  the  point  of  intersection 
of  the  three  lines.  Changes  in  center  of  mass  on  removal 
of  varices  were  estimated  in  the  same  way,  except  that 
hanging  positions  were  adjusted  depending  on  which  varix 
was  being  removed. 


FUNCTION  OF  CER.4TOSTOMA  VARICES 


63 


Morphometry 

The  following  morphometric  measurements  on  perfect, 
uneroded  C.foliatum  shells  were  made:  areas  of  the  three 
main  varices  and  left-remnant  varix  (described  below), 
aperture  area,  aperture  length,  total  shell  length  including 
siphon,  labial  spine  (tooth)  length,  and  live  body  weight 
(measured  in  air  with  the  animal  retracted  into  its  shell). 
The  left-remnant  varix  constitutes  a  remnant  of  the  pre- 
vious left  varix  that  abuts  on  the  current  right  varix  to 
form  a  combined  right-shelf  area  of  larger  dimension  (see 
Fig.  1,  bottom).  This  abutment  is  often  not  perfect,  yet 
visual  analysis  of  many  shells  suggests  that  the  animal 
may  use  this  remnant  as  a  guide  to  where  to  terminate 
growth  of  each  new  varix.  The  end  result  of  the  juxta- 
position of  these  two  varices.  and  the  surface  area  of  the 
left  varix,  is  a  broad  shelf  surrounding  the  aperture. 

The  labial  spine  projects  downward  from  the  anterior- 
right  aperture  margin.  It  is  an  extension  of  a  shallow  collar 
that  borders  the  aperture  on  the  right-hand  side.  It  is  this 
collar  that  ultimately  extends  during  growth  to  form  the 
new  right  varix.  and  the  spine  remnant  can  be  seen 
embedded  in  the  upper  surface  of  each  preceding  varix. 
Its  function  may  be  to  anchor  the  snail  during  feeding, 
as  surmised  for  other  gastropods  (Paine,  1966).  Spine 
length  was  denned  as  the  magnitude  of  its  extension  above 
the  rim  of  the  collar. 

Varix  and  aperture  areas  were  determined  by  drawing 
their  outlines  at  8-power  magnification  using  a  Leitz 
drawing  tube,  then  analyzing  with  a  SIGMA-SCAN  area- 
measurement  software  system  (Jandel  Scientific,  Cali- 
fornia). 

Allometric  or  isometric  relationships  between  the  mor- 
phological features  were  investigated  with  standard  least- 
squares  linear  regressions  calculated  on  log-transformed 
values  for  several  combinations  of  variables.  The  slope 
(/>)  of  a  regression  is  often  underestimated  due  to  error  in 
measurements  of  the  independent  variable  (LaBarbera, 
1989).  To  compensate  for  this  underestimation,  reliability 
ratios  (A.)  were  calculated  (as  described  below)  and  the 
log-log  transformed  slope  (h)  was  multiplied  by  A:"1  to 
produce  a  corrected  log-log  slope  (ft)  (Fuller,  1987;  Harvey 
and  Pagel,  1991;  Johnson  and  Koehl,  1994).  To  calculate 
k  for  length  measurements,  the  lengths  of  10  shells  were 
measured  three  times.  The  first  set  of  values  was  regressed 
on  the  second  set  of  values,  the  first  on  the  third,  and  the 
second  on  the  third.  The  mean  r  for  these  regressions  was 
used  as  the  value  of  A:.  To  calculate  A:  for  area  measure- 
ments, the  areas  of  10  circles  were  calculated  in  two  ways: 
( 1 )  from  measured  radii,  and  (2)  using  the  SIGMA-SCAN 
software  system  described  above.  The  values  of  the  two 
sets  of  data  were  regressed  and  the  resulting  r  was  used  as 
the  value  of  A:,  ft  was  then  tested  against  the  predicted 
slope  for  isometry  for  each  scaling  relationship  using  a  / 
test. 


Results 

Field  tests  of  falling  distances  of  1 00  snails  encountered 
in  random  sampling  of  rocky  subtidal  habitats  showed 
that  1 8  resided  on  horizontal  surfaces  from  which  falling 
did  not  occur,  28  resided  on  inclines  from  which  a  bump- 
and-roll  response  without  free-fall  occurred,  28  resided 
on  inclines  from  which  bump-and-roll  responses  followed 
by  free-fall  occurred,  and  26  resided  on  vertical  slopes 
from  which  only  free-fall  occurred.  Of  the  54  animals  that 
experienced  free-fall.  40  (74%)  fell  less  than  5  body  lengths, 
while  the  remaining  14  (26%)  fell  more  than  5  body 
lengths.  However,  no  animal  fell  further  than  8.4  body 
lengths.  Thus,  most  of  the  specimens  sampled  occupied 
rocky  habitats  from  which  only  a  short  unimpeded  vertical 
fall  was  possible,  while  the  rest  did  not  experience  free- 
fall. 

Righting  times  from  the  right-side  (hard)  orientation 
were  significantly  greater  than  from  the  left-side  (easy) 
orientation  (W  =  681,  p  <  0.001,  Wilcoxon  signed-rank 
test;  Fig.  3).  A  10-g  animal  took  6.3  min  to  right  from  the 
hard  orientation  and  2.9  min  from  the  easy  orientation. 

Significant  differences  were  shown  between  all  I  'O2  rates 
(p  <  0.001,  Friedman  repeated  measures  ANOVA)  and 
the  data  segregated  into  three  statistically  homogeneous 
subgroups  representing  each  activity  (Newman-Keuls  test, 
p  <  0.05;  Fig.  4). 

Righting  costs  (mJ)  from  the  hard  orientation  were  4- 
fold  greater  than  from  the  easy  orientation  (expressed  in 


100 


10  - 


1  - 


0.1 


.     '         RIGHT 


LEFT 


10 
Weight  (g) 


100 


Figure  3.  Righting  times  from  right  ("hard")  and  left  ("easy")  landing 
orientations  in  Ceratostoma  foliation  as  a  function  of  body  weight  (in- 
cluding shell).  Regression  statistics  are,  for  RIGHT:  log  Y  =  0.0626 
+  0.7382  log  A',  r2  =  0.494,  n  =  43  and,  for  LEFT:  log  Y  =  -0.1830 
+  0.6495  log  A',  r  =  0.494.  n  =  43. 


64 


T.  H.  CAREFOOT  AND  D.  A.  DONOVAN 


1000 


a.    100  - 


Q- 


10  - 


10 
Weight  (g) 


100 


Figure  4.  Rates  of  oxygen  consumption  during  righting  from  the 
right  ("hard")  and  left  ("easy")  orientation  compared  with  that  in  the 
upright  (resting)  orientation  as  a  function  of  body  weight  (including  shell) 
in  Ceratosloma  foliation.  Regression  statistics  are  for  RIGHT:  log  )' 
=  1.381  +  0.665  log  X,  r  =  0.522,  n  =  32;  for  LEFT:  log  Y  =  1.092 
+  0.814  log  A",  r2  =  0.529,  n  =  32;  and  for  UPRIGHT:  log  }'  =  0.688 
+  0.919  log  A',  r2  =  0.349,  /;  =  33. 


Table  I  for  an  equivalent  10-g  animal).  The  higher  costs 
from  the  right  side  are  explained  by  the  greater  angle 
(130°)  through  which  the  foot  must  traverse  to  gain  a 
purchase  on  the  substratum,  as  compared  with  that  of  the 
left  side  (110°).  These  angles  are  constant  through  a  wide 
weight  range  (1-41  g.  n  =  88)  and  variances  are  small: 
right-side  angle  =  130°  ±  4  SD,  left-side  angle  =110° 
±  5  SD.  It  should  be  noted  that  these  righting  angles  do 
not  correspond  directly  with  the  varix  angles  depicted  in 
Figure  2a  because,  as  the  shell  lies  on  its  side,  the  angle 
is  determined  by  the  relative  heights  of  the  varix-pair  and 
the  different  balance  points  of  the  shell  on  these  varices. 
A  summary  of  landing  frequencies  when  dropped 
through  seawater  of  depths  of  five  body  lengths  (Table  II) 


indicates  that  the  animals  landed  in  a  pattern  that  differed 
significantly  from  that  expected  based  on  the  proportion 
of  circumference  delineated  by  each  varix-pair  (X2  =  20.9, 
p  <  0.001).  Thus,  although  the  right-side  varix-pair  oc- 
cupied 31%  of  the  circumference  (see  Fig.  2a),  the  animals 
landed  on  it  only  1 5%  of  the  time.  The  left-side  varix- 
pair,  from  which  righting  was  easiest,  occupied  only  19% 
of  the  circumference,  but  was  landed  on  37%  of  the  time. 
The  aperture-down  landing  position  occurred  48%  of  the 
time,  conforming  closely  to  the  50%  circumference  that 
it  represented. 

The  reason  for  the  disproportionate  landings  on  the 
left-side  varix-pair  becomes  clear  when  the  snail's  center 
of  mass  is  known.  When  representative-sized  snails  were 
suspended  from  threads  attached  to  their  individual  var- 
ices, points  of  intersection  were  located  within  the  largest, 
most  recent  whorl,  slightly  to  the  left  of  the  longitudinal 
axis  of  the  snail,  and  about  midway  between  the  aperture 
and  the  top  of  the  main  body  whorl  (see  Fig.  1 ).  Thus, 
the  tendency  during  falling  was  for  the  shell  to  rotate  to 
its  most  stable  orientation,  with  the  center  of  mass  down- 
wards. This  falling  orientation  was  probably  aided  and 
further  stabilized  by  the  broad  right  varix,  which  presum- 
ably acted  as  a  rudder  as  it  trailed.  Thus,  most  animals 
not  landing  aperture  down  actually  landed  on  the  left- 
side varix  pair  from  which  subsequent  righting  was  easiest. 

Removal  of  the  varices  affected  this  landing  pattern  as 
follows  (Table  III).  Right-varix  removal  caused  the  ani- 
mals to  land  significantly  more  on  their  right  sides  and 
significantly  less  on  their  left  sides  and  upright.  Middle- 
varix  removal  led  to  a  similar  pattern  of  landing,  but  with 
even  greater  frequency  of  landings  on  the  right  side.  In 
contrast,  left-varix  removal  greatly  increased  the  proba- 
bility of  landing  upright  at  the  expense  of  both  left-side 
and  right-side  landings.  Varix  removal  was  accompanied 
by  shifts  in  center  of  mass:  right-varix  removal  produced 
a  slight  shift  dorsally,  middle-varix  removal  produced  a 
slight  shift  ventrally,  and  left-varix  removal  produced  a 
slight  shift  to  the  right. 

There  was  no  relationship  between  landing  orientation 
and  length  in  C.foliatum  (t  values  all  <  1.52,  p  values  all 
>  0.135,  /  test  of  significance  of  regression;  Zar,  1984). 


Table  I 

Righting  cmtx  n/"Ceratostoma  foliatum  (n  =  3-1)  from  the  right-side  ("hard")  and  left-aide  ("easy")  orientations 

Righting  cost  (mJ) 
Orientation  Angle  (°)  Regression  equation  r  for  10-g  animal 


Right-side  ("hard") 
Left-side  ("easy") 


log)' =  0.969  +  1.447  logA 
log}'  =  0.557  +  1.265  log.V 


0.703 
0.606 


261 
66 


The  angle  is  vhe  degree  through  which  the  snail's  foot  must  traverse  during  righting  from  each  orientation.  Righting  cost  is  the  product  of  rate  of 
energy  expenditure  and  total  righting  time.  The  regression  equations  were  generated  by  regressing  the  log  transformation  of  energy  needed  to  right 
(mJ)  against  the  log  transformation  of  body  weight  including  shell  (g).  Righting  costs  were  calculated  fora  10-g  animal  from  the  regression  equations. 


FUNCTION  OF  CER.ATOSTOMA  VARICES 


65 


Table  II 

Landing  frequencies  <>l  Ceratostoma  foliatum  ("n  =  137)  onto  the 
right  side,  the  left  side,  and  the  normal  aperture-down  position 

Mean  landing        %  circumference 

frequency  occupied  by 

Orientation  %  ±  1  SD  each  varix-pair 


Aperture-down 

(right  and  left  varix  pair) 

48  ±  19 

50 

Right-side 

(right  and  middle  varix  pair) 

1  5  ±  16 

31 

Left-side 

(left  and  middle  varix  pair) 

37  ±  17 

14 

Each  snail  was  dropped  10  times  in  an  aquarium  tank  with  a  seawater 
depth  of  5  body  lengths.  These  data  were  combined  into  a  grand  aggregate 
for  all  animals,  from  which  the  landing  percentage  values  were  calculated. 
Percentage  circumference  data  were  taken  from  Figure  2. 


Regression  analyses  on  log-log  transformed  data  showed 
that  there  were  significant  relationships  between  most 
shell-parts  as  well  as  body  weight  and  shell-parts  (Table 
IV).  Right-,  middle-,  and  left-varix  areas  scaled  allometri- 
cally  to  shell  length  (corrected  log-log  slopes  equal  to  2. 1 5. 
2.38.  and  2.38  respectively;  all  significantly  greater  than 
2.0,  /  =  2.72,  5.78,  and  5.22,  respectively,  p  all  <  0.01), 
as  did  aperture  area  (ft  =  2.24,  significantly  different  than 
2.0,  /  =  6.24.  p  <  0.001).  Of  interest  was  the  fact  that 
right-varix  area  scaled  to  shell  length  with  a  constant  log- 
log  slope  of  2.15  (Fig.  5a).  Thus,  animals  of  the  largest 
size  recorded  here  (82  mm  shell  length)  were  producing 
new  varices  in  a  constant  allometric  proportion  to  length. 
Combined  varix  area  (right,  left,  and  left-remnant  varices) 
increased  linearly  with  aperture  area.  The  log-log  slope  of 
the  regression  of  right-varix  area  compared  to  aperture 
area  (ft  =  0.93.  r2  =  0.918)  differed  significantly  from  1.0 
(/  =  2.24,  p  <  0.05).  but  when  the  left-varix  and  left  varix- 
remnant  areas  were  successively  added  to  the  right-varix 


area  (the  three  components  combine  to  produce  a  flat 
shelf  surrounding  the  aperture),  progressive  improvements 
in  fit  were  seen:  right  +  left  vs.  aperture:  0  =  0.96,  r2 
=  0.93 1  and  right  +  left  +  left-remnant  vs.  aperture:  ft 
=  0.98,  r  =  0.941.  The  addition  of  the  left  varix  and  the 
left-remnant  varix  also  changed  the  log-log  slopes  of  the 
lines  in  a  progressive  manner  such  that  they  no  longer 
differed  significantly  from  1.0  (right  +  left  vs.  aperture:  l 
=  1.58,  p  >  0. 10  and  right  +  left  +  left-remnant  vs.  ap- 
erture: t  =  0.8 1 ,  p  >  0.20).  This  shows  that  as  the  aperture 
grows  in  size  in  C.  foliatum.  so  the  shelf  surrounding  it 
grows  in  proportional  scale,  with  the  best  fit  being  realized 
when  all  three  shelf  components  are  included. 

To  ensure  that  our  two  populations  from  Barkley  Sound 
and  Telegraph  Cove  did  not  differ  in  any  respect  of  mor- 
phometry,  we  compared  log-log  slopes  and,  if  necessary, 
intercepts  of  regression  lines  for  each  morphometric  com- 
parison generated  independently  for  each  population.  In 
no  instance  was  a  significant  difference  shown  (all  com- 
parisons: /  <  1.96,  p  >  0.05,  tests  of  slope  and  intercept 
differences:  Zar,  1984). 

Labial-spine  length  scaled  allometrically  to  body  length 
((3  =  1.87,  significantly  different  than  1.0,  /  =  12.67,  p 
<  0.001;  Table  IV).  There  was  no  indication  in  our  data 
of  any  break  in  this  relationship,  at  least  over  the  size 
range  represented  by  our  collection  (25-82  mm  length). 

Discussion 

Several  notable  findings  have  arisen  from  this  study. 
First,  aerobic  righting  costs  from  the  right-side,  or  hard, 
orientation  are  significantly  greater  than  aerobic  costs 
from  the  left-side,  or  easy,  orientation.  This  was  expected 
in  view  of  the  greater  angle  that  the  foot  must  traverse, 
requiring  a  greater  period  of  time  and  a  greater  absolute 
need  for  oxygen.  Also  in  accordance  with  our  prediction 
was  that  landings  after  short  vertical  falls  were  preferen- 


Table  III 

Landing  frequencies  ol  Ceratostoma  foliatum  alter  removal  of  single  varices 


Landing  orientation  (%) 


Following  removal  of 

Upright 

Right 

Left 

Upright  v.s.  upright 

Right  vy  right 

Left  is  left 

Right  varix 

33 

44 

23 

t=  3.16 

t  =  11.21 

t  =  5.65 

Control 

44 

16 

40 

p  =  0.003 

/><  0.001 

p  <  0.001 

Middle  varix 

16 

73 

1  1 

t=  12.03 

t  =  20.93 

/  =  8.96 

Control 

48 

13 

39 

P  <  0.001 

/><  0.001 

p  <  0.001 

Left  varix 

85 

6 

9 

t  =  12.05 

H  =  431 

t  =  13.59 

Control 

50 

13 

37 

/'  <  0.001 

P  <  0.001 

p  <  0.001 

Drops  were  in  seawater  of  5  body-length  depth.  .V  =  50  snails  for  each  treatment,  with  20  drops  for  each  snail  before  (control)  and  after  removal 
of  varix.  Values  presented  are  mean  percentage  landings  in  each  orientation  for  20  drops  for  each  of  50  animals,  tested  intact  (control)  and  then 
following  removal  of  a  single  varix.  /:  paired  /  test;  H":  Wilcoxon  signed-rank  test. 


66  T.  H.  CAREFOOT  AND  D.  A.  DONOVAN 

Table  IV 

Sailing  relationships  of  shell  and  body  pans  of  Ceratostoma  foliatum  (n  =  88) 


Relationship 
(  }'  vs.  \) 

log" 

b 

r2 

k 

n 

Predicted  slope 
for  isometry 

weight  v.s.  shell  length 

-3.829 

2.83 

0.984 

0.999 

2.83* 

3 

aperture  area  vs.  shell  length 

-3.599 

2.24 

0.975 

0.999 

2.24* 

2 

labial  spine  length  vs.  shell  length 

-2.808 

1.87 

0.882 

0.999 

1.87* 

1 

right-varix  area  v.s.  shell  length 

-3.099 

2.15 

0.946 

0.999 

2.15* 

2 

middle-varix  area  v.v.  shell  length 

-3.707 

2.38 

0.938 

0.999 

2.38* 

2 

left-varix  area  v.s.  shell  length 

-3.860 

2.38 

0.925 

0.999 

2.38* 

2 

nght-varix  area  v.s  aperture  area 

0.358 

0.93 

0.918 

0.999 

0.93* 

1 

nght  +  left  varix  area  vs.  aperture  area 

0.511 

0.96 

0.931 

0.999 

0.96 

I 

right  +  left  +  left  remnant  varix  area  vs.  aperture  area 

0.550 

0.98 

0.441 

0.999 

0.98 

1 

Regression  statistics  are  for  the  equation  log)'  =  loga  +  b  log.V.  ft  is  the  corrected  slope  calculated  by  multiplying  b  by  the  reliability  ratio,  k  (see 
text  for  explanation). 

*  Indicates  that  corrected  log-log  slope  (fi)  differs  significantly  from  the  predicted  slope  for  isometry,  p  all  <  0.05. 


tially  on  the  left-side  varix-pair,  which  offered  the  smaller 
angle  for  the  foot  to  traverse. 

Our  data  suggest  that  it  is  the  presence  of  the  right  varix 
that  most  contributes  to  this  result.  Its  effects  are  both 
through  its  potential  vanelike  influence  and  through  its 
large  size  (weight).  In  the  absence  of  the  right  varix  the 
animal  lands  more  on  its  right,  or  more  vulnerable,  side, 
probably  because  the  trailing-edge  stabilizing  function  of 
the  broad  right  varix  is  missing  (see  Table  III).  Now  the 
animal  falls  with  the  middle  and  left  varices  trailing  up- 
wards in  winglike  fashion,  but  rotated  slightly  clockwise 
by  an  accompanying  dorsalward  shift  in  center  of  mass. 
This  produces  a  bias  towards  a  right-side  landing.  In  the 
absence  of  the  middle  varix  the  animal  falls  with  center 
of  gravity  down  (aperture  up),  with  right  and  left  varices 
extending  outwards,  and  lands  on  the  main  body  whorl. 
On  impact,  the  imbalanced  weight  of  the  right  varix  tends 
to  roll  the  animal  onto  its  right  side,  even  from  landing 
orientations  that,  in  the  presence  of  the  middle  varix, 
would  have  resulted  in  a  left-side  posture.  Thus,  73%  of 
landings  with  the  middle  varix  absent  are  on  the  right 
side.  The  slight  ventral  shift  in  center  of  mass  on  removal 
of  the  middle  varix  was  not  enough  to  affect  this  falling 
orientation.  A  similar  falling  orientation  is  produced  in 
the  absence  of  the  left  varix.  Now  the  animal  falls  with 
center  of  mass  downwards,  stabilized  by  the  right  and 
middle  varices,  which  project  out  in  winglike  fashion. 
However,  because  of  the  disproportionate  weight  of  the 
right-varix,  this  falling  orientation  is  not  perfectly  sym- 
metrical: rather,  it  is  skewed  somewhat  to  a  right-side- 
down  orientation,  perhaps  aided  by  a  slight  shift  to  the 
right  in  center  of  mass  on  removal  of  the  varix.  Thus, 
mostly  aperture-down  landings  result.  Where  this  skewing 
is  less,  landing  is  still  mostly  on  the  main  body  whorl 
previously  occupied  by  the  left  varix.  and  the  heavy  weight 
of  the  right  varix  rolls  the  animal  onto  its  aperture.  Thus, 


85%  of  landings  in  the  absence  of  the  left  varix  lead  to  an 
upright  position.  In  all  cases  where  a  varix  was  absent, 
subsequent  landing  orientation  was  greatly  influenced  by 
the  two  trailing  varices,  especially  the  right  one  in  its  pres- 
ence. Whereas  Palmer  (1977)  attributed  a  slight,  but  sig- 
nificant, destabilizing  effect  of  the  middle  varix,  leading 
to  more  upright  landings  from  falls  of  10  body  lengths  or 
greater,  we  show  that  the  right  varix  is  mainly  responsible 
for  the  type  of  "destabilizing"  event  recorded  here.  Ob- 
viously these  functions  are  inter-related,  as  the  right  varix 
ultimately  becomes  the  middle  one. 

Based  on  these  considerations,  then,  the  optimal  shell 
design  (ignoring  other  possible  varix  functions)  would  be 
to  have  only  right  and  middle  varices,  and  not  a  left  varix. 
But,  because  of  the  way  the  snails  grow,  varices  can  only 
be  partially  removed  after  they  are  laid  down.  Thus,  the 
method  of  growth  constrains  C.  foliatum  to  a  less-than- 
optimal  shell  shape  with  respect  to  the  feature  of  landing 
orientation  after  falling. 

Our  morphometric  analyses  failed  to  indicate  the  dim- 
inution of  growth  that  was  noted  for  San  Juan  Island, 
Washington,  populations  of  C.  foliatum  by  Spight  and 
Lyons  (1974)  and  Spight  el  al.  ( 1974).  These  authors  sug- 
gested that  growth  stops  in  mature  snails,  with  the  animals 
subsequently  appearing  to  shrink  in  size  as  the  varices, 
siphon,  and  spire  erode.  Our  data  indicate  that  animals 
are  still  adding  undiminished  right  varices  up  to  82  mm 
length;  but,  as  this  is  also  equivalent  to  the  largest  size 
recorded  by  Spight  and  colleagues,  it  may  represent  a 
maximum  for  the  species  in  this  geographical  area.  Fur- 
thermore, the  allometry  of  labial  spine  size  to  body  length 
was  consistent  over  the  complete  size  range  (25-82  mm 
length)  found  in  our  populations.  There  was  no  break 
discernible  in  the  relationship  corresponding  to  the  spine 
reaching  its  "mature"  form  at  60  mm  body  length;  a  break 


FUNCTION  OF  CERATOSTOMA  VARICES 


67 


10- 


5- 


1- 


0.5- 


Q 


20 


~~ r~ 
30 


40 
Length   (mm) 


T — 
50 


100 


20- 


10- 


5- 


1- 


RIGHT+LEFT+ 

LEFT  REMNANT 
RIGHT+LEFT 

RIGHT 


— I —    — I — 
0.5  1  5 

Aperture  area   (cm 


— r~ 
10 


Figure  5.  (a)  Right-varix  area  as  a  function  of  shell  length  in  Cera- 
tostoma  foliaium.  (b)  Areas  of  various  varix  combinations  around  the 
aperture  plotted  against  aperture  area  in  C.  foliaium.  The  combined 
right  +  left  +  left-remnant  varix  area  represents  the  flat  shelf  that  sur- 
rounds the  aperture.  Regression  statistics  for  the  relationships  are  given 
in  Table  IV.  A'  =  88  for  each  regression. 


was  noted  for  San  Juan  populations  of  C  foliaium  by 
Spight  and  Lyons  (1974). 

Despite  the  attractiveness  of  a  dual  destabilizing-effect 
theory  for  the  function  of  the  varices  during  falls— that 
is,  that  the  right  one  leads  to  preferential  landing  on  the 
easy  side  during  short  falls  and  the  middle  one  to  pref- 


erential upright  landings  during  long  falls — the  truth  is 
probably  that  the  varices  serve  multiple  functions.  Our 
inclination  on  viewing  C.  foliaium.  and  from  our  mor- 
phometry  data,  is  to  believe  that  the  extended  platform 
surrounding  the  aperture  formed  from  the  combined  right 
and  left  varices  could  serve  for  protection  of  the  soft  parts 
during  feeding  and  locomotion  or,  as  suggested  by  Palmer 
( 1977),  for  providing  a  stable  platform  during  drilling  of 
its  prey.  These  ideas  are  supported  by  the  juxtaposition 
of  the  previous  left-varix  remnant  with  the  present  right 
varix,  which  increases  this  area.  In  fact,  we  showed  that 
the  combined  right-,  left-,  and  left  remnant-varix  areas 
scaled  isometrically  with  aperture  area  (0  =  0.98),  sug- 
gesting a  related  function. 

Such  hypotheses  of  protection  and  stability  are  testable, 
and  could  provide  provocative  areas  for  future  work,  es- 
pecially if  incorporated  into  a  larger  comparative  study. 
Several  other  muricid  species  (in  the  genera  Ceratostoma 
and  Pteropurpitra)  on  Pacific  Ocean  coasts  have  tri-varix 
morphology  similar  to  that  ofC.foliatum.  Most  are  large 
(4-8  cm)  and  all  are  carnivorous,  but  data  on  their  habitat 
preferences  and  other  aspects  of  their  biology  are  scanty. 
A  comparison  of  varix  morphometries  of  a  few  of  these 
species  relative  to  aperture  areas,  combined  with  tests  of 
their  susceptibility  to  nipping  or  enveloping  predators  and 
their  stability  in  currents,  could  lead  to  further  insights 
on  optimal  shell  design  in  gastropod  molluscs. 

Acknowledgments 

We  thank  Jo  Wieruszewski  for  help  with  the  morpho- 
metric  measurements;  Patricia  Lee,  Joseph  West,  and  Jef- 
frey Fleming  for  other  technical  help;  Steve  Land  for  as- 
sistance in  scuba  collections;  and  Andy  Spencer,  Director 
of  the  Bamfield  Marine  Station,  and  his  staff  for  logistical 
support  during  collection  of  animals.  Barbara  Taylor, 
Steve  Pennings,  and  A.  Richard  Palmer  gave  helpful 
comments  on  the  manuscript.  The  work  was  supported 
by  a  Natural  Science  and  Engineering  Research  Council 
(NSERC)  grant  to  T.  Carefoot  and  a  University  Graduate 
Fellowship  to  D.  Donovan. 

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Elliott,  J.  M.,  and  \V.  Davison.  1975.  Energy  equivalents  of  oxygen 
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Fuller,  \V.  A.  1987.  Measurement  Error  Models.  John  Wiley  and  Sons. 
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NJ. 


Marine 

Biological 

Laboratory 

Woods  Hole 

Massachusetts 


Ninety-Seventh  Report 

for  the  Year  1994 

One-Hundred  and  Sixth  Year 


Officers  of  the  Corporation 


Sheldon  J.  Segal,  Chairman  of  the  Board  of  Trustees 
Robert  E.  Mainer,  1 7ft'  Chairman  of  the  Board  of 

Trustees 

James  D.  Ebert,  President  of  the  Corporation 
John  E.  Burris,  Director  and  Chief  Executive  Officer 
Robert  D.  Manz,  Treasurer 
Neil  Jacobs,  Clerk  of  the  Corporation 


Contents 

Report  of  the  Director  R 1 

Report  of  the  Treasurer  R9 

Financial  Statements  ..RIO 

Report  of  the  Librarian  ...    R20 
Educational  Programs 

Summer  Courses  ...     R23 

Short  Courses  .  .  .  R26 

Summer  Research  Programs 

Principal  Investigators  ...    R31 

Other  Research  Personnel  R32 

Library  Readers   .  R34 

Institutions  Represented  .  .  .    R35 

Year-Round  Research  Programs  R39 

Honors  R47 

Board  of  Trustees  and  Committees  R53 

Laboratory  Support  Staff  .  ....    R56 

Members  of  the  Corporation 

Life  Members  .  R58 

Members  R59 

Associate  Members  R70 

Certificate  of  Organization  R73 

Articles  of  Amendment  R73 

Bylaws  R73 


Report  of  the  Director 
and  Chief  Executive  Officer 


In  1994,  the  Decennial  Review  Committee,  which  has 
met  even.'  decade  since  1924  to  comprehensively 
evaluate  the  Marine  Biological  Laboratory,  examined 
the  full  range  of  Laboratory  activities,  including 
summer  and  year-round  research  and  education 
programs.  I  am  pleased  to  say  that  its  final  report, 
prepared  under  the  chairmanship  of  Malcolm  Steinberg 
of  Princeton  University,  was  thorough  and  positive.  It 
affirms  that  the  MBL  continues  to  be  a  valuable  part  of 
biological  research  in  the  U.S.  and  the  world. 

"The  socioscientific  ethos  generated  by  the  amalgam  of 
knowledge,  drive,  and  free  scientific  exchange  that 
continues  to  characterize  it  has  served  to  make  the  MBL 
the  most  important  center  of  its  kind  in  the  world.  This 
spectacularly  successful  enterprise  is  the  product  of  a 
unique  blend  of  historical  accidents  and  good  sense  .  .  . 
the  ultimate  scientific  watering  hole,  bringing  together 
the  most  distinguished  minds  in  the  biological  sciences 
in  an  environment  where  free  interaction  is  possible.  " 
— 1994  Report  of  the  Decennial  Review  Committee 


Year-Round  Scientific  Activities 

The  Ecosystems  Center  is  one  of  many  scientific 
"watering  holes"  at  the  Marine  Biological  Laboratory. 
The  largest  of  the  year-round  laboratories,  the  Center  is 
one  of  the  premier  facilities  in  the  world  for 
investigating  the  functioning  of  terrestrial  and  aquatic 
ecosystems  and  the  effects  of  human  activities  on  those 
systems.  Research  projects  currently  underway  include 
the  study  of  global  change  and  the  carbon  balance  of 
Arctic  ecosystems,  land  use  change  and  soil  processes  in 
the  Amazon  River  Basin,  and  a  study  on  the  effects  of 
land  use  and  the  introduction  of  organic  matter  to 
estuarine  ecosystems.  Some  of  the  estuarine  research 
was  conducted  locally  in  Plum  Island  Sound  north  of 
Boston  and  in  Waquoit  Bay  here  on  Cape  Cod. 


In  1994.  researchers  at  the  Ecosystems  Center 
pioneered  the  use  of  a  stable  isotope  of  nitrogen  (15N) 
to  follow  the  pathway  of  this  important  element  in 
undisturbed  ecosystems.  Senior  Scientist  Bruce  Peterson 
added  ammonium-'5N  to  an  Arctic  river  continuously 
for  three  weeks  to  trace  how  fast  nitrogen  moved  from 
the  water  into  algae,  then  into  grazing  insects  and 
eventually  into  predatory  fish.  These  rates  were  then 
used  to  test  a  mathematical  model  of  the  entire  stream 
system.  Associate  Scientist  Knute  Nadelhoffer  used 
both  ammonium-and  nitrate-15N  to  study  what 
happened  to  the  nitrogen  added  to  forests  of  New 
England  from  acid  rain.  Most  of  the  added  nitrogen 
remains  in  the  soil.  Scientists  from  the  Ecosystems 
Center  incorporate  results  from  these  and  other  studies 
into  ecosystem  models  of  carbon  and  nitrogen 
dynamics.  One  of  these  models,  called  the  Terrestrial 
Ecosystem  Model,  is  now  part  of  an  MIT-centered 
integrated  assessment  model  that  also  deals  with  the 
atmosphere,  the  oceans,  and  the  global  economy.  This 
large  model  is  used  to  address  global  change  issues. 

Investigators  in  the  Architectural  Dynamics  in  Living 
Cells  Program  conduct  research  at  the  molecular  and 
cellular  level  at  the  interface  of  anatomy  and 
physiology.  They  use  advanced  instrumentation  to 
image  real-time  cellular  events  occurring  in  living  cells. 
The  report  of  the  Decennial  Review  Committee  stated 
that  "MBL  Distinguished  Scientist  Dr.  Shinya  Inoue's 
work  has  produced  a  true  revolution  in  the  analysis  of 
the  living,  functioning  cell,  marrying  the  best  available 
light-microscopic  techniques  with  the  most 
sophisticated  image  analysis  approaches,  utilizing  both 
video  and  computer  graphics  at  an  exquisite  level  of 
performance."  During  the  summer  of  1994.  Program 
investigators  collaborated  with  Rand  Fellow  Ted 
Salmon  (University  of  North  Carolina,  Chapel  Hill) 
and  Nikon  Fellow  Andrew  Murray  (University  of 
California.  San  Francisco).  While  in  Woods  Hole. 
Salmon  and  Murray  successfully  attached 


Rl 


R2   Annual  Report 


chromosomes  (derived  from  sperm  in  the  egg  extract) 
to  microtubules  assembled  in  frog  egg  extract,  and.  for 
the  first  time,  experimentally  got  them  to  go  through 
anaphase  movement  outside  of  a  living  cell.  These 
exciting  events  were  recorded  as  4-D  sequences  in 
collaboration  with  Ted  Inoue  of  Universal  Imaging 
Corporation.  In  the  fall  of  1 994,  the  Program  also 
welcomed  two  visiting  investigators  from  Japan,  Dr. 
Rieko  Arimoto  of  Nikon,  Inc.,  and  Dr.  Keisuke  Suzuki 
of  the  Olympus  Corporation. 

The  Molecular  Evolution  Program,  directed  by  Dr. 
Mitchell  Sogin,  is  another  resident  laboratory  where 
scientists  gather,  often  over  a  computer  monitor,  to 
share  information.  The  Program  has  helped  to  advance 
the  field  of  molecular  evolution  by  using  molecular 
techniques  to  calibrate  the  evolution  of  organisms  and 
their  genes.  Using  measures  of  similarity  for  ribosomal 
RNAs,  investigators  measure  genetic  differences 
between  members  of  the  same  species  or  reconstruct 
phylogenies  for  organisms  that  span  the  largest 
evolutionary  distances.  Drs.  Sogin,  Hinkle,  and 
collaborators'  studies  on  the  stable  co-evolution  of 
attine  ants  and  their  fungi  over  50  million  years 
suggests  that  preservation  of  biodiversity  may  require 
maintenance  of  both  members  of  a  symbiotic 
partnership.  This  work  was  published  in  1994  in  the 


Postdoctoral  Research  - )  \\ociulc  Hilary  Morrison  in  the  Molecular  Evo- 
lution laboratory  (photo  hy  Richard  Ilimanl). 


Senior  Scientist  Osaiuu  Shimomura  (photo  by  Linda  Golder/MBL). 


journal  Science,  and  featured  subsequently  in  The  New 
York  Times  and  the  local  press. 

The  Calcium  Patterning  Program,  under  the 
direction  of  Senior  Scientist  Lionel  F.  Jaffe  and 
Assistant  Scientist  Andrew  L.  Miller,  continues  to 
advance  the  study  of  the  role  played  by  calcium  in  a 
wide  range  of  fundamental  cell  processes.  The  Program 
uses  a  group  of  bioluminescent  proteins,  aequorins,  that 
emit  light  when  in  contact  with  calcium  for  their 
studies.  Aequorin,  which  was  first  purified  by  MBL 
Senior  Scientist  Osamu  Shimomura  in  the  1960s,  is 
produced  in  a  species  of  jellyfish.  Throughout  1994, 
Program  investigators  continued  their  work  with  the 
slime  mold  Diclyostc/ium,  which  they  use  as  a  simple 
model  for  studying  the  development  of  multicellular 
organisms.  In  a  paper  just  published  by  the  journal 
Development,  Miller  and  Jaffe  and  their  colleagues 
documented  the  cellular  calcium  patterns  throughout 
the  life  cycle  of  the  slime  mold.  This  is  an  important 
first  step  to  understanding  the  role  that  calcium  plays  in 
switching  on  or  off  various  genes  within  the  organism. 
Collaborations  are  currently  underway  with  scientists  at 
Massachusetts  General  Hospital,  the  University  of 
Pennsylvania,  and  Cornell  University  to  pursue  this 
research  in  other  animal  and  plant  models. 

The  National  Vibrating  Probe  Facility,  directed  by 
Associate  Scientist  Peter  J.  S.  Smith,  develops  and 
makes  available  techniques  for  measuring,  non- 
invasively,  the  movement  of  ions  across  cell 
membranes.  In  1994,  Facility  investigators  were 
successful  in  their  attempts  to  measure  the  small  steady- 
state  calcium  fluxes  from  isolated  neurons  in  culture. 
This  has  opened  up  new  areas  of  research  into  the 
regulation  of  calcium  and  second  messengers.  Two  new 
probes  are  currently  in  development  at  the  Facility.  The 
BioKelvin  probe  is  being  designed  to  measure  the  weak 


Report  of  the  Director  and  CEO   R3 


ion  fields  that  exist  around  tissue  in  a  gaseous 
environment.  Investigators  plan  to  use  this  new 
machine  in  the  study  of  skin.  The  second  instrument 
currently  in  development  is  a  vibrating  oxygen  probe, 
which  Facility  investigators  intend  to  use  in  the  study  of 
cell  respiration. 

In  September  of  1994.  the  Boston  University  Marine 
Program  kicked  off  a  year-long  celebration  of  its  25th 
anniversary.  Since  it  began  in  1969,  BUMP  has  grown 
from  a  small  graduate  program  into  one  of  the  strongest 
undergraduate  and  graduate  programs  in  marine 
biology  in  the  country.  In  1994  BUMP  enrolled  60 
undergraduates  and  33  masters  and  doctoral  candidates 
who  studied  with  8  teaching  and  9  research  faculty. 

The  year-round  research  program  at  the  MBL 
expanded  in  1994  with  the  addition  of  two  new 
programs.  In  the  fall  the  MBL  welcomed  the 
Laboratory  of  Cell  Communication,  led  by  Drs.  Werner 
Loewenstein  and  Birgit  Rose.  Investigators  in  this 
Laboratory  study  the  membrane  channel  built  into 
junctions  between  cells  that  provides  one  of  the  most 
basic  forms  of  intercellular  communication  in  organs 
and  tissues.  They  also  examine  feedback  loops  in 
normal  and  abnormal  cell  proliferation,  which  is 
important  in  cancer  research.  The  MBL  also  received 
an  award  from  NASA  to  establish  the  Center  for 
Advanced  Studies  in  the  Space  Life  Sciences.  This  new 


BL'MP celebrates  ils  25th  Anniversary  (photo  hv  Boston  I'niwrsitv  Photo 
Services). 


Center  will  review  and  study  a  variety  of  life  science 
areas,  with  special  attention  on  how  gravity  influences 
biological  processes. 

The  Marine  Resources  Center 

The  Marine  Resources  Center  (MRC),  now  in 
operation  for  two  years,  continues  to  supply  healthy 
animals  for  biological  and  biomedical  research  and 
education.  The  seawater  system  is  monitored  daily  to 
provide  readings  on  temperature,  nutrients,  and 
dissolved  gases.  A  variety  of  research  projects  involve 
the  use  of  this  facility. 

•  The  clam,  Mulinia,  is  being  raised  in  the  MRC's 
mariculture  room.  This  clam,  a  close  relative  of  the 
extensively  studied  surf  clam.  Spisulii,  is  easy  to 
maintain  and  culture.  It  is  also  useful  as  a  model  in  the 
study  of  the  cellular  and  molecular  biology  of  cell 
division.  These  studies  are  important  for  learning  more 
about  diseases  such  as  cancer,  where  cells  divide 
without  adequate  control. 

•  Ecosystems  Center  scientists  are  analyzing 
oxygen  uptake  from  the  water  overlying  sediments  to 
measure  bacterial  and  animal  respiration  in  subtidal 
muds  from  Boston  Harbor  and  Massachusetts  Bay.  The 
data  are  part  of  an  environmental  impact  assessment 
on  the  proposed  sewage  outfall  pipe.  Center  scientists 
moved  their  project  from  the  Homestead  building  to 
the  MRC  to  take  advantage  of  the  running,  filtered,  and 
temperature-controlled  seawater  available  there. 

•  In  a  cooperative  effort  with  a  local  commercial 
scallop  producer,  approximately  250,000,000  scallop 
"spat"  were  produced  from  about  1,200  mature 
scallops  conditioned  by  overfeeding,  an  accelerated 
light/dark  cycle,  and  exposure  to  a  computer-controlled 
regime  of  increasing  seawater  temperatures.  Animals 
spawned  early  in  the  year  gained  more  than  two 
months  additional  growing/maturation  time  ahead  of 
feral  stocks. 

•  This  past  year.  MBL  scientists  began 
collaborating  with  the  Nantucket  Research  and 
Education  Foundation  to  improve  the  health  of 
cultured  scallops  and  to  develop  genetic  markers  to 
trace  their  dispersion  patterns  in  order  to  gauge  the 
success  of  mariculture  efforts. 

In  1994  the  MRC  continued  to  win  accolades.  In 
May  the  project  received  "High  Honors"  in  R  &  D 
Magazine's  Laboratory  of  the  Year  competition;  in 
September.  Facilities  Manager  Richard  Cutler  accepted 
the  American  Institute  of  Plant  Engineers'  FAME 
Award  of  Excellence  on  behalf  of  the  MBL;  and  in 
October  Tsoi/Kobus,  the  architectural  firm  that 
designed  the  MRC,  was  awarded  one  of  eleven  awards 


R4  Annual  Report 


Associate  Scientist  Alan  Kuzirian  shows  Congressman  Gerry  Slitdds 
Mulinia  cultures  in  the  AfRC  (photo  by  Mark  Domblaser/MBL). 


in  the  Top  Quality  Urban  Waterfront  Projects  category 
sponsored  by  the  Waterfront  Center  organization  in 
Washington,  DC.  Most  recently,  the  MBL  received  the 
Construction  Industry  Liaison  Group  "Owner 
Recognition  Award"  at  the  Build  Boston  Design  and 
Construction  Industry  Convention. 

The  MBL  was  fortunate  to  receive  a  total  of  $1 
million  from  the  Clowes  Fund  and  the  Lakian 
Foundation  to  help  recruit  a  leading  scientist  to 
establish  an  independent  research  program  and  direct 
research  services  provided  by  the  MRC.  As  I  prepare 
this  report,  a  group  of  applicants  for  this  position  is 
being  interviewed  by  our  Search  Committee. 

Summer  Research  Activities 

From  August  15  to  17,  MBL  scientists  gathered  in 
the  Lillie  Auditorium  for  the  Laboratory's  annual 
General  Scientific  Meetings.  Last  year  more  than  60 
investigators  and  students  presented  their  summer's 
research  at  the  meeting.  Topics  ranged  from  cell 
division  to  comparative  biology  to  neurobiology  to 
phytoplankton  dynamics.  Fifty-one  of  those 
presentations  were  published  as  Short  Reports  in  the 
October  issue  of  the  MBL's  journal.  The  Biological 
Bulletin. 

The  two  short  reports  submitted  by  Peter  Armstrong 
(University  of  California,  Davis)  and  James  Quigley 
(SUNY,  Stony  Brook)  were  recognized  by  The 
Biological  Bulletin  as  the  issue's  Featured  Articles. 
During  the  summer  of  1994,  Armstrong  and  Quigley 
and  their  co-workers  found  evidence  that  two  protein 
molecules  found  in  the  blood  of  horseshoe  crabs  — 
limulin  and  «:-macroglobulin —  play  key  roles  in  the 
innate  immune  systems  of  invertebrates.  Their  work 


suggests  that  limulin  both  recognizes  and  destroys 
invading  microbes,  and  that  «2-macroglobulin  appears 
to  be  the  regulator  of  that  cell-destruction  system. 

Research  performed  by  Yale  University  Professor 
Walter  Boron  and  colleagues  last  summer  at  the  MBL 
was  recently  described  in  an  article  published  in  the 
journal  Nature.  In  that  article.  Boron  and  colleagues 
documented  a  novel  technique  that  they  developed  for 
studying  the  transport  of  bicarbonate  across  cell 
membranes  using  the  squid  giant  axon.  This  technique 
involves  rapidly  mixing  two  solutions  to  produce  an 
out-of-equilibrium  solution  with  virtually  any 
combination  of  pH,  carbon  dioxide,  and  bicarbonate 
levels.  Boron's  work  resulted  in  the  identification  of  a 
new  mechanism  for  bicarbonate  transport,  a  potassium/ 
bicarbonate  transporter.  This  work  could  be  significant 
for  understanding  human  diseases  in  which  acid-base 
balance  is  important,  including  epilepsy  and  stroke, 
respiratory  and  renal  disorders,  cancer,  and  heart 
disease. 

The  results  of  research  from  many  summer 
laboratories  saw  publication  in  a  number  of  other 
journals.  The  1994  work  of  Rodolfo  Llinas  (NYU)  and 
his  colleagues  was  published  in  the  December  issue  of 
the  Proceedings  oj  the  National  Academy  of  Science;  an 
article  by  Antionio  Giuditta  (University  of  Naples)  and 
co-authors  appeared  in  the  Journal  of  Neurochemistry; 
and  Robert  Paul  Malchow  (University  of  Illinois), 
Haohua  Qian  (Harvard  University),  and  Harris  Ripps 
(University  of  Illinois)  published  an  article  in  the 
December  1994  issue  of  the  Journal  of  General 
Physiology.  Many  other  articles  based  on  MBL  summer 
research  appeared  in  The  Journal  of  Biological 
Chemistry.  Cell  Motility  and  the  Cytoskeleton.  the 
Journal  of  Cell  Science,  the  Journal  of  Cell  Biology,  the 
Jounicil  of  Neurophysiology,  Neuroscience  Letters, 


Joe  DeGiorgi^  i 


a  squid  giant  axon. 


Rt-port  of  the  Director  and  CEO  R5 


Summer  inveMifnitor  Robert  Pala::o  examines  a  surf  clam. 


Biophysical  Journal.  Biomembrane  Electrochemistry, 
the  Journal  of  Neuroscience,  Glia,  and  Brain  Research 
Bulletin.  A  partial  list  of  articles  published  in  1994  by 
year-round  and  summer  MBL  scientists  appears  at  the 
end  of  this  Annual  Report. 

Educational  Programs 

The  MBL's  strong  commitment  to  teaching  the 
scientific  method  in  an  interdisciplinary  and  highly 
motivated  research  environment  represents  the  apex  of 
training  efforts  in  the  life  sciences. 

"Long  regarded  as  jewels  in  the  crown  of  U.S.  graduate 
education  in  the  biological  sciences.  [MBL]  courses  have 
retained  their  superb  quality.  Many  outstanding 
scientists  pay  tribute  to  them  as  landmarks  in  their  own 
education  .  .  .  Students  in  these  courses  receive  state-of- 
the-art  training  that  cannot  be  obtained  at  their  home 
institution — or  indeed  any  other  institution  of  higher 
learning  in  the  world.  This  is  made  possible  by  the 
intense,  total  immersion-in-scienee  approach  that  results 
from  having  a  large  faculty,  drawn  from  around  the 
world,  who  interact  on  an  a/most  24  hour-a-day  basis 
providing  the  student  with  conceptual  and  practical 
training  .  .  .  the  additional  availability  of  the  most  up- 


to-date  equipment  provides  access  to  the  latest  and  even 

breaking  technologies. " 

—  1994  Report  of  the  Decennial  Review  Committee 

While  preparing  this  report,  1  learned  that  the 
Howard  Hughes  Medical  Institute  will  award  the  MBL 
$2  million  to  support  four  years  of  summer  courses, 
from  1996  to  1999.  "The  courses  at  the  MBL  are  so 
consistently  excellent  that  scientists  are  always  eager  to 
enroll,"  said  Joseph  G.  Perpich.  M.D.,  J.D.,  HHMI's 
vice  president  for  grants  and  special  programs.  The 
MBL  is  indeed  grateful  to  the  Howard  Hughes  Medical 
Institute  for  its  continued  support  of  these  remarkable 
courses. 

The  summer  of  1994  marked  the  final  year  of  a  five- 
year  tenure  as  directors  for  John  Breznak  and  Martin 
Dworkin  of  the  Microbial  Diversity  course,  Ron 
Calabrese  and  Martha  Constantine-Paton  of  the  Neural 
Systems  &  Behavior  course,  and  Irwin  Levitan  and 
Leonard  Kaczmarek  of  the  Neurobiology  course.  The 
efforts  of  these  retiring  course  directors  are  greatly 
appreciated;  each  director  served  his  or  her  course  and 
the  Laboratory  with  dedication  and  enthusiasm.  Ron 
Calabrese  and  Irwin  Levitan  will  continue  to  serve  the 
Laboratory  as  co-chairs  of  the  newly  formed  Education 
Committee,  which  replaces  the  MBL's  Instruction 
Committee.  The  new  Committee  reports  directly  to  the 
Science  Council  on  all  educational  matters. 

Summer  Fellowships  and  Scholarships 

The  MBL  Summer  Research  Fellowship  and 
Scholarship  Programs  provided  funding  for  150 
individuals  last  summer.  Fellowship  awards  to  support 
independent  research  totaled  nearly  $157.000,  a 


Senior  Sciential  Mnche/l  Sogin  reaching  the  Molecular  Evolution  U'urk- 
shup  at  the  MBL 


R6  Annual  Report 


$26,000  increase  over  1993.  Students  attending  MBL 
courses  were  awarded  more  than  $357,000  in 
scholarships  last  summer. 

Twelve  young  scientists  were  awarded  Grass 
fellowships  in  Neurophysiology  at  the  MBL  last 
summer.  The  Program,  which  is  sponsored  by  the  Grass 
Foundation,  has  been  held  at  the  MBL  for  over  40 
years,  offering  young  neuroscientists  an  opportunity  to 
do  independent  research,  often  for  the  first  time  in  their 
careers. 

Nine  minority  graduate  students  from  around  the 
country  participated  in  the  7th  annual  Minority 
Fellowship  Program  (MFP)  at  the  MBL  during  the 
summer  of  1994.  The  Program,  sponsored  by  a  grant 
from  the  National  Institute  of  Mental  Health,  is 
designed  to  unite  pre-doctoral  neuroscience  students 
from  underrepresented  ethnic  groups  with  postdoctoral 
fellows  and  members  of  the  MFP  scientific  board. 
Students  participating  in  this  month-long  program  gain 
scientific  "survival  skills."  including  increased  skills  in 
writing,  delivering  oral  scientific  presentations,  and 
career  development  techniques. 

The  Science  Writing  Fellowships  Program  completed 
its  ninth  successful  year,  enrolling  13  journalists  from  a 
variety  of  print  and  broadcast  media  last  summer.  The 
Program,  which  fosters  a  better-informed  and  more 
interactive  dialogue  between  scientists  and  science 
writers  to  provide  the  public  with  improved 
information  about  the  life  sciences,  has  now  trained 
over  100  journalists. 


MBL  Trustees 


We  were  fortunate  to  welcome  three  new  Trustees  to 
the  Board  in  1994.  They  are:  Dr.  Darcy  Brisbane  Kelley 
of  the  Department  of  Biological  Sciences  at  Columbia 
University,  Dr.  Laurie  Landeau  of  Listowel,  Inc.,  and 
Dr.  Burton  J.  Lee,  III  of  Intracel  Corporation. 

Dr.  Kelley  is  a  former  co-director  of  the  MBL's 
Neural  Systems  &  Behavior  Course.  She  is  also 
professor  and  chair  of  the  Department  of  Biological 
Sciences  at  Columbia  University.  Dr.  Kelley  received 
her  A.B.  from  Barnard  College  and  her  Ph.D.  from 
Rockefeller  University. 

Dr.  Lee  is  a  member  of  the  MBL's  Council  of 
Visitors.  He  received  his  B.A.  from  Yale  University  and 
his  M.D.  from  Columbia  LIniversity.  Dr.  Lee  served  as 
President  Bush's  physician  during  Mr.  Bush's  term  in 
the  White  House,  and  was  a  member  of  the  White 
House  Task  Force  on  Infant  Mortality  and  the 
Presidential  Drug  Advisory  Council.  He  now  serves  as 


Chairman  of  the  Board  of  Intracel  Corporation  in 
Cambridge,  MA. 

Dr.  Landeau  is  also  a  member  of  the  MBL's  Council 
of  Visitors  and  serves  as  an  associate  director  of  the 
AQUAVET  Program  held  annually  at  the  Laboratory. 
She  is  General  Manager  of  Listowel,  Inc.,  and  the 
President  of  the  Marinetics,  Inc.,  a  firm  that  researches 
and  develops  disease-resistant  shellfish  for  culture.  Dr. 
Landeau  holds  an  MBA  and  a  VMD  from  the 
University  of  Pennsylvania. 

MBL  Trustee  and  Harvard  University  neurobiologist 
Edward  Kravitz  recently  completed  his  term  on  the 
MBL  Board.  Ed  has  served  several  terms  on  the  Board 
and  has  been,  and  continues  to  be,  a  strong  supporter 
of  the  Laboratory's  programs  for  young  scientists.  His 
contributions  to  the  Laboratory  were  recognized  with 
appreciation  at  the  February  1995  meeting  of  the  MBL 
Board  of  Trustees. 


The  MBL/WHOI  Library 

The  Library  continued  its  evolution  from  the 
traditional  role  of  exclusively  providing  information  in 
print  into  electronic  document  delivery  services.  We 
were  the  nation's  first  library  to  mount  commercial 
CD-ROMs  on  the  Internet,  and  we  negotiated  various 
novel  distribution  arrangements  for  our  collection 
through  the  National  Library  of  Medicine,  Elsevier 
Publishing  Corporation,  and  Readmore  Subscription 
Agency.  Much  of  this  development  has  been  possible 
through  grants  from  Howard  Hughes  Medical  Institute, 
the  Andrew  W.  Mellon  Foundation  and  the  Bay 
Foundation.  The  Library  remains,  however,  a  vital 


.-I  student  examine*  journals  in  the  .MBL/W  'HOI  Library  (photo  by  Rich- 
ard I  Inward) 


Report  of  the  Director  and  C'KO  R7 


resource  for  access  to  a  complete  and  up-to-date  book 
and  print  journal  collection.  Roger  Stoddard  of  the 
Houghton  Library-  at  Harvard  University  reviewed  the 
collection  in  1994  and  stated  that  it  was  "breath-taking 
in  its  comprehensiveness  of  coverage  and  completeness 
of  holdings." 


Renovations,  Restorations,  and  Recycling 


The  renovation  of  the  Crane  wing  of  the  Lillie 
Building  was  completed  in  1994.  Renovations  included 
the  removal  of  asbestos  and  the  installation  of  an 
HVAC  system  throughout  the  wing,  which  has  resulted 
in  a  much  improved  working  environment  for  our 
Lillie  Laboratory  scientists  and  staff. 

If  the  MBL's  1994  water  and  sewer  bill  is  any 
indication,  our  conservation  efforts  are  paying  off. 
Thanks  to  fewer  aspirators  in  the  laboratories,  the 
conversion  of  the  Whitman  seawater  chiller  from  fresh 
to  salt  water,  the  installation  of  water-saver  toilets  and 
shower  heads  in  the  dorms,  better  facility  maintenance, 
and  fewer  coldwave  room  chillers  in  the  Lillie  building, 
the  MBL's  water  and  sewer  bill  dropped  dramatically— 
from  $152.630  in  1991  to  $86.917  in  1994. 

All  MBL  renovation  projects,  whether  large  or  small, 
are  first  evaluated  with  an  eye  towards  conservation.  A 
good  example  of  this  effort  is  the  replacement  last  year 
of  windows  in  the  Homestead  building,  funded  at  a  cost 
of  $23,000  by  the  local  Colonial  Gas  company.  The 
installation  of  those  energy-efficient  windows  has  netted 
the  MBL  an  annual  savings  of  approximately  $5,000  in 
gas  usage.  Ecosystems  Center  personnel  are  also 
enjoying  a  more  comfortable  working  environment. 


Special  Events,  Symposia,  and  Conferences 

The  MBL  held  its  first  annual  Poster  Session  last 
June  to  promote  scientific  exchange  among  students, 
faculty,  fellows,  and  established  summer  and  year- 
round  MBL  investigators.  The  event  was  a  great 
success:  45  posters  were  presented  by  year-round  and 
summer  scientists  on  topics  ranging  from  the  control  of 
the  cell  cycle  in  early  embryos  to  protein  synthesis  in 
the  squid  axon  and  nerve  endings.  Over  250  members 
of  the  MBL  community  gathered  to  review  the  posters 
and  attend  a  reception  that  followed  in  the  Meigs 
Room.  Another  Poster  Session  is  scheduled  for  the 
summer  of  1995. 

The  Tokyo  String  Quartet  returned  to  the  MBL  last 
July  for  another  spectacular  Lillie  Auditorium  concert. 
The  quartet  played  to  a  full  house,  and  the  elegant  post- 
concert  candlelight  dinner  on  the  Swope  Terrace  made 
for  a  memorable  evening.  The  Quartet  will  perform 
again  at  the  MBL  this  coming  summer. 

In  September  the  MBL  sponsored  its  first  annual 
symposium  in  marine  biotechnology  titled 
"Biomaterials  from  the  Sea."  The  symposium  was 
designed  to  provide  scientific  and  technical  insights  into 
the  exciting  area  of  marine  biomaterials.  Seventy-five 
individuals  from  the  worlds  of  science,  business, 
investment,  and  policy  participated  in  the  meeting.  The 
second  annual  marine  biotechnology  symposium  will 
be  held  on  September  22.  1995. 

The  Marine  Biological  Laboratory  also  provided 
conference  and  housing  services  for  over  40  scientific 
meetings,  departmental  retreats,  and  conferences  last 
year,  which  attracted  an  additional  3800  scientists  and 
students  to  Woods  Hole.  For  the  first  time  in  the 
history  of  the  Laboratory,  MBL  housing  and  dining 
revenues  surpassed  the  $1  million  mark  in  1994. 


MBL  Head  Mail  Clerk  Bob  Illgen  recycle-. 


Center  Co-Director  .lerry  Melillo  greets  participants  in  the 
(H  II:  conference  held  at  the  MRl-  lasl  spring. 


R8  Annual  Report 


In  Closing 

As  I've  become  more  familiar  and  involved  with  the 
MBL's  multi-faceted  research  and  educational 
programs  during  my  tenure  here,  I  marvel  at  this 
century-old  Laboratory's  ability  to  reinvent  itself  in  the 
face  of  rapid  and  broad-based  change.  I  close  with  a 
statement  from  a  student  in  one  of  last  summer's 
courses.  It  resonates  with  my  feelings  about  the  MBL: 

"I  feel  like  Woods  Hole  is  to  scientists  what  Paris  is 
to  artists.  It 's  a  special  place  where  people  with 


similar  interests  roam  together  to  feed  and  grow  off 
of  other  people's  ideas  and  ultimately  create 
something  greater  than  anyone  could  ever  do  alone. 
It  was  wonderful  to  be  in  such  a  community,  to  be 
able  to  discuss  ideas  with  some  of  the  greatest  minds 
.  .  .  For  a  student  starting  out  in  science  it  was  the 
best  thing  I  could  ever  do.  And  it  seemed  for  the  old 
pros  a  great  opportunity  to  remember  why  they  got 
into  the  field  and  to  get  a  little  enthusiasm  from  the 
students.  " 

— John  E.  Burris 


Report  of  the  Treasurer 


In  1994  our  financial  performance  was  marked  by  con- 
tinued success  in  operating  funds,  in  financial  stabilization 
of  the  MBL's  physical  plant,  and  in  a  decreased  value  of 
our  invested  portfolios. 


Operating  Funds 

The  MBL's  Unrestricted  Operating  Funds  ended  the 
year  with  a  surplus  of  $5 1 3,900,  the  General  Fund 
showed  a  surplus  of  $347,81 1,  and  the  Housing  and 
Dining  Auxiliary  Fund  had  a  surplus  of  $166,089. 
Mandatory  transfers  to  repay  debt  principal  amounted 
to  $69,010;  $104,984  was  used  to  fund  capital 
acquisitions;  and  $359,000  was  transferred  to  the 
renewals  and  replacement  fund  for  future  capital 
investment  ($235,000  to  the  General  Renewals  and 
Replacement  Fund  and  $124,000  to  the  Housing 
Renewals  and  Replacement  funds).  While  the  amount 
of  the  transfers  to  the  Renewals  and  Replacement  funds 
was  slightly  less  than  in  1993,  it  is  still  a  significant 
amount  and  demonstrates  the  Laboratory's 
commitment  to  one  of  the  Trustees'  long-standing 
goals:  Reinvesting  in  the  physical  plant  infrastructure  of 
the  MBL. 

In  1994  funding  of  research  and  education  programs 
both  increased  over  1993.  The  former  grew  from 
$5,727,323  to  $5,900,614,  and  the  latter  from 
$1,538,190  to  $1,702,345.  Equally  encouraging  is  the 
stabilization  of  the  summer  research  program  rentals, 
which  have  increased  almost  5%  over  1993.  The  Boston 
University  Marine  Program  has  expanded  as  well,  and 
has  provided  increased  revenues  for  the  MBL. 

The  financial  operating  environment  of  the  MBL 
continues  to  improve,  and  as  we  look  forward  to  1995, 


we  have  every  reason  to  believe  we  will  build  on  that 
record  of  stability  and  measured  growth. 

Endowment  Funds 

The  success  that  we  enjoyed  in  operating  funds  was 
not  duplicated  in  our  endowment  funds.  In  fact,  the 
endowment  fund  market  values  decreased  in  1994, 
primarily  due  to  the  poor  performance  of  our 
investment  portfolios.  This  matter  has  concerned  us  for 
the  past  several  years,  and  the  Finance  and  Investment 
Committee  spent  much  of  the  past  year  reviewing  and 
evaluating  our  current  investment  strategies  and 
management.  In  December,  the  Committee  decided  to 
change  radically  our  approach  and  diversify  our 
investments  and  investment  managers  (this  was 
implemented  in  early  1995). 

In  making  this  change,  we  have  concluded  that  the 
investment  climate  for  the  future  is  far  more  complex 
than  it  has  been  in  the  recent  past.  We  therefore  need 
to  diversify  our  endowment  by  using  a  balance  of 
specialized  managers  if  we  are  to  improve  our 
performance  for  long-term  growth. 

For  the  future,  the  task  remains  clear:  Build  the 
necessary  overall  financial  support  base  that  will 
provide  stability  and  enhance  the  MBL's  research  and 
education  programs.  During  the  past  several  years,  we 
have  done  so  in  our  operating  funds.  For  1995  and  the 
years  ahead,  we  will  concentrate  our  efforts  in 
establishing  a  system  of  investment  management  that 
will  duplicate  our  recent  success  in  our  operating 
monies  and  provide  additional  financial  support  for  our 
research  and  education  programs. 

— Robert  D.  Manz 


R9 


Financial  Statements 


&Lybrand 


certified  public  accountants 


REPORT  OF  INDEPENDENT  ACCOUNTANTS 


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

We  have  audited  the  accompanying  balance  sheet  of  Marine  Biological  Laboratory  (the  "Laboratory") 
as  of  December  31,  1994  and  the  related  statement  of  support,  revenues,  expenses  and  changes  in  fund 
balances  for  the  year  then  ended.  We  previously  audited  and  reported  upon  the  financial  statements  of  the 
Laboratory  for  the  year  ended  December  31,  1993,  for  which  condensed  statements  are  presented  for 
comparative  purposes  only.  These  financial  statements  are  the  responsibility  of  the  Laboratory's 
management.  Our  responsibility  is  to  express  an  opinion  on  these  financial  statements  based  on  our  audit. 

We  conducted  our  audit  in  accordance  with  generally  accepted  auditing  standards.  Those  standards 
require  that  we  plan  and  perform  the  audit  to  obtain  reasonable  assurance  about  whether  the  financial 
statements  are  free  of  material  misstatement.  An  audit  includes  examining,  on  a  test  basis,  evidence 
supporting  the  amounts  and  disclosures  in  the  financial  statements.  An  audit  also  includes  assessing  the 
accounting  principles  used  and  significant  estimates  made  by  management,  as  well  as  evaluating  the 
overall  financial  statement  presentation.  We  believe  that  our  audit  provides  a  reasonable  basis  for  our 
opinion. 

In  our  opinion,  the  financial  statements  referred  to  above  present  fairly,  in  all  material  respects,  the 
financial  position  of  Marine  Biological  Laboratory  at  December  31,  1994,  and  its  support,  revenues, 
expenses  and  changes  in  fund  balances  for  the  year  then  ended  in  conformity  with  generally  accepted 
accounting  principles. 

Our  audit  was  conducted  for  the  purpose  of  forming  an  opinion  on  the  basic  financial  statements  taken 
as  a  whole.  The  supplemental  schedules  of  support,  revenues,  expenses  and  changes  in  fund  balances  for 
current  funds  (Schedule  I),  endowment  funds  (Schedule  II)  and  plant  funds  (Schedule  III)  as  of  December 
31,  1994  are  presented  for  purposes  of  additional  analysis  and  are  not  a  required  part  of  the  basic  financial 
statements.  Such  information  has  been  subjected  to  the  auditing  procedures  applied  in  the  audit  of  the 
basic  financial  statements  and,  in  our  opinion,  is  fairly  stated,  in  all  material  respects,  in  relation  to  the 
basic  financial  statements  taken  as  a  whole. 

Boston,  Massachusetts  f*  H      i  f) 

April  20,  1995  U^rCtoaAO  <r    *J*±MWrv/ 


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Financial  Statements  R13 


Marine  Biological  Laboratory 


Notes  to  Financial  Statements 

A.  Purpose  of  the  Laboratory: 

The  purpose  of  Marine  Biological  Laboratory  (the  "Laboratory")  is  to  establish  and  maintain  a  laboratory  or  station  for  scientific  study  and 
investigations,  and  a  school  for  instruction  in  biology  and  natural  history. 

B.  Significant  Accounting  Policies. 

Basis  o!  Presentation — Fund  Accounting 

In  order  to  ensure  observance  of  limitations  and  restrictions  placed  on  the  use  of  resources  available  to  the  Laboratory,  the  accounts  of  the 
Laboratory  are  maintained  in  accordance  with  the  principles  of  fund  accounting.  This  is  the  procedure  by  which  resources  are  classified  into 
separate  funds  in  accordance  with  specified  activities  or  objectives.  Separate  accounts  are  maintained  for  each  fund;  however,  in  the  accompanying 
financial  statements,  funds  that  have  similar  characteristics  have  been  combined  into  fund  groups.  Accordingly,  all  financial  transactions  have 
been  recorded  and  reported  by  fund  group. 

Externally  restricted  funds  may  only  be  utilized  in  accordance  with  the  purposes  established  by  the  donor  or  grantor  of  such  funds.  However, 
the  Laboratory  has  full  control  over  the  utilization  of  unrestricted  funds.  Restricted  gifts,  grants,  and  other  restricted  support  are  accounted  for 
in  the  appropriate  restricted  funds.  Restricted  current  funds  are  reported  as  revenue  as  the  related  costs  are  incurred  (see  Note  G). 

Endowment  funds  are  subject  to  restrictions  which  require  that  the  principal  be  invested  in  perpetuity.  Related  investment  income  is  available 
for  use  tor  restricted  or  unrestricted  purposes  by  the  Laboratory  depending  on  donor  restrictions.  Quasi-endowment  funds  have  been  established 
by  the  Laboratory  for  the  same  purposes  as  endowment  funds;  however,  the  principal  of  these  funds  may  be  expended  for  various  restricted  and 
unrestricted  purposes  at  the  direction  of  the  Trustees. 

Fixed  Assets 

Land,  buildings  and  equipment  purchased  by  the  Laboratory  are  recorded  at  cost.  Donated  fixed  assets  are  recorded  at  fair  market  value  at  the 
date  of  the  gift.  Depreciation  is  computed  using  the  straight-line  method,  beginning  the  month  after  the  asset  is  placed  in  service,  over  the  asset's 
estimated  useful  life.  Estimated  useful  lives  are  generally  three  to  five  years  for  equipment  and  20  to  40  years  for  buildings  and  improvements. 
When  assets  are  sold  or  retired,  the  cost  and  accumulated  depreciation  are  removed  from  the  accounts  and  any  resulting  gain  or  loss  is  included 
in  income  for  the  period. 

Contracts  and  Grants 

Revenues  associated  with  contracts  and  grants  are  recognized  in  the  statement  of  support,  revenues,  expenses  and  changes  in  fund  balances  as 
the  related  costs  are  incurred  (see  Note  G).  Reimbursement  of  indirect  costs  relating  to  government  contracts  and  grants  is  based  on  negotiated 
indirect  cost  rates.  Any  over  or  underrecovery  of  indirect  costs  is  recognized  through  future  adjustments  of  indirect  cost  rates. 

Investments 

Investments  purchased  by  the  Laboratory  are  carried  at  market  value.  Money  market  securities  are  carried  at  cost  plus  accrued  interest,  which 
approximates  market  value.  Donated  investments  are  recorded  at  fair  market  value  at  the  date  of  the  gift.  For  determination  of  gain  or  loss  upon 
disposal  of  investments,  cost  is  determined  based  on  the  first-in,  first-out  method. 

The  Laboratory  is  the  beneficiary  of  certain  investments  reported  in  the  endowment  funds  which  are  held  in  trust  by  others.  The  Laboratory's 
continuing  right  to  these  funds  is  subject  to  review  every  10  years  by  an  independent  committee.  The  committee  met  in  1994  and  determined 
that  MBL  was  still  eligible  to  remain  as  beneficiary  of  the  trusts  for  another  10  years.  The  market  values  of  such  investments  are  $4.595.615  and 
$4.873.242  at  December  31,  1994  and  1993,  respectively.  The  income  on  these  investments  totaled  $193.359  and  $226,702  in  1994  and  1993, 
respectively. 

Investment  Income  and  Distribution 

The  Laboratory  follows  the  accrual  basis  of  accounting  except  that  investment  income  is  recorded  on  a  cash  basis.  The  difference  between  such 
basis  and  the  accrual  basis  does  not  have  a  material  effect  on  the  determination  of  investment  income  earned  on  a  year-to-year  basis. 

Investment  income  includes  income  from  a  pooled  investment  account,  which  income  is  allocated  to  the  participating  funds  on  the  market  value 
unit  basis  (Note  D). 

Annuities  Payable 

Amounts  due  to  donors  in  connection  with  gift  annuities  are  determined  based  on  remainder  value  calculations,  as  of  December  31,  1994  with 
varied  assumptions  of  rates  of  return  and  payout  terms. 

Tax-Exempt  Status 

The  Laboratory  is  exempt  from  federal  income  tax  under  Section  501(c)3  of  the  Internal  Revenue  Code 


RM   Annual  Report 


Professional  Standards 

For  the  fiscal  year  ending  December  31.  1995.  the  Laboratory  will  adopt  Statement  of  Financial  Accounting  Standards  (SFAS)  No.  1 16.  Accounting 
for  Contributions  Received  and  Made,  and  SFAS  No.  1 1 7.  Financial  Statements  of  Not-for-Protit  Organizations,  and  will  apply  these  standards 
on  a  retroactive  basis.  SFAS  No.  117  establishes  standards  for  external  financial  reporting  by  not-for-profit  organizations  and  requires  that 
resources  be  classified  for  accounting  and  reporting  purposes  into  three  net  asset  categories  (unrestricted,  temporarily  restricted  and  permanently 
restricted)  according  to  externally  (donor)  imposed  restrictions.  SFAS  No.  I  16  requires  that  unconditional  promises  to  give  (pledges)  be  recorded 
as  receivables  and  revenues  and  requires  the  organization  to  distinguish  between  contributions  received  for  each  net  asset  category  in  accordance 
with  donor  imposed  restrictions.  Outstanding  pledges  as  of  December  31.  1994  are  disclosed  in  Note  I. 


C.   Investments: 

The  following  is  a  summary  of  the  cost  and  market  value  of  investments  at  December  31,  1994  and  1993: 

Market 


Certificates  of  deposit 
Money  market  securities 
U.S.  Government  securities 
Corporate  fixed  income 
Common  stocks 
Real  estate 

Total  investments 


1994 


$ 


50.173 
1.877.731 
1.057.616 
12.248.905 
7.732.931 

13.247 

22.980.603 


1993 


$        48.483 
4.822.875 

9.706.380 
9,093.522 

13.247 

$23.684,507 


1994 


$        50.173 

1.877,731 

1.047,615 

12.598.467 

7.428.618 

13.247 

$23.015.851 


Cost 


1993 


$        48,483 
4.822,875 

8.824,824 

7,052,496 

13.247 

$20.761.925 


Investments  by  fund  group  and  related  portfolios  for  the  years  ended  December  31.  1994  and  1993  are  as  follows: 


Market 


Cost 


Current  Funds 
Certificates  of  deposit 
Money  market  securities 
S.T.A.R.  Fund 
Instruction  Fund 

Total 


1994 


50.173 

1.000,000 

1,019.462 

795.097 

2,864.732 


199.1 


48.483 
2.000.000 

1.378.838 
3.427.321 


1994 


50,173 

1,000,000 

1.041,641 

886.250 

2,978.064 


1993 


48.483 
2.000.000 

1.339.557 
3.388.040 


Li»ii;-7'erni  Funds 

Endowment  and  quasi-endowment 

General  endowment  trust  fund 

Library  endowment  trust  fund 

Ecosystem  funds 

Pooled  funds 
Other  Funds: 

Annuity  Fund 

Real  Estate 

Total 

Total  investments 


3,636.845 

958.770 

4,388.463 

9,782.000 

1.336,546 
13.247 

20.1  15.871 
522.980,603 


3,855,724 

1.017,518 

4,829,277 

10,343.724 

197.696 

1 3.247 

20.257.186 
$23.684.507 


3.445.228 

923.305 

4.363.838 

9.949.252 

1.342.917 

13.247 

20.037.787 
523.015.851 


3.135,652 

807,175 

4.029.966 

9,187,655 

200,190 

13.247 

17.373.885 
$20.761.925 


D.  Accounting  for  Pooled  Investments: 

Certain  endowment  fund  assets  are  pooled  for  investment  purposes.  Investment  income  from  the  pooled  investment  account  is  allocated  on  the 
market  value  unit  basis,  and  each  endowment  fund  subscribes  to  or  disposes  of  units  on  the  basis  of  the  market  value  per  unit  at  the  beginning 
of  the  calendar  quarter  within  which  the  transaction  takes  place.  The  unit  participation  of  the  funds  at  December  31.  1994  and  1993  is  as  follows: 


Quasi-endowment  unrestricted 
Quasi-endowment  restricted 
Endowment,  income  for  restricted  purposes 
Endowment,  income  for  unrestricted  purposes 

lotal 


1994 

4.342 

8.773 

65,524 

229 

76,868 


1993 

4.342 

8.771 

62.158 

152 

75.423 


Financial  Statements  R15 


Pooled  investment  activity  on  a  per-unit  basis  was  as  follows: 

Unit  value  at  beginning  of  year 
Unit  value  at  end  of  year 

Increase  in  realized  and  unrealized  appreciation 
Net  income  earned  on  pooled  investments 

Total  return  on  pooled  investments 


1994 

$137.18 

127.43 

(9.57) 

5.53 

$  (4.22) 


1993 

$128.66 
137.18 

8.52 
4.52 

$   13.04 


E.    Commitment  and  Contingencies: 
Capital  Leases 

As  of  December  31.  1994  the  Laboratory  had  capital  leases  for  office  equipment.  Interest  rates  on  the  obligation  are  between  1.55%  and  6.63%. 
The  future  minimum  lease  payments  as  of  December  31,  1994  are  as  follows: 


1995 
1996 
1997 
1998 


$28.014 

26,789 

14.458 

3.440 

$72,701 


F.    Long-Term  Debt: 

Long-term  debt  at  December  31.  1994  amounted  to  $2.475,681.  The  aggregate  amount  of  principal  due  for  each  of  the  next  five  fiscal  years  and 
thereafter  is  as  follows: 


1995 
1996 
1997 
1998 
1999 
Thereafter 

Less  current  portion 
Total 


$  79,010 
76,671 
80.000 
85.000 
90,000 
2.065.000 

2.475.681 
79.010 

$2,396.671 


In  1992.  the  Laboratory  issued  $1,100.000  Massachusetts  Industrial  Finance  Authority  (MIFA)  Series  1992A  Bonds  and  $1.500,000  MIFA  Series 
1992B.  These  bonds  pay  varying  annual  interest  rates  ranging  from  3.48%  to  6.63%.  Interest  expense  on  this  debt  totaled  $151.354  for  the  year 
ended  December  31.  1994.  The  Series  1992  A  and  B  Bonds  mature  on  December  I,  2012  and  are  collateralized  by  a  first  mortgage  on  certain 
Laboratory  property. 

The  agreements  related  to  these  Bonds  subject  the  Laboratory  to  certain  covenants  and  restrictions.  Under  the  most  restrictive  covenant  of  this 
debt,  the  Laboratory's  operating  surplus  (before  transfers),  interest,  expense  and  transfers  from  the  quasi-endowment  for  debt  service  must  equal 
or  exceed  all  debt  service  payments.  The  Laboratory  was  in  compliance  with  these  covenants  and  restrictions  at  December  31.  1994. 


G.  Restricted  Current  Funds  Deferred  Support: 

The  Laboratory'  defers  revenue  on  current  restricted  funds  until  the  related  costs  are  incurred.  Amounts  received  in  excess  of  expenses  are  recorded 
as  deferred  support.  The  following  summarizes  the  activity  of  the  deferred  support  account: 


1994 


1993 


Balance  at  beginning  of  year 
Additions: 

Gifts,  endowment  income  and  grants  received 

Net  unrealized  gains  (losses) 

Net  realized  gains 

Transfers 
Deductions: 

Funds  expended  under  gifts  and  grants 

Transfers 

Balance  at  end  of  year 


$3,796.864 

7,983,471 
(143.698) 
93,184 
59,862 

8.798,213 
$2,991,470 


$3,518,263 

8,509,929 

(58.026) 

155.460 

21,223 

8,349.985 
$3,796,864 


Deferred  restricted  gifts  of  $559,476  and  $527,945  were  expended  in  1994  and  1993,  respectively,  for  the  support  of  indirect  costs  attributable 
to  the  Laboratory's  instruction  programs. 


R16   Annual  Report 


H.  Retirement  Plan: 

The  Laboratory  participates  in  the  denned  contribution  pension  plan  of  TIAA-CREF  (the  "Plan").  The  Plan  is  available  to  permanent  employees 
that  have  Completed  two  years  of  service.  Under  the  Plan,  the  Laboratory  contributes  10%  of  total  compensation  for  each  participant.  Contributions 
amounted  to  $525,918  in  1994  and  $507.324  in  1993. 

I.     Pledges: 

As  of  December  31.  1994.  the  Laboratory  has  outstanding  pledges  of  $1.746.331  of  which  $1,713,731  is  restricted  (unaudited).  These  pledges  are 
scheduled  to  be  paid  over  the  next  three  years  in  the  amounts  of  $1,016,600.  $434,731.  and  $295.000,  respectively.  As  required  by  SFAS  No. 
1 16,  pledges  will  be  included  in  the  financial  statements  for  the  year  ended  December  31,  1995  (Note  B). 

J.    Interlnnd  Borrowings: 

Current  unrestricted  fund  interfund  borrowings  at  December  31  are  as  follows: 

1994  1993 


Due  from  restricted  education  funds  $    3 1 ,098 

Due  from  (to)  restricted  endowment  fund  $      15.126  (6,445) 

Due  from  restricted  quasi-endowment  funds  125.000 

Total  $      15.126  $  149.653 

K.  Postretin-menl  Benefits: 

On  November  20.  1993.  the  Laboratory  adopted  Statement  No.  106.  "Employers'  Accounting  for  Postretirement  Benefits  Other  Than  Pensions," 
for  the  year  beginning  January  1 ,  1 994.  This  new  standard  requires  employers  to  accrue,  during  the  years  that  the  employee  renders  the  necessary 
service,  the  expected  cost  of  benefits  to  be  provided  during  retirement. 

The  Laboratory's  policy  is  that  all  current  retirees  and  certain  eligible  employees  who  retire  prior  to  June  1,  1994  will  continue  to  receive 
postretirement  health  benefits.  The  remaining  current  employees  will  receive  benefits;  however,  those  benefits  will  be  limited  as  denned  by  the 
Plan.  Employees  hired  on  or  after  January  1.  1995  will  not  be  eligible  to  participate  in  the  postretirement  medical  benefit  plan. 

Accumulated  postretiremen!  benefit  obligations  at  the  date  of  adoption: 

1993  accumulated  postretirement  benefit  obligation 
Net  postretirement  benefits  for  1994  include: 

Service  cost  (benefits  earned  during  period)  $      54.494 

Interest  cost  (on  projected  benefit  obligation)  135,459 

Actual  return  on  plan  assets  (3,032) 

Net  amortization  and  deferral  86,918 

Net  postretirement  benefits  cost  $273.839 


Below  is  a  reconciliation  of  the  funded  status  of  the  Plan  at  December  31.  1994: 
Accumulated  benefit  obligation 

Retirees  and  dependents  $  1 . 1 95,739 

Fully  eligible  active  participants  242,279 

Other  active  participants  401.951 

Total  1,839,969 

Market  value  of  plan  assets  190,601 

Assets  less  than  obligations  1.649.368 
Unrecognized  prior  service  cost  (credit) 

Unrecognized  net  (gain)  loss  96 

Unrecognized  transition  obligation  1.649,625 

Prepaid  postretirement  benefit  cost  $           161 


The  health  care  cost  trend  rate  assumptions  used  in  determining  the  projected  benefit  obligation  begins  at  10.0%  in  1994  and  gradually  decreases  to 
6%  in  the  year  2004  and  thereafter.  The  effect  of  raising  the  assumed  health  care  cost  trend  rate  by  one  percentage  point  in  each  year  would  be  to 
increase  the  accumulated  postretirement  benefit  obligation  as  of  December  31,  1994  by  $167,767  and  to  increase  the  aggregate  of  the  service  and 
interest  cost  components  of  net  periodic  postretirement  benefit  cost  for  the  year  then  ended  by  $15.780.  The  discount  rate  used  in  determining  the 
accumulated  postretirement  benefit  obligation  is  8.0%.  and  the  expected  return  on  plan  assets  was  8.0%>.  During  1994,  the  Laboratory  contributed 
$274.000  to  fund  the  Trust  for  these  postretirement  benefits. 


MARINE  BIOLOGICAL  LABORATORY 

STATEMENT  OF  SUPPORT,  REVENUES,  EXPENSES  AND  CHANGES  IN  FUND  BALANCES 

CURRENT  FUNDS 
for  the  vear  ended  December  31.  1994 


SCHEDULE  I 


Current  I  'nrestricted  Fund* 


SUPPORT  AND  REVENUE: 

Grant  reimbursements  of  direct  costs 

Recover,1  of  indirect  costs 

Tuition 

Fees  for  services: 

Dormitories 

Dining  hall 

Library 

Scientific  journals 

Research  services 

Marine  resources 
Investment  income 
Miscellaneous  revenue 
Gifts 
Change  in  deferred  support 

Total  support  and  revenues 
EXPENSES: 
Research 
Instruction 

Scholarships,  fellowships  and  stipends 
Services: 

Dormitories 

Dining  hall 

Library1 

Scientific  journals 

Research  services 

Marine  resources 
Administration: 

Administration 

Sponsored  projects  administration 
Plant  operations 
Other 

Total  expenses 

Excess  (deficit)  of  support  and  revenues 
over  expenses  before  gain  on 
investments 

Net  unrealized  gain  (loss)  on  investments 

Net  realized  (loss)  on  investments 

Net  gain  on  investments 

TRANSFERS  AMONG  FUNDS: 
Debt  services 
Acquisition  of  fixed  assets 
Repairs  and  replacement 
Endowment  transfer 
Capitalization  of  income 
Other 

Total  transfers  among  funds 
Net  change  in  fund  balances 
Fund  balances,  beginning  of  year 
Fund  balances,  end  of  vear 


Auxiliary 

Current 

Operating 

Enterprises 

Restricted 

l-'itnd 

Fund          Total 

Fund 

Total 

$5.388,844 

$5.388.844 

$3,700.412 

$3,700.412 

3.700,412 

577.365 

577,365 

$1,077,395       1,077,395 

1.077,395 

1,030,324       1.030,324 

1,030,324 

486,287 

486.287 

486,287 

241,169 

241,169 

44.500 

285,669 

430.341 

430.341 

56.719 

487.060 

184,957 

1X4,957 

184,957 

453,507 

453,507 

496,870 

950,377 

116,867 

116.867 

658,864 

775,731 

581,554 

581,554 

1,328.616 

1,910,170 

559,476 

559.476 

237.087 

796,563 

6,754.570 

2.107,719       8,862.289 

8,788.865 

17.651,154 

5.900.614 

5,900.614 

1.702,345 

1.702.345 

465,824 

465,824 

831.866        831,866 

831,866 

924,942        924,942 

924,942 

837.480 

837.480 

120.711 

958.191 

192.549 

192,549 

48,282 

240,831 

540.055 

540.055 

153,026 

693,081 

443,007 

443,007 

9,086 

452,093 

2,013.705 

184,822       2,198,527 

2.198.527 

456.108 

456,108 

456,108 

1.923,855 

1.923,855 

156,592 

2,080,447 

— 

241.733 

241,733 

6,406.759 

1,941,630       8.348.389 

8,798.213 

17,146.602 

347,811 


(29,010) 
(102,895) 
(235,000) 


22.440 

(344.465) 

3.346 

32.097 

$      35.443 


166.089 


(40.000) 

(2,089) 

(124.000) 


(166.089) 


5 1 3,900 


(69,010) 
(104,984) 
(359,000) 


22.440 

(510.554) 

3.346 

32.097 

$      35,443 


(9,348) 


93,184 

(143.698) 

(50,514) 


150,000 
(180,916) 

90.778 

59,862 


504.552 

93.184 
(143.698) 

(50.514) 


(69,010) 
(104.984) 
(359.000) 

150,000 
(180.916) 

113.218 

(450.692) 
3.346 
32.097 
35.443 


SCHEDULE  II 


MARINE  BIOLOGICAL  LABORATORY 

STATEMENT  OF  SUPPORT,  REVENUES,  EXPENSES  AND  CHANGES  IN  FUND  BALANCES 

ENDOWMENT  FUNDS 
for  the  year  ended  December  31,1 994 


Unrestricted 


Quasi- 
Endcmment 


SUPPORT  AND  REVENUES: 
Gifts 

Total  support  and  revenues 

Net  realized  gain  on  investments 

Net  unrealized  gain  (loss)  on  investments 

Net  gain  (loss)  on  investments 

TRANSFERS  AMONG  FUNDS: 
Capitalization  of  income 
Endowment  transfers 
Other  transfers 

Total  transfers  among  funds 
Net  change  in  fund  balances 
Fund  balances,  beginning  of  year 
Fund  balances,  end  of  year 


33,689 
(76.004) 

(42,315) 


0 

0 

0_ 

0_ 

(42.315) 

595.694 

$     553,379 


Restricted 

Income  for 
Unrestricted 
Purposes 

Income  for 
Restricted 
Purposes 

Quasi- 
Endowment 

Total 
Restricted 

$        10,000 

$      165,380 

$            250 

$      175,630 

10,000 

165.380 

250 

175.630 

310,755 
(531.119) 

599,012 
(1.270.791) 

497,650 
(929,847) 

1,407,417 
(2.731,757) 

(220.364) 

(671.779) 

(432.197) 

(1,324,340) 

0 
0 
0 

0 
0 
13,623 

180,916 
(  1  50,000) 
0 

180,916 
(  1  50.000) 
13,623 

0 

13.623 

30,916 

44,539 

(210,364) 

(492,776) 

(401.031) 

(1,104,171) 

3,876,604 

9,547,900 

5.907,490 

19,331,994 

$  3,666,240 

$  9,055,124 

$  5,506,459 

$18,227,823 

Total 


$      175.630 
175.630 

1,441,106 
(2.807.761) 

(1.366.655) 


180.916 

(150,000) 
13.623 

44.539 
(1.146.486) 
19,927.688 
$18,781,202 


R18 


SCHEDULE  III 


MARINE  BIOLOGICAL  LABORATORY 
STATEMENT  OF  SUPPORT,  REVENUES,  EXPENSES  AND  CHANGES  IN  FUND  BALANCES 

PLANT  FUNDS 
for  the  year  ended  December  31,  1994 


SUPPORT  AND  REVENUES: 
Grant  for  capital  additions 
Investment  income 
Gifts 
Other  revenue 

Total  support  and  revenues 

EXPENSES: 
Depreciation 

Plant  operations 

Total  expenses 

Excess  (deficit)  of  support  and  revenues  over  expenses 

TRANSFERS  AMONG  FUNDS: 
Debt  service 

Acquisition  of  fixed  assets 
Capital  additions 
Other  transfers 

Total  transfers  among  funds 
Net  change  in  fund  balances 
Fund  balances,  beginning  of  year 
Fund  balances,  end  of  year 


Unrestricted 


$   1,224,994 


1.224,994 
(1.224.994) 

69,010 

104.984 

634.303 

0 


(416.697) 
19,869,320 


Unrestricted 


Repairs  and 

Replacements 

Reserve 


$  215.260 
215.260 
(215,260) 


333.000 
333.000 
117.740 
546,838 


Total 
Unrestricted 


!   1,224,994 

215,260 

1.440.254 

(1,440.254) 


69,010 
104,984 
634,303 
333.000 

1.141.297 

(298.957) 

20,416,158 


Restricted 


185,087 

1.566 

58,000 

244,653 


p_ 

244.653 


(634,303) 
(100.841) 

(735.144) 
(490.491) 
539.878 


Total 


5   185,087 

1.566 

58,000 

244.653 

1,224,994 
215.260 

1.440.254 
(1.195.601) 


69,010 
104.984 

232,159 

406.153 

(789.448) 

20,956.036 


$19.452.623   $   664,578   $20,117,201    $   49,387   $20,166,588 


R19 


Report  of  the  Librarian 


During  1994  the  MBL/WHOI  Library  implemented 
systems  to  protect  our  holdings.  Recognizing  the 
Library's  need  to  provide  information,  service,  and  a 
comfortable  and  safe  working  environment  for  its  users, 
the  Trustees  and  the  members  of  the  Joint  Library 
Committee  agreed  that  it  must  adopt  ways  to  protect 
both  the  collection  and  its  users.  Traditionally,  the 
Library's  doors  have  been  open  24  hours  a  day,  365 
days  a  year.  Beginning  in  1995,  patrons  will  still  be 
allowed  that  same  access  to  the  collection  but  by  means 
of  a  card  access  security  system  during  non-staffed 
hours.  I  am  pleased  to  report  that  the  system  was 
successfully  installed,  tested  and  placed  on-line  on 
February  1,  1995.  The  new  MBL/WHOI  Library  access 
card  is  available  free  of  charge  to  all  MBL  corporation 
members  and  year-round  staff.  Others  wishing  to  use 
the  Library  may  purchase  readerships  which  will  assure 
them  access  to  the  collection  after  hours. 

The  Journal  Collection 


the  Library  is  terribly  overcrowded.  In  the  coming  year 
we  will  begin  planning  for  off-site  storage  of  a  portion 
of  the  collection. 

The  combined  forces  of  subscription  costs,  space 
needs,  and  a  large  increase  in  our  student  user 
population  have  led  to  the  creation  of  a  new  service 
model  for  article  delivery.  Easy  access  article  delivery 
services  are  now  available  from  the  Library's  electronic 
menu,  table  of  contents,  and  on-line  document 
ordering  and  delivery.  Interlibrary  photo  duplication 
via  the  Internet  or  Fax  are  also  available. 

During  the  past  year  the  Library  automated  serial 
management  and  binding  and  negotiated  a  favorable 
agreement  with  a  new  subscription  agency,  the 
Readmore  Company.  Joe  DeVeer,  Serials  Librarian, 


Monograph  and  Serial  Costs  in  ARL  Libraries, 
1986-1993 


We  were  able  to  maintain  our  level  of  journal 
subscriptions  in  1994.  However,  next  year  we  will  again 
need  to  reduce  the  number  of  journals  to  which  we 
subscribe  to  allow  us  to  purchase  new  journals  that 
meet  changing  research  needs.  The  cost  of  journals  has 
skyrocketed  in  recent  years.  During  the  past  eight  years, 
journal  subscription  rates  have  increased  108%,  while 
our  serials  budget  has  increased  only  36%  during  that 
same  time.  The  current  forecast  for  journal  subscription 
prices  in  FY96  is  an  overall  increase  of  13.1%  based  on 
a  33%  North  American  67%  European  title  split. 
Because  most  of  the  MBL's  journals  come  from 
European  publishers,  our  increase  will  be  even  higher, 
meaning  that  it  will  not  be  financially  possible  to 
maintain  our  collection  at  the  current  level. 

Overcrowded  stacks  are  another  area  of  concern.  Six 
years  ago,  surveys  showed  that  the  Library  would  be 
filled  to  capacity  in  1995.  Although  this  will  not  occur. 


'(ft 

0) 


6 


Serial  Lmii  Price  <  +  !08?c) 


Serial  Expenditures 
(+927C> 


Monograph  Urut  Price 


Monograph  Expenditures 


.Serials  Purchased  (- 


Monographs 

Purchased  (-23%) 


Fiscal  year 


R20 


Report  of  the  Librarian  R2I 


and  Maggie  Rioux.  Systems  Librarian,  were 
instrumental  in  effecting  a  smooth  transition. 

A  contract  was  signed  with  UnCover,  a  multi- 
disciplinary  organization  supplying  journal  articles  and 
tables  of  contents.  This  service  makes  accessible 
19,000+  journal  titles  on-line.  The  system  is 
customized  such  that  users  can  easily  identify  articles  in 
journals  owned  by  either  MBL/WHOI  Library  or  the 
Boston  Library  Consortium.  An  automatic  table  of 
contents  service  initiated  with  this  same  company 
provides  up  to  50  different  table  of  contents  on  the  day 
of  publication  from  titles  selected  by  the  user.  All  of 
these  transactions  take  place  over  the  Internet 
minimizing  delivery  time  and  costs. 

The  Book  Collection 

Over  the  last  year  a  new  gift  policy  that  was 
developed  in  collaboration  with  both  WHOI  and  MBL 
development  offices  enabled  us  to  acquire  resources 
that  added  value  to  our  collection  and  reduced  costs 
and  processing  time.  Detailed  lists  and  bibliographies 
supplied  by  the  donors  enable  Library  staff  to  assess  the 
collection  before  handling. 

The  donation  of  a  major  gift  collection,  the  success 
of  the  Book  Fair,  and  the  guidance  of  the  Library 
Coordinators  for  Book  Acquisitions  resulted  in 
enhanced  book  acquisitions  in  subject  areas,  including 
molecular  biology  and  environmental  sciences,  that 
required  strengthening  and  additional  support. 

The  I  'olunteers 

Last  year  former  WHOI  Research  Librarian  Carol 
Winn.  who  retired  in  1994,  joined  Dr.  and  Mrs.  Robert 
Huettner  as  a  volunteer  in  the  Archives.  A  portion  of 
the  rare  book  collection  is  now  available  for  local  and 
national  access  due  to  the  automation  efforts  of  this 
trio.  Programs  for  groups  and  visitors  to  the  Archives 
were  conducted,  and  more  than  25  rare  books — some 
dating  back  to  the  1600s — were  restored,  rebound,  and 
deacidified.  The  Huettners  also  began  a  program  to 
raise  funds  for  the  restoration  of  rare  books  in  memory 
of  departed  members  of  the  MBL  community.  This 
year  20  books  were  rebound  as  part  of  this  program.  In 
the  main  collection,  Arthur  Voorhis  continues  to  care 
for  and  mend  the  monographs. 

In  the  Data  Library  at  McLean.  Mr.  William 
Dunkle.  who  retired  as  Data  Librarian/Archivist  in 
1994.  provided  assistance  in  the  Data  Library  and 
Archives  while  focusing  primarily  on  the  Map/Chart 
and  Photo  collections.  In  particular,  he  has  been  largely 
responsible  for  WHOFs  acquisition  of  the  Bowdoin 
Collection  of  historical  maps.  These  maps,  circa  1 840- 


1870.  will  be  a  valuable  addition  to  the  existing 
historical  map  and  chart  collection.  Mr.  Garfield  Arthur 
continues  his  volunteer  role  in  evaluating  the  dive 
videos  including  generating  machine-searchable  log 
shots  which  are  w  idely  referenced  by  members  of  the 
community. 

The  Archives.  Preservation,  and  Rare  Books  Collect/on 

Last  March,  at  the  request  of  the  Joint  MBL/WHOI 
Library  Trustees  Committee,  Dr.  Robert  Stoddard 
(Houghton  Library  Harvard  University)  provided  us 
with  an  assessment  of  the  Archive  and  rare  book 
collections.  He  wrote  "This  [collection]  is  a  powerful 
arsenal  for  historical  studies  and  programs  that  could 
not  be  duplicated  anywhere."  Mr.  Stoddard's  report 
was  shared  with  the  trustees  of  both  institutions.  The 
Committee  agreed  that  his  assessment  "forces  us  to 
develop  a  plan  to  fulfill  our  shared  accountability  and 
responsibility  for  providing  access  to  this  national 
treasure  and  for  its  preservation  and  conservation."  As 
a  first  step  in  developing  such  a  plan  the  Librarian  has 
engaged  Ralph  Titcomb,  rare  book  appraiser,  to  assess 
the  financial  value  of  the  rare  book  collection. 

A  new  exhibit,  Women  of  Science  at  Woods  Hole — 
The  Middle  Years,  was  prepared  and  is  now  available 
on  the  MBL/WHOI  Web  server  along  with  its 
predecessor,  Women  of  Science  at  Woods  Hole — The 
Early  Years.  The  Leuckart  Charts  have  all  been 
scanned  and  loaded  on  the  Library's  Web  Home  Page 
and  are  proving  to  be  a  popular  resource.  The  Library 
also  supplied  research  papers  and  co-sponsored  a  talk  at 
the  Clark  Laboratory  last  November  by  Dr.  Gary  Weir, 
Naval  Historian,  Navy  Museum.  "Finding  a  Niche, 
Columbus  Iselin.  and  mobilizing  Oceanography  for 
War." 

Cooperative  Relationships 

Two  new  reciprocal  borrowing  and  photo  duplication 
agreements  with  the  Scripps  Institution  of 
Oceanography  in  California  and  the  Institute  of  Ocean 
Science  in  the  UK  give  us  access  to  new  journal 
resources  oceans  apart  and  bring  the  number  of 
resource  sharing  agreements  we  have  to  82  libraries.  To 
assure  the  continued  support  of  core  journals  in 
chemistry  and  neuroscience.  the  Library  has  entered 
into  an  agreement  with  members  of  the  Boston  Library 
Consortium  to  maintain  selected  titles.  These 
cooperative  arrangements  will  become  increasingly 
important  as  the  economic  climate  and  copyright  rules 
for  scientific  publications  become  progressively  more 
restrictive. 


R22  Annual  Report 


The  Library  entered  into  a  second  agreement  with 
the  UnCover  company  to  become  a  supplier  of 
documents.  Our  unique  captive  collection  of  journals 
makes  us  a  perfect  candidate  for  this  type  of  resource 
sharing.  UnCover  financed  the  establishment  of  Library 
office  space  and  personnel  to  meet  the  demands  of  the 
service,  and  the  Library  receives  compensation  for  each 
article  shared  from  our  collection. 

The  Electronic  Library 

The  MBL/WHOI  Library  is  on  the  Internet  via  its 
World  Wide  Web  home  page  at  html://www. mbl.edu/ 
html/LIBRARY/libweb.html  and  through  the  Library 
gopher.  New  CD-ROMS  on  fish,  water  resources,  Arctic 
literature,  and  marine  research,  and  our  UnCover 
Reveal  Table  of  Contents  service  as  well  as  automated 
article  request  and  delivery  at  a  reduced  rate  are 
available  on  the  Library  network. 

The  physical  relocation  of  our  local  library  utility 
and  on-line  catalog,  CLAMS,  from  WHOI  to  Hyannis 
has  resulted  in  a  continuing  disruption  in  access  and  a 
crisis  in  service  for  both  patrons  and  staff.  The  Library 
is  currently  seeking  a  permanent,  reliable  long-term 
solution  to  our  predicament. 

WHOI  Branch  Libraries 

The  WHOI  branch  libraries  underwent  a  re- 
engineering  process  by  a  team  of  scientists  charged  "to 
examine  the  current  WHOI  library  services  and  to 
make  recommendations  for  improvement  in  these 
services  while  maintaining  or  reducing  overall  costs." 
The  time  and  effort  expended  by  the  team  and  Library 
staff  were  enormous  and  the  results  are  currently  under 
consideration  by  the  WHOI  Directorate.  The  Joint 
Library  Committee,  staff  and  department  heads  have  all 
been  invited  to  comment  on  the  final  report,  and  the 
results  will  be  available  in  the  spring  of  1995.  The 
Library  has  also  changed  its  reporting  schedule  and 
now  reports  to  the  Associate  Director  for  Education 
and  Dean  of  Graduate  Studies,  John  Farrington. 


Library  Instruction 

New  classes  were  developed  to  introduce  the 
scientific  community  to  the  World  Wide  Web  browsing 
tools.  Classes  were  conducted  on  a  weekly  basis  in  the 
National  Library  of  Medicine  (NLM)  computer  Lab  in 
Loeb,  and  Connecting  Point  (a  local  computer  vendor) 
rented  space  for  teaching  basic  courses  on  Windows, 
Word,  Excel,  and  Lotus  to  the  scientific  community. 
The  NLM's  Medical  Informatics  course  was  held  here 
again  in  1994  and  numerous  sessions  were  conducted 
throughout  the  year.  Four  groups  of  Elderhostel  classes 
were  taught  how  to  "surf  the  Internet  and  search  the 
National  Library  of  Medicine's  resources  using  Grateful 
Med.  David  Remsen  and  Cathy  Norton  again 
participated  in  the  cooperative  teaching  effort  with 
Northern  Arizona  University's  Summer  of  Science 
Program,  which  brought  20  Native  Americans  and 
minority  students  and  teachers  to  the  MBL  for  a  two- 
week  experience  using  local  marsh  environmental 
models  and  the  computer  lab  to  enhance  their  science 
skills. 

Library  Trustees 

The  Joint  MBL/WHOI  Trustees'  Library  Planning 
Committee  delivered  a  second  report  to  the  Trustees  of 
the  MBL  in  February  and  to  WHOI  in  May  on 
management's  progress  on  their  earlier 
recommendations  and  proposed  inter-institutional 
development  priorities  to  address  goals  that  cannot  be 
met  without  new  funding.  The  Library  Trustees' 
priorities  for  capital  outlay  include  ( 1 )  the  installation 
of  a  heating,  ventilation,  and  air-conditioning  system 
and  improved  lighting  in  the  stacks,  (2a)  the  purchase 
of  computer  equipment  for  the  library  network,  (2b) 
rare  book  restoration,  and  (3)  digitization  of  the  video/ 
film  archives.  The  development  of  policies  and 
procedures  for  a  joint  development  effort  of  the  two 
institutions  was  also  highlighted  in  the  report. 

— Catherine  Norton 


Educational  Programs 


Summer  Courses 

Biology  of  Parasitism  (June  12-August  13) 

Director 

Steven  Hajduk,  University  of  Alabama.  Birmingham 

Course  Faculty 

John  Boothroyd.  Stanford  University 

Jean  Feagin.  Seattle  Biomedical  Research  Institute 

Fred  Finkelman,  Uniformed  Services  University  of  the  Health 

Service 
Patricia  Johnson,  University  of  California  School  of  Medicine,  Los 

Angeles 

Keith  Joiner.  Yale  University  School  of  Medicine 
Richard  Komuniecki.  University  of  Toledo 
Richard  Locksley.  University  of  California.  San  Francisco 
Steven  Reiner,  University  of  California.  San  Francisco 
Buddy  Ullman.  Oregon  Health  Sciences  University- 
Joseph  F.  Urban.  United  States  Department  of  Agriculture 

Teaching  Assistants 

Thomas  Allen.  Seattle  Biomedical  Research  Institute 
Con  Beckers,  Yale  University  School  of  Medicine 
Peter  Bradley.  University  of  California.  Los  Angeles 
Francisca  Diaz,  University  of  Toledo 
Mark  Drew.  Seattle  Biomedical  Research  Institute 
Michele  Klmgbeil.  University  of  Toledo 
Allen  J.  LeBlanc,  Jr..  University  of  Alabama.  Birmingham 
Suzanne  Morris.  Uniformed  Services  University  of  the  Health 

Sciences 
Anthony  Sinai.  Yale  University  School  of  Medicine 

Laboratory  Assistants 

Curtis  Maier.  University  of  Alabama.  Birmingham 
Victoria  Pollard,  University  of  Alabama,  Birmingham 

Students 

Billy  Apola,  National  Museums  of  Kenya,  Kenya 
Hani  Atamna.  Hebrew  University.  Israel 
Leonard  Basco,  University  of  Paris,  France 
Barbara  Davids,  University  of  Wisconsin.  Madison 
Socrates  Herrera  Valencia,  Umversidad  del  Valle.  Colombia 


Laura  Knoll.  Washington  University- 
Susan  Little,  University  of  Georgia 
Stephen  Manale.  Louisiana  State  University- 
Deborah  Schechtman,  Weizmann  Institute  of  Science.  Israel 
Andrea  Smith.  University  of  Alabama.  Birmingham 
Marline  Soete.  INSERM,  France 
Andrea  Torres-Perez,  Stanford  University 
Henri  van  der  Heyde.  University  of  Wisconsin,  Madison 
Fred  van  Leeuwen,  Netherlands  Cancer  Institute.  The  Netherlands 
Ulrike  Zelck,  University  of  Hamburg,  Germany 
Xiaonong  Zhou.  Jiangsu  Institute  of  Parasitic  Diseases,  China 


Embryology  (June  15- July  29) 


Directors 

Eric  H.  Davidson,  California  Institute  of  Technology 
Michael  Levine.  University  of  California.  San  Diego 
David  McClay.  Duke  University 

Course  Faculty 

Mariann  Bienz,  Medical  Research  Council,  Cambridge 
Marianne  Bronner-Fraser,  University  of  California,  Irvine 
R.  Andrew  Cameron,  California  Institute  of  Technology- 
Lois  Edgar,  University  of  Colorado 
Scott  E.  Fraser.  California  Institute  of  Technology 
Janet  Heasman.  University  of  Minnesota  School  of  Medicine 
Alexander  D.  Johnson.  University  of  California.  San  Francisco 
Andrew  McMahon,  Harvard  University 
Noriyuki  Satoh,  Kyoto  University,  Japan 
Christopher  C.  Wylie.  University  of  Minnesota  School  of  Medicine 

Teaching  Assistants 

Kristin  B.  Artinger,  University  of  California.  Irvine 
Andres  Collazo,  California  Institute  of  Technology 
Susan  Gray.  University  of  California.  San  Diego 
Carmen  Kirchhamer,  California  Institute  of  Technology 
Carole  LaBonne,  Harvard  University 
Catriona  Logan,  Duke  University 
Yusuke  Mankawa,  Kyoto  University,  Japan 
Jorg  Muller.  Medical  Research  Council.  Cambridge.  UK 
Jos  Raats,  The  Wellcome/CRC  Institute,  UK 
Kimberly  Van  Auken,  University  of  Colorado 
Robert  Zeller.  California  Institute  of  Technology 


R23 


R24   Annual  Report 


Administrator 

Jane  Rigg.  California  Institute  of  Technology 

Course  Assistants 
Courtney  Herman.  Wesleyan  University 
Aaron  Sloboda.  Skidmore  College 

Course  Coordinator 
Linda  Huffer,  Marine  Biological  Laboratory 

Students 

Catherine  Brennan,  University  of  Southern  California 

Melchiorre  Cervello,  Institute  di  biologia  dello  Sviluppo,  Italy 

Anna  Di  Gregorio,  Stazione  Zoologica  A.  Dohrn,  Italy 

Shigeki  Fujiwara,  Kochi  University,  Japan 

Martin  Garcia-Castro.  University  of  Cambridge,  UK 

Wendy  Gerber,  University  of  Texas,  Austin 

Xiaohua  Gong,  Scnpps  Research  Institute 

Maneesha  Inamdar,  Tata  Institute  of  Fundamental  Research,  India 

Boris  Kablar,  University  of  Pisa,  Italy 

Karla  Knobel,  University  of  Utah 

Matthew  Kourakis.  University  of  Chicago 

Julie  Kuhlman.  Cornell  Medical  College 

Elizabeth  Laxson.  University  of  Wisconsin.  Madison 

Radma  Mahmood,  Guy's  Hospital,  London,  UK 

Daniel  Martinez,  University  of  California,  Irvine 

Michael  McGrew,  Boston  University 

Ivan  Moskowitz,  University  of  Wisconsin,  Madison 

Craig  Nelson.  Harvard  University 

Annette  Neubuser,  Max-Planck-Institute  of  Immunobiology, 

Germany 

Sandra  Nicola,  Carol  Davila  University  of  Medicine.  Romania 
Lennart  Olsson,  Uppsala  University,  Sweden 
Kevin  Peterson,  University  of  California,  Los  Angeles 
Stephan  Schneider,  Max-Planck-lnstitute  for  Developmental 

Biology.  Germany 
Eliza  Shah,  Harvard  University 
Melissa  Shirley,  Case  Western  Reserve  University 

Microbial  Diversity  (June  12-July  28) 

Directors 

John  Breznak,  Michigan  State  University 
Martin  Dworkin,  University  of  Minnesota 

Faculty 

Yehuda  Cohen,  Hebrew  University,  Israel 

Jorg  Overmann,  Universitat  Oldenburg,  Germany 

Teaching  Assistants 
Joseph  P.  Calabrese,  West  Virginia  University 
Susan  Childers,  University  of  Connecticut 
John  D'Elia,  University  of  Illinois 

Magdalena  Martinez-Canamero.  University  of  Southern  California 
Michael  Renner,  Michigan  State  University 

Course  Coordinator 

Richard  M.  Behmlander.  Michigan  State  University 

Laboratory  Assistant 
Jessica  L.  Breznak 


Students 

Frederic  Ampe,  INSA.  France 

Michael  Cerio,  University  of  Connecticut 

Mark  DeSouza,  University  of  South  Carolina 

Ilka  Faath,  University  of  Bonn.  Germany 

Daniel  Ferber.  University  of  Illinois 

Georg  Jander.  Harvard  Medical  School 

Kathleen  Londry,  University  of  Oklahoma 

Sarah  McHatton.  University  of  California.  Davis 

Ute  Muh.  Philipps  Universitat  Marburg 

Julie  Olson,  University  of  North  Carolina.  Chapel  Hill 

Mary  Rothermich,  University  of  Massachusetts 

James  Scott,  Center  for  Great  Lakes  Studies 

Antonius  Suwanto,  Bogor  Agricultural  University,  Indonesia 

Debra  Tumbula,  University  of  Georgia 

Marc  van  der  Maarel,  University  of  Groningen,  Germany 

Paula  van  Schie,  Rutgers  L'niversity 

Madeline  Vargas,  University  of  Connecticut 

Shiri  Venezia,  Tel  Aviv  University,  Israel 

David  Westenberg.  Dartmouth  College 

Dinesh  Vernool,  Rutgers  University 

Neural  Systems  &  Behavior  (June  12-August  5) 

Directors 

Ronald  L.  Calabrese.  Emory  University 
Martha  Constantine-Paton,  Yale  University 

Faculty 

Larry  Abott,  Brandeis  University 

Alexander  Borst,  Max-Planck-lnstitiit  fur  Biologische,  Germany 

Thomas  Carew,  Yale  University 

Holly  Cline,  Cold  Spring  Harbor  Laboratory 

Elizabeth  Debski.  University  of  Kentucky 

Patsy  Dickinson,  Bowdoin  College 

Robert  Douglas,  University  of  British  Columbia,  Canada 

Douglas  L.  Falls,  Harvard  Medical  School 

Cole  Gilbert,  Cornell  University 

Richard  Levine.  University  of  Arizona 

Christine  Li,  Boston  University 

Robert  Malinow,  Cold  Spring  Harbor  Laboratory 

Pierre  Meyrand,  University  of  Bordeaux,  France 

Michael  Nusbaum.  University  of  Alabama,  Birmingham 

Bruce  O'Gara,  Barnard  College 

Martin  Shankland,  Harvard  Medical  School 

Darrell  R.  Stokes,  Emory  University 

Janis  Weeks.  University  of  Oregon 

Angela  Wenning,  LIniversitat  Konstanz,  Germany 

Sc/iolars- in- Residence 
Ron  Hoy.  Cornell  University 
Darcy  Kelley,  Columbia  University 
Eduardo  Macagno,  Columbia  University 

Teaching  Assistants 

Syd  Cash,  Columbia  University 

Melissa  Coleman.  LIniversity  of  Alabama,  Birmingham 
Jennifer  Cummings,  University  of  California.  San  Francisco 
John  F.  Dalton,  Emory  University 
Yang  Dan,  Columbia  University 

Juergen  Haag,  Max-Planck-lnstitiit  fur  Biologische  Kybernetik. 
Germany 


Kducational  Programs    R25 


Neal  A.  Hessler.  Cold  Spring  Harbor  Laboratory 

Zachary  Mainen,  University  of  California,  San  Diego 

Farzan  Nadim,  Emory  University 

Laurie  Nelson.  Boston  University 

Andrea  Novicki,  University  of  Oregon 

Oystein  Olsen.  Emory  University 

Glen  Prusky,  University  of  Lethbridge.  Canada 

James  Weimann,  Stanford  Llniversity 

Anne  West,  Harvard  Medical  School 

Sonia  Wine.  Cold  Spring  Harbor  Laboratory 

Students 

John  Allison,  Vanderbilt  Medical  School 
Curtis  Anderson,  Northern  Arizona  University 
Michael  Bern.  Harvard  University 
James  Contos.  University  of  California.  San  Diego 
Amanda  Edmiston.  University  of  Colorado 
Ruth  Empson.  University  of  Koln,  Germany 
Sarah  Farris,  University  of  Illinois.  Urbana-Champaign 
Lynn  Hodges.  University  of  California.  Los  Angeles 
Patricio  Huerta,  Brandeis  Llniversity 
Douglas  Ikelheimer.  Columbia  University 
Patricia  Janak,  University  of  California,  Berkeley 
Ole  Kjaerulff,  Copenhagen  University.  Denmark 
Dawn  Konrad,  University  of  Washington 
Mildred  Morales,  Albert  Einstein  College  of  Medicine 
Sowmyalakshmi  Rasika,  Rockefeller  University 
Kimberly  Scearce.  Columbia  University- 
Stefan  Schuster.  Max-Planck-Institiit  fur  Biologische  Kybernetik, 

Germany 

Karel  Svoboda.  Harvard  University 
James  Williams.  University  of  California.  San  Diego 
Bettina  Winckler.  Columbia  Llniversity 

Neurobiology  (June  12-AugustI3) 


Directors 

Leonard  Kaczmarek,  Yale  University  School  of  Medicine 
Irwin  Levitan.  Brandeis  University 

Course  Faculty 

Hannelore  Asmussen.  University  of  Virginia  Medical  School 

Gary  Banker.  University  of  Virginia  Medical  School 

Judith  Drazba.  National  Institutes  of  Health 

Keith  Elmslie.  Tulane  University  Medical  Center 

Richard  Horn,  Jefferson  Medical  College 

Stephen  Jones.  Case  Western  Reserve  University 

Bechara  Kachar.  National  Institutes  of  Health 

Julie  Kauer.  Duke  University  School  of  Medicine 

Richard  Kramer.  University  of  Miami 

Diane  Lipscombe.  Brown  University 

John  Marshall,  Yale  University  School  of  Medicine 

Carol  Ann  Mason.  Columbia  University  College  of  Physicians  & 

Surgeons 

Andrew  1.  Matus.  Fnedrich  Miescher  Institute.  Germany 
Sally  Moody,  The  George  Washington  University 
Angus  Nairn,  Rockefeller  University 
Marina  Picciotto.  The  Pasteur  Institute.  France 
Thomas  Reese.  National  Institutes  of  Health 
Peter  Reinhart.  Duke  University  Medical  Center 
Talvinder  Sihra.  Royal  Free  Hospital  School  of  Medicine,  UK 
Carolyn  Smith.  National  Institutes  of  Health 
Leslie  Vosshall.  Columbia  University 


Course  Assistant 

Ethan  Treistman.  University  of  North  Carolina.  Chapel  Hill 

Students 

Max  Boakye,  National  Institutes  of  Health 

Michale  Fee.  AT&T  Bell  Laboratories 

Jonathan  Gale.  Bristol  University,  UK 

Paul  Huynh.  Albert  Einstein  College  of  Medicine 

Peter  Kloppenburg.  ARLDN,  Tucson 

Carol  Koenigsberger.  Mayo  Graduate  School 

Zhixin  Lin.  Brown  University 

David  Molea.  University  of  Washington 

Kazunori  Nakajima.  RIKEN,  Japan 

Teresa  Nick,  Yale  LJniversity 

Christophe  Pouzat,  Laboratoire  de  Neurobiologie,  ENS.  France 

Claudia  Wiedemann.  Friedrich-Miescher  Institut.  Germany 

Physiology  (June  12- July  23) 


Director 

Mark  S.  Mooseker.  Yale  University 

Karen  Yeow,  University  of  Manitoba.  Canada 

Course  Faculty 
Steven  Block.  Princeton  University 
William  Busa.  Johns  Hopkins  University 
Richard  Cheney.  Yale  University 
Laura  Davis.  Duke  University  Medical  Center 
Stuart  Feinstein,  University  of  California.  Santa  Barbara 
Kathleen  Foltz,  Llniversity  of  California.  Santa  Barbara 
Mary  Lou  Guerinot.  Dartmouth  College 
Leah  Haimo.  Llniversity  of  California.  Riverside 
C.  Robertson  McClung,  Dartmouth  College 
Michael  Mendelsohn,  New  England  Medical  Center 
Robert  E.  Palazzo,  University  of  Kansas 
Roger  D.  Sloboda.  Dartmouth  College 
Margaret  A.  Titus.  Duke  University  Medical  Center 
Joseph  S.  Wolenski.  Yale  Llniversity 

Teaching  Assistants 
Ken  Belanger.  Duke  University 
Mary  Lynn  Benka.  Oregon  State  University 
Linda  Ferrans.  Johns  Hopkins  University 
Margaret  Kenna,  Duke  University 
Koen  Visscher,  Princeton  University 

Course  Assistants 

Caroline  Day,  Yale  Llniversity 

Raymond  Murray.  County  College  of  Morris 

Students 

ShaAvhree  Buckman.  Washington  University  School  Medicine 

Smaranda  Burlacu.  Howard  Hughes  Medical  Institute 

Laura  Cole.  Ohio  University 

Ana  DePina.  Dartmouth  College 

Prabha  Dias,  Scnpps  Research  Institute 

Suzanne  Gaudet.  Harvard  University 

Aaron  Granger.  Yale  School  of  Medicine 

Robert  Grant.  University  of  California.  San  Francisco 

Amanda  Hayward-Lester.  Texas  Tech  LJniversity 

Sher  Karki.  University  of  Pennsylvania 


R26   Annual  Report 


Matthew  Lee.  University  of  Southern  California 

Christi  Magrath.  Tulane  University  Medical  Center 

Suzanne  (Stovall)  Mann.  Bowman  Gray  School  of  Medicine 

Oana  Marcu,  University  of  Western  Ontario 

Sandra  Marques.  George  Washington  University 

Andre  Nussenzweig.  Memorial  Sloan-Kettering  Cancer  Center 

Adam  Pack.  SUNY  Health  Science  Center.  Syracuse 

Peter  Piepenhagen,  Stanford  University 

Manisha  Raje.  University  of  Kansas 

Samara  Reek-Peterson.  University  of  Pennsylvania 

Wendy  Reed,  Johns  Hopkins  University 

Frederick  Reitz,  University  of  Washington 

Fabrice  Roegiers.  Station  Zoologique,  France 

Sheree  Rybak,  Carnegie  Mellon  University 

Adrian  Salic,  Harvard  University 

Eric  Scarfone.  University  of  Montpellier 

Galen  Schneider.  University  of  North  Carolina 

Erik  Schultes,  University  of  California.  Los  Angeles 

Jennifer  Smith-Hall,  Indiana  University  School  of  Medicine 

Viktor  Stole.  Yale  University 

Luis  Vidali,  University  of  Massachusetts.  Amherst 

James  Walker.  University  of  Cambridge.  UK 

Jennifer  Waters,  University  of  North  Carolina 

Naoyuki  Yamamoto.  Nippon  Medical  School.  Japan 

Judith  Yanowitz,  Princeton  University 

Karen  Yeow.  University  of  Manitoba,  Canada 

Short  Courses 

Analytical  &  Quantitative  Light  Microscopy 

(May  12-20) 


Directors 

Greenfield  Sluder,  Worcester  Foundation  for  Experimental  Biology 
David  Wolf,  Worcester  Foundation  for  Experimental  Biology 

Course  Faculty  and  Lecturers 

William  B.  Amos,  Medical  Research  Council.  UK 
Richard  Cardullo,  University  of  California.  Riverside 
Frederick  Fay,  University  of  Massachusetts  Medical  School 
Shinya  Inoue,  Marine  Biological  Laboratory 
Edward  Salmon.  University  of  North  Carolina.  Chapel  Hill 
Randi  Silver,  Cornell  University  Medical  College 
Kenneth  Spring.  National  Institutes  of  Health 
D.  Lansing  Taylor,  Carnegie  Mellon  University 

Teaching  Assistants 

Christine  McKinnon,  Worcester  Foundation  for  Experimental 

Biology 

Frederick  Miller,  Worcester  Foundation  for  Experimental  Biology 
Elizabeth  Thompson,  Worcester  Foundation  for  Experimental 

Biology 

Students 

Eiki  Adachi.  Medical  Research  Council.  UK 

Paul  Bianco,  University  of  California.  Riverside 

Daniel  Cordova.  Marine  Biological  Laboratory 

Robert  Davis,  Worcester  Foundation  for  Experimental  Biology 

Ed  Devlin.  Hampden-Sydney  College 

Joseph  Di  Salvo.  University  of  North  Carolina,  Chapel  Hill 

Seth  Fraden.  Cornell  University  Medical  College 


Yue  Hu.  Worcester  Foundation  for  Experimental  Biology 

Marty  Jacobson,  National  Institutes  of  Health 

Maria  Jure-Kunkel,  Worcester  Foundation  for  Experimental 

Biology 

Linda  McMeekin,  Worcester  Foundation  for  Experimental  Biology 
Robert  Monette,  UMass  Medical  School 
Adam  Myerov.  Carnegie  Mellon  University 
Thomas  Pitta,  Rowland  Institute  for  Science 
Karl  Richter,  Marine  Biological  Laboratory 
Angeliki  Rigos.  Marine  Biological  Laboratory 
Lauren  Robertson,  Marine  Biological  Laboratory 
Laura  Romberg,  University  of  California,  San  Francisco 
Clifford  Slayman,  Yale  School  of  Medicine 
Robert  Specian,  Louisiana  State  University  Medical  Center 
Sandra  Spence,  Noran  Instruments 
Jennifer  Waters,  University  of  North  Carolina 
Simon  Watkins,  University  of  Pittsburgh 
James  Wilhelm.  University  of  California,  San  Francisco 
Ping  Xia.  National  Institutes  of  Health 
Yang  Zeng,  Worcester  Foundation  for  Experimental  Biology 


Fundamental  Issues  in  Vision  Research  (August 

14-27) 


Director 

David  S.  Papermaster,  University  of  Texas  Health  Science  Center, 
San  Antonio 

Course  Faculty 

Bob  Barlow,  Syracuse  University 

Robert  Baughman,  Harvard  Medical  School 

David  Beebe.  Uniformed  Services  University  of  the  Health  Sciences 

George  Benedek.  Massachusetts  Institute  of  Technology 

Eliot  Berson.  Massachusetts  Eye  and  Ear  Infirmary 

Richard  Brubaker,  Mayo  Clinic 

Connie  Cepko,  Harvard  Medical  School 

John  Dowling.  Harvard  University 

Judah  Folkman.  Harvard  Medical  School 

Daniel  Goodenough.  Harvard  Medical  School 

Robert  Grainger,  University  of  Virginia 

Paul  Hargrave.  University  of  Florida 

John  Hassel,  Eye  &  Ear  Institute  of  Pittsburgh 

Fielding  Hejtmancik,  National  Eye  Institute.  NIH 

Jonathan  Horton,  University  of  California 

Joseph  Horwitz,  Jules  Stein  Eye  Institute 

Douglas  Johnson,  Mayo  Clinic 

Ehud  Kaplan,  Rockefeller  University 

Carl  Kupfer,  National  Eye  Institute,  NIH 

Wen-Hwa  Lee,  University  of  Texas  Health  Science  Center 

Robert  Malchow,  University  of  Illinois 

Richard  Masland,  Harvard  Medical  School 

Anthony  Movshon,  New  York  University 

Jeremy  Nathans.  Johns  Hopkins  University  School  of  Medicine 

James  Nathanson.  Massachusetts  General  Hospital 

Eric  Newman,  University  of  Minnesota 

Krzysztof  Palczewski.  University  of  Washington 

Joram  Piatigorsky,  National  Eye  Institute.  NIH 

Haohua  Qian,  Harvard  University 

Robert  Rando.  Harvard  Medical  School 

Elio  Raviola,  Harvard  Medical  School 

Julia  Richards,  University  of  Michigan 

Barry  Rouse,  University  of  Tennessee 


Kducational  Programs   R27 


Dwight  Stambolian,  University  of  Pennsylvania 
Henry  Sun.  New  York  University  Medical  Center 
Charles  Zucker,  University  of  California.  San  Diego 

Lab  Coordinator 

Nancy  Ransom.  University  of  Texas  Health  Science  Center.  San 
Antonio 

Course  Administrator 

Carol  Masch.  University  of  Texas  Health  Science  Center,  San 
Antonio 

Course  Assistant 
Zera  Herskovits.  Yale  University 

Students 

Rent  Anderson,  Baylor  College  of  Medicine 
Brian  Brooks.  University  of  Pennsylvania 
Jinghua  Tsai  Chang.  Johns  Hopkins  University 
Victoria  Connaughton.  University  of  Texas.  Houston 
Dorette  Ellis-Ihidapo.  University  of  South  Florida 
Anna  Francesconi.  Dyson  Vision  Research  Institute 
Lin  Gan.  MD  Anderson  Cancer  Center 
Kim  Gottshall,  University  of  California,  San  Diego 
Abigail  Jensen.  University  College  London.  UK. 
Simon  John.  University  of  North  Carolina 
Brian  Link.  Oregon  Health  Sciences  University- 
Andrew  Magnet,  University  of  California,  San  Diego 
James  Marrs,  Stanford  University 
Judith  Mays.  MGH/Harvard  Medical  School 
Scott  McPherson,  University  of  Minnesota 
Linda  Musil,  Harvard  Medical  School 
Sandra  Ryeom.  Cornell  University  Medical  College 
Kathenne  Stnssel.  Massachusetts  General  Hospital 
Daniel  Sullivan.  National  Eye  Institute,  NIH 
John  Torseth,  University  of  Minnesota 
Robert  Wordinger.  University  of  North  Texas 

Medical  Informatics  (May  31 -June  7) 


Director 

Homer  Warner,  University  of  Utah  School  of  Medicine 

Course  Faculty 

Paul  Clayton.  Columbia  Presbyterian  Medical  Center 
Peter  Haug,  University  of  Utah  School  of  Medicine 
Donald  D.A.B.  Lindberg.  National  Library  of  Medicine 
David  Lipman,  National  Library1  of  Medicine 
Daniel  Masys,  Listerhill  Center  for  Biomedical  Communications 
Carol  Newton,  University  of  California  School  of  Medicine.  Los 

Angeles 

Catherine  Norton.  Marine  Biological  Laboratory 
David  Remsen,  Marine  Biological  Laboratory 
Rick  Rodgers.  National  Library  of  Medicine 
Robert  Sideh.  Columbia  Presbyterian  Medical  Center 

Lab  Coordinator 

Sylvia  Jessen.  University  of  Utah  School  of  Medicine 

Students 

Elizabeth  Alger,  UMDNJ-New  Jersey  Medical  School 
Jeroan  Allison.  University  of  Alabama.  Birmingham 


Michael  Altman.  Northwestern  University  Medical  School 

Ralph  Arcari.  University  of  Connecticut 

Donald  Boudreau.  Louisiana  State  University 

Lavonda  Broadnax,  DC  General  Hospital 

Holly  Buchanan,  Medical  College  of  Georgia 

William  Casey,  Nassau  County  Medical  Center 

Christine  Chastam-Warheit.  Medical  Center  of  Delaware 

William  Cordell,  Methodist  Hospital 

Stanley  Freedman.  Scripps  Clinic 

Sarah  Garrison,  Lincoln  Hospital.  Bronx,  NY 

Stephen  Grund.  Massachusetts  General  Hospital 

Beverly  Hill,  Indiana  University 

Frank  Keary,  U.S.  Department  of  State 

Emmet  Kenney,  Yale  Primary  Care  Research  Program 

Michele  Klein.  Children's  Hospital  of  Michigan 

Anthony  Kwak.  LIniversity  of  California,  Los  Angeles 

James  Legler.  University  Texas  Health  Science  Center 

Maria  Lenaz.  St.  Margaret's  Center  for  Women/Infants 

Elizabeth  Like,  Countway  Library/Harvard  Medical  School 

Catherine  MacLeod,  Rush  Medical  College 

Ellen  Marks,  Wayne  State  University 

Paul  McKinney.  Dallas  VAMC 

Leon  Moore,  University  of  Maryland,  Baltimore 

Kathleen  Oliver.  NIH  Library 

Miranda  Pao,  University  of  Michigan 

Gail  Persily,  University  of  California,  San  Francisco 

Valerie  Summers,  University  of  Kentucky 

Kenneth  Williams,  University  of  Massachusetts 

Methods  in  Computational  Neuroscience  (July 
31- August  27)  


Directors 

David  Kleinfeld.  AT&T  Bell  Laboratories 
David  W.  Tank,  AT&T  Bell  Laboratories 

Course  Faculty 

Lawrence  Abbott.  Brandeis  University 

Joseph  Atick.  Rockefeller  University 

Mark  Bear.  Brown  LIniversity 

William  Bialek,  NEC  Research  Institute. 

Ronald  Calabrese,  Emory  University 

Carmen  Canavier,  Baylor  College  of  Medicine 

Kerry  Delaney,  Simon  Fraser  University 

Rodney  Douglas,  MRC,  UK 

Bard  Ermentrout,  University  of  Pittsburgh 

Apostolos  Georgopoulos.  Veterans  Administration  Medical  Center 

Charles  Gray.  University  of  California,  Davis 

John  Hopfield,  California  Institute  of  Technology 

Christof  Koch,  California  Institute  of  Technology 

Nancy  Kopell,  Boston  University 

Stephen  Kosslyn,  Harvard  University 

Terry  Kovacs,  AT&T  Bell  Laboratories 

John  Lisman.  Brandeis  University 

Rodolfo  Llinas,  New  York  University  Medical  Center 

Kevin  Martin.  University  of  North  Carolina 

John  Maunsell.  Baylor  College  of  Medicine 

David  McCormick.  Yale  University  School  of  Medicine 

Alan  Peters.  Boston  University  School  of  Medicine 

John  Rinzel.  National  Institutes  of  Health 

Terrance  Sejnowski,  Salk  Institute 

H.  Sebastian  Seung,  AT&T  Bell  Laboratories 


R28  Annual  Report 


Shihab  Shamma.  University  of  Maryland 
Arthur  Sherman,  National  Institutes  of  Health 
Boris  Shraiman,  AT&T  Bell  Laboratories 
Karen  Sigvardt,  University  of  California,  Davis 
Frederick  Sigworth,  Yale  School  of  Medicine 
Haim  Sompolinsky,  Hebrew  University,  Israel 
Ben  Stowbridge,  AT&T  Bell  Laboratories 
Roger  Traub.  IBM  Corporation 
Michael  Vanier,  California  Institute  of  Technology 
John  White,  University  of  Iowa 
Matthew  Wilson,  University  of  Arizona 

Lab  Instructors 

Michael  Hines,  Duke  University  Medical  Center 
Roderick  Jensen.  Wesleyan  University 

Course  Assistant 
Joy  Langford 

Students 

John  Anderson,  University  of  Cambridge 
Joshua  Berke.  Harvard  University 
Dana  Cohen,  Hebrew  University.  Jerusalem 
Gennady  Cymbalyuk.  Institute  of  Mathematical  Problems  of 

Biology,  Russia 

Akira  Date,  Tokyo  University  of  Agriculture  &  Technology 
Christopher  deCharms.  University  of  California,  San  Francisco 
Opher  Donchin,  Hebrew  University,  Israel 
Stacia  Friedman-Hill,  University  of  California,  Davis 
Christopher  Hickie.  Yale  University  School  of  Medicine 
John  Kebabian,  Research  Biochemicals  International 
InSong  koh,  Boston  University 
Jiirgen  Kupper,  Ecole  Normale  Superieure,  France 
Peter  Latham,  University  of  Man  land 
Heather  Lennox,  Carleton  University,  Canada 
Anita  Luethi,  Brain  Research  Institute,  University  of  Zurich, 
Nicholas  Poolos,  Harvard  Medical  School 
Ramnarayan  Ramachandran,  Johns  Hopkins  University 
Lawrence  Saul,  Massachusetts  Institute  of  Technology 
Eric  Schwartz,  University  of  Chicago 
Akaysha  Tang.  Harvard  University 
Toby  Velte,  University  of  Minnesota 
Jun  Zhu,  University  of  Wisconsin  Medical  School 


Students 

Sandra  Baksi,  Environmental  Protection  Agency 
Peter  Bannerman.  Children's  Hospital  of  Philadelphia 
David  Cole,  Utah  State  University 
Robert  Donahue.  National  Institutes  of  Health 
Carol  Gregorio,  Scnpps  Research  Institute 
David  Hessinger,  Loma  Linda  Llniversity  School  of  Medicine 
David  Leaf.  Western  Washington  University 
Terry  McCann.  Babraham  Institute 
Lesley  Mills,  University  of  Rhode  Island 
Alexander  Minin,  Protein  Research  Institute,  Russia 
Stephen  Pasquale,  Washington  University 
Ulrich  Schaible,  Washington  Llniversity 
William  Schuyler,  Atlanta  Veterans  Affairs  Medical  Center 
Ching-hwa  Sung.  Johns  Hopkins  University  School  of  Medicine 

Rapid  Measurement  of  Neurotransmitter 

Signals  in  the  Central  Nervous  System  (Session 
I:  August  19-22;  Session  II:  August  24-28) 

Director 
Greg  Gerhardt.  University  of  Colorado  Health  Science  Center 

Course  Faculty 

Kate  Bowenkamp,  University  of  Colorado  Health  Sciences  Center 
Michael  Doherty,  Douglas  Hospital  Research  Center 
Marilyn  Friedemann,  Llniversity  of  Colorado  Health  Sciences 

Center 

Don  Gash.  Lexington.  KY 

Alain  Gratton.  Douglas  Hospital  Research  Center 
Harold  Haul,  Medical  Systems  Corporation 
Alex  Hoffman,  University  of  Colorado  Health  Sciences  Center 
Michael  Palmer.  University  of  Colorado  Health  Sciences  Center 
Michael  Parnsh,  University  of  Colorado  Health  Sciences  Center 
William  Proctor,  Llniversity  of  Colorado  Health  Sciences  Center 
Scott  Robinson,  Llniversity  of  Colorado  Health  Sciences  Center 
Steve  Robinson,  Llniversity  of  Colorado  Health  Sciences  Center 
Craig  Van  Horn,  Brigham  &  Women's  Hospital 

Course  Coordinator 
Laura  Lee  Lamothe,  University  of  Colorado  Health  Sciences  Center 


Microinjection  Techniques  (May  24-31) 


Director 
Robert  B.  Silver,  Cornell  University 

Course  Faculty 

Suzanne  Chandler,  Cornell  University 
Karen  Kindle,  Cornell  University 
Douglas  Kline,  Kent  State  University 
Paul  McNeil.  The  Medical  College  of  Georgia 
Jeb  Oblek,  Cornell  University 
Eric  Shelden.  University  of  Connecticut 

Course  Assistants 

Gwendolyn  Jeun,  Cornell  Llniversity 
Lisa  Mehlmann,  Kent  State  University 


Students,  Session  I 

Rodrigo  Andrade,  St.  Louis  Llniversity  School  of  Medicine 
Karen  Bach,  University  of  Wisconsin,  Madison 
Tilmann  Brotz,  Fnedrich-Miescher-Laboratory,  Germany 
Dipanjan  Chakravarty.  Southern  Illinois  Llniversity  School  of 

Medicine 

Deborah  Cory-Slechta,  Llniversity  of  Rochester 
Subimal  Datta,  Harvard  Medical  School 
Lyn  Daws,  University  of  Texas  Health  Science  Center 
Bromheld  Hine,  University  of  Puerto  Rico 
Susan  Hochstenbach,  University  of  Western  Ontario,  Canada 
Rodrigo  Iturriaga,  Catholic  Llniversity  of  Chile,  Chile 
Sathasiva  Kandasamy,  Armed  Forces  Radiobiology  Research 

Institute 

Prakash  Kara.  University  of  Alabama,  Birmingham 
Sergei  Kirov.  National  Institutes  of  Health  Gerontology  Research 

Center 

Gordon  Mitchell,  University  of  Wisconsin 
David  Mogul,  Northwestern  Llniversity 


Educational  Programs   R29 


Shoji  Nagatani.  Nagoya  University.  Japan 

Vladimir  Parpura.  Iowa  State  University 

Patricia  Rosas-Arellano,  University  of  Western  Ontario.  Canada 

Philip  Shea.  Geo  Centers 

Dennison  Smith,  Oherlin  College 

Philip  Starr.  Children's  Hospital.  Boston 

Vishnu  Suppiramaniam.  Tuskegee  University 

Lidia  Szczupak,  University  of  California.  San  Diego 

Fletcher  Wason.  Cambridge  Neuroscience.  Inc. 

Christina  Zuch.  University  of  Rochester 

Students.  Session  II 

Sonia  Connaughton.  Cambridge  Neuroscience 

Carlos  Cream,  Dartmouth  Medical  School 

Adrian  Dunn,  Louisiana  State  University  Medical  Center, 

Shreveport 

Daniel  Feller,  VA  Medical  Center.  Portland 
Dwayne  Godwin,  State  University  of  New  York.  Stony  Brook 
Laszlo  Harsing.  Institute  for  Drug  Research.  Budapest.  Hungary 
Bettye  Hollins.  Medical  College  of  Georgia 
Michael  Kramer.  Pennsylvania  Hospital 
Lauren  Liets,  University  of  California,  Davis 
Anthony  Lombardino,  Rockefeller  University 
Janea  Mack,  Meharry  Medical  College 
Yong-Gou  Park,  Yonsei  University  College  of  Medicine,  Seoul. 

Korea 

David  Smith,  University  of  Pennsylvania 
Andrew  Spielman,  New  York  University 
Robert  Stingele.  Johns  Hopkins  University 
Anthony  Stretton,  University  of  Wisconsin,  Madison 
Artur  Swiergiel,  LSLI  Medical  Center 
Chuanyao  Tong.  Bowman  Gray  School  of  Medicine 
Timothy  Turner,  Tufts  University 
Kaido  Viik,  Burroughs  Wellcome 
Carol  Watkins.  Massachusetts  Institute  of  Technology 
Yun  Zagvazdin,  University  of  Tennessee 

Optical  Microscopy  and  Imaging  in  the 
Biomedical  Sciences  (October  15-22) 


Director 

Colin  S.  Izzard.  State 


Universitv  of  New  York.  Albany 


Course  Faculty  and  Lecturers 
Steven  M.  Block.  Rowland  Institute  for  Science 
Gary  R.  Bright,  Case  Western  Reserve  University 
Fredric  S.  Fay,  University  of  Massachusetts  Medical  School 
Robert  Hard,  State  University  of  New  York.  Buffalo 
Shinya  Inoue,  Marine  Biological  Laboratory 
Ernst  Keller.  Carl  Zeiss.  Inc. 
Greta  M.  Lee.  University  of  North  Carolina 
John  M.  Murray.  University  of  Pennsylvania 
Kenneth  R.  Spring.  National  Institutes  of  Health,  NHLBI 

Teaching  Assistants 

Joseph  A.  DePasquale.  New  York  State  Department  of  Health 
Loretta  M.  Memmo.  State  University  of  New  York,  Albany- 
Gerald  Rupp.  State  LIniversity  of  New  York.  Buffalo 


Students 

Reiko  Arimoto,  Nikon  Corporation 


Gary  Bassell,  Harvard  Medical  School 

Hilary  Beggs.  University  of  North  Carolina.  Chapel  Hill 

Judith  Berman.  University  of  Minnesota 

Solange  Brown.  Harvard  Medical  School 

Kirk  Czymmek,  DuPont  Company 

Fiona  Doetsch.  Rockefeller  University 

Daniel  Erb.  Miami  Project,  LIniversity  of  Miami 

Michael  Esterman,  Lilly  Research  Labs 

James  Gordon,  Poloroid  Corporation 

Joseph  Italiano.  Florida  State  University 

Mane-Helene  Jouvin,  National  Institutes  of  Health 

Li  Ma,  Columbia  University 

Fraser  McDonald,  University  of  London,  UK 

Gero  Miesenboeck,  Sloan-Kettering  Institute 

Luiz  Monteiro-Leal.  Federal  University  of  Rio  de  Janeiro 

Andrew  Nechkin,  The  Johns  Hopkins  University 

Robert  Scott,  Georgia  Institute  of  Technology 

Daniella  Steel.  McGill  University,  Canada 

Edith  Suss-Toby,  National  Institutes  of  Health 

Keisuke  Suzuki,  Olympus  Optical  Co.,  Ltd. 

Douglas  Taatjes,  University  of  Vermont 

Tomomi  Tani.  University  of  Tokyo,  Japan 

Barry  Ticho,  Children's  Hospital.  Boston 

Workshop  on  Molecular  Evolution 

(August  7-19) 

Director 

Mitchell  L.  Sogin.  Marine  Biological  Laboratory 

Course  Faculty 

Dan  Davison,  University  of  Houston 
Joseph  Felsenstein,  University  of  Washington 
Walter  Fitch,  University  of  California.  Irvine 
George  Fox,  University  of  Houston 
Robin  Guttell.  University  of  Colorado 
David  Hillis.  University  of  Texas 
Rick  Hudson,  University  of  California,  Irvine 
Laura  Landweber,  Harvard  University 
David  Maddison.  LIniversity  of  Arizona 
Roger  Milkman.  University  of  Iowa 
Catherine  Norton.  Marine  Biological  Laboratory 
Stephen  O'Brien.  National  Cancer  Institute 
Gary  Olsen.  University  of  Illinois 
Norman  Pace.  Indiana  University- 
Margaret  Riley,  Yale  University 
Monica  Riley.  Marine  Biological  Laboratory 
Terry-  Speed,  University  of  California.  Berkeley 
David  Swofford.  Smithsonian  Institution 
Peter  Waddell,  Massey  University 
Bruce  Walsh,  University  of  Arizona 

Teaching  Assistant 
Saira  Mian.  University  of  California.  Santa  Cruz 

Course  Consultant 

Brendan  Reilly.  Software  Editing  Corporation 

Students 

Joe  Bernardo.  University  of  Texas,  Austin 
Robert  Browne.  Wake  Forest  University 


R30  Annual  Report 


John  Burke,  University  of  Houston 

Dana  Campbell,  Harvard  University 

Carol  Casavant,  University  of  Idaho 

Joby  Chesnick,  Lafayette  College 

Mary  Crabtree,  Centers  for  Disease  Control.  Colorado 

Richard  Davis.  San  Francisco  State  University 

Alison  Davis.  University  of  Southern  California 

Art  Edison,  University  of  Wisconsin,  Madison 

Jonathan  Eisen.  Stanford  University 

Anjay  Elzanowski.  National  Center  for  Biotechnology  Information 

Melinda  Pagan.  Stanford  University 

Marcia  Fisher,  Cornell  University 

Paul  Flook,  Basel  University.  Switzerland 

James  Fogleman.  University  of  Denver 

Silvana  Gaudieri,  University  of  Western  Australia.  Australia 

Angela  Gawthrop,  University  of  Glasgow,  UK. 

Yvonne  Gra'ser,  Institute  of  Microbiology,  Berlin,  Germany 

Bill  Hahn,  Smithsonian  Institution 

Aaron  Halpern,  Los  Alamos  National  Laboratory 

Healy  Hamilton,  University  of  California,  Berkeley 

Phillip  Harris,  Oregon  State  University 

Daniel  Haydon,  LIniversity  of  Oxford.  UK 

Catherine  Jones,  Oxford  University,  UK 

Brian  Kinkle.  University  of  Cincinnati 

Anne-Mette  Krabbe-Pedersen.  University  of  Aarhus,  Germany 

Armand  Leroi.  Albert  Einstein  College  of  Medicine 

Marc  Lipsitch,  University  of  Oxford,  UK 

Stephen  Lougheed.  Queen's  University,  Canada 

Russell  Malmberg.  University  of  Georgia 

Magdalena  Martinez-Canamero.  University  of  Granada.  Spain 


Lynn  Messinger,  University  of  Wisconsin 

Weiland  Meyer,  Duke  University  Medical  Center 

Andrew  Mitchell,  University  of  Maryland 

Jon  Norenburg,  Smithsonian  Institution 

Jane  Norman.  University  of  Illinois,  Chicago 

Diana  Northup,  University  of  New  Mexico 

Fred  Opperdoes.  ICP-Brussels.  Belgium 

Tom  Quinn.  University  of  Denver 

Allen  Rogerson,  St.  Lawrence  University 

Shane  Sarver,  University  of  Miami/RSMAS 

Christian  Schlotterer,  Zoologisches  Institut.  Germany 

Karl  Schmid,  University  of  Munich,  Germany 

Barathi  Sethuraman.  University  of  California,  Berkeley 

Andrew  Shedlock,  University  of  Washington 

Gerald  Shields.  University  of  Alaska,  Fairbanks 

Janet  Siefert,  University  of  Houston 

Joana  Silva,  University  of  Arizona 

Pedro  Silva,  University  of  Lisbon,  Uppsala.  Sweden 

Felipe  Soto-Adames.  University  of  Illinois,  Urbana-Champaign 

Birgit  Stache,  University  of  California.  Santa  Cruz 

Eleanor  Steinberg.  University  of  Washington 

Randall  Terry.  University  of  Wyoming 

Miranda  von  Dornum,  Harvard  University 

Mary  White,  Southeastern  Louisiana  University 

Birgitta  Winnepenninckx.  University  of  Antwerp.  Belgium 

Grace  Wyngaard.  James  Madison  University 

Anne  Voder,  Harvard  University 

Sarah  Zehr,  Harvard  University 

Giuseppe  Zuccarello,  University  of  California,  Santa  Cruz 


Summer  Research 


Principal  Investigators 

Alkon.  Daniel  L..  National  Institutes  of  Health 
Allen.  Nina  S..  Wake  Forest  University 
Armstrong.  Clay.  University  of  Pennsylvania 
Armstrong.  Peter  B..  University  of  California.  Davis 
Augustine.  George  J..  Duke  University  Medical  Center 

Barlow.  Jr..  Robert  B..  Syracuse  University  Institute  for  Sensory 

Research 

Bearer.  Elaine.  Brown  University 
Beauge,  Luis.  Institute  M.  y  M.  Ferreyra.  Argentina 
Bennett.  Michael  V.  L..  Albert  Einstein  College  of  Medicine 
Berbenan.  Graciela  Elso  de.  Institute  M.  y  M.  Ferreyra.  Argentina 
Berlin.  Joshua.  Graduate  Hospital 
Bloom.  George  S.,  The  University  of  Texas  Southwestern  Medical 

Center.  Dallas 

Bodznick.  David.  Wesleyan  University 
Borgese.  Thomas  A..  Lehman  College.  CUNY 
Boron.  Walter  F..  Yale  University  Medical  School 
Brady.  Scott  T.,  The  University  of  Texas  Southwestern  Medical 

Center.  Dallas 

Burdick.  Carolyn  J.,  Brooklyn  College.  CUNY 
Burger.  Max  M..  Friedrich  Miescher  Institut.  Switzerland 

Cardell.  Robert  R..  University  of  Cincinnati 

Chaet.  A.  B..  University  of  West  Florida 

Chang.  Donald  C.  Hong  Kong  University  of  Science  &  Technology. 

Hong  Kong 

Chappel,  Richard  L..  Hunter  College.  CUNY 
Charlton.  Milton.  University  of  Toronto.  Canada 
Clay.  John.  National  Institutes  of  Health 
Cohen.  Lawrence  B.,  Yale  University  School  of  Medicine 
Cohen.  William  D.,  Hunter  College,  CUNY 
Crutcher.  Keith  A..  University  of  Cincinnati 

D'Avanzo.  Charlene.  Hampshire  College 

Dan.  Yang.  Columbia  University 

Davis.  Graeme  W..  University  of  Massachusetts 

De  Weer,  Paul.  Universiu  of  Pennsylvania  School  of  Medicine 

Di  Polo.  Reinaldo.  IVIC,  Venezuela 

Ehrlich.  Barbara.  University  of  Connecticut 

Fay.  Richard,  Parmly  Hearing  Institute 

Feng.  Guoping.  State  University  of  New  York.  Buffalo 


Finch.  Elizabeth  Ann.  Duke  University  Medical  Center 
Fishman,  Harvey  M.,  The  University  of  Texas  Medical  Branch, 
Galveston 

Gadsby.  David.  The  Rockefeller  University 

Gainer,  Harold,  National  Institutes  of  Health 

Garcia-Blanco,  Mariano  A.,  Duke  University  Medical  Center 

Garrick.  Rita  Anne,  Fordham  University  College.  Lincoln  Center 

Giuditta,  Antonio.  University  of  Naples.  Italy 

Goldman.  Robert  D..  Northwestern  University  Medical  School 

Gould,  Robert,  New  York  State  Institute  of  Basic  Research  in 

Developmental  Disabilities 
Gyoeva,  Fatima,  Russian  Academy  of  Sciences,  Institute  of  Protein 

Research 

Haimo,  Leah,  University  of  California.  Riverside 

Hall,  Zach  W.,  University  of  California  School  of  Medicine.  San 

Francisco 

Halvorson.  Harlyn  O..  University  of  Massachusetts,  Dartmouth 
Hardin,  John,  Medical  College  of  Georgia 
Henry.  Jonathan.  University  of  Illinois 

Highstein.  Steven  M..  Washington  University  School  of  Medicine 
Holmgren.  Miguel.  The  Chicago  Medical  School 
Holz,  IV,  George  G.,  Harvard  Medical  School.  Massachusetts  General 

Hospital 

Hoskin,  Francis  C.  G..  Illinois  Institute  of  Technology 
Humphreys.  Tom,  University  of  Hawaii 

Jaffe.  Laurinda.  University  of  Connecticut  Health  Center 
Johnston,  Daniel.  Baylor  College  of  Medicine 
Johnston,  Rebecca,  University  of  Arizona 

Kaneshiro,  Edna.  University  of  Cincinnati 
Kaplan.  Barry,  Western  Psychiatric  Institute  &  Clinic 
Kuhns,  William,  The  Hospital  for  Sick  Children,  Canada 
Kumar,  Ajit,  George  Washington  University  School  of  Medicine 
Kuznetsov,  Sergei.  University  of  Rostock.  Germany 

Landowne,  David.  University  of  Miami 

Lane,  Mary  C.,  University  of  California,  Berkeley 

Langford.  George.  Dartmouth  College 

Laskin,  Jeffrey.  University  of  Medicine  and  Dentistry  of  New  Jersey 

Laufer.  Hans.  University  of  Connecticut 

Lemon.  William  C.,  University  of  Arizona 

Lester.  Roger.  University  of  Arkansas  Medical  Sciences 

Lipicky,  Raymond  J.,  Food  and  Drug  Administration 

Llinas.  Rodolfo  R..  New  York  L'niversity  Medical  Center 


R3I 


R32   Annual  Report 


Principal  Invexligator  Rohcri  Barlow.  Jr.  (Syracuse  University). 


Makarenko.  Vladimir.  Russian  Academy  of  Sciences,  Russia 
Malchow,  Robert  Paul.  University  of  Illinois  College  of  Medicine 
Maranto.  Anthony.  St.  Elizabeth's  Hospital 
Martindale.  Mark.  University  of  Chicago 
Metuzals.  Janis.  University  of  Ottawa,  Canada 
Miyakawa.  Hiroyoshi.  Tokyo  College  of  Pharmacy 
Moore,  Lisa,  Albert  Einstein  College  of  Medicine 
Moorman,  Stephen,  UNT  Health  Science  Center.  Fort  Worth 
Moreno,  Alonso  P..  State  University  of  New  York,  Buffalo 
Murray,  Andrew.  University  of  California.  San  Francisco 

Nasi,  Enrico,  Boston  University  School  of  Medicine 
Nierhaus,  Knud,  Max  Planck  Institute  for  Molecular  Genetics. 
Germany 

Palazzo,  Robert  E..  University  of  Kansas 

Pant.  Harish.  National  Institutes  of  Health 

Pozzo-Miller.  Lucas  D.,  Roche  Institute  of  Molecular  Biology 

Qian.  Haohua.  Harvard  University 

Quigley,  James  P..  State  L'niversity  of  New  York,  Stony  Brook 

Rakowski.  Robert  F.,  University  of  Health  Sciences/The  Chicago 

Medical  School 

Rasmussen.  Howard.  College  of  Georgia 
Ratner.  Nancy,' University  of  Cincinnati 
Reese,  Thomas  S..  National  Institutes  of  Health 
Rieder,  Conlv  L..  Wadsvvorth  Center  for  Labs  &  Research 


Ripps.  Harris.  University  of  Illinois  College  of  Medicine 
Rome.  Lawrence.  University  of  Pennsylvania 
Ross.  William,  New  York  Medical  College 
Ruderman,  Joan  V.,  Harvard  Medical  School 
Russell.  John  M.,  Medical  College  of  Pennsylvania 

Saitoh.  Setsuo.  Hokkaido  Central  Fisheries  Experimental  Station 

Salmon,  Edward,  University  of  North  Carolina.  Chapel  Hill 

Sarda,  Rafael,  Consejo  Superior  Investigaciones  Cientificas.  Spain 

Schweizer,  Felix  E.,  Duke  University 

Sharp.  Andrew  A.,  Brandeis  University 

Silver,  Robert  B.,  Cornell  University 

Sloboda,  Roger  D..  Dartmouth  College 

Sluder,  Greenfield.  Worcester  Foundation  for  Experimental  Biology 

Sobel,  Erik  C.,  AT&T  Bell  Laboratories,  Inc. 

Steinacker,  Antoinette.  University  of  Puerto  Rico  Medical  Sciences 

Sugimori.  Mutuysuki,  New  York  University  Medical  Center 

Swenson,  Katherine  I.,  Duke  LIniversity  Medical  Center 

Telzer,  Bruce.  Pomona  College 
Trinkaus.  John  P..  Yale  University 
Troll,  Walter.  New  York  University  Medical  Center 
Tytell.  Michael.  Bowman  Gray  School  of  Medicine  of  Wake  Forest 
University 

Weiss.  Dieter.  University  of  Rostock,  Germany 

Wylie.  Douglas  R.,  New  York  University  Medical  Center 

Yamoah,  Ebenezer,  University  of  Texas  Medical  School 
Yoshioka,  Tohru.  Waseda  University,  Japan 

Zago,  Cristina,  National  Research  Council 

Zignian,  Seymour,  University  of  Rochester  School  of  Medicine  and 

Dentistry 

Zottoli,  Steven  J..  Williams  College 
Zuazago  de  Ortiz,  Conchita,  University  of  Puerto  Rico 


Other  Research  Personnel 

Altamirano.  Anibal  A.,  Medical  College  of  Pennsylvania 
Alvarez.  Guillermo,  University  of  Seville,  Spain 
Andreu-Sanchez.  Maria  E.,  University  of  Alicante,  Spain 
Andrews,  S.  Brian,  National  Institutes  of  Health 
Araneda,  Ricardo,  Albert  Einstein  College  of  Medicine 
Armstrong.  Clara.  University  of  Pennsylvania 

Backskai,  Brian,  University  of  California,  La  Jolla 

Bau,  Mu-Yeh,  University  of  Texas  Southwestern  Medical  Center 

Benech,  Juan  Claudio,  Instituto  de  Investigaciones  Biologicas. 

LIruguay 

Bittner,  George  D.,  University  of  Texas,  Austin 
Boakye.  Maxwell,  National  Institutes  of  Health 
Boyle,  Richard,  Oregon  Health  Science  University 
Breitwieser,  Gerda  E.,  Johns  Hopkins  School  of  Medicine 
Brown.  Joel  E..  Albert  Einstein  College  of  Medicine 
Burns,  Marie.  Duke  LIniversity 
Busch.  Karina.  Miami  University.  Ohio 

Calhoun.  Benjamin.  Medical  College  of  Georgia 
Callaway.  Joseph,  New  York  Medical  College 
Cameron,  Mark,  Harvey  Mudd  College 
Carroll,  David  J..  University  of  Connecticut  Health  Center 
Chen,  Barrv,  National  Institutes  of  Health 


Summer  Research  Programs   R33 


Chludzinski.  John.  National  Institutes  of  Health 
Christofi.  Geri.  Universit>  College  London 
Chun.  Jong  Tai.  University  of  Pittsburgh 
Cohen.  Avrum.  Yale  University 
Cohen.  Darien.  Dartmouth  College 
Collin.  Carlos.  National  Institutes  of  Health 
Coughlin.  David.  University  of  Pennsylvania 
Crispino.  Marianna.  University  of  Naples 

Dadacay.  Alma  Villa,  Hunter  College 
Dawson.  Timothy  Charles.  University  of  Kansas 
DeBello.  William.  Duke  University 

Dehnbostel.  Denise.  UNT  Health  Science  Center.  Fort  Worth 
Dodge.  Frederick,  Syracuse  University- 
Dodge.  Susan.  University  of  North  Carolina.  Chapel  Hill 
Donaghy.  Brenda.  University  of  Iowa 
Dopp.  Hike.  University  of  Rostock,  Germany 
Drazba,  Judy.  National  Institutes  of  Health 
Dresbach.  Thomas.  Max  Planck  Institute  for  Brain  Research. 
Germany 

Eddleman.  Chns.  University  of  Texas.  Austin 
Eilers.  Jems.  Universitat  des  Saarlandes.  Germany 
Ellis-Thipado.  Doretta.  University  of  South  Florida 
Escalona  de  Motta.  Gladys.  University  of  Puerto  Rico 

Favit.  Antonella.  National  Institutes  of  Health 
Fernandez-Busquets,  Xavier,  Frednch  Miescher  Institut.  Switzerland 
Flucher.  Bernhard.  National  Institutes  of  Health 

Galbraith.  James.  University  of  California,  San  Diego 

Gallant.  Paul  E.,  National  Institutes  of  Health 

Gerosa.  Daniela,  Friedrich  Miescher  Institut,  Switzerland 

Giraud.  Lisette.  University  of  Puerto  Rico 

Godell,  Chris,  University  of  Texas.  Galveston 

Goldman.  Anne  E..  Northwestern  University  Medical  School 

Gomez.  Maria.  Boston  University  School  of  Medicine 

Grant.  Philip.  National  Institutes  of  Health 

Greenfield.  Benjamin.  Brown  University 

Guerra.  Ernesto.  American  Psychological  Association 

Guillfoyle.  Kerry  Jo..  Hampshire  College 

Hammes.  Michelle.  Hampshire  College 

Harris-Collazo,  Raul,  University  of  California,  San  Diego 

Heck.  Diane.  University  of  Medicine  and  Dentistry  of  New  Jersey 

Hershko,  Avram.  Israel  Institute  of  Technology,  Israel 

Hershko.  Judith.  Israel  Institute  of  Technology,  Israel 

Hogan.  Emilia  M.,  Yale  University  Medical  School 

Horoyan,  Marianne.  National  Institutes  of  Health 

Huerta.  Julio.  Hunter  College,  CUNY 

Hunt.  James.  Duke  University 

Jarchow.  Janina,  Friedrich  Miescher  Institut.  Switzerland 
Johnson.  Donald.  Lehman  College.  CUNY 
Jones.  James.  American  Psychological  Association 
Jones.  Kevin.  Duke  University 
Jue,  Renata,  Dartmouth  College 

Kaech.  Stefanie.  National  Institutes  of  Health 

Kaftan.  Edward.  University  of  Connecticut  Health  Center 

Kamino.  Kohtaro.  Tokyo  Medical  and  Dental  University  School  of 

Medicine,  Japan 

Kaplan.  Ehud.  Rockefeller  University 
Kaplan,  Ilene  M..  Union  College 


Principal  Investigator  Sergei  Kiiznetsov  (University  of  Rostock). 


Kawahara.  Shigenori.  University  of  Tokyo.  Japan 

Kelly.  Mary.  State  University  of  New  York  Health  Science  Center 

Kelman.  Elise  S..  Montefiore  Medical  Center 

Keynan,  Alex,  Hebrew  University.  Israel 

Khan.  Sohaib.  University  of  Cincinnati 

Khodakah.  Kamran.  University  of  Pennsylvania 

Khuon.  Satya.  Northwestern  University  Medical  School 

Kinno.  Yutaka,  University  of  Tokyo,  Japan 

Knudsen,  Knud  D..  Food  and  Drug  Administration 

Konnerth.  Arthur,  Universitat  des  Saarlandes,  Germany 

Kudo,  Yoshihisa,  Mitsubishi  Kasei  Life  Science  Institute,  Japan 

Lahav.  Shirley.  Israel  Institute  of  Technology,  Israel 

Landau.  Matthew,  Stockton  State  College 

Lang.  Eric,  New  York  University  Medical  Center 

Lasser-Ross,  Nechama.  New  York  Medical  College 

Liu.  Lei.  University  of  Connecticut 

Lopez,  Veronica.  University  of  California,  Berkeley 

Lu.  Jin,  University  of  Texas  Medical  Branch 

Lyddone.  Clay.  University  of  Kansas 

Martinez.  Jr..  Joe  L..  University  of  California.  Berkeley 

Master,  Viraj,  University  of  Chicago 

Matheisz.  Katherine.  Syracuse  University 

McNeil.  Paul.  Georgia  Medical  College 

Melchior,  Ralph,  University  of  California,  Davis 

Meng,  Chunling.  Hong  Kong  University  of  Science  and  Technology. 

Hong  Kong 

Mensinger,  Allen  F.,  Washington  University 
Minkoff,  Charles,  Duke  University  Medical  Center 
Miyaguchi,  Katsuyuki,  National  Institutes  of  Health 
Montgomery.  John  C.  University  of  Auckland.  New  Zealand 
Moreira,  Jorge  E..  National  Institutes  of  Health 

Necela.  Brian,  University  of  West  Florida 

Ogielski.  Andrew,  Bell  Communications  Research 
Olds.  James  L..  National  Institutes  of  Health 

Palos.  Teresa,  University  of  California.  Los  Angeles 

Parker.  Libbie  Lynn,  Duke  University 

Passaglia.  Christopher,  Syracuse  University 

Perez.  Reynaldo.  University  of  Puerto  Rico 

Pfister,  K.  Kevin,  University  of  Virginia  School  of  Medicine 


R34   Annual  Report 


Poenie,  Martin,  University  of  Texas,  Austin 

Porcello,  Darrell  M.,  Bowdoin  College 

Powell,  Angela,  Spelman  College 

Powell,  Angella  M.,  Spelman  College 

Powers,  Maureen,  Vanderbilt  University 

Pumplin,  David  W.,  University  of  Maryland  School  of  Medicine 

Qu,  Xiangdong,  New  York  University  Medical  Center 
Quinn,  Kerry.  University  of  Connecticut  Health  Center 

Rabbitt,  Richard,  University  of  Utah 

Radominska,  Anna,  University  of  Arkansas  for  Medical  Sciences 

Rayos,  Nancy,  Hunter  College 

Reed.  Robyn,  Wake  Forest  University 

Reyes,  Rosario,  University  of  Oregon 

Romero,  Michael,  University  of  Washington 

Rotllant.  Guiomar,  Institute  de  Ciencias  del  Mar,  Spain 

Rule,  Randall,  University  of  California,  Berkeley 

Russell.  Joshua  C,  Medical  College  of  Pennsylvania 

Sakakibara,  Manabu,  Tokai  University,  Japan 

Saver,  Michelle,  University  of  Calgary,  Canada 

Schauer,  Stephen,  Franklin  and  Marshall  College 

Schiffmann.  Dietmar,  University  of  Rostock,  Germany 

Shih,  John,  California  Institute  of  Technology 

Shrier.  Alvin,  McGill  University 

Sparks,  Christopher.  University  of  North  Texas 

Spencer,  C.  Ian,  Graduate  Hospital 

Srimal,  Subita.  Indian  Institute  of  Science,  India 

Sterkenburg,  Cynthia,  University  of  Texas.  Austin 

Stockbridge,  Norman,  Food  and  Drug  Administration 

Sudakin,  Valery,  Israel  Institute  of  Technology.  Israel 

Swank,  Douglas,  University  of  Pennsylvania  School  of  Medicine 

Syme,  Doug,  University  of  Pennsylvania 

Tabares,  Lucia,  University  of  Seville  School  of  Medicine.  Spain 
Takac,  Peter,  Institute  of  Zoology  &  Ecozoology,  Slovak  Republic 
Takahashi,  Megumi,  Yokohama  City  University  School  of  Medicine, 

Japan 

Terasaki,  Mark.  National  Institutes  of  Health 
Todora,  Michael,  University  of  Texas,  Austin 
Treat,  Susan,  University  of  Arkansas  for  Medical  Sciences 

Umbach,  Joy,  University  of  California,  Los  Angeles 

Valdes,  Lexia,  Barry  University 

Vargas,  Fernando,  Food  and  Drug  Administration 

Vogel,  Jackie,  University  of  Kansas 

Vojta,  Beth,  University  of  Pittsburgh 

Walton,  Peggy  L.,  Washington  University  School  of  Medicine 
Warner,  Anne,  Dartmouth  College 

Xie.  Ping,  Hong  Kong  University  of  Science  and  Technology,  Hong 
Kong 

Yang.  Tsau.  Yale  University  School  of  Medicine 

Ye,  Jane,  Dartmouth  College 

Yeh,  Jennifer,  Medical  College  of  Georgia 

Zakevicius,  Jane  M.,  University  of  Illinois  at  Chicago  College  of 

Medicine 

Zavilowitz,  Joseph,  Albert  Einstein  College  of  Medicine 
Zecevic,  Dejan,  Yale  University  School  of  Medicine 


Zecevic.  Nada,  University  of  Connecticut  Health  Center 
Zigman.  Bunnie  R..  University  of  Rochester  Medical  Center 
Zukin,  R.  Suzanne,  Albert  Einstein  College  of  Medicine 


Library  Readers 


Alkon,  Daniel,  National  Institutes  of  Health 

Allen,  Garland.  Washington  University 

Alliegro,  Mark  C..  Louisiana  State  University  Medical  Center 

Anderson.  Everett,  Harvard  Medical  School 

Baccetti,  Baccio,  Siena,  Italy 

Benjamin.  Thomas,  Harvard  Medical  School 

Bernhard,  Jeffrey.  University  of  Massachusetts  Medical  Center 

Bernheimer,  Alan,  NYU  Medical  Center 

Breinin.  Goodwin,  NYU  Medical  Center 

Candelas,  Graciela,  University  of  Puerto  Rico 
Cariello,  Lucio.  Stazione  Zoologica,  Italy 
Clark,  Arnold,  Woods  Hole,  MA 
Clarkson,  Kenneth,  AT&T  Bell  Labs 
Cohen.  Leonard.  American  Health  Foundation 
Cohen,  Seymour,  Woods  Hole,  MA 
Collier,  Marjorie,  St.  Peters'  College 
Copeland.  D.  Eugene.  Woods  Hole,  MA 
Corliss,  Bruce,  Duke  University 
Corwin,  Jeffrey,  University  of  Virginia 
Cowling,  Vincent.  SUNY,  Albany 

D'Alessio,  Guiseppe,  Stazione  Zoologica,  Italy 
Dixon.  Keith,  Hinders  University 
Duncan,  Thomas,  Nichols  College 

Eisen,  Herman  N..  Massachusetts  Institute  of  Technology 
Epstein.  Herman,  Woods  Hole,  MA 

Federici,  Celine.  Woods  Hole,  MA 

Feldman,  Susan,  New  Jersey  Medical  School 

Fitzpatnck.  Thomas,  Woods  Hole,  MA 

Frenkel,  Krystyna.  NYU  Medical  Center 

Friedler,  Gladys,  Boston  University  School  of  Medicine 

Galatzer-Levy,  R.,  University  of  Illinois 

Goldfarb,  Ronald,  University  of  Pittsburgh  Medical  School 

Goldstein,  Moise.  Johns  Hopkins  University 

Grossman,  Albert,  NYU  Medical  Center 

Gruner,  John.  Cephalon  Inc. 

Guttenplan,  Joseph.  NYU  Dental  Center 

Hall,  Valerie.  Nantucket  High  School 
Hepler,  Peter,  University  of  Massachusetts 
Herskovits.  Theodore,  Fordham  University 
Hill,  Richard,  Michigan  State  University 
Hines,  Michael.  Duke  University  Medical  Center 

Hans,  Joseph,  Case  Western  Reserve  University 
Inoue,  Sadyuki.  McGill  University.  Canada 

Jacobson,  Allan.  University  of  Massachusetts  Medical  School 
Josephson,  Beth,  Ocean  Arks  International 

Kaltenbach,  Jane,  Mount  Holyoke  College 

Kammer,  Benjamin.  Boston  University  School  of  Medicine 


Summer  Research  Programs   R35 


Brvoioan  book  collection  in  the  MBL  Rare  Books  Room. 


Karhn.  Arthur.  Columbia  University 

Kelly.  Robert.  University  of  Illinois 

Kramer.  F.  R..  Public  Health  Research  Institute.  NY 

Krane.  Stephen.  Massachusetts  General  Hospital 

King.  Kenneth.  Falmouth,  MA 

Laderman.  Aimlee.  Yale  University 

Landsberg.  Joseph.  CSIRO.  Australia 

Laster.  Joseph,  University  of  Massachusetts  Medical  Center 

Lee.  John.  City  College  of  CUNY 

Leighton.  Joseph.  Aeron  Biotechnology.  Inc. 

Leonard.  Chris.  New  York  University 

Linck.  Richard,  University  of  Minnesota 

Lorand.  Laszlo.  Northwestern  University  Medical  School 

Luponm.  P..  MCA,  Camerino,  Italy 

Martin.  Donald  C..  Woods  Hole.  MA 

Mauzerall.  Das  id.  The  Rockefeller  University 

Michaelson.  James.  Massachusetts  General  Hospital 

Mizell,  Merle.  Tulane  University 

Moore.  John.  Duke  University  Medical  Center 

Morrell.  F..  Rush  Medical  Center 

Mounier.  Franc.  University  of  Paris.  France 

Narahashi.  Toshio.  Northwestern  University 
Naugle.  John,  North  Falmouth.  MA 
Nickerson.  Peter,  SUNY.  Buffalo 
Nierhaus.  Knud.  Max  Planck  Institute.  Germany 

Ohki.  Shinpei.  State  University 

Olds.  James.  National  Institutes  of  Health 

Pappas.  George.  University  of  Illinois 
Person.  Philip.  Sloan  Kettering  Institute 


Peirce.  Sidney,  University  of  Maryland 
Plummer-Cobb,  Jewel,  California  State  University 
Prusch,  Robert  D.,  Gonzaga  University 

Rabmowitz,  Michael,  Marine  Biological  Laboratory 

Ravetch,  Robert  T..  Sloan  Kettering  Institute 

Reynolds,  George.  Princeton  University 

Rose.  Birgit.  University  of  Miami  Medical  School 

Rosenbluth.  Jack.  NYU  Medical  Center 

Rosenbluth.  Raja,  Simon  Fraser  University 

Rosenkranz,  Herbert,  University  of  Pittsburgh 

Roth,  Lorraine,  Brookline,  MA 

Ryan.  Terrance,  Regeneron  Pharmaceuticals 

Sanger,  Jean  M.,  University  of  Pennsylvania 

Sanger,  Joseph,  University  of  Pennsylvania 

Schifellite.  Carmen,  Atkinson  College 

Schippers,  Jay,  Jacksonville.  FL 

Sears.  James.  University  of  Massachusetts.  Dartmouth 

Segal.  Sheldon.  Rockefeller  Foundation 

Shanklin,  Douglas.  University  of  Tennessee 

Sheetz.  Michael,  Duke  University  Medical  Center 

Shepard.  Frank,  Deep  Sea  Research 

Shepro,  David.  Boston  University 

Sonnenblick.  B.  P.,  Rutgers  University 

Spector,  Abraham,  Columbia  University 

Spiegel.  Evelyn,  Dartmouth  College 

Spiegel,  Melvin.  Dartmouth  College 

Spotte.  Stephen.  University  of  Connecticut 

Stephenson.  William,  Earlham  College 

Stuart,  Ann,  University  of  North  Carolina 

Sundquist,  Eric.  US  Geological  Survey 

Sweet.  Frederick.  Washington  University 

Trager.  William.  The  Rockefeller  University 
Troll,  Walter,  NYU  Medical  Center 
Tweedell,  Kenyon,  University  of  Notre  Dame 
Tykocinski.  Judith,  Case  Western  Reserve  University 

Van  Holde,  Kensal.  Oregon  State  University 

Walton,  Alan  John,  Cavendish  Lab 

Wangh,  Lawrence.  Brandeis  University 

Warren.  Leonard.  Wistar  Institute 

Webb,  Marguerite,  Woods  Hole,  MA 

Weidner.  Earl.  Louisiana  State  University 

Weir,  Gary,  US  Historical  Center 

Weissmann.  Gerald.  NYU  Medical  Center 

Whittaker,  J.  R..  University  of  New  Brunswick,  Canada 

Wilber.  Charles.  Colorado  State  University 

Wittenberg.  Beatrice,  Albert  Einstein  College 

Wittenberg,  Jonathan,  Albert  Einstein  College 

Wolfteich,  Jonathan.  Woods  Hole  Oceanographic  Institution 

Yevick.  George,  Stevens  Institute  of  Technology 


Domestic  Institutions  Represented 


Alabama.  University  of.  Birmingham 
Alaska,  University  of.  Fairbanks 
Albert  Einstein  College  of  Medicine 
American  Psychological  Association 
Arizona.  University  of 


Arkansas,  University  of.  Medical  Sciences 

ARLDN,  Tucson 

Armed  Forces  Radiobiology  Research 

Institute 
AT&T  Bell  Laboratories 


Atlanta  Veterans  Affairs  Medical  Center 

Barnard  College 
Barry  University 
Baylor  College  of  Medicine 


R36  Annual  Report 


Bell  Communications  Research 

Boston  University 

Bowdoin  College 

Bowman  Gray  School  of  Medicine 

Brandeis  University 

Brooklyn  College.  CUNY 

Brown  University 

Burroughs  Wellcome 

California  Institute  of  Technology 
California  Institute  of  Technology,  Beckman 

Institute 

California,  University  of,  Berkeley 
California,  University  of,  Davis 
California,  University  of,  Irvine 
California,  University  of.  La  Jolla 
California,  University  of,  Los  Angeles 
California,  University  of.  Riverside 
California,  University  of.  San  Diego 
California,  University  of,  San  Francisco 
California,  University  of.  Santa  Barbara 
California,  University  of,  Santa  Cruz 
California.  University  of.  School  of  Medicine 
Cambridge  Neuroscience,  Inc. 
Carleton  University 
Carnegie  Mellon  University 
Case  Western  Reserve  University 
Center  for  Great  Lakes  Studies 
Centers  for  Disease  Control,  Colorado 
Chicago,  University  of 
Children's  Hospital  of  Michigan 
Children's  Hospital  of  Philadelphia 
Children's  Hospital,  Boston 
Cincinnati,  University  of 
Cold  Spring  Harbor  Laboratory 
Colorado,  University  of 
Columbia  University 
Columbia  University  College  of  Physicians  & 

Surgeons 

Connecticut.  University  of.  Health  Center 
Connecticut.  University  of 
Cornell  University 
Cornell  University  Medical  College 

Dallas  VAMC 

Dartmouth  College 

Dartmouth  Medical  School 

Denver,  University  of 

DC  General  Hospital 

Duke  University 

Duke  University  Medical  Center 

Duke  University  School  of  Medicine 

DuPont  Company 

Dyson  Vision  Research  Institute 


Emory  University 
Environmental  Protection  Agency 

Florida  State  University 

Food  and  Drug  Administration 

Fordham  University  College.  Lincoln  Center 

Franklin  and  Marshall  College 


George  Washington  University 
Georgia  Institute  ol  Technology 
Georgia  Medical  College 
Georgia.  College  of 
Georgia,  University  of 

Hampden-Sydney  College 
Hampshire  College 
Harvard  Medical  School 
Harvard  University 
Harvey  Mudd  College 
Houston.  University  of 
Howard  Hughes  Medical  Institute 
Hunter  College,  CUNY 

Idaho,  University  of 

Illinois  Institute  of  Technology 

Illinois,  University  of 

Illinois,  University  of,  Chicago  College  of 

Medicine 

Illinois,  University  of,  Urbana-Champaign 
Indiana  University 

Indiana  Llniversity  School  of  Medicine 
Iowa  State  University 
Iowa,  University  of 

James  Madison  University 
Jefferson  Medical  College 
Johns  Hopkins  Llniversity 
Johns  Hopkins  University  School  of 
Medicine 

Kansas,  University  of 
Kentucky.  University  of 
Kewalo  Laboratory,  Pacific  Biomedical 
Research  Center 

Lafayette  College 

Lehman  College,  CUNY 

Lilly  Research  Labs 

Lincoln  Hospital.  Bronx.  NY 

Loma  Linda  University  School  of  Medicine 

Los  Alamos  National  Laboratory 

Louisiana  State  University 

Louisiana  State  University  Medical  Center 

Loyola  University  of  Chicago 

Maryland.  University  of.  Baltimore 

Maryland,  University  of.  School  of  Medicine 

Massachusetts  General  Hospital 

Massachusetts  Institute  of  Technology 

Massachusetts,  Llniversity  of 

Massachusetts.  University  of,  Amherst 

Massachusetts,  Llniversity  of.  Dartmouth 

Massachusetts.  University  of.  Medical  School 

Mayo  Graduate  School 

MD  Anderson  Cancer  Center 

Medical  Center  of  Delaware 

Medical  College  of  Georgia 

Medical  College  of  Pennsylvania 

Meharry  Medical  College 

Memorial  Sloan-Kettering  Cancer  Center 

Methodist  Hospital 

Miami  Project.  University  of  Miami 


Miami  University 

Miami,  University  of.  School  of  Medicine 

Miami.  University  of.  RSMAS 

Michigan  State  University 

Michigan.  University  of 

Minnesota.  University  of 

Minnesota.  University  of.  School  of  Medicine 

Montefiore  Medical  Center 

Montpellier,  University  of 

Nassau  County  Medical 
National  Center  for  Biotechnology 

Information 

National  Institutes  of  Health 
National  Institutes  of  Health  Gerontology 

Research  Center 

National  Institutes  of  Health,  NINDS 
National  Institutes  of  Health.  NEI 
New  England  Medical  Center 
New  Jersey,  University  of  Medicine  and 

Dentistry  of 

New  Mexico,  University  of 
New  York  Medical  College 
New  York  State  Institute  of  Basic  Research  in 

Developmental  Disabilities 
New  York  University 
New  York  University  Medical  Center 
Nikon  Corporation 
Noran  Instruments 

North  Carolina,  University  of.  Chapel  Hill 
Northern  Arizona  University 
North  Texas,  University  of 
North  Texas,  University  of.  Health  Science 

Center,  Fort  Worth 
Northwestern  Llniversity 
Northwestern  University  Medical  School 

Oberlm  College 
Ohio  University 
Oklahoma.  University  of 
Olympus  Optical  Co..  Ltd. 
Oregon  Health  Sciences  University 
Oregon  State  University 
Oregon.  Llniversity  of 

Pennsylvania  Hospital 
Pennsylvania,  University  of 
Pennsylvania,  University  of.  School  of 

Medicine 

Pittsburgh,  University  of 
Pittsburgh.  University  of.  School  of  Medicine 
Poloroid  Corporation 
Pomona  College 
Princeton  University 
Puerto  Rico.  University  of 
Puerto  Rico.  University  of.  Medical  Sciences 

Research  Biochemicals  International 
Rhode  Island.  University  of 
Roche  Institute  of  Molecular  Biology 
Rochester.  University  of.  Medical  Center 
Rochester,  University  of.  School  of  Medicine 

&  Dentistry 
Rockefeller  University 


Summer  Research  Programs    R37 


Rowland  Institute  for  Science 
Rush  Medical  College 
Rutgers  University 

San  Francisco  State  University 
Scnpps  Clinic 
Scnpps  Research  Institute 
Seattle  Biomedical  Research  Institute 
Skidmore  College 
Sloan- Keltering  Institute 
Smithsonian  Institution 
South  Carolina,  University  of 
Southeastern  Louisiana  University- 
Southern  California,  University  of 
Southern  Illinois  University  School  of 

Medicine 

South  Florida.  University  of 
Spelman  College 
St.  Elizabeth's  Hospital 
St.  Lawrence  University 
St.  Louis  University  School  of  Medicine 
St.  Margaret's  Center  for  Women/Infants 
Stanford  University 
State  University  of  New  York  Health  Science 

Center 

State  University  of  New  York.  Buffalo 
State  University  of  New  York,  Stony  Brook 
Stockton  State  College 
Syracuse  University 


Syracuse  University  Institute  for  Sensory 
Research 


Tennessee.  University  of 

Texas  Tech  University  HSC 

Texas.  University  of.  Austin 

Texas,  University  of.  Galveston 

Texas.  University  of.  Health  Science  Center. 

San  Antonio 
Texas.  University  of.  Medical  Branch, 

Galveston 

Texas.  University  of.  Houston 
Texas.  University  of.  Southwestern  Medical 

Center.  Dallas 

The  George  Washington  LIniversity 
Toledo.  University  of 
Tufts  University 

Tulane  University  Medical  Center 
Tuskegee  University 


U.  S.  Department  of  Agriculture 

U.  S.  Department  of  State 

Uniformed  Services  University  of  the  Health 

Sciences 
Union  College 
Utah  State  University 
Utah.  University  of 


VA  Medical  Center.  Portland 

Vanderhilt  School  of  Medicine 

Vanderhilt  University 

Vermont.  University  of 

Virginia.  University  of.  School  of  Medicine 

Wadsworth  Center  for  Labs  and  Research 
Wake  Forest  University 
Washington  University 
Washington,  University  of 
Washington,  University  of.  School  of 

Medicine 

Washington  University  School  Medicine 
Wayne  State  University 
Wesleyan  University 
West  Florida,  University  of 
West  Virginia  University 
Western  Washington  University 
Williams  College 
Wisconsin.  University  of 
Wisconsin,  University  of.  Medical  School 
Worcester  Foundation  for  Experimental 

Biology 
Wyoming,  University  of 

Yale  Primary  Care  Research  Program 
Yale  University- 
Yale  University  School  of  Medicine 


Foreign  Institutions  Represented 


Alicante,  University,  Spain 
Auckland,  University  of.  New  Zealand 

Babraham  Institute.  UK 

Basel  University.  Switzerland 

Bogor  Agricultural  University.  Indonesia 

Brain  Research  Institute  University  of 

Zurich.  Switzerland 
Bristol  University.  UK 

Calgary.  University  of.  Canada 
Cambridge.  University  of.  UK 
Carol  Davila  University  of  Medicine. 

Romania 

Catholic  University  of  Chile.  Chile 
CNRS,  France 
Consejo  Superior  Investigaciones  Cientificas. 

Spain 
Copenhagen  University.  Denmark 

Ecole  Normale  Superieure.  France 

Federal  University  of  Rio  de  Janeiro.  Brazil 
Frednch  Miescher  Institut.  Switzerland 

Guy's  Hospital.  London 

Hebrew  University.  Israel 
Hokkaido  Central  Fisheries  Experimental 
Station.  Japan 


Hong  Kong  University  of  Science  & 
Technology.  Hong  Kong 

ICP-Brussels.  Belgium 

I.V.I.C..  Venezuela 

INSA.  France 

INSERM.  France 

Institute  for  Drug  Research.  Budapest. 

Hungary 
Institute  of  Mathematical  Problems  of 

Biology,  Russia 

Institute  of  Microbiology,  Berlin 
Instituto  di  biologia  dello  Sviluppo,  Italy 
Indian  Institute  of  Science,  India 
Institute  of  Protein  Research.  Russia 
Instituto  de  Ciencias  del  Mar.  Spain 
Instituto  de  Investigaciones  Biologicas. 

Uruguay 

Instituto  M.  y  M.  Ferreyra.  Argentina 
Israel  Institute  of  Technology.  Israel 

Jiangsu  Institute  of  Parasitic  Diseases.  China 

Kochi  University.  Japan 
Kyoto  University.  Japan 

Laboratoire  de  Neurobiologie.  ENS 
London.  University  College  of.  England 


Max  Planck  Institute  for  Biological 

Cybernetics.  Germany- 
Max  Planck  Institute  for  Developmental 

Biology,  Germans 
Max  Planck  Institute  of  Immunobiology. 

Germany 
Max  Planck  Institute  for  Molecular  Genetics, 

Germany 

Medical  Research  Council,  UK 
McGill  University,  Canada 
Mitsubishi  Kasei  Life  Science  Institute.  Japan 
MRC  Laboratory'  of  Molecular  Biology 

Naples.  University  of,  Italy 
Nagoya  University,  Japan 
National  Museums  of  Kenya,  Kenya 
National  Research  Council.  Italy- 
Netherlands  Cancer  Institute.  The 

Netherlands 
Nippon  Medical  School.  Japan 

Ottawa,  University  of,  Canada 
Oxford  University,  UK 

Pasteur  Institute,  France 

Philipps  Universitat  Marburg.  Germany 

Protein  Research  Institute.  Russia 

Queen's  University.  Canada 


R38   Annual  Report 


RIKEN,  Japan 

Rostock,  University  of,  Germany 

Royal  Free  Hospital  School  of  Medicine,  UK 

Russian  Academy  of  Sciences,  Russia 

Seville,  University  of,  Spain 

Slovak  Republic,  Institute  of  Zoology  & 

Ecozoology,  Slovak  Republic 
Station  Zoologique,  France 
Stazione  Zoologica  A.  Dohrn.  Italy 

Tata  Institute  of  Fundamental  Research, 

India 

Tel  Aviv  University,  Israel 
The  Hospital  for  Sick  Children,  Canada 
Tokai  University.  Japan 
Tokyo  College  of  Pharmacy.  Japan 
Tokyo  Medical  and  Dental  University  School 

of  Medicine,  Japan 
Tokyo,  University  of,  Japan 


Tokyo,  University  of  Agriculture  & 

Technology,  Japan 
Toronto,  University,  Canada 

Universitat  des  Saarlandes.  Germany 
Universidad  del  Valle,  Columbia 
Universitat  Konstanz,  Germany 
Universitat  Oldenburg,  Germany 
University  College  London,  UK 
University  of  Aarhus,  Denmark 
University  of  Antwerp,  Belgium 
University  of  Bonn,  Germany 
University  of  British  Columbia,  Canada 
University  of  Cambridge,  UK 
University  of  Glasgow,  UK 
University  of  Granada,  Spain 
University  of  Groningen,  Germany 
University  of  Hamburg,  Germany 
University  of  Koln,  Germany 
University  of  Lethbridge,  Canada 
University  of  Lisbon.  Portugal 


University  of  London,  UK 

University  of  Manitoba,  Canada 

University  of  Munich.  Germany 

University  of  Pisa.  Italy 

University  of  Oxford,  UK 

University  of  Paris.  France 

University  of  Tokyo,  Japan 

University  of  Western  Australia,  Australia 

University  of  Western  Ontario,  Canada 

Uppsala  University.  Sweden 

Waseda  University.  Japan 
Weizmann  Institute  of  Science,  Israel 
Wellcome/CRC  Institute,  UK 

Yokohama  City  University  School  of 

Medicine,  Japan 
Yonsei  University  College  of  Medicine  Seoul, 

Korea 

Zoologisches  Institut.  Germany 


Year-Round  Research  Programs 


Architectural  Dynamics  in  Living  Cells 
Program 

Established  in  1992,  this  program  focuses  on  architectural  dynamics 
in  living  cells — the  timely  and  coordinated  assembly  and  disassembly 
of  macromolecular  structures  essential  for  the  proper  functioning, 
division,  motility,  and  differentiation  of  cells;  the  spatial  and  temporal 
organization  of  these  structures;  and  their  physiological  and  genetic 
control.  The  program  is  also  devoted  to  the  development  and 
application  of  powerful  new  imaging  and  manipulation  devices  that 
permit  such  studies  directly  in  living  cells  and  functional  cell-free 
extracts.  The  Architectural  Dynamics  in  Living  Cells  Program 
promotes  interdisciplinary  research  and  consists  of  resident  core 
investigators  and  a  cadre  of  adjunct  members. 


Resident  Core  Investigators 

Inoue.  Shinya.  Distinguished  Scientist 
Mei.  Guang.  Research  Associate 
Oldenbourg.  Rudolf,  Associate  Scientist 
Stemmer.  Andreas.  Visiting  Assistant  Scientist 


Staff 

Knudson,  Robert,  Instrument  Development  Engineer 
Leighton.  Jane.  Executive  Assistant 


Visiting  Investigators 

Rieko  Arimoto,  Nikon  Corporation.  Tokyo.  Japan 

Theodore  D.  Inoue,  Universal  Imaging  Corporation,  West  Chester. 

PA 

Andrew  Murray.  University  of  California.  San  Francisco 
Fabrice  Roegiers,  Station  Zoologique,  Villefranche-sur-Mer.  France 
Edward  D.  Salmon.  University  of  North  Carolina,  Chapel  Hill 
Keisuke  Suzuki.  Olympus  Corporation.  Hachioji.  Japan 
Phong  Tran.  University  of  North  Carolina.  Chapel  Hill 


Boston  University  Marine  Program 

Facultv 

Atema,  Jelle,  Professor  of  Biology,  Director 
Dionne,  Vincent.  Professor  of  Biology 
Humes,  Arthur  G.,  Professor  of  Biology  Emeritus 
Kaufman.  Les,  Associate  Professor  of  Biology 
Lobel.  Phillip,  Associate  Professor  of  Biology 
Tamm,  Sidney  L.,  Professor  of  Biology 
Valiela,  Ivan.  Professor  of  Biology 
Voigt.  Rainer,  Research  Associate  Professor 

Staff 

Guilfoyle,  Kerry.  Course  Coordinator 

Hahn.  Dorothy,  Senior  Administrative  Secretary 

O'Brien,  Todd,  Staff  Assistant 

Pedersen.  Jennifer.  Program  Assistant 

Schillizzi,  Cynthia,  Program  Manager 

Visiting  Faculty  and  Investigators 

D'Avanzo,  Charlene,  Hampshire  College 

Hinkle,  Greg,  MBL 

Kremer,  James,  USC 

Margulis,  Lynn,  UMass-Amherst 

Mulsow,  Sandor.  Bedford  Institute  of  Oceanography 

Rietsma,  Carol,  SUNY  New  Paltz 

Sardet,  Christian,  Villefranche  Zoological  Station 

Simmons.  William.  Visiting  Lecturer.  Boston  University 

Wainwright.  Norman,  MBL 

Ward,  Nathalie,  Center  for  Coastal  Studies 

Research  Staff 

Basil,  Jennifer.  Postdoctoral  Investigator 
Breithaupt.  Thomas,  Postdoctoral  Investigator 
Collins,  Glynnis,  Visiting  Research  Assistant 
Delay.  Rona.  Postdoctoral  Investigator 
Dennison,  William,  University  of  Sydney 


R39 


K40   Annual  Report 


Duhin,  Adrienne,  Postdoctoral  Investigator 
Eisthen,  Heather,  Postdoctoral  Investigator 
Foreman,  Kenneth.  Postdoctoral  Investigator 
Gerardo.  Hortense.  Postdoctoral  Investigator 
Grasso,  Frank,  Postdoctoral  Investigator 
McConneli.  Joanne,  Postdoctoral  Investigator 
Ni.xon.  Jennifer,  Research  Assistant 
Soucy,  Lori,  Research  Assistant 
Seely.  Brad.  Visiting  Research  Assistant 
Tamm,  Signhild,  Senior  Research  Associate 

Graduate  Students 
PhD  students 

Balint,  Claire 
Batjakas,  loannis 
Behr,  Peter 
Bushmann,  Paul 
Dale,  Jonathan 
Farley,  Lynda 
Gomez.  George 
Hauxwell.  Jennifer 
Hersh,  Douglas 
Karavanich.  Christy 
LaMontagne,  Michael 
Lowe,  Bnan 
Loynes,  Janet 
Ma,  Diana 
McClelland,  James 
Oliver,  Steven 
Portnoy,  John 
Tamse,  Armando 
Usup,  Gires 
Zhou,  Qiao 

MA  students 

Ashcraft,  Susan 
Bayha,  Keith 
Booking,  Beatrice 
Brazik,  David 
Burkhalter.  Brenda 
DiNunno,  Paul 
Economakis,  Alistair 
Fricke,  Julie 
Goldstein,  Jennifer 
Kerr,  Lisa 
Ludlow,  Amanda 
Maglic,  Boris 
Nathan,  Miselis 
Philibotte,  Jason 
Pinto-Torres,  Sonia 
Rader,  Lauren 
Schreiber.  Suzanne 
Tomasky.  Gabrielle 
Wittenberg,  Kim 

Summer  1994  Undergraduate  Interns 

Bartholomew,  Aaron 
Bertrand,  Virginie 
Boxhill,  Jessica 
Chalfoun,  Anna' 
de  Maries.  Axe 
Ellis,  Melissal 


Han,  Tina 
Harrison,  Timothy 
Home,  C.  Ashton 
Hurlburt.  Peter 
Leonard,  Ann 
McDonnell.  Kristin 
Monti.  Jill 
Rudy,  Michelle 
Tolly,  Krystal 
Tyndale.  Libby 
Vazquez,  Lory  Sandiago 
White,  Brad 

Undergraduate  Students.  Fall  1994 

Barneby,  Sebastian 
Beaudette,  Britte 
Bhatt,  Sonal 
Bielawski,  April 
Bota,  Dalena 
Boyle,  Bridget 
Canizio.  Casey 
Cappa,  Aimie 
Cardinale.  Nicole 
Carlson.  Daphne 
Dean,  Tony 
Downing,  Amy 
DeGrenier,  Jennifer 
DeSantis.  Krystal 
Elsasser,  Emily 
Ewell,  Cara 
Gibbons,  Lynn 
Haeuber,  Elaina 
Hammel,  Scott 
Henchar,  Teresa 
Hoddinott,  Jennifer 
Hooper,  Brian 
Horal.  Melissa 
Forne,  C.  Ashton 
Kelly,  Christopher 
Kulawiak,  Karen 
Levin,  Use 
Mastaitis,  Jason 
Mela.  Alexandra 
Mendoza,  Daniel 
Mihkow,  Davis 
Mohammadian,  Marlon 
Mosconi,  Christopher 
Palmer,  Robin 
Pollard,  Amina 
Portante,  Gerald 
Richards,  Kyle 
Sadler.  Theo 
Scarfo.  Julie 
Schaffhauser.  Lori 
Schrader,  Heather 
Sikorski,  Kristan 
Sleigh,  Kimberly 
Steinert.  Cara 
Tauber,  Julia 
Tschaepe.  Nikol 
Uhlenhopp.  Amy 
Vincent,  Lara 
Wey,  Patricia 


Year-Round  Research  Programs  R41 


Williams.  Josh 

\\  itkop.  kimberlee 

Yung.  Angela 


Laboratory  ofJelle  Aicnui 

Many  organisms  use  chemical  signals  as  their  main  source  of 
information  about  the  environment.  These  signals  are  transported  in 
the  marine  environment  by  turbulent  currents,  viscous  flow,  and 
molecular  diffusion.  Receptor  organs  extract  signals  through  various 
physical  &  biological  filtering  processes.  Currently,  the  lobster  with  its 
exquisite  sense  of  taste  and  smell,  is  our  major  model  to  study  the 
signal  filtering  capabilities  of  the  whole  animal  and  the  tuning 
properties  of  its  receptor  cells.  Research  focuses  on  food  signals  and 
pheromones  used  in  courtship  and  dominance,  neurophysiology  of 
receptor  cells,  behavior  guided  or  modulated  by  chemical  signals, 
computational  models  of  odor  plumes  and  neural  filters,  and 
underwater  robotics. 

Laboratory  uf]  lucent  Dionne 

Odors  are  powerful  stimuli.  They  can  focus  the  attention,  elicit 
behaviors  (or  misbehaviors),  and  even  resurrect  forgotten  memories. 
These  actions  are  directed  by  the  central  nervous  system,  but  they 
depend  upon  the  initial  transduction  of  chemical  signals  by  olfactory 
receptor  neurons  in  the  nasal  passages.  More  than  just  a  single  process 
appears  to  underlie  odor  transduction.  and  the  intracellular  pathways 
that  are  used  are  far  more  diverse  than  once  thought.  Hundreds  of 
putative  odor  receptor  molecules  have  been  identified  that  work 
through  several  different  second  messengers  to  modulate  the  activity 
of  various  types  of  membrane  ion  channels.  Our  studies  are  being 
conducted  with  aquatic  salamanders  using  amino  acids  and  other 
soluble  chemical  stimuli  which  these  animals  perceive  as  odors.  Using 
electrophysiological  and  molecular  approaches,  the  research  examines 
how  these  cellular  components  produce  odor  detection,  and  how 
odors  are  identified  and  discriminated. 

Laboratory  of  Arthur  G.  H  nines 

Research  interests  include  systematics.  development,  host 
specificity,  and  geographical  distribution  of  copepods  associated  with 
marine  invertebrates.  Current  research  is  on  taxonomic  studies  of 
copepods  from  invertebrates  in  the  tropical  Indo-Pacific  area,  and 
poecilostomatoid  and  siphonostomatoid  copepods  from  deep-sea 
hydrothermal  vents  and  cold  seeps. 

Laboratory  of  Philip  Label 

This  laboratory  is  developing  a  complete  model  of  the  interactions 
of  man  and  nature  in  the  Johnston  Atoll  lagoon.  Johnston  Atoll  has 
been  occupied  continuously  by  the  military  since  the  1930s  and 
provides  a  unique  opportunity  for  assessing  the  biological  impacts  of 
pollution.  Unlike  a  city  harbor,  chemical  spills  at  the  atoll  are 
documented  as  to  location,  date,  and  amount. 

Laboratory  of  Sidney  Tani/u 

Marine  model  systems  offer  unique  experimental  advantages  for 
solving  basic  problems  in  cell  biology  and  physiology.  In  particular, 
comb  jellies  (ctenophoresl.  important  members  of  the  marine 
zooplankton.  possess  the  largest  cilia  and  smooth  muscles  in  nature,  a 
simple  nervous  system,  and  interesting  feeding  and  locomotory 
behaviors.  We  use  ctenophores  to  investigate  the  mechanism  of  ciliary 


movement  and  ciliary  coordination,  the  neural  and  ionic  control  of 
cilia  (particularly  stimulus-evoked  intraciliary  calcium  transients  and 
distribution  of  ciliary  calcium  channels),  geotaxis  and  mechanosensory 
transduction  by  motile  cilia  statocyst.  structure  and  function  of 
smooth  muscle,  double-modality  sensory1  receptors  and  the 
cytoskeleton.  and  evolution  of  neurotransmitters.  and  a  new  type  of 
reversible  cell-cell  adhesion  that  closes  the  mouth  of  Berne,  a 
voracious  predator  of  other  ctenophores.  In  addition,  we  use  a  termite 
protozoan  with  a  continuously  rotating  head  to  investigate  novel  types 
of  cell  motility.  the  fluid  nature  of  cell  membranes,  and  remarkable 
prokaryotic-eukaryotic  motility  symbioses. 

Laboratory  of  Ivan  \  'a/ic/a 

Our  major  research  activity  involves  the  Waquoh  Bay  Land  Margin 
Ecosystems  Research  Project.  This  work  examines  how  human 
activity  in  coastal  watersheds  (including  landscape  use  and 
urbanization)  increases  nutrient  loading  to  groundwater  and  streams. 
Nutrients  in  groundwater  are  transported  to  the  sea.  and,  after 
biogeochemical  transformation,  enter  coastal  waters.  There,  increased 
nutrients  bring  about  a  series  of  changes.  The  Waquoit  Bay  LMER  is 
designed  to  help  understand  and  model  the  coupling  of  land  use  and 
consequences  to  receiving  waters,  to  study  the  processes  involved,  and 
to  assess  consequences  and  opportunities  for  coastal  management. 

A  second  long-term  research  topic  is  the  structure  and  function  of 
salt  marsh  ecosystems,  including  the  processes  of  predation.  herbivory. 
decomposition,  and  nutrient  cycles. 

Calcium  Patterning  Program 

This  laboratory  investigates  the  role  played  by  calcium  ions  in  a 
wide  range  of  fundamental  cell  processes:  in  developing  eggs,  in 
differentiated  tissues,  and  in  cell  extracts.  This  is  possible  through  the 
use  of  aequorin,  a  bioluminescent  protein  complex.  Aequonn  can 
either  be  microinjected  into  cells  or  transgenically  expressed  without 
disturbing  function  or  development.  The  pattern  of  luminescence  that 
is  emitted  by  an  aequorin-loaded  cell  reveals  changing  patterns  and 
levels  of  free  calcium  within  the  cell  (or  its  progeny).  Photons  are 
collected  and  correlated  with  dynamic  cellular  events  by  an  imaging 
system  developed  in  our  laboratory.  This  technique  has  some 
substantial  advantages  over  other  methods  of  imaging  intracellular 
calcium  and  as  a  result  supports  an  extensive  collaborative  research 
effort.  The  laboratory  is  currently  studying  cytokinesis  in  frog  and  fish 
eggs;  cell  cycle  control  in  sea  urchin  and  surf  clam  eggs;  polarity 
expression  in  frog  eggs;  tip  growth  in  pollen  tubes;  injury  and 
degeneration  in  neurons;  mechanisms  of  fertilization  in  sea  urchins; 
differentiation  in  slime  molds;  and  calcium  release  in  cell  extracts 
from  frog  eggs.  The  laboratory  is  supported  by  the  NSF  to  both  pursue 
biological  questions  and  to  develop  the  aequorin-based  imaging 
technique.  Great  emphasis  is  being  placed  on  the  development  of 
transgenic  strains  of  both  animals  and  plants  that  express  the 
transfected  apo-aequorin  gene.  We  are  currently  working  on  slime 
molds,  zebraftsh,  mice  and  tobacco  seedlings  that  all  express  the 
jellyfish  gene. 


Staff 

Miller.  Andrew  L..  Assistant  Scientist 
Jaffe,  Lionel  F..  Senior  Scientist 

I  Isiiing  Investigators 

Azhar,  Mohamed,  Indian  Institute  of  Science,  Bangalore.  India 
Bozhkova.  Valentina,  Russian  Academy  of  Sciences.  Russia 


R42  Annual  Report 


Browne,  Carole.  Wake  Forest  University 

Chauhan.  Amnt,  Macalester  College 

Creton,  Robert,  University  of  Utrecht,  The  Netherlands 

Cubitt.  Andrew,  C.  UCSD 

Denegre.  Jim,  University  of  California,  Irvine 

Eckberg.  Bill,  Howard  University 

Fluck,  Richard,  A.,  Franklin  and  Marshal  College 

Galione,  Antony,  Oxford  University.  UK 

Huebner,  Envin,  University  of  Manitoba.  Canada 

Rammer,  Benny,  Boston  University 

Machesky,  Laura,  MRC.  Cambridge,  UK 

McWilliams,  Harry 

Miller,  Brent.  Purdue  University 

Sardet,  Christian,  Villefranche-sur-Mer,  France 

Speksnijder,  Johanna.  E.,  Hubrecht  Laboratory,  The  Netherlands 


The  Ecosystems  Center 

The  Center  carries  out  research  and  education  in  ecosystems 
ecology.  Terrestrial  and  aquatic  scientists  work  in  a  wide  variety  of 
ecosystems  ranging  from  the  streams,  lakes,  and  tundra  of  the  Alaskan 
Arctic  (limits  on  plant  primary  production!  to  sediments  of 
Massachusetts  Bay  (controls  of  nitrogen  cycling),  to  forests  in  New 
England  (effects  of  soil  warming  on  carbon  and  nitrogen  cycling)  and 
South  America  (effects  on  greenhouse  gas  fluxes  of  conversion  of  rain 
forest  to  pasture)  and  to  large  estuaries  in  the  Gulf  of  Maine  (effects 
on  the  plankton  and  benthos  of  nutrients  and  organic  matter  in 
stream  runoff).  Many  projects,  such  as  those  dealing  with  carbon  and 
nitrogen  cycling  in  forests,  streams,  and  estuaries,  use  the  stable 
isotopes  I3C  and  I5N  to  investigate  natural  processes.  A  mass 
spectrometer  facility  is  available  at  the  Center.  Data  from  field  and 
laboratory  research  are  used  to  construct  mathematical  models  of 
whole-system  responses  to  change.  Some  of  these  models  are 
combined  with  geographically  referenced  data  to  produce  estimates  of 
how  environmental  changes  affect  key  ecosystem  indexes  such  as  net 
primary  productivity  and  carbon  storage  throughout  the  world's 
terrestrial  biosphere.  The  results  of  the  Center's  research  are  applied, 
wherever  possible,  to  the  questions  of  the  successful  management  of 
the  natural  resources  of  the  earth.  In  addition,  the  ecological  expertise 
of  the  staff  is  made  available  to  public  affairs  groups  and  government 
agencies  who  deal  with  problems  such  as  acid  rain,  coastal 


Aerial  view  ol.Pliim  Island  Sound  Estuary,  where  the  Ecosystems  Center 
is  studying  carbon  and  nitrogen  dynamics  ami  food  »'eb  structure. 

IPholo  by  Bruce  Peterson) 


eutrophication,  and  possible  carbon  dioxide-caused  climate  change. 
There  are  opportunities  for  postdoctoral  fellows  and  graduate 
students. 

Staff 

Hobbie,  John  E..  Co-Director 

Melillo.  Jerry  M.,  Co-Director 

Bahr.  Michele 

Buffam.  Ishi 

Castro,  Mark 

Castro,  Nancy 

Catricala,  Christina 

Deegan,  Linda 

Donovan,  Suzanne 

Dornblaser.  Mark 

Downs,  Martha 

Drummey.  Todd 

Dugan,  Deirdre 

Fry,  Brian 

Garritt,  Robert 

Giblin.  Anne 

Giehtbrock.  David 

Harvey.  Christopher 

Helfnch.  John 

Hopkinson,  Charles 

Jones.  David 

Kicklighter,  David 

Kwiatkowski.  Bonnie 

Laundre,  James 

McGuire,  A.  David 

McKane,  Robert 

Monahan,  Jean 

Murray,  Georgia 

Nadelhoffer,  Knute 

Neill.  Christopher 

Newkirk.  Kathleen 

Nolin.  Amy 

Pennington,  Susan 

Peterson,  Bruce 

Rastetter.  Edward 

Redmond.  Leslie 

Regan,  Kathleen 

Ricca,  Andrea 

Scanlon.  Deborah 

Seifert,  Mary  Ann 

Shaver.  Gaius 

Steudler.  Paul 

Tholke.  Kristin 

Tucker,  Jane 

Wollheim,  Wilfred 

Postdoctorals 

Fernandes.  David 
Johnson,  Loretta 
Pan.  Yude 
Vallino.  Joseph 
Williams,  Mathew 
Xiao,  Xiangming 

Consultants 

Bowles.  Francis 
Bowles.  Margaret 
Schwarzman,  Elisabeth 
Thomson,  Lee 


Year-Round  Research  Programs   R43 


Laboratory  for  Marine  Animal  Health 

The  laboratory  provides  diagnostic,  consultative  research,  and 
educational  services  to  the  institutions  and  scientists  of  the  Woods 
Hole  community  concerned  with  marine  animal  health.  Diseases  of 
wild,  captive,  and  cultured  animals  are  investigated. 


Staff 

Abt.  Donald  A..  Director  and  The  Robert  R.  Marshak  Term  Professor 

of  Aquatic  Animal  Medicine  and  Pathology,  School  of  Veterinary 

Medicine.  University  of  Pennsylvania 
Bullis.  Robert  A..  Research  Assistant  Professor  of  Microbiology, 

University  of  Pennsylvania 
Leibovitz.  Louis.  Director  Emeritus 
McCafferty,  Michelle.  Histology  Technician.  University  of 

Pennsylvania 

Moniz.  Priscilla  C.,  Secretary 
Smolowitz.  Roxanna  M.,  Research  Associate  in  Pathology,  University 

of  Pennsylvania 
Wadman,  Elizabeth  A..  Microbiology  Technician,  University  of 

Pennsvlvania 


Laboratory  of  Aquatic  Biomedicine 

This  laboratory  investigates  leukemias  of  soft  shell  clams. 
Monoclonal  antibodies  developed  by  this  laboratory  and  techniques  in 
molecular  biology  are  used  to  investigate  the  differences  between 
normal  and  leukemic  cells  and  their  ontogeny.  The  impact  of 
pollutants  on  leukemogenesis  is  currently  being  studied  with  an 
emphasis  on  regional  superfund  sites. 


Staff 

Reinisch.  Carol  L.,  Investigator.  MBL.  and  Chairperson,  Department 
of  Comparative  Medicine,  Tufts  University  School  of  Veterinary 
Medicine 


Laboratory  of  Cell  Biochemistry 

This  laboratory  uses  cell  and  molecular  biological  methods  to  study 
the  regulation  of  gene  expression  in  marine  fish.  Current  emphasis  is 
on  gene  products  involved  in  heme  metabolism  including:  (a| 
aminolevulmate  synthase,  the  first  and  rate  determining  enzyme  of 
heme  production:  (b)  cytochrome  P450.  a  heme-requiring  catalyst  for 
oxidation  of  hydrophobic  chemicals;  and  (c)  heme  oxygenase,  a  stress- 
induced,  microsomal  enzyme  that  catalyzes  the  first  reaction  of  heme 
degradation.  The  expression  of  all  three  enzymes  is  affected  by 
endogeneous  and  pharmacological  agents  as  well  as  xenobiotics  and 
carcinogens.  We  have  cloned  and  sequenced  cDNAs  for  both  the 
erthyroid  and  housekeeping  forms  of  aminolevulinate  synthase,  have 
developed  specific  probes  for  cytochrome  P450.  and,  by  RT-PCR,  are 
generating  a  homologous  probe  for  heme  oxygenase.  When  that  is 
completed,  we  will  have  a  battery  offish-specific  molecular  biological 
reagents  that  can  be  used  to  monitor  environmental  effects  on  heme 
biosynthesis,  utilization,  and  degradation.  It  is  expected  that  such 
simultaneous  analyses  will  be  much  more  informative  than 
measurements  made  on  only  one  aspect.  We  recently  have  shown  that 
isolated  fish  hepatocytes  regulate  heme  biosynthesis  in  a  manner 
resembling  that  in  terrestrial  vertebrates  (including  humans),  and  we 
are  using  primary  cultures  of  fish  hepatocytes  to  address  some  long- 
standing biomedical  questions  regarding  the  mechanisms  of  that 
regulation.  Because  preliminary  sequence  alignments  indicate  that 


aminolevulinate  synthase  has  an  interesting  evolutionary  history  that 
bears  on  the  endosymbiont  hypothesis  for  the  origin  of  animal 
mitochondria,  these  studies  will  be  extended  to  include  comparative 
molecular  biology  of  aminolevulinate  synthases  from  invertebrates 
and  lower  eukaryotes. 

Staff 

Cornell,  Neal  W..  Senior  Scientist 
Faggart.  Maura  A..  Research  Assistant 
Martin,  Holly  A..  Research  Assistant 
Macarro,  Jackie,  Laboratory  Assistant 

Visiting  Scientists 

Fox,  T.  O.,  Harvard  Medical  School 
Schaffer,  Walter  T..  NIH 

Laboratory  of  Cell  Communication 

Established  in  1994,  this  laboratory  is  devoted  to  the  study  of 
intercellular  communication.  The  research  focuses  on  the  cell-to-cell 
channel,  a  membrane  channel  built  into  the  junctions  between  cells. 
This  channel  provides  one  of  the  most  basic  forms  of  intercellular 
communication  in  organs  and  tissues.  The  work  is  aimed  at  the 
molecular  physiology  of  this  channel,  in  particular,  at  the  mechanisms 
that  regulate  the  communication.  Electrophysiological-,  fluorescent- 
tracer-,  and  molecular  biological  techniques  are  used  to  this  end.  As 
was  recently  discovered  in  this  laboratory,  the  channel  is  the  conduit 
of  growth-regulating  signals.  It  is  instrumental  in  a  basic  feedback  loop 
whereby  cells  in  organs  and  tissues  control  their  number:  in  a  variety 
of  cancer  forms  it  is  crippled.  Work  is  aimed  now  at  the  mechanisms 
of  growth  control  and  at  correcting  cancer  growth  by  transferring  the 
gene  for  the  cell-to-cell  channel  protein  from  normal  cells  into  the 
cancer  cells.  Molecular  genetic  techniques  are  used  in  this  endeavor. 

Staff 

Werner  Loewenstein.  Senior  Scientist 
Birgit  Rose.  Senior  Scientist 
Tracy  Jillson.  Research  Assistant 


R44   Annual  Report 


Laboratory'  ofShinya  Inoue 

Study  of  the  molecular  mechanism  and  control  of  mitosis,  cell 
division,  cell  motility.  and  cell  morphogenesis,  with  emphasis  on 
biophysical  studies  made  directly  on  single  living  cells,  especially 
developing  eggs  in  marine  invertebrates.  Development  of  biophysical 
instrumentation  and  methodology,  such  as  polarization  optical  and 
video  microscopy  and  digital  image  processing  techniques,  and 
exploration  of  their  underlying  theory  are  an  integral  part  of  the 
laboratory's  effort. 

Staff 

Inoue,  Shinya,  Distinguished  Scientist 

Knudson,  Robert,  Instrument  Development  Engineer 

Leighton,  Jane,  Executive  Assistant 

Maccaro,  Jackie,  Laboratory  Assistant 

Mei.  Guang,  Research  Associate 

Stemmer.  Andreas,  Visiting  Assistant  Scientist 

Woodward.  Bertha  M..  Laboratory  Manager 

Laboratory  of  Alan  M.  Kuzirian 

Research  in  this  laboratory  explores  the  functional  morphology  and 
ultrastructure  of  various  organ  systems  present  in  opisthobranch 
mollusks.  The  program  includes  mariculture  of  the  nudibranch. 
Hermissenda  crassicarnis.  with  emphasis  on  developing  reliable 
culture  methods  for  rearing  and  maintaining  this  animal  as  a  research 
resource.  Studies  include  optimization  of  adult  and  larval  nutrition, 
control  of  facultative  pathogens  and  disease,  development  of 
morphologic  criteria  for  staging  larvae  and  juveniles,  and 
metamorphic  induction.  Morphologic  studies  stress  the  ontogeny  of 
neural  and  sensory  structures,  and  neurochemicals  associated  with  the 
photic  and  vestibular  systems  which  have  been  used  as  models  systems 
in  learning  and  memory  studies. 

Concurrent  with  these  studies  is  the  development  of  a  new 
technique  to  obtain  and  reconstruct  serial  block  face  images  (SBFI)  ot 
epoxy-embedded  or  cryoprepared  tissues  sectioned  or  freeze-fractured/ 
freeze-etched  inside  an  SEM  by  an  in  situ  miniature  ultramicrotome. 

Collaborative  research  includes  histochemical  investigations  on 
strontium's  role  in  initiating  calcification  in  molluscan  embryos  (shell 
and  statoliths),  as  well  as  immunocytochemical  labelling  of  cell-surface 
and  secretory  product  antigens  using  monoclonal  and  polyclonal 
antibodies  on  Hermissenda  sensory  and  neurosecretory  neurons  ;/; 
situ,  and  in  cell  culture.  Toxicity  studies  on  heavy  metal  effects  on 
Hermissenda  learning  and  physiology  of  cultured  neurons  are  also 
being  conducted. 

Additional  collaborative  research  includes  DNA  fingerprinting  of 
Hermissenda  using  RAPD-PCR  techniques  in  preparation  for  genetic 
strain  development,  as  well  as  chemical  ecological  studies  of  the  roles 
natural  products  play  in  larval  metamorphosis  and  predator-prey 
recognition  and  defense  mechanisms.  Systematic  and  taxonomic 
studies  of  nudibranch  mollusks  are  also  of  interest. 

Staff 

Kuzirian.  Alan  M..  Associate  Scientist 
Tamse.  Catherine  T..  Research  Assistant 

I  '/siting  Investigators 

Avila,  Conxita,  Postdoctoral  Associate,  Centre  d'Estudis  Avancats  de 

Blanes,  Blanes.  Spain 

Chikarmane.  Hemant.  Assistant  Scientist.  MBL 
Leighton.  Stephen  B..  Biomed.  Engineering/Instrumen.  Branch. 

NCRR-NIH 


Laboratory  of  Rudolf  Oldenbourg 

The  laboratory  develops  advanced  instrumentation  in  light 
microscopy  and  investigates  physical  optics  relevant  to  microscope 
imaging  for  high  resolution  studies  of  architectural  dynamics  in  living 
cells  and  cell  components.  The  current  focus  of  the  laboratory  is  the 
development  of  a  new  polarized  light  microscope  that  combines 
microscope  optics  with  new  electro-optical  components,  video,  and 
digital  image  processing  for  fast  analysis  of  specimen  birefringence 
over  the  entire  viewing  field  at  the  highest  resolution  of  the  light 
microscope.  Biological  systems  currently  investigated  with  the  new 
pol-scope  are  microtubule-based  structures  (mitotic  spindles,  asters, 
single  microtubules),  striated  muscles  (myofibril),  and  virus  liquid 
crystals. 

Staff 

Rudolf  Oldenbourg,  Associate  Scientist 

Guang  Mei,  Research  Associate 

Robert  Knudson,  Instrumentation  Engineer 

Laboratory  of  Nancy  Rafferty 

This  laboratory  investigates  the  role  of  the  lens  cytoskeleton  and  its 
associated  proteins  in  the  maintenance  of  lens  shape,  in  lens 
accommodation  and  development  of  cataract  when  the  cytoskeleton  is 
disrupted.  Studies  include  an  assessment  of  the  role  of  cytosolic  free 
calcium  on  homeostasis  of  the  lens  cytoskeleton,  the  localization  of 
various  cytoskeletal  proteins  in  lens  epithelium,  and  determination  of 
the  relative  amounts  of  soluble  actin  to  filamentous  actin  in  lens  cells 
during  aging.  Most  of  these  studies  employ  an  elasmobranch  fish  and 
rabbit  model  using  primary  cultures  of  lens  epithelium  and  electron 
and  immunofluorescence  microscopy. 

Staff 

Rafferty,  Nancy  S.,  Scientist,  Northwestern  University 
Rafferty,  Keen  A.,  Research  Associate 

Laboratory  of  Sensory  Physiology 

Since  1973,  the  Laboratory  has  conducted  research  on  various 
facets  of  vision.  Current  investigations  focus  on  structural,  functional. 
and  mechanistic  aspects  of  visual  pigments.  The  chemical  basis  of 
color  vision  is  investigated  principally  with  light-microscope-based 
absorption  spectroscopy.  In  addition  to  fresh  preparations  from  fish 
and  amphibians,  in  vitro  model  systems  are  studied  with  infrared  and 
other  spectroscopic  techniques.  The  aim  is  a  thorough  understanding 
of  the  chemistry  that  underlies  spectral  tuning. 

Staff 

Harosi,  Ferenc  I.,  Associate  Scientist,  MBL,  and  Boston  L'niversity 
School  of  Medicine 


ng  Investigator 
Sandorfy,  C,  Universite  de  Montreal.  Canada 

Laboratory  ofOsamu  Shimomura 

Biochemical  mechanisms  involved  in  the  bioluminescence  of 
various  luminous  organisms  are  investigated.  Based  on  the  results 
obtained  in  this  laboratory,  improved  forms  of  bioluminescent  probes 
are  designed  and  produced  for  the  measurements  of  intracellular  free 
calcium  and  superoxide  anion. 


Year-Round  Research  Programs   R45 


The  tiiadfi\h. 


Staff 

Shimomura.  Osamu,  Senior  Scientist,  MBL,  and  Boston  University 

School  of  Medicine 
Shimomura.  Akcmi.  Research  Assistant 

Laboratory  of  Raquel  Sussman 

We  investigate  the  molecular  mechanism  of  DNA  damage-inducihle 
functions  in  E.  aili.  Present  studies  deal  with  novel  genes  that  affect 
radiation-induced  mutagenesis  and  analysis  of  RecA  functions.  In 
addition,  we  have  been  developing  techniques  for  genomic  mapping 
and  collaborating  in  the  isolation  of  neuronal  genes  in  squid. 

Staff 

Sussman.  Raquel.  Associate  Scientist 

nwiiigators 

Gwen  Szent-Gyorgyi 

Berbenan.  Graciela.  Institute  de  Investigacion  Medica.  Cordoba. 
Argentina 

Molecular  Evolution  of  Genomes 

Research  in  this  laboratory  focuses  on  the  molecular  evolution  and 
gene  expression  in  the  bacterium  Eschmcliia  ct'li.  In  a  collaborative 
effort,  a  database  containing  information  on  the  intermediary 


metabolism  and  biochemical  pathways  off-  coli  is  being  developed. 
When  completed,  this  database  is  expected  to  contain  information  on 
each  metabolic  reaction,  the  enzyme,  the  reactants,  products, 
cofactors,  activators,  inhibitors,  kinetics,  equilibrium  constants, 
binding  constants,  etc. 

Related  research  is  on  the  evolution  of  the  E  coli  DNA  and 
organization  of  the  genes  in  the  chromosome.  Comparative  nucleotide 
and  amino  acid  sequence  data  provide  information  on  the 
evolutionary  relationships  of  E.  coli  genes  to  other  genes  in  the  E.  coli 
genome  and  to  homologous  genes  in  related  bacteria. 

Staff 

Riley.  Monica.  Senior  Scientist 
Pellegrino-Toole,  Alida,  Research  Assistant 

Molecular  Evolution  Program 

Molecular  biology  has  shifted  studies  of  early  evolution  and 
biodiversity  from  the  arena  of  hypothetical  scenario  to  one  of 
experimental  science.  The  comparison  of  genetic  elements  that  have 
been  transmitted  from  generation  to  generation  makes  possible  the 
measurement  of  genetic  differences  between  members  of  populations, 
species,  and  even  between  kingdoms  of  organisms.  These 
measurements  permit  inference  of  the  very  same  evolutionary 
framework  in  which  morphological  and  biochemical  differences 
among  organisms  arose  and  provides  a  practical  metric  to  assess 
biodiversitv 


R46  Annual  Report 


This  laboratory  relies  upon  structural  studies  of  ribosomal  RNAs 
and  actin  genes  to  measure  genetic  differences  between  divergent  taxa. 
As  a  result  our  understanding  of  the  universal  tree  of  life  is  very 
different  from  the  plant-animal  dichotomy  articulated  by  early 
systematists  or  today's  text  book  standard,  the  "Five  Kingdoms" 
(plants,  animals,  fungi,  protists  and  bacteria).  Instead  of  being 
relatively  recent  biological  inventions,  eukaryotes  appear  to  represent 
a  discrete  lineage  that  may  be  as  old  as  the  archaebacterial  and 
eubacterial  lines  of  descent.  The  earliest  branching  lineages  are 
represented  by  protists  that  lack  mitochondria  and  live  in  the  near 
absence  of  oxygen.  Compared  to  more  derived  eukaryotes,  these 
organisms  have  simple  cytoskeletons  and  membrane  networks  instead 
of  a  well-organized  Golgi  apparatus.  In  addition  to  the  early  branching 
patterns,  boundaries  separating  major  eukaryotic  groups  have  been 
redefined.  The  three  "higher"  kingdoms  (Fungi,  Plantae,  and 
Animalia)  are  now  joined  by  two  novel  complex  evolutionary 
assemblages,  the  "Alveolates"  (ciliates,  apicomplexans,  and 
dinoflagellates)  and  the  "Stramenopiles"  (oomycetes.  labyrinthulids, 
xanthophytes,  phaeophytes,  chrysophytes,  and  diatoms).  Both 
assemblages  include  numerous  marine  organisms  of  ecological  and 
economical  importance;  their  phenotypic  diversity  is  roughly 
equivalent  to  that  seen  in  the  other  eukaryotic  kingdoms.  More 
remarkably,  fungi  and  animals  must  have  shared  a  recent  common 
ancestry  exclusive  of  any  other  eukaryotic  groups. 

Recently  we  have  integrated  our  studies  of  rRNA  evolution  into 
microbial  ecology  and  biodiversity  projects.  We  have  demonstrated  a 
co-evolution  between  fungal  symbionts  and  leaf-cutting  ants  that  has 
lasted  for  more  than  80  million  years.  In  collaboration  with  the 
Ecosystems  Center  we  are  using  molecular  techniques  to  catalog 
prokaryotic  populations  in  Toolik  Lake  in  Alaska.  Other  efforts  are 
directed  towards  population  and  systematic  studies  of  brachiopods 
and  lobsters. 


the  added  advantage  of  measuring  the  net  flux  of  individual  ions  and, 
being  based  on  commercially  available  lonophores.  is  broadly 
applicable.  Further,  it  is  one  of  the  few  methods  available  for 
measuring  the  movements  of  ions  involved  in  non-electrogenic 
transport,  for  example  the  activity  of  pumps  and  porters. 

The  weak  voltages  associated  with  relatively  steady-state  currents 
reflect  numerous  aspects  of  cell  physiology  important  in  normal 
conditions.  Frequently  these  currents  are  perturbed  by  disease  or 
damage.  Our  current  applications  reflect  the  diversity  of  function. 
Single  cell  studies  are  numerous,  and  include  research  on  proton  and 
potassium-linked  regulation  in  oxyntic  cells  of  the  stomach  wall,  free- 
radical-induced  perturbation  of  neuronal  calcium  homeostasis,  2nd 
messenger  regulation  of  trans-membrane  ion  flux,  lead  toxicity  and 
developmental  currents  from  oocytes  and  embryos.  Ion  regulation  at 
the  tissue  and  organismal  level  are  also  being  studied,  particularly  with 
reference  to  the  regulation  of  the  brain  microenvironment,  hearing, 
and  tissue  regeneration. 

Two  new  systems  are  in  development.  The  first  is  the  vibrating 
BioKelvin  probe  which  will  measure  the  weak  fields  around  tissue  in  a 
gaseous  environment.  Our  specific  goal  is  to  use  this  new  machine  in 
the  study  of  skin.  Also  under  development  is  a  vibrating  oxygen  probe 
which  will  be  used  in  the  study  of  cell  respiration. 

Staff 

Jaffe,  Lionel  F.,  Director  Emeritus 

Smith,  Peter  J.  S.,  Director 

Hammar,  Katherine,  Research  Assistant 

Land,  Stephen  C.  Lakian  Fellow 

McLaughlin,  Jane  A.,  Research  Assistant 

Sanger,  Richard  H.,  Senior  Electronics  Technician 

Shipley,  Alan  M.,  Research  Associate 


Staff 

Sogin,  Mitchell  L.,  Director  and  Senior  Scientist 
Hinkle,  Greg,  Postdoctoral  Research  Associate 
Leipe,  Detlev,  Postdoctoral  Research  Associate 
Mormon,  Hilary  G..  Postdoctoral  Research  Associate 
Silberman.  Jeffrey,  Postdoctoral  Research  Associate 

Visiting  Investigator 

Barnhisel,  Rae,  Postdoctoral  Sloan  Fellow 

National  Vibrating  Probe  Facility 

This  Facility  develops  and  makes  available  techniques  for  the  non- 
invasive  measurement  of  trans-membrane  ion  flux.  Two  types  of 
systems  are  now  housed  in  the  Facility  for  general  use;  they  are  the 
non-invasive  voltage  probe  (NVPPD)  and  the  non-invasive  ion- 
selective  probe  (NVP,).  In  both  cases  the  vibration  of  the  probes 
results  in  a  highly  sensitive  self-referencing  electrode  with  vibration- 
coherent  signals  being  averaged  and  abstracted  from  noise.  Both 
techniques  are  primarily  utilized  in  the  study  of  steady-state  currents. 
The  NVPPD  is  the  more  sensitive,  measuring  nanovolt  fields  relating  to 
net  current  flow  across  membranes  of  tissues  and  cells.  The  NVP,  has 


Sabbatical  I  'isitors 

Kunkel,  Joseph  G.,  University  of  Massachusetts,  Amherst 
Ryan.  James,  Hobart  and  William  Smith  College 

Visiting  Investigators 

Allen.  Nina,  Wake  Forest 

Aloulou,  Amine,  Institut  National  Agronomique.  Paris-Gnguon 

Baikie,  Iain,  Aberdeen,  UK 

Brown.  Dennis.  Harvard  Medical  School/Mass  General  Hospital 

Demarest,  Jeffery,  Juniata  College 

Devlin,  Leah,  Penn  State  University 

Estee  Lauder  Co. 

Fishman.  Harvey,  University  of  Texas,  Galveston 

Hill,  Susan,  Michigan  State 

Hill,  Robert  B.,  University  of  Rhode  Island 

Huebner,  Erwin.  University  of  Manitoba,  Winnipeg 

Keefe,  David,  Yale  University  School  of  Medicine 

Leech.  Colin.  Harvard  Medical  School/Mass  General  Hospital 

Rahemtulla,  Firoz,  University  of  Alabama 

Tytell,  Michael,  Wake  Forest 

Wall,  Betty.  Independent  Investigator 

Yamoah.  Ebenezer,  Johns  Hopkins  University.  School  of  Medicine 


Honors 


Friday  Evening  Lectures 


Jon  Miller.  Chicago  Academy  of  Sciences.  June  24.  "The  Public  Understanding  of  Basic  Biomedical  Concepts" 

George  Langford.  Dartmouth  College.  July  1.  "Actm-Dependenl  Movement  ofOrganelles  in  the  Squid  Giant  Axon" 

Walter  Freeman.  University  of  California.  Berkeley.  July  8  (Lang  Lecturer).  "Some  Thoughts  on  a  Role  for  Music  in  the  Neurobiology  of 

Learning" 

Thomas  Eisner.  Cornell  University.  July  15.  "The  Hidden  I'ahie  of  Nature" 
Zach  Hall.  University  of  California.  San  Francisco.  July  21,  22  (Forbes  Lecturer).  "How  Nerves  Talk  to  Muscles  During  Synapse  Function:  the 

Role  ofAgrin "  and  "The  Nicolinic  Acetylcholine  Receptor:  Putting  it  all  Together" 

Carla  Shatz,  University  of  California.  Berkeley,  July  29  (Monsanto  Lecturer).  "Order  From  Disorder  in  \'isual  System  Development" 
Harold  Varmus.  National  Institutes  of  Health.  August  5  (Glassman  Lecturer).  "New  Direction  at  the  NIH" 

Stephen  O'Bnen.  National  Cancer  Institute.  August  1 2.  "Retracing  the  Natural  History  of  Endangered  Species:  Lessons  From  the  Big  Cats" 
Evelyn  Fox  Keller.  Massachusetts  Institute  of  Technology.  August  19.  "Gender.  Language,  and  Science" 


Fellowships  and  Scholarships 


In  1994,  the  MBL  awarded  research  fellowships  amounting  to  $156,922  to  17  scientists  from  around  the  world  who 
investigated  topics  ranging  from  the  physiological  regulation  of  muscle  contraction  to  the  biochemical  analysis  of 
ion  exchange  in  nerves  to  the  effect  of  nutrient  loading  on  fish  production  in  local  estuaries.  Scholarship  support 
amounting  to  $357,162  was  awarded  to  131  students  in  the  MBL's  six  summer  courses. 

Donors  who  made  a  gift  to  the  Fellowship  and  Scholarship  Programs  during  1994  are  noted  below.  Those 
individuals  who  received  fellowships  and  scholarships  follow. 


Robert  Day  Allen  Fellowship 

Drs.  Jean  M.  and  Joseph  W.  Sanger 

American  Society  for  Cell  Biology  Scholarships 

Dr.  Elizabeth  Marincola 

Frederik  B.  Bang  Fellowship  Fund 

Mrs.  Betsy  G.  Bang 

Charles  R.  Crane  Fellowship  Fund 

Ms.  Judith  E.  Casey 

The  Jean  and  katsuma  Dan  Fellowship  Fund 

Dr.  and  Mrs.  Teru  Hayashi 
Mrs.  Eleanor  Steinbach 


Bernard  Davis  Fellowship  and  Scholarships 

Mr.  and  Mrs.  Harold  Abrams 

Dr.  Porter  W.  Anderson.  Jr. 

Mrs.  Ann  S.  Butler 

Drs.  Herman  N.  Eisen  and  Natalie  A.  Aronson 

Mr.  and  Mrs.  Robert  H.  Silsbee 

Dr.  and  Mrs.  Edward  O.  Wilson 


Aline  D.  Gross  Scholarship  Fund 

Mr.  and  Mrs.  Alfred  Weisberg 

Ann  E.  ka miner  Memorial  Fellowship  Fund 

Mr.  Richard  M.  Eakin 
Ms.  Jean  G.  Malamud 
Ms.  Jane  E.  Schroeder 


R47 


R48   Annual  Report 


Stephen  W.  Kuffler  Fellowship 

Drs.  Clay  M.  and  Clara  F.  Armstrong 

Lakian  Postdoctoral  Scholar 

Lakian  Foundation 

Lakian  Summer  Fellowships 

Lakian  Foundation 

MBL  Research  Fellowships 

Dr.  and  Mrs.  Shinya  Inoue 
Dr.  and  Mrs.  J.  P.  Trinkaus 

James  A.  and  Faith  Miller  Fellowship  Fund 

Dr.  Gwynn  C.  Akin 

Drs.  Madeline  P.  and  William  D.  Burbanck 

Mr.  and  Mrs.  Hubert  W.  Burden 

Dr.  E.  Robert  Burns 

Prof,  and  Mrs.  Donald  Eugene  Copeland 

Mr.  and  Mrs.  James  L.  Culberson 

Dr.  and  Mrs.  Rolland  Golden 

Dr.  and  Mrs.  Richard  G.  Hibbs 

Mr.  and  Mrs.  Felix  Inigo 

Dr.  Kenneth  H.  Jones 

Mr.  and  Mrs.  Andrew  Lees 

Mrs.  Charles  Levie 

Ms.  Barbara  Baker  Louden 

Dr.  and  Mrs.  John  J.  Martinek 

Mr.  and  Mrs.  Dennis  J.  McLane 

Mr.  and  Mrs.  David  A.  Miller 

Mr.  and  Mrs.  Edward  A.  Miller 

Cdr.  and  Mrs.  James  P.  Miller 

Mr.  and  Mrs.  John  Oberpnller 

Dr.  John  E.  Pauly 

Dr.  and  Mrs.  Dwight  E.  Phillips 

Mr.  and  Mrs.  Richard  D.  Rink 

Dr.  and  Mrs.  S.  Meryl  Rose 

Ms.  Helen  M.  Rosenthal 

Ms.  Susan  L.  Rosenthal 

Dr.  and  Mrs.  Charles  M.  Roser.  Sr. 

Mr.  and  Mrs.  Herbert  Shanker 

Dr.  and  Mrs.  Philip  Sieg 

Mr.  Robert  G.  Summers,  Jr. 

Mr.  and  Mrs.  John  Valois 

Mr.  and  Mrs.  William  P.  Wood 

Dr.  and  Mrs.  Rizkalla  Zakhary 

Mountain  Memorial  Fund 

Mr.  and  Mrs.  Dean  C.  Allard.  Jr. 

Mr.  Scott  M.  Allard 

Dr.  Garland  E.  Allen 

Ms.  Brenda  J.  Bodian 

Ms.  Elinor  W.  Bodian 

Ms.  Helen  Bodian  &  Mr.  Roger  Alcaly 

Mr.  and  Mrs.  Donald  Carroll 

Ms.  Mildred  S.  Carson 

Mr.  and  Mrs.  Robert  W.  Cavenagh 

Dr.  and  Mrs.  Gary  S.  Cohen 

Mr.  and  Mrs.  Brewstcr  H.  Gere.  Jr. 


Mr.  and  Mrs.  R.  G.  Gillette 

Ms.  Elizabeth  J.  Goulett 

Dr.  and  Mrs.  Harlyn  O.  Halvorson 

Ms.  Lois  Hoffman 

Mr.  and  Mrs.  Alan  J.  Jacobsen 

Mrs.  Virginia  Stokes  Jones 

Dr.  and  Mrs.  Benjamin  Rammer 

Ms.  Anne  C.  Kimball 

Mr.  Kenneth  H.  Lange 

Ms.  Charlotte  Z.  LeMay 

Ms.  Kathryn  H.  Miller 

Mr.  and  Mrs.  James  E.  Milligan 

Dr.  Isabel  Mountain 

Ms.  Helen  T.  North 

The  Grace  Jones  Richardson  Trust 

Mr.  and  Mrs.  Thomas  H.  Roberts 

Mr.  and  Mrs.  Henry  J.  Rose 

Dr.  Joel  L.  Rosenbaum 

Dr.  and  Mrs.  Robert  G.  Savarese 

Dr.  and  Mrs.  R.  Walter  Schlesinger 

Mr.  and  Mrs.  Herbert  G.  Sparrow 

Mr.  and  Mrs.  John  W.  Stewart 

Dr.  and  Mrs.  William  N.  Thomas 

Mrs.  Roberta  Tracy 

Mr.  and  Mrs.  George  E.  Webster 

Mr.  and  Mrs.  Andrew  Yen 


Nikon  Fellowship 


Nikon.  Inc. 


Science  Writing  Fellowships  Program 

The  American  Academy  of  Neurology 

The  American  Association  of  Immunologists 

The  American  Federation  for  Clinical  Research 

The  American  Society  for  Biochemistry  and  Molecular  Biology 

The  American  Society  for  Cell  Biology 

The  American  Society  for  Investigative  Pathology 

American  Society  for  Microbiology 

The  American  Society  for  Neurochemistry,  Inc. 

Association  of  Anatomy,  Cell  Biology,  and  Neurobiology 

Chairpersons 

Association  for  Research  in  Vision  and  Ophthalmology 
Burroughs  Wellcome  Fund 
Foundation  for  Microbiology 
Friendship  Fund 

John  S.  and  James  L.  Knight  Foundation 
Merck  &  Company,  Inc. 
Society  for  Neuroscience 

He  uiv  <//MI  grateful  to  the  Charles  A  Dana  Foundation  lor  its 
continued  support  of  this  program. 


The  Moshe  Shilo  Memorial  Scholarship  Fund 

Dr.  and  Mrs.  Harlyn  O.  Halvorson 
Dr.  and  Mrs.  J.  Woodland  Hastings 
Dr.  and  Mrs.  Laszlo  Lorand 


Howard  A.  Schneiderman  Scholarship 

Mrs.  Howard  Schneiderman 


Honors  R49 


The  Evelyn  and  Melvin  Spiegel  Fellowship  Fund 

Drs.  Jean  and  Joseph  W.  Sanger 
Drs.  Evelyn  and  Melvin  Spiegel 

H.  B.  Steinbach  Fellowship 

Mrs.  H.  Burr  Steinbach 

Mr.  and  Mrs.  Volker  Ulbrich 


Marjorie  \V.  Stetten  Scholarship  Fund 

Dr.  and  Mrs.  W.  Redwood  Wright 

Horace  W.  Stunkard  Fellowship 

Dr.  and  Mrs.  Albert  J.  Stunkard 


Mrs.  Marian  Rigaumont 
Dr.  Jean  Wilson 
Mr.  and  Mrs.  Ross  A.  Wilson 
Mr.  and  Mrs.  Wavne  V.  Wilson 


Young  Scholars/Fellows  Program 

Dr.  Frank  M.  Child,  III 

Mr.  and  Mrs.  Leonard  D.  Friedman 

Dr.  and  Mrs.  Laszlo  Lorand 

Dr.  and  Mrs.  Jerry  M.  Melillo 

Mr.  and  Mrs.  John  R.  Peterson 


Philip  H.  Presley  Memorial  Scholarships 

Carl  Zeiss.  Inc. 


The  Walter  L.  Wilson  Endowed  Scholarship  Fund 

Mr.  and  Mrs.  Lam,  McLean 
Mr.  and  Mrs.  Raymond  Rapaport 


Post-Course  Research  Support  Provided  by 

Carl  Zeiss  Inc. 

Universal  Imaging  Corporation 


Fellowships  Awarded 


MBL  Summer  Research  Fellows 


•  Joshua  R.  Berlin,  a  Lakian  Research  Fellow,  is  a  Research 
Scientist  at  the  Bockus  Research  Institute  in  Philadelphia.  PA.  Berlin 
looked  at  local  calcium  transients  during  muscle  excitation- 
contraction  coupling. 

•  Graciela  Elso  de  Berbenan.  a  Frederik  B  Bang  Fellow,  is  a 
Research  Associate  at  the  Institute  M  y  M.  Ferreyra.  Argentina.  Elso 
de  Berbenan  studied  the  biochemical  characterization  of  Na+/Ca+* 
exchanger  in  nerve  cells. 

•  Mariano  Garcia-Blanco.  a  H  'illiain  Townsend  Porter  Fellow. 
is  an  Assistant  Professor  at  Duke  University  Medical  Center.  Garcia- 
Blanco  examined  rRNA  trafficking. 

•  Fatima  Gyoeva,  funded  by  the  Lucy  B  Lemann  Fellowship 
Fund  ant!  the  MBl.  Research  Fellowship  Fund,  is  an  investigator  at 
the  Russian  Academy  of  Sciences  Institute  of  Protein  Research. 
Gyoeva  used  biochemical  and  immunochemical  methods  to  identify 
kinesin  receptors  in  fish  melanophores. 

•  Valerie  A.  Hall,  supported  by  the  Frank  A  Brown  Memorial 
Fund  and  the  John  O  Crane  Fellowship  Fund,  is  a  high  school  teacher 
in  Nantucket.  MA.  Hall  spent  her  summer  writing  a  high  school 
oceanography/marine  biology  textbook. 

•  Jonathan  J.  Henry,  an  MBL  Associates  Fellow,  is  an  Assistant 
Professor  at  the  University  of  Illinois.  Henry  analyzed  cell  lineage  of 
the  nemertean,  Cerebratuhts  lacleau.  Specifically,  he  considered  the 
evolution  of  spiralian  development. 

•  Mary  Constance  Lane,  supported  by  the  Evelyn  and  Melvin 
Spiegel  Fellowship  Fund,  the  H.  B.  Steinbach  Fellowship  Fund,  the 
MBL  Associates  Fellowship  Fund,  the  James  A.  and  Faith  Mi  Her 
Fellowship  Fund,  and  the  MBL  Research  Fellowship  Fund,  is  a 
Postdoctoral  Fellow  from  the  University  of  California,  Berkeley.  Lane 
looked  at  the  role  of  microtubules  in  morphogenesis. 

•  William  Lemon,  a  Lakian  Research  Fellow,  is  a  Postdoctoral 
Fellow  from  the  University  of  Arizona.  Lemon  optically  recorded 
magnetically  induced  neural  activity  in  the  brain  of  the  honeybee, 
Apis  mellifera. 


•  Vladimir  I.  Makarenko.  a  James  S  McDonnell  Foundation 
Fellow,  is  a  Physicist  from  the  Russian  Academy  of  Sciences  Institute 
of  Mathematical  Problems  of  Biology.  Laboratory  of  Neural  Networks 
in  Russia.  While  at  the  MBL.  Makarenko  used  computers  to  model 
neural  networks. 

•  Anthony  Maranto,  supported  by  the  Frank  R  Li/lie  and  the 
MBL  Fellowship  Funds,  is  an  Assistant  Professor  at  Tufts  University 
Medical  School.  Maranto  examined  the  characterization  and  function 
of  biochemical  receptors  in  surf  clams  and  sea  urchins. 

•  Stephen  Moorman,  funded  by  the  Stephen  II'  Kuffler  and  the 
Lucy  B  Lemann  Fellowship  Funds,  is  an  Assistant  Professor  at  the 
UNT  Health  Science  Center  at  Fort  Worth,  TX.  Moorman  studied 
oligodendrocyte  interactions  during  development. 

•  Andrew  Murray,  a  Nikon  Inc.  Fellow,  is  an  Assistant 
Professor  at  the  University  of  California.  San  Francisco.  Murray  used 
high  resolution  microscopy  to  study  mitosis  in  extracts  from  frog  eggs 
and  yeast. 

•  Rnud  Nierhaus,  funded  by  the  MBL  Associates  and  the 
Herbert  H"  Rand  Fellowship  Funds,  is  a  Professor  at  the  Max  Planck 
Institute  for  Molecular  Genetics  in  Germany.  He  worked  on  his  book, 
Translation  of  the  Genetic  Message,  which  deals  with  protein 
biosynthesis. 

•  Haohua  Qian,  a  Bernard  Davis  Fellow,  is  a  Postdoctoral 
Fellow  at  Harvard  University.  Qian,  a  neurobiologist.  looked  at  the 
properties  of  GABA  receptors  on  the  glial  cells  of  the  skate  retina. 

•  Edward  Salmon,  a  Herbert  M'.  Rand  Fellow,  is  a  Professor  at 
the  University  of  North  Carolina.  Chapel  Hill.  Salmon,  a  cell 
biologist,  used  high  resolution  microscopy  to  study  mitosis  in  extracts 
of  frog  eggs  and  yeast. 

•  Rafael  Sarda,  an  MBL  Associates  Fellow,  is  a  scientist  at  the 
Centra  de  Estudios  Avanzados  de  Blanes.  Spain.  Sarda.  an  ecologist. 
studied  nutrient  loading  in  the  watersheds  of  Waquoit  Bay. 
Specifically,  he  looked  at  the  effect  of  nutrient  loading  on  benthic 
invertebrate  assemblages. 


R50  Annual  Report 


•  Cristina  Zago,  an  MBL  Associates  Fellow,  is  a  researcher  at 
the  Institute  per  lo  Studio  della  Dinamica  delle  Grandi  Masse  in 
Venice.  Italy.  Zago  analyzed  heavy  metal  distribution  in  porewaters 
using  various  chemical  equilibrium  computer  models. 

Grass  Fellows 

•  Yang  Dan,  Columbia  University.  Dan  engaged  in  optical 
studies  of  electrical  activity  in  a  circuit  of  neurons. 

•  Graeme  W.  Davis,  University  of  Massachusetts,  Amherst. 
Davis  examined  functional  synaptic  specificity  and  the  activity  of  the 
calcium-activated  potassium  channel  at  the  Drosophila  neuromuscular 
junction. 

•  Guoping  Feng,  State  University  of  New  York,  Buffalo.  Feng 
performed  a  functional  analysis  of  tipE,  a  mutation  affecting  sodium 
channels  in  Drosophila. 

•  Elizabeth  Ann  Finch,  Harvard  Medical  School.  Finch  studied 
molecular  mechanisms  of  synaptic  plasticity. 

•  Miguel  Holmgren,  Chicago  Medical  School.  Holmgren 
examined  charge  translocation  by  the  NA+/K+  pump  in  internally 
perfused  squid  giant  axon. 

•  Rebecca  M.  Johnston,  University  of  Arizona.  Johnston 
looked  at  the  developmental  fate  and  modulation  of  rhythmic  motor 
patterns  in  the  hawkmoth.  Manduca  se.xla. 

•  Lucas  D.  Pozzo  Miller,  Roche  Institute  of  Molecular  Biology. 
Miller  examined  integrative  properties  of  the  first  order  giant  neurons 
from  the  ganglion  magnocellularis  of  the  squid,  Loligo  pealei. 

•  Lisa  K.  Moore,  Albert  Einstein  College  of  Medicine.  Moore 
studied  horizontal  cell  gap  junctional  communication  in  teleost  retina, 
and,  specifically,  modulation  by  retinoic  acid. 


•  Felix  E.  Schweizer.  Stanford  University  Medical  Center. 
Schweizer  examined  the  role  of  interactions  between  a-SNAP  and 
synaptotagmm/p65  for  neurotransmitter  release  at  the  giant  synapse  of 
the  squid.  Loligo  pealei. 

•  Andrew  A.  Sharp,  Brandeis  University.  Sharp  studied  the  role 
of  synaptic  inhibition  and  Ih  in  controlling  the  heartbeat  oscillator  in 
the  leech,  llinulo  medicinalis. 

•  Eric  C.  Sobel.  AT&T  Bell  Laboratories.  Sobel  looked  at  the 
anatomical  and  physiological  characterization  of  visual  space 
constancy  neurons  in  the  crayfish. 

•  Ebenezer  N.  Yamoah,  University  of  Texas  HSC  at  Houston. 
Yamoah  examined  calcium  fluxes  in  the  photoreceptors  and  cultured 
neurons  of  the  sea  slug,  Hermissenda  crassicorni.i. 

Science  Writing  Fellows 

Pallava  Bagla,  Publications  &  Information  Directorate,  India 

Yvonne  Baskin,  Freelance 

Cara  Bimttieri.  New  England  Cable  News 

Giovanna  Breu,  People  magazine 

Matthew  Crenson.  Dallas  Morning  News 

Dian  Duthie,  CBC-TV  Newsday.  Canada 

Alberto  Enriquez,  Mail  Tribune 

S.  Paul  Gasek.  Stony  Brook  Films 

Scott  LaFee,  San  Diego  Union-Tribune 

Ed  Regis.  Freelance 

Richard  Saltus,  The  Boston  Globe 

Richard  Stone,  Science  magazine 

Mutsumi  Yoshida,  Newton  magazine.  Japan 

David  Zimmerman,  PROBE 


Scholarships  Awarded 


Bernard  Davis  Scholarship  Fund 

Michael  Ceno,  University  of  Connecticut 
Ilka  Faath,  University  of  Bonn,  Germany 
Lite  Muh.  Philhpps-Universitat  Marburg.  Germany 


Daniel  S.  Grosch  Scholarship  Fund 

Paula  van  Schie,  Rutgers  LIniversity 

Frank  R.  Lillie  Scholarship  Fund 

Smaranda  Burlacu,  University  of  Texas 
Martin  Garcia-Castro,  Wellcome/CRC  Institute,  UK 
Akemi  Hanamura,  Cold  Spring  Harbor  Laboratory 
Ole  Kjaerulff,  Copenhagen  University,  Denmark 

William  Townsend  Porter  Scholarship  Fund 

Max  Boakye,  National  Institutes  of  Health 

ShaAvhree  Buckman,  Washington  University  School  of  Medicine 

Ana  DePina.  Dartmouth  College 

Mildred  Morales,  Albert  Einstein  College  of  Medicine 

Wendy  Reed,  Johns  Hopkins  University 

James  Scott.  Center  for  Great  Lakes  Studies 

Andrea  Torres-Perez,  Stanford  LIniversity 

Madeline  Vargas,  University  of  Connecticut 


Herbert  W.  Rand  Scholarship  Fund 

Ruth  Empson.  University  of  Koln.  Germany 

Martin  Garcia-Castro.  Wellcome/CRC  Institute,  UK 

Patricio  Huerta.  Brandeis  University 

Boris  Kablar,  University  of  Pisa,  Italy 

Ole  Kjaerulff,  Copenhagen  University,  Denmark 

Peter  Kloppenburg,  LIniversity  of  Arizona 

Oana  Marcu,  University  of  Western  Ontario,  Canada 

Christophe  Pouzat,  Ecole  Normale  Superieure,  France 

Fabrice  Roegiers,  Station  Zoologique,  France 

Eric  Scarfone,  University  of  Montpellier.  France 

Dinesh  Vernool.  Rutgers  University 

Karen  Yeow.  University  of  Manitoba.  Canada 

American  Society  for  Cell  Biology  Scholarships 

ShaAvhree  Buckman.  Washington  LIniversity  School  of  Medicine 

Ana  DePina,  Dartmouth  College 

Mildred  Morales,  Albert  Einstein  College  of  Medicine 

Wendy  Reed,  Johns  Hopkins  University 

James  Scott,  Center  for  Great  Lakes  Studies 

Andrea  Torres-Perez.  Stanford  University 

Madeline  Vargas,  University  of  Connecticut 

Biology  Club  of  the  City  of  New  York  Scholarship  Fund 

Sowmyalakshmi  Rasika,  Rockefeller  LIniversity 


Honors   R51 


Father  Arsenius  Boyer  Scholarship  Fund 

Akemi  Hanamura.  Cold  Spring  Harbor  Laboratory 
Stefan  Schuster,  Max  Plank  Institute.  Germans 


C.  Lalor  Burdick  Scholarship  Fund 

Boris  Kablar.  University  of  Pisa.  Italy 

Gary  N.  Calkins  Scholarship  Fund 

Melchiorre  Cervello,  Institute  di  Biologia  dello  Sviluppo  del 
Consiglio.  Italy 

Frances  S.  Claff  Scholarship  Fund 

Melchioore  Cervello.  Institute  di  Biologia  della  Sviluppo  del 
Consiglio.  Italy 

Edwin  Grant  Conklin  Scholarship  Fund 

Melchiorre  Cervello,  Institute  di  Biologia  della  Sviluppo  del 

Consiglio,  Italy 
Daniel  Martinez.  University  of  California.  Irvine 

Lucretia  Crocker  Scholarship  Fund 

Peter  KJoppenburg.  University  of  Arizona 
Adrian  Salic,  Harvard  University 

\Villiam  F.  and  Irene  C.  Diller  Scholarship  Fund 

Xiaohua  Gong.  Scnpps  Research  Institute 
Boris  Kablar.  University  of  Pisa.  Italy 

Caswell  Grave  Scholarship  Fund 

Frederic  Ampe,  Laboratoire  de  Physiologie,  INSA.  France 
Max  Boakye.  National  Institutes  of  Health 
Ruth  Empson,  University  of  Koln.  Germany 
Paula  van  Schie,  Rutgers  University 

Aline  D.  Gross  Scholarship  Fund 

Ruth  Empson.  University  of  Koln.  Germany 
Sowmyalakshmi  Rasika.  Rockefeller  University 

William  Randolph  Hearst  Educational  Endowment 
Scholarships 

John  Allison.  Vanderbilt  Medical  School 
Catherine  Brennan.  University  ol  Southern  California 
Dawn  Konrad.  University  of  Washington 
Elizabeth  Laxson,  University  of  Wisconsin.  Madison 
Chnsti  Magrath.  Tulane  University  Medical  Center 
Sarah  McHatton.  University  of  California,  Davis 
Julie  Olson.  University  of  North  Carolina 
Debra  Tumbula,  University  of  Georgia 


Howard  Hughes  Medical  Institute  Educational  Program 
Scholarship  Funding 

Mark  DeSouza.  University  of  Southern  California 
Patncio  Huerta.  Brandeis  University 


Radma  Mahmood,  Guy's  Hospital.  UK 
Sandra  Marques.  George  Washington  University 
Annette  Neubuser.  Max  Planck  Institute.  Germany 
Sandra  Nicola,  Carol  Davila  University  of  Medicine 
Christophe  Pouzat.  Ecole  Normale  Superieure,  France 
Manisha  Raje,  University  of  Kansas 
Stefan  Schuster.  Max  Planck  Institute.  Germany 
Shiri  Venezia.  Tel  Aviv  University,  Israel 
James  Walker,  University  of  Cambridge,  UK 

Merkel  H.  Jacobs  Scholarship  Fund 

James  Walker,  University  of  Cambridge,  UK 

Arthur  Klorfein  Scholarship  Fund 

Anna  Di  Gregorio.  Anton  Dohrn  Stazione  Zoologica.  Italy 
Martin  Garcia-Castro,  Wellcome/CRC  Institute.  UK 
Xiaohua  Gong,  Scripps  Research  Institute 
Daniel  Martinez,  University  of  California.  Irvine 
Stefan  Schuster.  Max  Planck  Institute,  Germany 

Jacques  Loeb  Founders'  Scholarship  Fund 

Luis  Vidali,  University  of  Massachusetts.  Amherst 

John  D.  and  Catherine  T.  MacArthur  Foundation 
Scholarships 

Billy  Apola,  National  Museums  of  Kenya,  Kenya 

Hani  Atamna.  Hebrew  University,  Israel 

Leonard  Basco,  University  of  Paris,  France 

Socrates  Herrera  Valencia,  Universidad  del  Valle,  Columbia 

Laura  Knoll.  Washington  University 

Susan  Little.  University  of  Georgia 

Stephen  Manale.  Louisiana  State  University 

Deborah  Schechtman,  Weizmann  Institute  of  Science,  Israel 

Andrea  Smith,  University  of  Alabama 

Martine  Soete,  INSERM.  France 

Andrea  Torres-Perez,  Stanford  University 

Henri  van  der  Heyde,  University  of  Wisconsin,  Madison 

Fred  van  Leeuwen.  Netherlands  Cancer  Institute 

Ulrike  Zelck,  University  of  Hamburg,  Germany 

Xiaonong  Zhou.  Jiangsu  Institute  of  Parasitic  Diseases.  China 

S.  O.  Mast  Memorial  Fund  Scholarships 

Amanda  Hayward-Lester,  Texas  Tech  University 
Luis  Vidali,  University  of  Massachusetts,  Amherst 

Allen  R.  Memhard  Memorial  Fund  Scholarships 

Melchiorre  Cervello.  Institute  di  Biologia  dello  Sviluppo  del 
Consiglio,  Italy 

Michigan  State  University  Center  for  Microbial 
Ecology  Scholarship 

James  Scott,  Center  for  Great  Lakes  Studies 

James  S.  Mountain  Memorial  Fund,  Inc.  Scholarships 

Robert  Grant,  University  of  California,  San  Francisco 
Amanda  Havvvard-Lester.  Texas  Tech  Universitv 


R52  Annual  Report 


Peter  Piepenhagen,  Stanford  University 

Samara  Reek-Peterson,  University  of  Pennsylvania 

Jennifer  Smith-Hall.  Indiana  University  School  of  Medicine 


Planetary  Biology  Institute  Scholarships 

Mary  Rothermich.  University  of  Massachusetts 

Marc  van  der  Maarel,  University  of  Groningen,  Germany 

Society  for  Developmental  Biology  Scholarships 

Julie  Kuhlman.  Cornell  Medical  College 
Elizabeth  Laxson,  University  of  Wisconsin,  Madison 
Ivan  Moskowitz,  University  of  Wisconsin,  Madison 
Kevin  Peterson,  University  of  California,  Los  Angeles 
Melissa  Shirley,  Case  Western  Reserve  University 


Society  of  General  Physiologists  Scholarships 

1993 

Joshua  Gold.  Stanford  University 

Richard  J.  Kollman.  University  of  Texas  Southwestern  Medical 

Center 

Richard  Mullins,  University  of  Kentucky 
Christopher  Rose.  Harvard  University 


1994 

Michael  S.  Fee,  AT  &  T  Bell  Laboratories 

Martin  Garcia-Castro.  University  of  Cambridge.  U.K. 

Frederick  B.  Reitz.  University  of  Washington 

Karel  Svoboda,  AT  &  T  Bell  Laboratories 

Moshe  Shilo  Memorial  Scholarship  Fund 

Shiri  Venezia.  Tel  Aviv  University,  Israel 

Marjorie  W.  Stetten  Scholarship  Fund 

Michael  Cerio,  University  of  Connecticut 
Stefan  Schuster,  Max  Planck  Institute,  Germany 

William  Morton  Wheeler  Family  Founders' 
Scholarships 

Max  Boakye.  National  Institutes  of  Health 
Christophe  Pouzat,  Ecole  Normale  Superieure.  France 

Philip  H.  Presley  Memorial  Scholarships 

Maneesha  Inamdar,  Tata  Institute  of  Fundamental  Research,  India 

Teresa  Nick,  Yale  University 

Antonius  Suwanto,  Bogor  Agricultural  University 


Board  of  Trustees 
and  Committees 


Corporation  Officers  and  Trustees 


Chairman  of  the  Board  of  Trustees.  Sheldon  J.  Segal.  The  Population 

Council.  New  York.  NY 
Vice  Chairman  of  the  Board  of  Trustees.  Robert  E.  Mainer,  The 

Boston  Company.  Boston.  MA 
President  of  the  Corporation.  James  D.  Ebert,  Johns  Hopkins 

University.  Baltimore.  MD 
Director  and  Chief  Executive  Officer.  John  E.  Burns.  Marine 

Biological  Laboratory.  Woods  Hole.  MA* 
Chair  of  the  Science  Council.  George  M.  Langford.  Dartmouth 

College,  Hanover.  NH* 

Treasurer.  Robert  D.  Manz.  Helmer  &  Associates,  Waltham.  MA* 
Clerk  of  the  Corporation.  Neil  Jacobs.  Hale  and  Dorr,  Boston,  MA 

Class  of  1998 

John  R.  Lakian.  The  Fort  Hill  Group.  Inc. 
Joan  V.  Ruderman.  Harvard  Medical  School 
Sheldon  J.  Segal.  The  Population  Council 
Alfred  Zeien.  The  Gillette  Company 

Class  of  1997 

Frederick  Bay.  Josephine  Bay  Paul  and  C.  Michael  Paul  Foundation.  Inc. 

Martha  W.  Cox.  Hobe  Sound.  FL 

Mary  J.  Greer.  Cambridge.  MA 

Thomas  D.  Pollard.  John  Hopkins  Medical  School 

William  C.  Steere.  Jr..  Pfizer  Inc. 

Gerald  Weissmann,  New  York  University  School  of  Medicine 

Class  of  1996 

Norman  Bernstein.  Norman  Bernstein  Management.  Inc. 

Alexander  W.  Clowes.  University  of  Washington  School  of  Medicine 

Eric  H.  Davidson.  California  Institute  of  Technology 

Robert  D.  Goldman.  Northwestern  University  Medical  School 

Paul  A.  Marks.  Memorial  Sloan-Kettenng  Cancer  Center 

Irving  W.  Rabb,  Stop  &  Shop  Company — retired 


*  E.\  ot/icin 


Class  of  1995 

Mary-Ellen  Cunningham.  Grosse  Pointe  Farms,  MI 
Neil  Jacobs.  Hale  and  Dorr 
Edward  A.  Kravitz,  Harvard  Medical  School 
Robert  E.  Mainer.  The  Boston  Company 


Honorary  Trustees 

William  T.  Golden,  New  York,  NY 
Ellen  R.  Grass.  The  Grass  Foundation 
Homer  P.  Smith.  Woods  Hole.  MAt 


Emeriti 

Edward  A.  Adelberg,  Yale  University.  New  Haven,  CT 

John  B.  Buck.  Sykesville.  MD 

Seymour  S.  Cohen.  Woods  Hole,  MA 

Arthur  L.  Colwin,  Key  Biscayne,  FL 

Laura  Hunter  Colwin.  Key  Biscayne.  FL 

D.  Eugene  Copeland.  Marine  Biological  Laboratory.  Woods  Hole.  MA 

Sears  Crowell.  Indiana  University.  Bloomington.  IN 

Alexander  T.  Daignault.  Boston,  MA 

Teru  Hayashi.  Woods  Hole.  MA 

Ruth  Hubbard,  Cambridge,  MA 

Lewis  KJeinholz.  Reed  College,  Portland,  OR 

Maurice  E.  Krahl.  Tucson.  AZ 

Charles  B.  Metz.  Miami.  FL 

Keith  R.  Porter,  University  of  Pennsylvania,  Philadelphia,  PA 

C.  Ladd  Prosser.  University  of  Illinois.  Urbana.  IL 
S.  Meryl  Rose.  Waquoit,  MAt 

W.  D.  Russell-Hunter.  Syracuse  University.  Syracuse.  NY 

John  W.  Saunders.  Jr..  Waquoit.  MA 

Man  Sears.  Woods  Hole.  MA 

David  Shepro,  Boston  University.  Boston,  MA 

D.  Thomas  Tngg,  Wellesley,  MA 
Walter  S.  Vincent.  Woods  Hole.  MA 
George  Wald.  Cambridge.  MA 

Science  Council 

George  M.  Langford.  Chairman 
Donald  A.  Abt 
John  E.  Bums* 


R53 


R54   Annual  Report 


Ronald  L.  Calabrese 

Neal  W.  Cornell  (thru  8/94) 

Barbara  E.  Ehrlich  (thru  8/94) 

John  E.  Hobbie  (thru  8/94) 

Shinya  Inoue 

Irwin  B.  Levitan 

Knute  Nadelhoffer  (from  8/94) 

Robert  E.  Palazzo 

Robert  B.  Silver  (from  8/94) 

Mitchell  Sogin  (from  8/94) 

Ann  E.  Stuart 


Executive  Committee  of  the  Board  of  Trustees 

Sheldon  J.  Segal.  Chairman 

Frederick  Bay 

John  E.  Bums* 

Mary-Ellen  Cunningham 

Robert  D.  Goldman 

George  Langford* 

Robert  E.  Mainer,  Vice-Chairman 

Robert  Manz* 

Thomas  D.  Pollard 

Gerald  Weissmann 


Standing  Committees  of  the  Board  of  Trustees 


Development 

Man-Ellen  Cunningham,  Chair 

Robert  Barlow 

Fred  Bay 

Martha  Cox 

James  Ebert 

Neil  Jacobs 

John  Lakian 

Franklin  Loew 

William  Speck 

William  Steere 


Finance  and  Investment 

Robert  Mainer.  Chairman 
Norman  Bernstein 
Alexander  Clowes 
Eric  Davidson 
Donald  DeHart 
Neil  Jacobs 


John  Lakian 
Werner  Lowenstem 
Robert  Manz 
Irving  Rabb 

Nominating 

Gerald  Weissmann.  Chairman 

Alexander  Clowes 

Martha  Cox 

Mary-Ellen  Cunningham 

Mary  Greer 

George  Langford 

Thomas  Pollard 

Sheldon  Segal 

William  Steere 

Facilities  and  Capital  Equipment 

Robert  Goldman,  Chairman 
Jelle  Atema 


Al  Chaet 
Frank  Loew 
Jerry  Melillo 
Joan  Ruderman 
Robert  Silver 


Long-Range  Planning 

Fred  Bay,  Co-Chair 
Thomas  Pollard.  Co-Chair 
Eric  Davidson 
John  Dowling 
Gerald  Fischbach 
Robert  Goldman 
Mary  Greer 
George  Langford 
Robert  Manz 
Joan  Ruderman 
Mitchell  Sogin 


Corporation  Standing  Committees 


Buildings  &  Grounds 

Alfred  B.  Chaet,  Chairman 
Barbara  Boyer 
Lawrence  B.  Cohen 
Richard  D.  Cutler* 
William  Eckberg 
Barry  Fleet* 
Ferenc  Harosi 
Joe  Hayes* 
Kenyon  Tweedel 

Fellowships 

Thoru  Pederson,  Chairman 
Kathleen  Dunlap 
John  Rummel* 

*  Ex  officio 


Ann  Giblin 
Jose  Lemos 
Eduardo  Macagno 
Carol  L.  Reinisch 


Housing,  Food  Service, 
and  Child  Care 

Stephen  Highstein,  Chairman 

Elaine  Bearer 

Donald  C.  Chang 

Milton  Charlton 

Richard  Cutler* 

Robert  Gould 

Stephen  Highstein 

LouAnn  King* 

Dar\l  Stokes 


Education 

Ronald  L.  Calabrese.  Co-Chair 
Irwin  Levitan,  Co-Chair 
Elaine  Bearer 
Donanne  Chrysler* 
Vincent  Dionne 
Janet  Heasman 
Holger  W.  Jannasch 
Michael  E.  Mendelsohn 
John  D.  Rummel* 
Steven  J.  Zottoli 

MBL/HHOI  Library'  Joint 
Advisory 

David  Shepro,  Chair 
Judith  Ashmore,  MBL* 
Susan  Berteaux,  WHOI* 


Trustees  and  Committees  R55 


Henry  Dick,  WHOI 

Kevin  Friedland.  NMFS 

Steve  Gegg.  WHOI* 

John  Hobbie.  MBL 

Mark  Kurz,  WHOI 

Catherine  Norton,  MBL/WHOI* 

Monica  Riley,  MBL 

Jim  Robb,  USGS 

Peter  J.  S.  Smith.  MBL 

Bruce  Warren.  WHOI 


E.\  officio 


Research  Services 

Peter  Armstrong,  Chairman 

Neal  Cornell 

Richard  Cutler* 

Kenneth  Foreman 

Louis  Kerr 

William  Kuhns 

Andy  Mattox* 

James  Quigley 

John  Rummel* 

Peter  J.  S.  Smith 

Paul  Steudler 

Michael  Tvtell 


Research  Space 

Hans  Laufer,  Chairman 
Paul  DeWeer 
David  Landowne 
Eduardo  Macagno 
Andy  Mattox* 
Jem,  M.  Melillo 
Robert  Silver 
Steven  Treistman 
Ivan  Valiela 
Richard  Vallee 


Laboratory  Support  Staff : 


Biological  Bulletin 

Clapp,  Pamela  L..  Managing  Editor 

Gibson,  Victoria  R. 

McCaffrey.  Karen 

Controller's  Office 
Speer,  John  W.,  Controller 

Accounting  Services 
Afonso,  Janis  E. 
Binda,  Ellen  F 
Campbell,  Ruth  B. 
Davis,  Dons  C. 
Ghetti,  Pamela  M. 
Gilmore,  Mary  F. 
Hobbs,  Roger  W..  Jr. 
Lancaster,  Cindy 
Poravas,  Maria 

Chem  Room 
Schorer.  Timothy  M. 
Shepherd.  Denise  M. 

Purchasing 

Hall,  Lionel  E..  Jr. 

Shepherd.  Denise  M. 

Director 's  Office 

Burns.  John  E..  Director  and  CEO 

Catania.  Didia 

Kaufmann,  Sandra  J. 

Nelson,  Mary  F. 

Leighton,  Jane  L. 


*  Including  persons  »:lm  joined  or  kit  the 
staff  during  1994, 


External  Affairs 
Carotenuto.  Frank  C..  Director 

Aspinwall,  Duncan  P. 

Berthel,  Dorothy 

Black,  Nancy  O. 

Faxon.  Wendy  P. 

Lessard,  Kelley  J. 

Nelson,  Mary  F. 

Associates  Program 
Armstrong.  Ellen  P..  Liaison 
Broun.  M.  Kathryn  S..  Gift  Shop 

Communications  Office 
Clapp.  Pamela  L..  Director 
Liles.  Beth  R. 
Moorhouse.  Laura  A. 

Housing 

King.  LouAnn  D.,  Conference  Center  and 
Housing  Manager 

Barry,  Maureen  J. 

Johnson,  Frances  N. 

Telephone  Office 
Baker.  Ida  M. 
Geggatt.  Agnes  L. 
Mayne.  Pamela 
Ridley.  Alberta  W. 

Human  Resources 
Goux.  Susan  P.,  Manager 
Donovan.  Marcia  H. 

Journal  of  Membrane  Biology 
Loewenstem.  Werner  R.,  Editor 

Fay,  Catherine  H. 

Howard,  Linda  L. 

Lynch.  Kathleen  F. 


MBL/U'HOI  Library 

Stonehill,  David  L.,  Director 
Norton,  Catherine  N.,  Head  Librarian 

Ashmore.  Judith  A. 

Costa.  Marguerite  E. 

Drury,  Eulalie  A. 

Mirra,  Anthony  J. 

Nelson.  Heidi 

Pratson,  Patncia  G. 

deVeer,  Joseph  M. 

Zuwallack,  Barbara  A. 

Zuwallack,  Raymond  J. 

Zuwallack,  Ronald  L. 


Copy  Service  Center 

Mountford.  Rebecca  J..  Supervisor 

Barry.  Maureen  J. 

Jesse.  Martha  V. 

LaPlante,  Robert  F. 

Mancini.  Mary  E. 

Tebeau,  Christopher 

I ' it/in 'ination  Systems  Division 

Norton,  Catherine  N.,  Director 

Hamre,  Lynne 

Remsen.  David 

Renna,  Denis  J. 

Space,  David  B. 

Tollios,  Constantme  D. 


Safety  Services 

Mattox,  Andrew  H.,  Environmental.  Health, 
and  Safety  Manager 


Sen'ices,  Projects,  and  Facilities 
Cutler.  Richard  D..  Manager 
Enos,  Jovce  B. 


R56 


Laboratory  Support  Staff  R57 


Apparatus 

Marine  Resource*  Center 

Bolton,  Jason  D. 

Barnes,  Franklin  D. 

Enos,  Edward  G.,  Jr.,  Superintendent 

Boyer,  Paul  L. 

Haskins,  William  A. 

Boucher.  Richard  L. 

Cameron,  Lawrence  M. 

Cipoletta,  Charles  D. 

Chappell,  Carol  L. 

Building  Services  unit  Grounds 

Fisher,  H.  Thomas,  Jr. 

Crabb,  Andrew  H. 

Hayes,  Joseph  N..  Superintendent 
Allen.  Wavne  D. 

Grossman.  William  M. 
Moniz.  Priscilla  C. 

Cutler,  Laura 
DeLinks,  Elisabeth 

Anderson,  Lewis  B. 

Sexton,  Andrew  W. 

DeMenil,  Ben 

Barnes.  Susan  M. 

Sullivan,  Daniel  A. 

Diachun,  Peter  J. 

Beaudoin   Helen 

Tassinan.  Eugene 

Fitton,  Robert  R. 

Boucher.  Richard  L. 

Greenfield.  Benjamin 

Callahan,  John  J. 

MRC  Life  Support  System 

Halfant.  Jennie  A. 

Collins,  Paul  J. 

Mebane,  William  N..  Systems  Operator 

Higham,  Benjamin  T. 

Dorris.  John  J. 

Hanley,  Janice  S. 

Home,  Joseph  M. 

Jearld,  Saba  A. 

Dutra.  Roger  S.,  Jr. 
Gibbons,  Roberto  G. 

Photolab 

Just,  Thomas  E. 

Gonsalves,  Nelson 

Colder,  Linda  M. 

Kagoyire,  Diane  B. 

lllgen.  Robert  F. 
Krajewski.  Viola  I. 
Luther,  Herbert 
Lynch.  Henry  L. 

Colder.  Robert  J. 
Research  Administration  &  Educational  Programs 

Kay,  Paul  A. 
Kilpatrick,  Andrew  J. 
Kilpatrick.  Brian 
Langford,  George  II 
Lewis,  Barry 

Rummel.  John  D.,  Director 

Mancini,  Man  E. 
McNamara,  Noreen  M. 

Chrysler,  Dorianne 
Hamel,  Carol  C. 

Lovell,  Lynne 
Luther,  Jonathan  A. 

Rattacasa.  Frank  D. 

Huffer,  Linda 

Melillo.  Edward  D. 

Serrano.  Robert  A. 

Hunt.  Sharon  L. 

Michael,  Samuel  A. 

K.autmann,  Sandra  J. 

Monteiro,  Salvatore 

Plain  Operations  and  Maintenance 

Lynch,  Kathleen  F. 

Moyer,  Lisa  A. 

Fleet.  Barry  M.,  Superintendent 

Nelson,  Beth 

Barnes,  John  S. 

Central  Microscopy  Facility 

O'Connor.  John  E. 

Blunt.  Hugh  F. 

Kerr,  Louis  M. 

Ordway,  Cheryl  C. 

Bourgom.  Lee  E. 

Pennington,  Marc 

Carini,  Robert  J. 
Carroll.  James  R. 
Fish,  David  L.,  Jr. 
Fuglister.  C.  Kurt 
Gonsalves.  Walter  W..  Jr. 
Hathaway.  Peter  J. 
Justason.  C.  Scott 
Lochhead.  William  M. 
Lunn,  Alan  G. 

Temporary  Employees 

Price.  Elizabeth  C. 
Rakowski,  Cara  E. 
Regan,  John  F. 
Richmond.  Hazel  E. 
Roache,  Kathryn  H. 
Sacks,  Audrey  B. 
Saintours,  Frederick  H.,  Jr. 
Shephard,  Jillian  M. 
Smith,  Mandy  L. 

Michael  Baptiste 
DeGiorgis,  Joseph  A. 
DiPasqua.  Andrew  K. 
Gilbrook.  Maggie  M. 
Mansfield,  Darren  P. 
O'Connor,  Patricia  M. 
Paris,  Susan  J. 

c»  1  1  L-.a\-     I  »t  1  \ 

McAdams,  Herbert  M.  Ill 
McHugh.  Michael  O. 

olUKcV,  JCUV 

Till.  Geoffrey  A. 

Snow,  Jason  M. 
Sonnabend,  Aaron  P. 

Mills.  Stephen  A. 

Stephen,  Michael  J. 

Olive,  Charles  W.,  Jr. 

Summer  Support  Staff 

Tong,  Cherry 

Schoepf.  Claude 

Andrews,  Ethan 

Ulbrich,  Ciona 

Serrano.  Robert  A. 

Andrews,  Mark 

Varao,  John 

Sexton,  Andrew 

Antonangeli,  Donald,  Jr. 

Welch,  Christine  E. 

Atwood,  Karl  M. 

Welenc.  Karen  P. 

Machine  Shop 

Baldic,  David  P. 

Wetzel,  Ernest  D. 

Sylvia,  Frank  E. 

Berrios.  Kelly  L. 

Woitkiewicz.  Mark  D. 

Members  of  the  Corporation* 


Life  Members 


Adelberg,  Edward  A..  Peabody  Museum,  Yale  University,  New 

Haven,  CT  06520 
Amatniek,  Ernest,  4797  Boston  Post  Road.  Pelham  Manor,  NY  10803 

Bang,  Betsy  G.,  76  F.R.  Lillie  Road.  Woods  Hole,  MA  02543 
Bartlelt,  James  II.,  Department  of  Physics,  University  of  Alabama, 

Box  870324.  Tuscaloosa.  AL  35487-0324 
Bernheimer,  Alan  \\ '.,  Department  of  Microbiology.  New  York 

University  Medical  Center,  550  First  Ave.,  New  York,  NY  10016 
Bertholf,  Lloyd  M.,  Westminster  Village  #2114.  2025  E.  Lincoln  St., 

Bloomington,  IL  61701-5995 

Bosch,  Herman  F.,  Box  617.  Woods  Hole,  MA  02543 
Bridgman,  A.  Josephine,  913  Wesley  Woods  Towers.  1825  Clifton 

Rd..  NE,  Atlanta,  GA  30329 

Buck,  John  B.,  7200  Third  Ave.,  #C020,  Sykesville,  MD  21784 
Burbanck.  Madeline  P.,  Box  15134.  Atlanta,  GA  30333 
Burbanek,  William  D.,  Box  15134.  Atlanta,  GA  30333 

Carlson,  Francis  D.,  Johns  Hopkins  University,  Biophysics 

Department.  Jenkins  Hall,  N.  Charles  Street,  Baltimore,  MD  21218 
Clark,  Arnold  M.,  53  Wilson  Rd.,  Woods  Hole,  MA  02543 
Cohen,  Seymour  S.,  10  Carrot  Hill  Rd..  Woods  Hole.  MA  02543-1206 
Colwin,  Arthur  L.,  320  Woodcrest  Rd.,  Key  Biscayne.  FL  33149 
Colwin,  l-aura  Hunter,  320  Woodcrest,  Key  Biscayne,  FL  33149 
Cooperstein,  Sherwin  J.,  Professor  Emeritus  and  Acting  Head. 
Department  of  Anatomy,  MC3405.  University  of  Connecticut 
Health  Center,  263  Farmington  Avenue,  Farmington,  CT  06030- 
3405 
Copeland,  D.  E.,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 

Corliss,  John  O.,  P.  O.  Box  53008.  Albuquerque,  NM  87153 
Costello,  Helen  M.,  Carolina  Meadows,  Villa  137.  Chape!  Hill.  NC 

27514 
Crouse,  Helen,  Rt  3.  Box  213.  Hayesville.  NC  28904 

Dudley,  Patricia  L.,  3200  Alki  Avenue.  SW.  #401,  Seattle,  WA  981 16 

Edwards,  Charles,  2244  Harbour  Court  Drive,  Longboat  Key.  FL 

34228 
Krulkar,  Solomon  I).,  318  Kent  Road.  Bala  Cynwyd.  PA  19004 

*  Including  action  of  the  1994  Annual  Meeting. 


Failla,  Patricia  M.,  2149  Loblolly  Lane,  Johns  Island.  SC  29455 
Ferguson,  James  K.  \V.,  56  Clarkehaven  St.,  Thornhill,  Ontario  L4J 
2B4  Canada 

Glusman,  Murray,  New  York  State  Psychiatric  Institute,  722  West 

168th  Street,  Unit  #70.  New  York.  NY  10032 
Goldman,  David,  63  Loop  Rd..  Falmouth.  MA  02540 
Graham,  Herbert,  36  Wilson  Rd.,  Woods  Hole,  MA  02543 
Green,  James  \V.,  409  Grant  Ave..  Highland  Park.  NJ  08904 

Hamburger,  Viktor,  Department  of  Biology.  Washington  University, 

St.  Louis,  MO  63 130 
Hamilton,  Howard  I,.,  Department  of  Biology.  Llniversity  of  Virginia, 

238  Gilmer  Hall,  Charlottesville,  VA  22901 

Harding,  Clifford  V.,  Jr.,  54  Two  Ponds  Road,  Falmouth,  MA  02540 
Haschemeyer,  Audrey  E.  V.,  2 1  Glendon  Road,  Woods  Hole,  MA 

02543 

Hauschka,  Theodore  S.,  FD1,  Box  781.  Damariscotta,  ME  04543 
Hisaw,  F.  L.,  5925  SW  Plymouth  Drive.  Corvallis.  OR  97330 
I  luskm,  Francis  C.  G.,  %  Dr.  John  E.  Walker.  United  States  Army 

Natick  RD  &  E  Center,  SAT  NC-YSM,  Kansas  Street.  Natick,  MA 

01760-5020 
llubhard,  Ruth.  Biological  Laboratories.  Harvard  University. 

Cambridge.  MA  02138 
Humes,  Arthur  G.,  Boston  University  Marine  Program,  Marine 

Biological  Laboratory.  Woods  Hole.  MA  02543 
Ilurwitz,  Charles,  Veterans  Administration  Hospital,  Basic  Science 

Research  Laboratory,  Albany.  NY  1 2208 

karush,  Fred,  Department  of  Microbiology,  Llniversity  of 

Pennsylvania  School  of  Medicine.  Philadelphia.  PA  19104-6076 

(deceased) 
Kingsbury,  John  M.,  Department  of  Plant  Biology,  Cornell 

Llniversity,  Plant  Science  Building,  Ithaca,  NY  14853 
Kit-mill,!/.  Lewis,  Department  of  Biology,  Reed  College,  3203  SE 

Woodstock  Blvd.,  Portland,  OR  97202 

I  .i.U  i  in. in,  Ezra,  Yale  University.  School  of  Music.  New  Haven,  CT 

06520 

LauH'er,  Max  A.,  Address  unknown 

LeFevre,  Paul  G.,  15  Agassiz  Road,  Woods  Hole,  MA  02543 
Levine,  Kachmiel,  City  of  Hope  Medical  Center,  Shapiro  Building. 

Duarte,  CA  91010 
Lochhead,  John  H.,  49  Woodlawn  Rd.,  London  SW6  6PS,  England, 

UK 


R58 


Members  of  the  Corporation    R59 


Loewus.  Frank  A.,  Washington  State  University,  Institute  of 

Biological  Chemistry,  Pullman,  WA  99164 
I.oftfield,  Robert  B..  Department  of  Biochemistry,  University  of  New 

Mexico  School  of  Medicine.  Albuquerque.  NM  87131 

Magruder.  Samuel  R.,  270  Cedar  Lane.  Paducah,  K.Y  42001 
Malkiel.  Saul,  Allergic  Diseases,  Inc..  130  Lincoln  St..  Worcester.  MA 

01609 

Martin,  Lowell  V.,  10  Buzzards  Bay  Avenue.  Woods  Hole.  MA  02543 
Mathews,  Rita  \V.,  Box  131.  Southfield.  MA  01259 
Moore,  John  A.,  Department  of  Biology.  University  of  California. 

Riverside.  CA  92521 
Moscona.  Arthur  A.,  University  of  Chicago.  Department  of  Molecular 

Genetics  and  Cell  Biology.  920  East  58th  Street.  Chicago.  IL  60637 
Mountain,  Isabel.  Arleigh  Burke  Pavilion.  1739  Kirby  Road,  McLean, 

VA  22101 
Musacchia,  Xavier  J.,  P.O.  Box  5054,  Delia  Vista,  AR  72714-0054 

Nasatir,  Maimon,  P  O  Box  379.  Ojai.  CA  93024-0379 

Pollister,  A.  \V..  8  Euclid  Ave..  Belle  Mead,  NJ  08502 

Porter,  Keith  R.,  74  Pasture  Lane,  #319  Beaumont,  Bryn  Mawr,  PA 

19010 
Prosser.  C.  Ladd,  Department  of  Physiology,  Burrill  Hall  524, 

University  of  Illinois.  Urbana.  IL  61801 
Prytz,  Margaret  McDonald.  Address  unknown 

Ratner.  Sarah,  Department  of  Biochemistry.  Public  Health  Research 

Institute,  455  First  Ave..  New  York.  NY  10016 
Renn,  Charles  E.,  Address  unknown 
Reynolds,  George.  Department  of  Physics.  Princeton  University, 

Jadwm  Hall.  Princeton.  NJ  08544 
Rice,  Robert  V.,  30  Burnham  Dr..  Falmouth,  MA  02540 
Richards,  A.  Glenn,  942  Cromwell  Ave..  St.  Paul,  MN  551 14 

(deceased) 
Rockstein,  Morris,  600  Biltmore  Way.  Apt.  805.  Coral  Gables,  FL 

33134 
Ronkin,  Raphael  R.,  3212  McKinley  St..  NW,  Washington.  DC 

20015-1635 
Rose,  S.  Meryl,  32  Crosby  Ln..  E.  Falmouth.  MA  02536  (deceased) 

Sanders,  Howard,  Woods  Hole  Oceanographic  Institution.  Woods 

Hole.  MA  02543 
Sato,  1 1  iik  mi.  Faculty  of  Social  Science.  Nagano  University. 

Shiminogo,  Ueda.  Nagano  386-12,  Japan 
Saz.  Arthur  K,  Georgetown  University  Medical  School.  Department 

of  Immunology,  Washington,  DC  20007 
Scharrer,  Berta,  Department  of  Anatomy.  Albert  Einstein  College  of 

Medicine.  1300  Morris  Park  Avenue.  Bronx.  NY  10461 
Schlesinger.  R.  \\  alter,  University  of  Medicine  and  Dentistry  of  New- 
Jersey,  Department  of  Molecular  Genetics  and  Microbiology. 

Robert  Wood  Johnson  Medical  School,  Piscataway,  NJ  08854-5635 
Schmitl,  F.  O.,  Room  16-512.  Massachusetts  Institute  of  Technology. 

Cambridge,  MA  02 1 39 

Scott,  Allan  C.,  Colby  College.  Waterville,  ME  04901 
Silverstein,  Arthur  M.,  The  Johns  Hopkins  Hospital.  Johns  Hopkins 

University,  Institute  of  the  History  of  Medicine.  1900  E.  Monument 

Street.  Baltimore.  MD  21205 

Smith,  Homer  P.,  8  Quissett  Ave..  Woods  Hole,  MA  02543  (deceased) 
Smith,  Paul  F.,  P.  O.  Box  264.  Woods  Hole,  MA  02543 
Sonnenblick,  B.  P.,  515A  Heritage  Hill  Village.  Southbury,  CT  06488 
Steinhardt,  Jacinto.  1  508  Spruce  St..  Berkeley.  CA  94709 
Stephens,  Groier  C.,  Department  of  Ecology  &  Evolutionary  Biology. 

School  of  Biological  Sciences.  University  of  California.  Irvine.  CA 

92717 


Taylor,  Robert  K.,  20  Harbor  Hill  Rd..  Woods  Hole,  MA  02543 
1  horndike,  W.  Nicholas,  Wellington  Management  Company,  200 

State  Street,  Boston.  MA  02109 
Trager.  \\  illiam.  The  Rockefeller  University,  1230  York  Ave..  New 

York.  NY  10021-6399 

Villee,  Claude  A.,  Harvard  Medical  School.  Carol  L.  Countway 

Library,  25  Shattuck  Street,  Boston,  MA  021  15 
Vincent,  \Valter  S.,  16  F.R.  Lillie  Rd..  Woods  Hole,  MA  02543 

\Vald.  George,  Harvard  University,  16  Divinity  Avenue,  Cambridge, 

MA  02 1 38 
\Vaterman,  T.  H.,  Yale  University.  210  OML  Biology  Department, 

Box  6666.  New  Haven.  CT  065 1 1 

\\iercinski,  Floyd  J.,  21  Glenview  Road,  Glenview,  IL  60025 
\\  igley,  Roland  L.,  35  Wilson  Rd..  Woods  Hole.  MA  02543 
\\ilber,  Charles  G.,  Department  of  Biology,  Forensic  Science  Lab. 

Colorado  State  University.  Fort  Collins,  CO  80523 

/.inn,  Donald  J.,  Department  of  Zoology.  University  of  Rhode  Island. 

Kingston.  RI  02881 
Zweifach,  Benjamin  \V.,  881  1  Nottingham  Place.  La  Jolla,  CA  92037 

Members 

Abt,  Donald  A.,  Marine  Biological  Laboratory.  Laboratory  for  Marine 

Animal  Health.  Woods  Hole.  MA  02543 

Acheson,  George  H.,  25  Quissett  Ave.,  Woods  Hole.  MA  02543 
Adams,  James  A.,  Florida  A  &  M  University.  Department  of  Biology, 

Tallahasee,  FL  32307-0077 

Adelman,  William  J.,  Jr.,  160  Locust  St.,  Falmouth,  MA  02540 
Afzelius,  Bjorn,  Wenner-Gren  Institute,  University  of  Stockholm. 

Stockholm.  Sweden 
Alberte.  Randall  S..  Department  of  Molecular  Genetics  and  Cell 

Biology,  University  of  Chicago.  1 103  E.  57th  Street,  Chicago.  IL 

60637 
Alkon,  Daniel,  Laboratory  of  Adaptive  Systems.  National  Institutes  of 

Health.  36  Convent  Drive.  36/4A21,  Bethesda.  MD  20892-4124 
Allen,  Garland  E.,  Department  of  Biology,  Washington  University, 

Box  1 137.  One  Brookings  Drive.  St.  Louis.  MO  63130-4899 
Allen,  Nina  S.,  Department  of  Biology.  Wake  Forest  University.  Box 

7325,  Winston-Salem.  NC  27109 
Anderson,  Everett,  Department  Cell  Biology,  Harvard  Medical  School. 

25  Shattuck  St.,  Boston.  MA  021 15 
Anderson,  J.  M.,  1 10  Roat  St..  Ithaca,  NY  14850 
Anderson.  Porter  W.,  6855  East  Edgewater  Drive.  #2A.  Coral  Gables, 

FL  33133 
Armett-Kibel,  Christine,  Dean  of  Science  Faculty,  University  of 

Massachusetts.  Boston.  MA  02125 
Armstrong,  Clay  M.,  Department  of  Physiology.  University  of 

Pennsylvania  Medical  School,  8701  Richards  Bldg..  Philadelphia. 

PA  19104-6085 
Armstrong,  Ellen  P..  Marine  Biological  Laboratory,  Woods  Hole.  MA 

02543 
Armstrong,  Peter  B.,  Section  of  Molecular/Cellular  Biology. 

University  of  California.  Davis,  CA  95616-8755 
Arnold,  John  M.,  Pacific  Biomedical  Research  Center,  209A  Snyder 

Hall,  University  of  Hawaii,  Honolulu.  HI  96822-2233 
Arnold,  \\  illiam  A.,  Biology  Division.  Oak  Ridge  National 

Laboratory.  Oak  Ridge.  TN  37830 
Ashton,  Robert  \V.,  Esq.,  Bay  Foundation.  18th  Floor.  99  Wall  St.. 

New  York.  NY  10005 
Atema,  Jelle,  Boston  University  Marine  Program.  Marine  Biological 

Laboratory.  Woods  Hole,  MA  02543 


R60  Annual  Report 


Augustine  Jr.,  George  J.,  Department  of  Neurobiology.  Duke 
University  Medical  Center,  PO  Box  3209,  Durham,  NC  27710 

Ayers,  Donald  E.,  4607  1/2  MacArthur  Blvd.,  NW  #B,  Washington. 
DC  20007-2533 

Baccetti,  Baccio,  Institute  of  Zoology,  University  of  Siena,  53100 

Siena,  Italy 

Baker,  Robert  G.,  Department  of  Physiology  and  Biophysics,  New 
York  University  Medical  Center,  550  First  Ave.,  New  York,  NY 

10016 
Baldwin,  Thomas  O.,  Department  of  Biochemistry  and  Biophysics, 

Texas  A&M  University,  College  Station,  TX  77843 
Barlow,  Robert  B.,  Jr.,  Institute  for  Sensory  Research,  Syracuse 

University.  Merrill  Lane.  Syracuse,  NY  13244-5290 
Barry,  Daniel  T.,  2415  Fairwind  Dr.,  Houston,  TX  77062-4756 
Barry,  Susan  R.,  Mount  Holyoke  College,  Dept.  of  Biological 

Sciences,  So.  Hadley.  MA  01075 
Bartell,  Clelmer  K.,  2000  Lake  Shore  Drive,  New  Orleans.  LA  70122 

(resigned) 
Bass,  Andrew  H.,  Seely  Mudd  Hall,  Department  of  Neurobiology. 

Cornell  University,  Ithaca,  NY  14853 
Battelle,  Barbara-Anne,  Whitney  Laboratory,  University  of  Florida, 

9505  Ocean  Shore  Blvd.,  St.  Augustine.  FL  32086 
Bay,  Frederick,  Bay  Foundation,  99  Wall  St.,  18th  Fl.,  New  York,  NY 

10005 

Baylor,  Martha  B.,  P.  O.  Box  93,  Woods  Hole,  MA  02543 
Bearer,  Elaine  L.,  Division  of  Biology  &  Medicine.  Department  of 

Pathology,  Brown  University.  Box  G.  Providence.  RI  029 1 2 
Beauge,  Luis  Alberto,  Department  of  Biophysics,  Instituto  M.y.M. 

Ferreyra,  Casilla  de  Correo  389,  5000  Cordoba,  Argentina 
Beck,  Lyle  V.,  2455  Tamarack  Trail,  Apt.  8,  Bloomington,  IN  47408 
Begenisich,  Ted,  Department  of  Physiology,  University  of  Rochester, 

Medical  Center,  Box  642.  601  Elmwood  Ave..  Rochester,  NY 

14642 
Begg,  David  A.,  Department  of  Anatomy  &  Cell  Biology,  University 

of  Alberta,  Edmonton,  Alberta  T6G  2H7,  Canada 
Bell,  Eugene,  305  Commonwealth  Avenue.  Boston,  MA  021 15 
Benjamin,  Thomas  L.,  Harvard  Medical  School.  Pathology,  D2-230, 

200  Longwood  Avenue,  Boston.  MA  02  1  1  5 
Bennett,  M.  V.  L.,  Albert  Einstein  College  of  Medicine,  Department 

of  Neuroscience.  1410  Pelham  Pkwy.  S..  Bronx.  NY  10461 
Bennett,  Miriam  E.,  Department  of  Biology,  Colby  College, 

Waterville,  ME  04901 

Berg,  Carl  J.,  Jr.,  P.  O.  Box  769.  Kilauea,  Kauai,  HI  96754-0769 
Berlin,  Suzanne  T.,  5  Highland  St..  Gloucester.  MA  01930 
Berne,  Robert  M.,  Department  of  Physiology,  University  of  Virginia, 

School  of  Medicine,  Box  116,  MR4,  Charlottesville,  VA  22903 
Bernstein,  Norman,  Diane  and  Norman  Bernstein  Foundation,  Inc.. 

5301  Wisconsin  Ave..  #600,  Washington,  DC  20015-2015 
Bezanilla,  Francisco,  Department  of  Physiology.  University  of 

California,  Los  Angeles,  CA  90024 
Biggers,  John  D.,  Department  of  Physiology,  Harvard  Medical 

School.  Boston.  MA  02 1 1 5 
Bishop,  Stephen  H.,  Department  of  Zoology,  Iowa  State  University, 

Ames,  I A  50010 
Blaustein,  Mordecai  P.,  Department  of  Physiology,  School  of 

Medicine,  University  of  Maryland.  655  W.  Baltimore  Street, 

Baltimore.  MD  21201 

Blennemann,  Dieter,  50  Old  Stone  Bridge  Road,  Cos  Cob.  CT  06807 
Bloom,  George  S.,  Department  of  Cell  Biology  and  Neuroscience.  The 

University  of  Texas  Southwestern  Medical  Center,  5223  Harry 

Hmes  Blvd.'.  Dallas.  TX  75235-9039 
Bloom,  Kerry  S.,  Department  of  Biology,  University  of  North 

Carolina.  623  Fordham  Hall,  Chapel  Hill,  NC  27516 


Bodznick,  David  A.,  Department  of  Biology,  Wesleyan  University, 

Lawn  Avenue,  Middletown.  CT  06457-0170 
Boettiger,  Edward  G.,  1 7  Eastwood  Road.  Storrs  Mansfield,  CT 

06268-2401 
Boolootian,  Richard  A.,  Science  Software  Systems,  Inc..  3576 

WoodcliffRd..  Sherman  Oaks,  CA  91403 
Borgese.  Thomas  A.,  Department  of  Biology.  Lehman  College. 

CUNY,  Bedford  Park  Blvd.,  West.  Bronx.  NY  10468 
Borisy,  Gary  G.,  Laboratory  of  Molecular  Biology,  University  of 

Wisconsin,  Madison,  WI  53706 
Borst,  David  \V.,  Jr.,  Department  of  Biological  Sciences.  Illinois  State 

University.  Normal,  IL  61790-4120 
Bowles,  Erancis  P.,  Ecosystems  Center.  Marine  Biological  Laboratory, 

Woods  Hole.  MA  02543 
Boyer,  Barbara  C.,  Department  of  Biology,  Union  College, 

Schenectady,  NY  12308 
Brandhorst,  Bruce  P.,  Institute  of  Molecular  Biology  and 

Biochemistry.  Department  of  Biological  Sciences,  Simon  Eraser 

University.  Burnaby,  BC  V5A  156.  Canada 
Brinley,  F.  J.,  Neurological  Disorders  Program.  NINCDS.  NIH.  812 

Federal  Building.  Bethesda.  MD  20892 
Brown,  Stephen  C.,  Department  of  Biological  Sciences,  SUNY, 

Albany.  NY  12222 
Brown,  William  L.,  Retired  Chairman.  Bank  of  Boston,  100  Federal 

St.  01-23-1  1.  Boston.  MA  02106-2016 
Browne,  Carole  L.,  Department  of  Biology,  Wake  Forest  University, 

Winston-Salem,  NC27109 
Browne,  Robert  A.,  Department  of  Biology,  Wake  Forest  University, 

Box  7325,  Winston-Salem,  NC  27109 
Bryant,  Shirley  H.,  Department  of  Pharmacology  and  Cell  Biophysics. 

ML  575,  University  of  Cincinnati,  Cincinnati,  OH  45267 
Bucklin,  Anne  C.,  University  of  New  Hampshire,  Ocean  Process 

Analysis  Lab,  142  Morse  Hall,  Durham,  NH  03824 
Bullis,  Robert  A.,  Marine  Biological  Laboratory.  LMAH,  Woods 

Hole,  MA  02543 
Burd,  Gail  Deerin,  Department  of  Molecular  and  Cell  Biology,  Life 

Sciences  South,  Rm  444,  University  of  Arizona,  Tucson,  AZ  85721 
Hindu  k.  Carolyn  J.,  Department  of  Biology,  Brooklyn  College,  2900 

Bedford  Avenue,  Brooklyn.  NY  1 1210 
Burger,  Max,  Freidrich  Miesner  Institut  Bau  1060  Postfach  2543, 

Basel  4002,  Switzerland 
Burgos,  Mario,  1HEM  Medical  School,  UNC  Conicet.  Casilla  de 

Correo  56,  5500  Mendoza.  Argentina 
Burky.  Albert.  Department  of  Biology.  University  of  Dayton.  Dayton. 

OH  45469 
Burris,  John  E.,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 
Burstyn,  Harold  I,.,  Morrison  Law  Firm,  The  Morrison  Building,  145 

North  Fifth  Avenue,  Mt.  Vernon.  NY  10550 
Bursztajn,  Sherry,  Harvard  Medical  School.  Mailman  Research 

Center,  115  Mill  St.,  Belmont.  MA  02178 
Busa,  \\illiam.  Department  of  Biology.  Johns  Hopkins  University. 

3400  N.  Charles  St..  Baltimore.  MD  21218 

Calabrese,  Ronald  E.,  Department  of  Biology,  Emory  University, 

1555  Pierce  Drive,  Atlanta,  GA  30322 
Callaway,  Joseph  C.,  Department  of  Physiology,  New  York  Medical 

College.  Basic  Sciences  Bldg..  Valhalla,  NY  10595 
Calvin,  Katherine  Graubard,  University  of  Washington.  Department 

of  Zoology.  NJ-15.  Seattle,  WA  98195 
Cameron,  Andrew,  Department  of  Biology.  California  Institute  of 

Technology.  Pasadena,  CA  91  125 
Campbell,  Richard  H.,  Bang-Campbell  Associates,  Box  402,  Eel  Pond 

Place,  Woods  Hole,  MA  02543 


Members  of  the  Corporation  R6I 


Candelas,  Graciela  C.,  Department  of  Biology,  University  of  Puerto 

Rieo,  PO  Box  23360.  San  Juan.  PR  00931-3360 
Carew,  Thomas  .1.,  Department  of  Psychology,  Vale  University.  P.  O. 

Box  1 1  A.  Yale  Station.  New  Haven.  CT  06520 
Cariello.  I.ucio.  Biochemistry  Department,  Stazione  Zoologica,  Villa 

Comunale.  80121  Naples.  Italy- 
Case,  James,  University  of  California,  Associate  Vice  Chancellor  of 

Research,  Santa  Barbara.  CA  93106 
Cassidy,  Rev.  J.  D.,  Providence  College,  Pnory  of  St.  Thomas 

Aquinas,  Providence,  RI  02918 
Cavanaugh,  Colleen  M.,  Harvard  University.  Biological  Laboratories. 

16  Divinity  Ave..  Cambridge,  MA  02138 
Cebra,  John  J.,  Department  of  Biology.  Leidy  Labs.  G-6,  University 

of  Pennsylvania,  Philadelphia.  PA  19174 
Chaet,  Alfred  B.,  University  of  West  Florida.  Department  of  Cell  & 

Molecular  Biology.  1 1000  University  Parkway.  Pensacola.  FL 

32514 
Chambers,  Edward  I..,  Department  of  Physiology  and  Biophysics. 

University  of  Miami.  School  of  Medicine.  P.  O.  Box  016430, 

Miami.  FL  33101 
Chang,  Donald  C..  Hong  Kong  University  of  Science  &  Technology, 

Department  of  Biology,  Clear  Water  Bay,  Kowloon.  Hong  Kong 
Chappell,  Richard  I,.,  Department  of  Biological  Sciences,  Hunter 

College,  Box  210.  695  Park  Ave.,  New  York,  NY  10021 
Chen,  Thomas  T.,  Center  for  Marine  Biotechnology.  University  of 

Man-land.  600  E.  Lombard  St..  Baltimore.  MD  21202 
Chikarmane,  Ilemant  M.,  Marine  Biological  Laboratory.  Woods  Hole. 

MA  02543 
Child,  Frank  M.,  HI,  28  Lawrence  Farm  Road,  Woods  Hole.  MA 

02543 
Chisholm,  Rex  L.,  Department  of  Cell  Biology.  Northwestern 

University  Medical  School.  Chicago.  IL  6061 1 
Citkowitz,  Elena.  Hospital  of  St.  Raphael,  Lipid  Disorders  Clinic, 

1450  Chapel  Street,  New  Haven,  CT  065 1 1 
Clark,  Eloise  E.,  Vice  President,  Bowling  Green  State  University. 

Bowling  Green.  OH  43403 

Clark,  Hays,  26  Deer  Park  Drive,  Greenwich,  CT  06830 
Clark,  James  M.,  210  Emerald  Lane,  Palm  Beach,  FL  33480 
Clark,  \\allis  H.,  Jr.,  7922  NW  71st  Street,  Gainsville.  FL  32606 
Claude.  Philippa,  University  of  Wisconsin,  Primate  Center.  1223 

Capital  Court.  Madison.  WI  53715 
Clay,  John  R.,  Laboratory  of  Biophysics.  NIH.  36/2C02.  Bethesda. 

MD  20892 
Clowes,  Alexander  \\  .,  Department  of  Surgery  RF-25.  University  of 

Washington  School  of  Medicine,  Seattle,  WA  98195 
Clutter,  Mary,  Office  of  the  Director.  Room  518,  National  Science 

Foundation,  Washington.  DC  20550 
Cobb,  Jewel  Plummer,  California  State  University,  5151  State 

University  Drive.  Los  Angeles.  CA  90032-8500 
Cohen,  Carolyn,  Rosenstiel  Basic  Medical  Sciences  Research  Center, 

Brandeis  University.  Waltham.  MA  02254 
Cohen,  Lawrence  B.,  Department  of  Physiology,  Yale  University 

School  of  Medicine,  333  Cedar  Street,  New  Haven,  CT  06510-8026 
Cohen,  Maynard,  Department  of  Neurological  Sciences,  Rush  Medical 

College,  600  South  Paulina,  Chicago.  IL  60612 
Cohen,  Rochclle  S.,  Department  of  Anatomy.  University  of  Illinois. 

808  W.  Wood  Street.  Chicago.  IL  60612 
Cohen,  William  D.,  Department  of  Biological  Sciences.  Hunter 

College,  695  Park  Ave.,  Box  79,  New  York,  NY  10021 
Coleman,  Annette  \V.,  Division  of  Biology  and  Medicine,  Brown 

University.  Providence.  RI  01912 
Collier,  Jack  R.,  Department  of  Biology.  Brooklyn  College.  Bedford  & 

Avenue  H.  Brooklyn.  NY  1 121(1 
Collier.  Marjorie  McCann.  Biology  Department.  Saint  Peter's  College. 

2641  Kennedv  Boulevard,  Jersev  City.  NJ  07306 


Collin,  C'arlos,  National  Institutes  of  Health,  Laboratory  of  Adaptive 

Systems,  Bldg.  36,  B30X,  Bethesda,  MD  20892-0226 
Cook,  Joseph  A.,  The  Edna  McConnell  Clark  Foundation,  250  Park 

Ave.,  New  York,  NY  10177-0026 
Cornell,  N'cal  W.,  Marine  Biological  Laboratory,  Woods  Hole.  MA 

02543 
Cornwall,  Melvin  C.,  Jr.,  Department  of  Physiology  L714.  Boston 

University  School  of  Medicine,  80  E.  Concord  St.,  Boston,  MA 

02118 
Corson,  David  Wesley,  Jr.,  Storm  Eye  Institute.  Room  537.  171 

Ashley  Avenue.  Charleston.  SC  29425 
Corwin,  Jeffrey  T.,  Department  of  Otolaryngology,  Health  Science 

Center.  University  of  Virginia  Medical  Center.  Box  396. 

Charlottesville.  VA  22908 
Costello,  Walter  J.,  Department  of  Zoology  Z/BS.  College  of 

Medicine,  Ohio  University.  Athens.  OH  45701 
Couch,  Ernest  F.,  Department  of  Biology,  Texas  Christian  University, 

Fort  Worth.  TX  76129 

Crane,  Sylvia  E.,  438  Wendover  Drive.  Princeton.  NJ  08540 
Cremer-Bartels,  Gertrud,  Universitats  Augenklinik.  44  Munster. 

Germany 
Crow,  Terry  J.,  Department  of  Neurobiology  and  Anatomy. 

University  of  Texas  Medical  School.  Houston,  TX  77225 
Crowell,  Sears,  Department  of  Biology.  Indiana  University. 

Bloomington.  IN  47405 
Crowther.  Robert,  Department  of  Biology,  University  of  New 

Brunswick.  BS  45 1 1 1 .  Fredericton,  NB.  Canada  E3B  6E 1 
Cunningham,  Mary-Ellen,  62  Cloverly  Road.  Grosse  Pointe  Farms, 

MI  48236 

Currier,  David  I..,  P.  O.  Box  2476,  Vineyard  Haven.  MA  02568 
Cutler,  Richard,  Marine  Biological  Laboratory.  Woods  Hole,  MA 

02543 

D'Alessio,  Giuseppe,  Department  of  Organic  &  Biological  Chemistry. 

University  of  Naples.  Via  Mezzocannone  16.  Naples.  Italy  80134 
D'Avanzo,  Charlene,  Department  of  Natural  Science.  Hampshire 

College.  Amherst.  MA  01002 
Daignault.  Alexander  T.,  29  Quisset  Harbor  Rd.,  Falmouth,  MA 

02540 
Dan,  katsuma,  Tokyo  Metropolitan  Union,  1-1  Minami-Osawa, 

Hachioji  City  192-03,  Tokyo.  Japan 
David,  John  R.,  Tropical  Public  Health.  Harvard  School  of  Public 

Health,  665  Huntington  Ave.,  Boston.  MA  021  15 
Davidson,  Eric  H.,  Division  of  Biology.  156-29,  California  Institute  of 

Technology.  1201  E.  California  Blvd.,  Pasadena.  CA  91 125 
Davis,  Joel  P.,  P.O.  Box  208,  Brooksville,  ME  04617 
Daw,  Nigel  W.,  5  Old  Pawson  Rd.,  Branford.  CT  06405 
Deegan,  Linda  A.,  The  Ecosystems  Center,  Marine  Biological 

Laboratory,  Woods  Hole,  MA  02543 

DeGroof,  Robert  C..  145  Water  Crest  Dr..  Doylestown,  PA  18901 
Dellaan,  Robert  I,.,  Department  of  Anatomy  and  Cell  Biology. 

Emory  University  School  of  Medicine,  Atlanta,  GA  30322 
DeLanney,  Louis  E.,  The  Parkinson's  Institute.  1  170  Morse  Avenue, 

Sunnyvale.  CA  94089 
Denkla,  Marth  B.,  Kennedy-Krieger  Institute.  Johns  Hopkins  School 

of  Medicine.  707  North  Broadway.  Baltimore.  MD  21205 
Dentler,  William  I..,  Department  of  Physiology  &  Cell  Biology, 

University  of  Kansas.  401 1  Haworth  Hall.  Lawrence.  KS  66044 
DePhillips.  Henry  A.,  Jr.,  Department  of  Chemistry.  Trinity  College. 

300  Summit  Street.  Hartford,  CT  06106 
DeSimone,  Douglas  \\  .,  Department  of  Anatomy  and  Cell  Biology. 

Box  439.  Health  Sciences  Center.  University  of  Virginia. 

Charlottesville.  VA  22908 
De loledo-Morrell.  Leyla,  Rush-Presbyterian-St.  Lukes  Medical 

Center.  1653  West  Congress  Parkway.  Chicago.  IL  60612 


R62  Annual  Report 


Dettbarn,  Wolf-Dietrich,  Department  of  Pharmacology.  School  of 

Medicine.  Vanderbilt  University,  Nashville.  TN  37232 
De  \Veer,  Paul  J.,  Department  of  Physiology.  University  of 

Pennsylvania  School  of  Medicine.  Philadelphia,  PA  19104-6085 
Dionne,  Vincent  E.,  Boston  University  Marine  Program.  Marine 

Biological  Laboratory,  Woods  Hole.  MA  02543 
Dixon,  Keith  E.,  School  of  Biological  Sciences,  Flinders  University. 

Bedford  Park.  5042,  South  Australia.  Australia 
Dowling,  John  E.,  The  Biological  Laboratories,  Harvard  LIniversity, 

16  Divinity  St..  Cambridge.  MA  02138 
DuBois,  Arthur  Brooks,  John  B.  Pierce  Foundation  Laboratory,  290 

Congress  Ave.,  New  Haven.  CT  06519 
Duncan.  Thomas  K.,  Department  of  Environmental  Sciences.  Nichols 

College,  Dudley,  MA  01571 
Dunham,  Philip  B.,  Department  of  Biology,  Syracuse  LIniversity, 

Syracuse.  NY  1 3244 
Dunlap,  Kathleen,  Department  of  Physiology,  Tufts  LIniversity 

Medical  School,  Boston,  MA  02 1 1 1 
Dunlap,  Paul  V.,  Department  of  Biology,  Woods  Hole  Oceanographic 

Institution,  Redfield  316,  Woods  Hole.  MA  02543 
Dvvorkin,  Martin,  Department  of  Microbiology.  University  of 

Minnesota,  1460  Mayo  Bldg.,  Box  196  UMHC.  Minneapolis,  MN 

55455-0312 

Ebert,  James  D.,  Department  of  Biology.  Homewood.  The  Johns 

Hopkins  University,  3400  No.  Charles  St..  Baltimore.  MD  21218 
Eckberg,  William  R.,  Howard  University.  Department  of  Zoology, 

P.O.  Box  887,  Admin.  Bldg..  Washington,  DC  20059 
Edds,  Kenneth  T.,  R&D  Systems,  Inc..  Hematology  Division,  614 

McKinley  Place.  NE,  Minneapolis,  MN  55413 
Eder,  Howard  A.,  Albert  Einstein  College  of  Medicine.  1300  Morris 

Park  Ave..  Bronx.  NY  10461 

Edstrom.  Joan,  2515  Milton  Hills  Dr.,  Charlottesville,  VA  22901 
Egyud,  Laszlo  G.,  Cell  Research  Corporation,  100  Inman  Street, 

Cambridge.  MA  02 139 
Ehrlich,  Barbara  E.,  Division  of  Cardiology.  University  of 

Connecticut  School  of  Medicine,  263  Farmington  Avenue, 

Farmington.  CT  06030 
Eisen,  Arthur  Z.,  Division  of  Dermatology.  Washington  University, 

St.  Louis.  MO  63 110 
Eisen,  Herman  N.,  Massachusetts  Institute  of  Technology,  Center  for 

Cancer  Research,  El 7- 128,  77  Massachusetts  Ave.,  Cambridge.  MA 

02139 
Elder,  Hugh  Young,  Institute  of  Physiology,  LIniversity  of  Glasgow. 

Glasgow,  Scotland  G12  8QQ 
Elliott,  Gerald  F.,  The  Open  University  Research  Unit.  Foxcombe 

Hall.  Berkeley  Rd..  Boars  Hill.  Oxford,  England  OX1  5HR 
Englund,  Paul  T.,  Department  of  Biological  Chemistry,  Johns 

Hopkins  University,  725  No.  Wolfe  St.,  Baltimore,  MD  21205 
Epel,  David,  Hopkins  Marine  Station.  Pacific  Grove.  CA  93950 
Epstein,  Herman  T.,  18  Lawrence  Farm  Road.  Woods  Hole.  MA 

02543 
Epstein,  Ray  L.,  1602  West  Olympia  St.,  Hernando.  FL  34442 

Farb,  David  H.,  Department  of  Pharmacology  L603.  Boston 

University  School  of  Medicine.  80  E.  Concord  St.,  Boston,  MA 

02118 
Farmanfarmaian,  A.,  Department  of  Biological  Sciences.  Nelson 

Biological  Laboratory.  Rutgers  University.  FOB  1059.  Piscataway, 

NJ  08855 
Feinman,  Richard  D.,  Box  8.  Department  of  Biochemistry.  SUNY 

Health  Science  Center.  450  Clarkson  Avenue,  Brooklyn,  NY  1 1203 
Feldman,  Susan  C.,  Department  of  Anatomy.  University  of  Medicine 

and  Dentistry  of  New  Jersey,  New  Jersey  Medical  School,  100 

Bergen  St.,  Newark,  NJ  07103 


Fessenden,  Jane,  225  Lakeview  Ave.,  Falmouth,  MA  02540 
Festoff,  Barry  W.,  Neurology  Service  (151),  Veterans  Administration 

Medical  Center,  4801  Linwood  Blvd.,  Kansas  City,  MO  64128 
Fink,  Rachel  D.,  Department  of  Biological  Sciences.  Clapp 

Laboratory.  Mount  Holyoke  College.  South  Hadley,  MA  01075 
Finkelstein,  Alan,  Albert  Einstein  College  of  Medicine,  1300  Morris 

Park  Ave..  Bronx,  NY  10461 
Fischbach,  Gerald,  Department  of  Neurobiology.  Harvard  Medical 

School,  220  Longwood  Ave.,  Boston,  MA  02 1 1 5 
Fishman,  Harvey  M.,  Department  of  Physiology  and  Biophysics. 

University  of  Texas  Medical  Branch.  301  Univ.  Blvd..  Galveston, 

TX  77555-0641 

Flanagan,  Dennis,  12  Gay  St..  New  York.  NY  10014 
Fluck,  Richard  Allen,  Department  of  Biology,  Franklin  &  Marshall 

College,  Box  3003,  Lancaster,  PA  1 7604-3003 
Foreman,  K.  H.,  Boston  LIniversity  Marine  Program.  Marine 

Biological  Laboratory.  Woods  Hole.  MA  02543 
Fox,  Thomas  Oren,  Division  of  Medical  Sciences.  Harvard  Medical 

School.  260  Longwood  Ave..  Boston.  MA  02 1 1 5 
Franzini-Armstrong,  Clara.  School  of  Medicine,  University  of 

Pennsylvania,  330  S.  46th  Street.  Philadelphia.  PA  19143 
Frazier,  Donald  T.,  Department  of  Physiology  and  Biophysics, 

University  of  Kentucky  Medical  Center.  Lexington,  KY  40536 
French,  Robert  J.,  Health  Sciences  Center,  University  of  Calgary, 

Calgary,  Alberta.  T2N  4N1.  Canada 
Friedler,  Gladys,  Boston  University  School  of  Medicine.  80  East 

Concord  Street.  Boston.  MA  021 18  (resigned) 
Fry,  Brian,  Department  of  Biology.  Florida  International  University, 

OE  Building.  Room  239,  Miami.  FL  33199-0001 
Fulton.  Chandler  M.,  Department  of  Biology,  Brandeis  University. 

Waltham.  MA  02254 
Furshpan.  Edwin  J.,  Department  of  Neurophysiology.  Harvard 

Medical  School.  220  Longwood  Ave.,  Boston.  MA  021 15 
Futrelle,  Robert  P.,  College  of  Computer  Science.  Northeastern 

LIniversity,  360  Huntington  Avenue.  Boston,  MA  021 15 


Gabriel,  Mordecai.  Department  of  Biology,  Brooklyn  College,  2900 

Bedford  Ave.,  Brooklyn.  NY  1 1210 
Gadsby,  David  C.,  Laboratory'  of  Cardiac  Physiology,  The  Rockefeller 

University.  1230  York  Avenue.  New  York.  NY  10021-6399 
Gainer,  Harold,  Lab  of  Functional  Neurochemistry,  NIH.  Bldg.  36. 

Room  4D-20,  Bethesda,  MD  20892 
Galatzer-Levy,  Robert  M.,  180  N.  Michigan  Avenue,  Chicago.  IL 

60601 
Gall,  Joseph  G.,  Carnegie  Institution.  1 15  West  University  Parkway, 

Baltimore.  MD  21210 
Garber,  Sarah  S.,  Department  of  Physiology.  Medical  College  of 

Pennsylvania,  2900  Queen  Ln..  Philadelphia.  PA  19129 
Gascoyne,  Peter,  Box  85E.  LIniversity  of  Texas.  M.  D.  Anderson 

Hospital  and  Tumor  Institute.  6723  Bertner  Avenue,  Box  85E, 

Houston,  TX  77030 
Gelperin,  Alan.  Department  of  Biophysics.  AT&T  Bell  Labs.  Room 

1C464,  600  Mountain  Avenue.  Murray  Hill.  NJ  07974 
German,  James  L.,  Ill,  Lab  of  Human  Genetics,  The  New  York 

Blood  Center,  310  East  67th  St.,  New  York.  NY  10021 
Gibbs,  Martin,  Institute  for  Photobiology  of  Cells  and  Organelles. 

Brandeis  University.  Waltham,  MA  02254 
Giblin.  Anne  E.,  Ecosystems  Center,  Marine  Biological  Laboratory. 

Woods  Hole,  MA  02543 
Gibson,  A.  Jane,  Department  of  Biochemistry.  Cornell  LIniversity, 

Ithaca.  NY  14850 

Gifford.  Prosser.  540  N  Street.  SW,  S-903.  Washington,  DC  20024 
Gilbert,  Daniel  I,.,  Clinical  Neuroscience  Branch,  NIH/NINDS.  Bldg. 

36,  Room  5A09A,  Bethesda,  MD  20892 


Members  of  the  Corporation    R63 


Giudice,  Giovanni,  Dipartimcnto  di  Biologiu  Cellulare  e  Dello 

Sviluppo,  1-90123,  Via  Archirafi  22,  Universita  di  Palermo, 

Palermo,  Italy 
Giuditta.  Antonio,  Department  of  General  Physiology.  University  of 

Naples,  Via  Mezzocannone  8.  Naples,  Italy  80134 
Glynn,  Paul,  2770  Beechwood  Blvd.,  Pittsburgh,  PA  15217 
Golden,  William  T.,  American  Museum  of  Natural  History,  40  Wall 

St..  Room  4201.  New  York.  NY  10005 
Goldman.  Robert  D.,  Department  of  Cell,  Molecular  and  Structural 

Biology,  Northwestern  University,  303  E.  Chicago  Ave.,  Chicago,  IL 

60611 
Goldsmith,  Paul  K.,  NIH.  Bldg.  10.  Room  9C-10I.  Bethesda.  MD 

20892 
Goldsmith,  Timothy  H.,  Department  of  Biology,  Yale  University.  New 

Haven,  CT  065 10 
Goldstein,  Moise  H.,  Jr.,  ECE  Department,  Barton  Hall,  Johns 

Hopkins  University,  Baltimore.  MD  21218 
Goodman,  Lesley  Jean,  Department  of  Biological  Sciences.  Queen 

Man  College.  Mile  End  Road.  London.  El  4NS.  England.  UK 
Gould,  Robert  Michael,  Institute  for  Basic  Research  in  Developmental 

Disabilities,  1050  Forest  Hill  Rd..  Staten  Island,  NY  10314 
Gould,  Stephen  J.,  Museum  of  Comparative  Zoology,  Agassiz 

Museum.  Harvard  University,  Cambridge,  MA  02138  (resigned) 
Govind,  C.  K.,  Life  Sciences  Division,  Scarborough  College.  1 265 

Military  Trail.  West  Hill.  Ontario.  MIC  1A4,  Canada 
Grace,  Dick,  Doreen  Grace  Fund,  The  Brain  Center.  Seanest  Drive. 

Promontory  Pt.,  New  Seabury.  MA  02649 
Graf,  Werner,  Rockefeller  University,  1230  York  Ave..  New  York. 

NY  10021 
Grant,  Philip,  2939  Van  Ness  Street,  N.W..  Apt.  302,  Washington. 

DC  20008 
Grass,  Ellen  R.,  The  Grass  Foundation.  77  Reservoir  Rd.,  Quincy. 

MA  02 170 
Grassle,  Judith.  Institute  of  Marine  &  Coastal  Studies.  Rutgers 

University,  Box  231.  New  Brunswick,  NJ  08903 
Greenberg,  Everett  Peter,  Department  of  Microbiology.  College  of 

Medicine.  University  of  Iowa.  Iowa  City.  IA  52242 
Greenberg,  Michael  J.,  Whitney  Laboratory,  University  of  Florida, 

9505  Ocean  Shore  Blvd..  St.  Augustine.  FL  32086-8623 
Greer,  Mary  J.,  16  Hillside  Ave..  Cambridge.  MA  02140 
Griffin,  Donald  R.,  Concord  Field  Station,  Harvard  University,  Old 

Causeway  Road.  Bedford.  MA  01730 
Gross,  Paul  R.,  Center  for  Advanced  Studies,  University  of  Virginia. 

444  Cabell  Hall,  Charlottesville,  VA  22903 
Grossman.  Albert,  New  York  University  Medical  Center,  550  First 

Ave.,  New  York,  NY  10016 
Grossman,  Lawrence,  Department  of  Biochemistry.  Johns  Hopkins 

University.  615  North  Wolfe  Street.  Baltimore.  MD  21205 
Gruner,  John,  Cephalon.  Inc.,  145  Brandy-wine  Parkway.  W.  Chester. 

PA  19380-4245 

Gunning.  A.  Robert,  P.  O.  Box  165.  Falmouth,  MA  02541 
Gwilliam,  G.  P.,  Department  of  Biology,  Reed  College,  Portland.  OR 

97202 


Haimo,  Leah,  Department  of  Biology.  University  of  California. 

Riverside,  CA  92521 
Hall,  Linda  M.,  Department  of  Biochemistry  and  Pharmacology. 

SUNY,  317  Hochstetter.  Buffalo.  NY  14260 
Hall,  7,ack  W.,  Department  of  Physiology.  University  of  California, 

P.O.  Box  0444.  San  Francisco.  CA  94143 
Halvorson,  Harlyn  O.,  26  Fay  Road.  P.O.  Box  81.  Woods  Hole.  MA 

02543 
I  l.tinli  1 1.  Nancy  V.,  Department  of  Biology.  Harvey  Mudd  College. 

301  E.  12th  St.,  Claremont.  CA  9171 1 


llaneji,  Tatsuji,  Kyushu  Dental  College,  Department  of  Anatomy,  2- 

6-1,  Manazuru,  Kokurakita-Ku.  Kitakyushu  803.  Japan 
llanlon,  Roger  T.,  University  of  Texas  Medical  Branch.  Marine 

Biomedical  Institute,  200  University  Boulevard,  Galveston.  TX 

77550-2772 
Manna,  Robert  B.,  Department  of  Environmental  Science  and 

Forestry,  SUNY,  Syracuse.  NY  13210 
llarosi,  Kerenc  I.,  Laboratory  of  Sensory  Physiology,  Marine 

Biological  Laboratory,  Woods  Hole,  MA  02543 
llarrigan,  June  F.,  7415  Makaa  Place,  Honolulu,  HI  96825 
Harrington,  Glenn  W.,  Weber  State  University,  Department  of 

Microbiology,  Ogden.  UT  84408 
Hastings,  J.  W.,  The  Biological  Laboratories,  Harvard  University,  16 

Divinity  Street,  Cambridge,  MA  02138-2020 
Hayashi,  Teru,  7105  SW  1 12  Place.  Miami.  FL  33173 
Ilaydon-Baillie,  Wensley  G.,  Porton  Int.,  2  Lowndes  Place,  London, 

SW1X  8DD.  England,  UK 
Hayes,  Raymond  L.,  Jr.,  Department  of  Anatomy,  Howard 

University.  College  of  Medicine,  520  W  St.,  NW.  Washington,  DC 

20059 
llepler.  Peter  K.,  Department  of  Botany.  University  of  Massachusetts. 

Amherst,  MA  01003 
Herndon,  Walter  R.,  University  of  Tennessee,  Department  of  Botany, 

Knoxville,  TN  37996-1100 
Herskovils,  Theodore  T.,  Department  of  Chemistry,  Fordham 

University,  John  Mulcahy  Hall.  Room  638.  Bronx.  NY  10458 
lliatt,  Howard  H.,  Department  of  Medicine.  Brigham  and  Women's 

Hospital.  75  Francis  Street.  Boston,  MA  021  I  5 
Highstein,  Stephen  M.,  Department  of  Otolaryngology,  Box  8115. 

Washington  University  School  of  Medicine.  St.  Louis,  MO  631 10 
Ilildebrand,  John  G.,  Arizona  Research  Laboratories,  Division  of 

Neurobiology,  603  Gould-Simpson  Science  Building.  University  of 

Arizona.  Tucson.  AZ  85721 
Hill,  Richard  W.,  Department  of  Zoology,  Michigan  State  University. 

E.  Lansing,  MI  48824 
Hill,  Susan  D.,  Department  of  Zoology,  Michigan  State  University.  E. 

Lansing,  MI  48824 
Hillis,  Llewellya,  Smithsonian  Tropical  Research  Institute,  Unit  0948 

APO-AA,  Miami,  FL  34002-0948 
Hillman,  Peter,  Department  of  Biology.  Life  Sciences  & 

Neurobiology,  Hebrew  University,  Jerusalem  91904.  Israel 
llinegardner,  Ralph  T.,  Division  of  Natural  Sciences.  University  of 

California.  Santa  Cruz.  CA  95064 
Mines,  Michael,  Department  of  Computer  Science.  Yale  University. 

P.O.  Box  208205.  New  Haven,  CT  06520-8285 
Minsch,  Gertrude,  W'.,  Department  of  Biology,  University  of  South 

Florida,  Tampa.  FL  33620 
Mobbie,  John  E.,  Ecosystems  Center.  Marine  Biological  Laboratory. 

Woods  Hole.  MA  02543 
Hodge,  Alan  J..  3843  Mt.  Blackburn  Ave..  San  Diego,  CA  921  I  I 

I  loll  IIKIII.  Joseph,  Department  of  Cellular  and  Molecular  Physiology, 

School  of  Medicine,  Yale  University,  New  Haven,  CT  06515 
llollyfield,  Joe  G.,  Baylor  School  of  Medicine.  Texas  Medical  Center. 

Houston.  TX  77030 
Holz,  IV,  George  G,  Massachusetts  General  Hospital,  Laboratory  of 

Molecular  Endocrinology,  Wellman  320,  50  Blossom  St..  Boston. 

MA  021 14 
llupkinson,  Charles  S.,  Jr.,  Ecosystems  Center.  Marine  Biological 

Laboratory,  Woods  Hole.  MA  02543 
Hoy,  Ronald  R.,  Section  of  Neurobiology  and  Behavior,  Cornell 

University.  Ithaca,  NY  14853 
Mufnagel-Zackrotf,  Linda  A.,  Department  of  Microbiology.  University 

of  Rhode  Island.  Kingston,  RI  02881 

I 1  miunon.  William  D.,  Department  of  Biological  Sciences.  Ohio 
University,  Athens,  OH  45701 


R64   Annual  Report 


Humphreys,  Susie  H.,  Food  and  Drug  Administration,  HFS-156 

Switzer,  200  C  Street,  SW,  Washington,  DC  20204-0001 
Humphreys,  Tom  D.,  University  of  Hawaii,  Kewalo  Marine  Lab.  41 

Ahui  St.,  Honolulu,  HI  96813 
Hunt,  Richard  T.,  ICRF,  Clare  Hall  Laboratories,  South  Mimms 

Potter's  Bar,  Herb  EN6-3LD,  England 
Hunter,  Robert  D.,  Department  of  Biological  Sciences.  Oakland 

University.  Rochester.  MI  48309-4401 
Huxley,  Hugh  E.,  Department  of  Biology,  Rosenstiel  Center,  Brandeis 

University,  Waltham,  MA  02154 
Hynes,  Thomas  J.,  Jr.,  Meredith  and  Grew,  Inc.,  160  Federal  Street, 

Boston,  MA  02 II 0-1 70 1 

I l:in.  Joseph,  Department  of  Developmental  Genetics  and  Anatomy. 

Case  Western  Reserve  University  School  of  Medicine.  Cleveland. 

OH  44 1 06 
Ingoglia,  Nicholas,  Department  of  Physiology,  New  Jersey  Medical 

School,  100  Bergen  St.,  Newark.  NJ  07103 
Inoue,  Saduyki,  Department  of  Anatomy.  McGill  University  Cancer 

Centre,  3640  University  St.,  Montreal,  PQ  H3A  2B2,  Canada 
Inoue,  Shinya,  Marine  Biological  Laboratory1.  Woods  Hole.  MA  02543 
Isselbacher,  Kurt  J.,  Massachusetts  Genera]  Hospital  Cancer  Center. 

Charlestown.  MA  02129 
Issidorides,  Marietta,  R.,  Department  of  Psychiatry,  University  of 

Athens.  Monis  Petraki  8.  Athens,  140  Greece 
Izzard,  Colin  S.,  Department  of  Biological  Sciences,  SUNY,  1400 

Washington  Ave.,  Albany,  NY  12222 

Jacobs,  Neil,  Hale  &  Dorr,  60  State  St.,  Boston,  MA  02109 

Jaffe,  Lionel,  Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 

Jannasch,  Holger  W.,  Department  of  Biology,  Woods  Hole 

Oceanographic  Institution.  Woods  Hole.  MA  02543 
Jeffery,  William  R.,  Bodega  Marine  Laboratory.  University  of 

California.  Box  247.  Bodega  Bay.  CA  94923 
Johnston,  Daniel,  Division  of  Neuroscience,  Baylor  College  of 

Medicine,  I  Baylor  Plaza,  Houston.  TX  77030 
Josephson,  Robert  K.,  Department  of  Psychobiology,  University  of 

California,  Irvine,  CA  92717 

Kaczmarek,  Leonard  K.,  Department  of  Pharmacology,  Yale 

University  School  of  Medicine.  333  Cedar  St..  New  Haven,  CT 

06520 
Kaley,  Gabor,  Department  of  Physiology,  Basic  Sciences  Building, 

New  York  Medical  College,  Valhalla,  NY  10595 
kaltenbach,  Jane,  Department  of  Biological  Sciences,  Mount  Holyoke 

College,  South  Hadley.  MA  01075 
Kaminer,  Benjamin,  Department  of  Physiology.  School  of  Medicine, 

Boston  University,  80  East  Concord  St.,  Boston.  MA  021 18 
Kaneshiro,  Edna  S.,  Department  of  Biological  Sciences,  University  of 

Cincinnati.  JL  006.  Cincinnati,  OH  45221-0006 
Kao,  Chien-yuan,  Department  of  Pharmacology,  Box  29,  SLINY, 

Downstate  Medical  Center.  450  Clarkson  Avenue.  Brooklyn.  NY 

11203 
Kaplan.  Ehud,  Department  of  Biophysics,  The  Rockefeller  University. 

1230  York  Ave..  New  York.  NY  10024 
Karakashian,  Stephen  J..  Apt.  16-F.  165  West  91st  St.,  New  York. 

NY  10024 
Karlin,  Arthur,  Department  of  Biochemistry.  Columbia  University, 

630  West  168th  St.,  New  York.  NY  10032 
Katz,  George  M.,  Fundamental  and  Experimental  Research  Labs, 

Merck  Sharp-  and  Dohme.  P.  O.  Box  2000,  Rahway,  NJ  07065 
Kelley,  Darcy  Brisbane,  Columbia  University,  Department  of 

Biological  Sciences,  91  1  Sherman  Fairchild  Ctr.,  New  York,  NY 

10032 


Kelly,  Robert  E.,  Department  of  Anatomy,  University  of  Illinois, 

P.  O.  Box  6998,  Chicago,  IL  60680 
Kemp,  Norman  E.,  Department  of  Biology,  University  of  Michigan, 

Ann  Arbor,  MI  48 109 
Kendall.  John  P.,  Faneuil  Hall  Associates.  176  Federal  Street.  2nd 

Floor,  Boston,  MA  02 110 
Kendall.  Richard  E.,  26  Green  Harbor  Road,  East  Falmouth,  MA 

02536 
Kerr,  Louis  M.,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 
Keynan,  Alexander,  Laboratory  for  Developmental  and  Molecular 

Biology.  Department  of  Biochemistry,  Hebrew  University  of 

Jerusalem,  Givat-Ram,  Jerusalem,  Israel 
Khan,  Shahid  M.  M.,  Department  of  Anatomy  &  Structural  Biology. 

Albert  Einstein  College  of  Medicine.  1300  Morris  Park  Ave.,  Bronx, 

NY  10461 
Kiehart,  Daniel  P.,  Department  of  Cellular  Biology.  Duke  University 

Medical  Center.  Box  3709.  307  Nanaline  Duke  Bldg.,  Durham.  NC 

27710 
Kirk,  Mark  D.,  Division  of  Biological  Sciences,  University  of 

Missouri,  Columbia,  MO  6521 1  (resigned) 
Klotz,  Irving  M.,  Department  of  Chemistry.  Northwestern  University, 

Evanston.  IL  60201 
Knudson,  Robert  A.,  Marine  Biological  Laboratory.  Instrument 

Development  Lab,  Woods  Hole,  MA  02543 
Koide,  Samuel  S.,  Population  Council,  The  Rockefeller  University. 

1230  York  Avenue,  New  York,  NY  10021 
Kornberg,  Sir  Hans,  The  Master's  Lodge,  Christ's  College,  Cambridge 

CB2  3BU,  England,  UK 
Kosower,  Edward  M.,  Department  of  Chemistry,  Tel  Aviv  University, 

Ramat-Aviv,  Tel  Aviv,  Israel  69978 
Krahl,  M.  E.,  2783  W.  Casas  Circle.  Tucson.  AZ  85741 
Krane,  Stephen  M.,  Arthritis  Unit.  Massachusetts  General  Hospital, 

Fruit  Street.  Boston.  MA  021 14 

Krauss,  Robert,  FASEB,  9650  Rockville  Pike,  Bethesda,  MD  20814 
Kravitz,  Edward  A.,  Department  of  Neurobiology.  Harvard  Medical 

School,  220  Longwood  Ave..  Boston.  MA  02 1 1 5 
Kriebel,  Mahlon  F...  Department  of  Physiology.  SLINY  Health  Science 

Center.  Syracuse,  NY  13210 
Kristan,  William  B.,  Jr.,  Department  of  Biology  B-022.  University  of 

California  San  Diego.  La  Jolla,  CA  92093 
Kropinski,  Andrew  M.  B.,  Department  of  Microbiology/Immunology. 

Queen's  University.  Kingston,  Ontario  K7L  3N6,  Canada 
Kuhns,  William  J.,  Hospital  for  Sick  Children,  Department  of 

Biochemistry  Research,  Toronto.  Ontario  M5G  1X8,  Canada 
Kusano,  Kiyoshi,  NIH.  Bldg.  36.  Room  4D-20,  Bethesda,  MD  20892 
Kuzirian,  Alan  M..  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 


Laderman,  Aimlee,  Yale  University  School  of  Forestry  and 

Environmental  Studies.  370  Prospect.  New  Haven.  CT  0651 1 
LaMarche,  Paul  H.,  Eastern  Maine  Medical  Center.  489  State  St.. 

Bangor.  ME  0440 1 
I  andis.  Dennis  M.  D.,  Department  ot  Developmental  Genetics  and 

Anatomy.  Case  Western  Reserve  University  School  of  Medicine. 

Cleveland.  OH  44106 
Landowne,  David,  Department  of  Physiology.  P.  O.  Box  01 6430. 

University  of  Miami  School  of  Medicine,  Miami,  FL  33101 
Langford,  George  M.,  Department  of  Biological  Sciences.  Dartmouth 

College.  6044  Gilman  Laboratory,  Hanover,  NH  03755 
Lasser-Ross,  Nechama,  Department  of  Physiology,  New  York 

Medical  College.  Valhalla.  NY  10595 
Laster,  Leonard,  University  of  Massachusetts  Medical  School.  55  Lake 

Avenue,  North.  Worcester.  MA  01655 


Members  of  the  Corporation   R65 


Laufer,  Hans,  Department  of  Biological  Science,  Molecular  and  Cell 

Biology,  Group  LI- 125,  University  of  Connecticut,  S'orrs,  CT 

06269-3125 
Lazarow,  Paul  B.,  Department  of  Cell  Biology  and  Anatomy.  Mount 

Sinai  Medical  School.  Box  1007,  5th  Avenue  &  100th  Street.  New 

York.  NY  10021 
Lazarus,  Maurice.  Federated  Department  Stores,  Inc..  Sears  Cresent, 

City  Hall  Plaza,  Boston,  MA  02108 
Leadbcttcr,  Edward  R.,  Department  of  Molecular  and  Cell  Biology, 

U-131.  Uni\ersity  of  Connecticut.  Storrs.  CT  06268 
Lederberg,  Joshua,  The  Rockefeller  University,  1230  York  Ave..  New 

York.  NY  10021 
Lee,  John  J.,  Department  of  Biology,  City  College  of  CUNY, 

Convent  Ave.  and  138th  St.,  New  York,  NY  10031 
Leh>,  Donald  B.,  35  Willow  Field  Dr.,  N.  Falmouth,  MA  02556 
Leibovitz,  Louis,  3  Kettle  Hole  Road.  Falmouth,  MA  02540 
Leighton,  Joseph.  Aeron  Biotechnology.  Inc.,  1933  Davis  Street, 

#310,  San  Leandro.  CA  94577 

Leighton,  Stephen,  NIH.  Bldg.  13  3W13.  Bethesda,  MD  20892 
Leintvand,  Leslie  Ann,  Department  of  Microbiology  and  Immunology, 

Albert  Einstein  College  of  Medicine.  1300  Morris  Park  Ave.,  Bronx. 

NY  10461 
Lerman,  Sidney,  Eye  Research  Lab,  Room  41,  New  York  Medical 

College.  100  Grasslands  Ave..  Valhalla.  NY  10595 
Lerner,  Aaron  B.,  Department  of  Dermatology.  PO  Box  3333.  Yale 

University.  School  of  Medicine.  New  Haven.  CT  06510 
Lester,  Henry  A.,  Division  of  Biology.  California  Institute  of 

Technology,  156-29.  Pasadena,  CA  91 125 
Levin,  Jack,  Veterans  Administration  Medical  Center,  1 1  1  H2,  4150 

Clement  St.,  San  Francisco,  CA  94 1 2 1 
Levine,  Richard  B.,  ARL.  Division  of  Neurobiology,  University  of 

Arizona,  61 1  Gould-Simpson  Bldg..  Tucson.  AZ  8572  I 
Le\inthal,  Krancoise.  Department  of  Biological  Sciences.  Columbia 

University,  Broadway  &  1 16th  Street,  New  York.  NY  10026 
Letitan,  Herbert,  National  Science  Foundation.  4201  Wilson 

Boulevard,  Room  835.  Arlington,  VA  22230 
Levitan,  Irwin  B.,  Center  for  Complex  Systems,  Brandeis  University, 

Waltham.  MA  02254 
Linck,  Richard  \V.,  Department  of  Cell  Biology  and  Neuroanatomy. 

University  of  Minnesota.  321  Church  Street.  S.  E.,  Minneapolis. 

MN  55455 
Lipicky,  Raymond  J.,  Food  &  Drug  Administration.  1451  Rockville 

Pike,  Room  5093,  Rockville.  MD  20852 
Lisman,  John  E.,  Department  of  Biology,  Brandeis  University. 

Waltham,  MA  02254 

Liuzzi,  Anthony,  320  Beacon  St..  Boston.  MA  021 16 
Llinas,  Rodolfo  R.,  Department  of  Physiology  and  Biophysics,  New 

York  University  Medical  Center.  550  First  Ave.,  New  York.  NY 

10016 
Loew,  Franklin  M.,  Tufts  University  School  of  Veterinary  Medicine. 

200  Westboro  Rd.,  N.  Grafton,  MA  01536-1895 
Loewenstein.  Birgit  R.,  Marine  Biological  Laboratory.  Woods  Hole, 

MA  02543 
Loewenstein,  \\erner  R.,  Marine  Biological  Laboratory.  Woods  Hole. 

MA  02543 

London.  Irving  M..  Massachusetts  Institute  of  Technology.  Harvard- 
MIT  Division.  E-25-551.  Cambridge.  MA  02139 
Longo,  Frank  J.,  Department  of  Anatomy,  University  oflowa,  Iowa 

City.  1A  52442 
Lorand,  Laszlo,  Northwestern  University  Medical  School.  CMS 

Biology.  Searle  4-555,  303  East  Chicago.  Chicago.  IL  6061 1-3008 
Luckenbill-Kdds,  Louise,  Department  of  Biological  Sciences.  Irvine 
Hall,  Ohio  Universitv.  Athens.  OH  45701 


Macagno,  Eduardo  R.,  I003B  Fairchild,  Department  of  Biosciences, 

Columbia  University,  New  York,  NY  10027 
MacNichol,  E.  F.,  Jr.,  Department  of  Physiology.  Boston  University 

School  of  Medicine.  80  E.  Concord  St..  Boston,  MA  021 18 
Maglolt-Duffield.  Donna  R.,  American  Type  Culture  Collection. 

12301  Parklawn  Drive,  Rockville.  MD  20852-1776 
Maienschein.  Jane  Ann,  Department  of  Philosophy.  Arizona  State 

University,  Tempe.  AZ  85287-2004 
Mainer,  Robert,  The  Boston  Company,  One  Boston  Place,  OBP-15-D, 

Boston,  MA  02 108 
M. ill". IL  Craig  Curtis,  Department  of  Pharmacology,  Health  Sciences 

Center.  SUNY,  Stony  Brook.  NY  1 1794-8651 
Manalis,  Richard  S.,  Department  of  Biological  Sciences.  Indiana 

University— Purdue  University  at  Fort  Wayne.  2101  Coliseum 

Blvd.,  E..  Fort  Wayne.  IN  46805 
Mangum,  Charlotte  P.,  Department  of  Biology,  College  of  William 

and  Mary,  Williamsburg,  VA  23187-8795 
Manz,  Robert  D.,  Helmer  and  Associates,  Suite  1310,  950  Winter  St.. 

Walthan.  MA  02 1 54 
Margulis,  Lynn,  Botany  Department.  University  of  Massachusetts, 

Morrill  Science  Center,  Amherst,  MA  01003 
Marinucci,  Andrew  C,  102  Nancy  Drive.  Mercerville.  NJ  08619 
Marsh,  Julian  B.,  Department  of  Biochemistry  and  Physiology, 

Medical  College  of  Pennsylvania.  3300  Henry  Ave.,  Philadelphia, 

PA  19129 
Martinez,  Jr.,  Joe  L.,  Division  of  Life  Sciences.  University  of  Texas, 

6900  North  Loop,  1604  West,  San  Antonio,  TX  78249-0662 
Martinez-Palomo,  Adolfo,  Seccion  de  Patologia  Experimental, 

Cinvesav-ipn.  07000  Mexico,  D.F.  A.P..  140740,  Mexico 
Maser,  Morton,  Woods  Hole  Education  Assoc.,  P.  O.  Box  EM, 

Woods  Hole,  MA  02543  (deceased) 
Mastroianni,  Luigi,  Jr.,  Department  of  Obstetrics  and  Gynecology. 

Hospital  of  the  University  of  Pennsylvania.  106  Dulles,  3400  Spruce 

Street.  Philadelphia.  PA  19104-4283 
Matteson,  Donald  R.,  Department  of  Biophysics.  University  of 

Maryland  School  of  Medicine,  660  West  Redwood  Street, 

Baltimore,  MD21201 
Mautner,  Henry  G.,  Department  of  Biochemistry,  Tufts  University 

School  of  Medicine,  136  Harrison  Ave.,  Boston,  MA  021  1 1 
Mauzerall,  David,  The  Rockefeller  University,  1230  York  Ave..  New 

York.  NY  10021 
McCann,  Frances.  Department  of  Physiology.  Dartmouth  Medical 

School,  Lebanon,  NH  03756 
McLaughlin,  Jane  A.,  Marine  Biological  Laboratory,  Woods  Hole. 

MA  02543 
McMahon,  Robert  F..  Department  of  Biology.  Box  19498,  University 

of  Texas.  Arlington.  TX  76019 
Meedel,  Thomas,  Biology  Department.  Rhode  Island  College.  600  Mt. 

Pleasant  Ave..  Providence.  RI  02908 
Meinertzhagen,  Ian  A.,  Department  of  Psychology,  Life  Sciences 

Center.  Dalhousie  University,  Halifax.  Nova  Scotia  B3H  4J1, 

Canada 
Meiss,  Dennis  E.,  Immunodiagnostic  Laboratories.  488  McCormick 

St.,  San  Leandro,  CA  94577 
Melillo.  Jerry  M.,  Ecosystems  Center,  Marine  Biological  Laboratory, 

Woods  Hole,  MA  02543 
Mellon,  DeForest,  Jr.,  Department  of  Biology.  Gilmer  Hall. 

University  of  Virginia.  Charlottesville,  VA  22903 
Mellon,  Richard  P.,  P.  O.  Box  187,  Laughlintown.  PA  15655 
Mendelsohn,  Michael  E.,  Cardiovascular  Division.  Harvard  Medical 

School.  75  Francis  Street.  Boston,  MA  02 1 1 5 
Metuzals,  Janis,  Department  of  Pathology.  University  of  Ottawa.  451 

Smythe  Road,  Ottawa.  Ontario  K1H  8M5.  Canada 
Metz,  Charles  B.,  7220  SW  124th  St.,  Miami,  FL  33156 


R66  Annual  Report 


Miledi,  Ricardo,  Department  of  Psychohiology.  University  of 

California.  2205  Biological  Science  II,  Irvine,  CA  92717 
Milkman,  Roger,  Department  of  Biology.  University  of  Iowa,  Iowa 

City,  IA  52242 
Miller,  Andrew  L.,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 
Mills,  Robert,  10315  44th  Avenue,  W  12  H  Street,  Bradenton.  FL 

34210 
Misevic,  Gradimir,  Department  of  Research,  University  Hospital  of 

Basel,  Mebelstrasse  20,  CH-4031,  Basel,  Switzerland 
Mitchell,  Ralph,  DAS.  Harvard  University.  29  Oxford  Street, 

Cambridge,  MA  02 138 
Miyakawa,  Hiroyoshi,  Tokyo  College  of  Pharmacy,  Laboratory  of 

Cellular  Neurobiology,  1432-1  Horinouchi,  Hachiouji,  Tokyo  192- 

03,  Japan 
Miyamoto,  David  M.,  Department  of  Biology,  Drew  University, 

Madison,  NJ  07940 
Mizell,  Merle,  Department  of  Cell  &  Molecular  Biology,  Tulane 

University,  New  Orleans,  LA  701 18 
Moore,  John  VV.,  Department  of  Neurobiology,  Box  3209,  Duke 

University  Medical  Center,  Durham,  NC  27710 
Moore,  Lee  E.,  Department  of  Physiology  and  Biophysics,  University 

of  Texas  Medical  Branch,  Galveston.  TX  77550 
Morin,  James  G.,  Department  of  Biology.  University  of  California, 

Los  Angeles,  CA  90024 
Morrell,  Frank,  Department  of  Neurological  Science,  Rush  Medical 

Center,  1653  W.  Congress  Parkway,  Chicago,  IL  60612 
Morse,  Patricia  M.,  University  of  Washington  Marine  Labs,  620 

University  Rd..  Friday  Harbor,  WA  98250 
Morse,  Stephen  Scott,  The  Rockefeller  University,  1230  York  Ave., 

Box  120,  New  York,  NY  10021-6399 
Mote,  Michael  I.,  Department  of  Biology,  Temple  University, 

Philadelphia.  PA  19122 
Muller,  Kenneth  J.,  Department  of  Physiology  and  Biophysics, 

University  of  Miami  School  of  Medicine,  Miami,  FL  33101 
Murray,  Andrew  \V.,  Department  of  Physiology,  University  of 

California,  Box  0444,  513  Parnassus  Ave.,  San  Francisco.  CA 

94143-0444 
Murray,  Sandra  Ann,  Department  of  Neurology,  Anatomy  and  Cell 

Science,  University  of  Pittsburgh  School  of  Medicine.  Pittsburgh. 

PA  15261 

Nabrit,  S.  M.,  686  Beckwhh  St.,  SW.  Atlanta,  GA  30314 
Nadelhoffer,  Knute,  Marine  Biological  Laboratory,  Ecosystems  Center. 

Woods  Hole.  MA  02543 

Naka,  Ken-ichi,  2-9-2  Tatsumi  Higashi,  Okazaki,  Japan  444 
Nakajima,  Shigehiro,  Department  of  Pharmacology  and  Cell  Biology. 

University  of  Illinois  College  of  Medicine  at  Chicago.  835  S. 

Wolcott  Ave..  Chicago,  IL  60612 
Nakajima,  Vasuko,  Department  of  Anatomy  and  Cell  Biology. 

University  of  Illinois  College  of  Medicine  at  Chicago,  M/C  512. 

Chicago,  IL60612 
Narahashi,  Toshio,  Department  of  Pharmacology,  Northwestern 

University  Medical  School,  303  East  Chicago  Ave.,  Chicago.  IL 

60611 
Nasi,  Enrico,  Department  of  Physiology,  Boston  University  School  of 

Medicine,  R-406,  80  E.  Concord  St.,  Boston,  MA  021 18 
Nealson,  Kenneth  H.,  Great  Lakes  Research  Center.  LIniversity  of 

Milwaukee,  600  E.  Greenfield  Ave.,  Milwaukee,  WI  53204 
Nelson,  Leonard,  Department  of  Physiology.  CSI0008,  Medical 

College  of  Ohio.  Toledo.  OH  43699 
Nelson,  Margaret  C.,  Section  of  Neurobiology  and  Behavior,  Cornell 

University.  Ithaca.  NY  14850 
Nicholls,  John  G.,  Biocenter,  Klingelbergstrasse  70,  Basel  4056, 

Switzerland 


Nickerson,  Peter  A..  Department  of  Pathology.  SUNY,  Buffalo,  NY 

14214 
Nicosia,  Santo  V.,  Department  of  Pathology,  University  of  South 

Florida.  College  of  Medicine,  Box  11.  12901  North  30th  St., 

Tampa,  FL  33612 
Noe,  Bryan  D.,  Department  of  Anatomy  and  Cell  Biology.  Emory 

University  School  of  Medicine,  Atlanta.  GA  30322 
Northcutt,  R.  Glenn,  University  of  California,  San  Diego. 

Neuroscience  0201,  9500  Gilman  Drive,  La  Jolla,  CA  92093-0201 
Norton,  Catherine  N.,  Marine  Biological  Laboratory.  Woods  Hole, 

MA  02543 
Nusbaum,  Michael  P.,  Department  of  Neuroscience,  University  of 

Pennsylvania  School  of  Medicine,  215  Stemmler  Hall,  Philadelphia, 

PA  19104-6074 

O'Herron,  Jonathan,  Jonathan  &  Shirley  O'Herron  Foundation,  One 

Rockefeller  Plaza,  New  York,  NY  10020 

O'Melia,  Anne  F.,  16  Evergreen  Lane.  Chappaqua,  New  York  10514 
Obaid,  Ana  Lia,  Department  of  Neuroscience.  LIniversity  of 

Pennsylvania  School  of  Medicine,  234  Stemmler  Hall.  Philadelphia. 

PA  19104-6074 
Ohki,  Shinpei,  Department  of  Biophysical  Sciences.  SUNY  at  Buffalo. 

224  Cary  Hall.  Buffalo.  NY  14214 
Oldenbourg,  Rudolf,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 

Olds,  James  L.,  N1H,  9/1W125,  Bldg.  9,  Bethesda.  MD  20892 
Olins,  Ada  L.,  University  of  Tennessee-Oak  Ridge,  Graduate  School 

of  Biomedical  Sciences,  Biology  Division  ORNL,  P.  O.  Box  2009, 

Oak  Ridge,  TN  37831-8077 
Olins,  Donald  E.,  LIniversity  of  Tennessee-Oak  Ridge,  Graduate 

School  of  Biomedical  Sciences,  Biology  Division  ORNL,  P.  O.  Box 

2009,  Oak  Ridge.  TN  37831-8077 
Oschman,  James  L.,  3 1  Whittier  Street.  Dover,  NH  03820 

Palazzo,  Robert  E.,  Department  of  Physiology  &  Cell  Biology, 

Haworth  Hall,  LIniversity  of  Kansas,  Lawrence,  KS  66045 
Palmer,  John  D.,  Department  of  Zoology,  University  of 

Massachusetts,  221  Morrill  Science  Center,  Amherst,  MA  01003 
Palti,  Yoram,  Rappaport  Institution.  Techmon.  POB  9697,  Haifa, 

31096  Israel  (resigned) 
Pant,  Harish  C.,  NINCDS/NIH,  Laboratory  of  Neurochemistry.  Bldg. 

36,  Room  4D-20.  Bethesda.  MD  20892 
Pappas,  George  D.,  Department  of  Anatomy,  College  of  Medicine, 

University  of  Illinois,  808  South  Wolcott  St.,  Chicago,  IL  60612 
Pardee,  Arthur  B.,  Dana-Farber  Cancer  Institute,  D810.  44  Binney 

Street.  Boston.  MA  02 1 1 5 
Pardy,  Roosevelt  I,.,  School  of  Life  Sciences,  University  of  Nebraska, 

Lincoln,  NE  68588 

Parmentier,  James  L.,  175  S.  Great  Road,  Lincoln,  MA  01773-41 12 
Passano,  Leonard  M.,  Department  of  Zoology,  Birge  Hall,  University 

of  Wisconsin.  Madison,  WI  53706 
Pearlman,  Alan  I..,  Cell  Biology,  Box  8228,  School  of  Medicine, 

Washington  LIniversity,  St.  Louis,  MO  631 10 
Pederson,  Thoru,  Worcester  Foundation  for  Experimental  Biology, 

Shrewsbury.  MA  01 545 

Perkins,  C.  D.,  400  Hilltop  Terrace,  Alexandria.  VA  22301 
Person,  Philip,  137-87  75th  Road.  Flushing.  NY  1 1367 
Peterson,  Bruce  J.,  Marine  Biological  Laboratory'.  Ecosystems  Center. 

Woods  Hole.  MA  02543 
Pethig,  Ronald,  School  of  Electronic  Engineering  Science.  University 

College  of  N.  Wales.  Dean  St.,  Bangor,  Gwynedd.  LL57  IUT.  UK 
Pfohl,  Ronald  J.,  Department  of  Zoology.  Miami  University,  Oxford, 

OH  45056 
Pierce,  Sidney  K.,  Jr.,  Department  of  Zoology,  University  of 

Maryland,  College  Park.  MD  20742 


Members  of  the  Corporation    R67 


Poindextor,  Jeanne  S..  Barnard  College,  Columbia  University.  3009 

Broadway.  New  York.  NY  10027-6598 
Pollard,  Harvey  B.,  NIH.  NIDDKD.  Lab  of  Cell  Biology  &  Genetics. 

Bldg.  8.  Rm.  401.  Bethesda.  MD  20892 
Pollard.  Thomas  I)..  Department  of  Cell  Biology  and  Anatomy.  Johns 

Hopkins  University.  725  North  Wolfe  St..  Baltimore.  MD  21205 
Porter,  Beverly  II.,  5542  Windysun  Ct..  Columbia.  MD  21045 
Porter,  Mary  E.,  Department  of  Cell  Biology  and  Neurology. 

University  of  Minnesota.  4-147  Jackson  Hall.  Minneapolis.  MN 

55455 
Potter.  1 1. 11  nl.  Department  of  Neurobiology,  Harvard  Medical  School. 

25  Shattuck  St..  Boston.  MA  021 15 
Potts,  William  T.,  Department  of  Biology.  University  of  Lancaster. 

Lancaster.  England.  UK 
Powers,  Dennis  A..  Hopkins  Marine  Station,  Stanford  University. 

Pacific  Grove.  CA  93950 
Powers,  Maureen  K.,  Department  of  Psychology,  301  Arts  &  Science 

Psych  Building,  Vanderbilt  University.  Nashville.  TN  37240 
Pratt,  Melanie  M.,  VITAS  Healthcare  Corporation.  100  S.  Biscayne 

Boulevard.  Miami.  FL  33101 
Prendergast,  Robert  A.,  Wilmer  Institute.  Johns  Hopkins  Hospital. 

601  N.  Broadway.  Baltimore,  MD  21287-9142 
Presley.  Phillip  H.,  Carl  Zeiss,  Inc..  1  Zeiss  Drive.  Thornwood,  NY 

10594  (deceased) 
Price,  Carl  A.,  Waksman  Institute  of  Microbiology,  Rutgers 

University ,  P.  O.  Box  759.  Piscataway,  NJ  08854 
Prior,  David  J.,  Department  of  Biological  Sciences.  NAU  Box  5640. 

Northern  Arizona  University.  Flagstaff.  AZ  8601 1 
Prusch,  Robert  D.,  Department  of  Life  Sciences.  Gonzaga  University. 

Spokane.  WA  99258 
Purves,  Dale,  Department  of  Neurobiology.  Duke  University  Medical 

Center,  Box  3209,  1011  Bryan  Research  Building.  Durham.  NC 

27710 

Quigley,  James,  Department  of  Pathology.  SUNY  Health  Science 
Center,  BHS  Tower  9.  Rm.  140.  Stony  Brook.  NY  1 1794-8691 

Rabb,  Irving  \\ ..  1010  Memorial  Drive,  Cambridge.  MA  02138 
Rabin.  Harvey,  DuPont  Merck  Pharmaceutical.  R&D  Division,  Exp. 

Station  328/358.  Wilmington.  DE  19880 
Rabinowitz.  Michael  B.,  Marine  Biological  Laboratory.  Woods  Hole, 

MA  02543 
Rafferty,  Nancy  S.,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 
Rakowski,  Robert  F.,  Department  of  Physiology  and  Biophysics. 

UHS/The  Chicago  Medical  School.  3333  Greenbay  Rd..  N. 

Chicago.  IL  60064 
Ramon,  Fidel.  Division  of  Post  Graduate  Studies  and  Investigation. 

Faculty  of  Medicine.  Universidad  Nacional  Autonoma  de  Mexico. 

CU.  Mexico.  D.  F.  04510 
Ranzi.  Silvio,  Sez  Zoologia  Scienze  Naturali.  Dip  di  Biologia.  Via 

Coloria  26,  20133,  Milano.  Italy 
Rastetter,  Edward  B.,  Ecosystems  Center,  Marine  Biological 

Laboratory.  Woods  Hole.  MA  02543 
Rebhun.  Lionel  I.,  Department  of  Biology.  Gilmer  Hall  43.  University 

of  Virginia.  Charlottesville.  VA  22901 
Reddan.  John  R.,  Department  of  Biological  Sciences.  Oakland 

University.  Rochester.  MI  48309-4401 
Reese,  Barbara  F.,  NINCDS/NIH.  Bldg  36,  Room  3B26.  9000 

Rockville  Pike.  Bethesda.  MD  20892  (resigned) 
Reese,  Thomas  S.,  NINCDS/NIH.  Bldg.  36,  Room  2A21.  9000 

Rockville  Pike.  Bethesda.  MD  20892 
Reinisch.  Carol  I..,  Department  of  Comparative  Medicine.  Tufts 

University  School  of  Veterinary  Medicine.  200  Westboro  Rd..  Bldg. 

20.  North  Grafton.  MA  01536 


Rich,  Alexander,  Department  of  Biology  16-735.  Massachusetts 

Institute  of  Technology.  Cambridge,  MA  02139 
Rickles,  Frederick  R.,  Center  for  Disease  Control.  MS-D02.  1600 

Clifton  Road.  NE.  Atlanta.  GA  30333 
Riley,  Monica,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 
Ripps,  Harris,  Department  of  Ophthalmology.  University  of  Illinois. 

1855  W.  Taylor  Street.  Chicago,  IL  6061 1 
Ritchie,  Murdoch,  Department  of  Pharmacology,  Yale  University 

School  of  Medicine,  333  Cedar  St.,  New  Haven,  CT  06510 
Robinson,  Denis  M.,  200  Ocean  Lane  Drive  #908,  Key  Biscayne.  FL 

33 149  (deceased) 
Rome,  Lawrence  C.,  Department  of  Biology.  University  of 

Pennsylvania.  Philadelphia.  PA  19104 
Rosenbaum.  Joel  I..,  Department  of  Biology.  310  Kline  Biology 

Tower.  Yale  University.  New  Haven,  CT  06520 
Rosenbluth,  Jack.  Department  of  Physiology.  New  York  University 

School  of  Medicine.  550  First  Ave.,  New  York,  NY  10016 
Rosenbluth.  Raja,  Simon  Eraser  University.  Institute  of  Molecular 

Biology  and  Biochemistry.  Burnaby.  BC.  Canada,  V5A  1S6 
Rosenfield,  Allan,  Columbia  Llniversity  School  of  Public  Health.  600 

West  168th  Street.  New  York.  NY  10032-3702 
Roslansky,  John,  Box  208.  26  Bar  Neck  Road,  Woods  Hole,  MA 

02543 
Roslansky.  Priscilla  F.,  57  Buzzards  Bay  Ave..  Woods  Hole.  MA 

02543 
Ross.  William  N.,  Department  of  Physiology.  New  York  Medical 

College.  Valhalla.  NY  10595 

Roth,  Jay  S.,  P.  O.  Box  692.  Woods  Hole,  MA  02543 
Rowland,  Lewis  P..  Neurological  Institute,  710  West  168th  St..  New 

York,  NY  10032 
Ruderman,  Joan  V.,  Department  of  Anatomy  and  Cell  Biology, 

Harvard  University  School  of  Medicine.  220  Longwood  Ave.. 

Boston,  MA  02 1 1 5 
Rushforth,  Norman  B.,  Department  of  Biology.  Case  Western  Reserve 

University.  Cleveland.  OH  44106 
Russell-Hunter,  W.  D.,  71 1  Howard  Street.  Easton.  MD  21601-3934 


Saffo,  Mary  Beth,  Life  Sciences  Department,  Arizona  State 

University.  West.  P.  O.  Box  37100.  Phoenix.  AZ  85069-7100 
Sager,  Ruth,  Dana  Farber  Cancer  Institute.  44  Binney  St.,  Boston. 

MA  02 1 1 5 
Sagi,  Amir,  Department  of  Life  Sciences.  Ben-Gunon  University  of 

the  Negev,  P.O.  Box  653.  Bee-Sheva.  Israel,  84105 
Salama,  Guy,  Department  of  Physiology,  Llniversity  of  Pittsburgh. 

Pittsburgh.  PA  15261 
Salmon,  Edward  D.,  Department  of  Biology,  Wilson  Hall.  CB3280, 

Llniversity  of  North  Carolina.  Chapel  Hill,  NC  27599 
Salzberg,  Brian  M.,  Department  of  Neuroscience.  University  of 

Pennsylvania.  School  of  Medicine.  234  Stemmler  Hall. 

Philadelphia,  PA  19104-6074 
Sanger,  Jean  M.,  Department  of  Anatomy.  University  of 

Pennsylvania.  School  of  Medicine.  36th  and  Hamilton  Walk. 

Philadelphia.  PA  19104-6058 
Sanger.  Joseph,  Department  of  Anatomy.  University  of  Pennsylvania, 

School  of  Medicine.  36th  and  Hamilton  Walk.  B-13.  Philadelphia. 

PA  19104-6058 
Saunders,  John  \\ '.,  Jr.,  P.  O.  Box  3381,  Waquoit  Station,  Waquoit. 

MA  02536 
Schachman,  Howard  K..  Department  of  Molecular  Biology.  L'niversity 

of  California.  229  Stanley  Hall  #3206.  Berkeley,  CA  94720-3206 
Schalten.  Gerald  P..  Integrated  Microscopy  Facility  for  Biomedical 

Research.  University  of  Wisconsin,  1 1 17  W.  Johnson  St..  Madison. 

WI  53706 


K68  Annual  Report 


Schatten,  Heide,  Department  of  Zoology,  University  of  Wisconsin. 

Madison,  WI  53706 
Schiff,  Jerome  A.,  Institute  for  Photobiology  of  Cells  and  Organelles. 

Brandeis  University,  Waltham.  MA  02254 
Schmeer,  Arlene  C.,  Mercenene  Cancer  Research  Institute.  790 

Prospect  Street.  New  Haven.  CT  065  1 1 
Schmidek,  Henry  H.,  Department  of  Neurosurgery,  St.  Luke's 

Hospital,  102  Page  St.,  New  Bedford,  MA  02740 
Schnapp,  Bruce  J.,  Department  of  Cellular  &  Molecular  Physiology, 

Harvard  Medical  School,  25  Shattuck  St.,  Boston,  MA  02 1  1 5 
Schuel,  Herbert,  Department  of  Anatomy  and  Cell  Biology.  SUNY, 

Buffalo,  Buffalo,  NY  14214 
Schwartz,  James  H.,  Center  for  Neurobiology  and  Behavior,  New 

York  State  Psychiatric  Institute— Research  Annex.  722  W.  168th 

St.,  7th  Floor,  New  York,  NY  10032 
Schweitzer,  A.  Nicola,  Department  of  Pathology.  Brigham  & 

Women's  Hospital,  221  Longwood  Ave.,  LMRC  521.  Boston,  MA 

02115 
Scofield,  Virginia  Lee,  Department  of  Microbiology  and  Immunology, 

University  of  California  School  of  Medicine,  Los  Angeles.  CA 

90024 
Sears,  Mary,  Woods  Hole  Oceanographic  Institution,  Woods  Hole, 

MA  02543 
Segal,  Sheldon  J.,  The  Population  Council,  One  Dag  Hammarskjold 

Plaza,  New  York,  NY  10036 
Selman,  Kelly,  Department  of  Anatomy  and  Cell  Biology,  Box 

100235,  College  of  Medicine,  University  of  Florida  College  of 

Medicine,  Gainesville,  FL  32601 
Shanklin,  Douglas  R.,  Department  of  Pathology.  Room  576, 

University  of  Tennessee  College  of  Medicine.  800  Madison  Avenue. 

Memphis,  TN  381  17 
Shashoua,  Victor  E.,  Ralph  Lowell  Labs,  Harvard  Medical  School. 

McLean  Hospital.  115  Mill  St..  Belmont.  MA  02178 
Shaver,  Gaius  R.,  Ecosystems  Center,  Marine  Biological  Laboratory, 

Woods  Hole,  MA  02543 
Shaver,  John  R.,  Department  of  Zoology.  Michigan  State  University. 

East  Lansing,  Ml  48824 
Sheetz,  Michael  P.,  Department  of  Cell  Biology,  Duke  University 

Medical  Center,  Box  3709.  385  Nanalme  Duke  Bldg..  Durham,  NC 

27710 

Shepard,  David  C.,  P.  O.  Box  44.  Woods  Hole,  MA  02543 
Shepro,  David,  Department  of  Microvascular  Research.  Boston 

University,  5  Cummington  St..  Boston,  MA  02215 
Sheridan,  William  F.,  Biology  Department,  University  of  North 

Dakota,  Box  8238.  University  Station.  Grand  Forks,  ND  58202- 

8238 
Sherman,  I.  W.,  Department  of  Biology.  University  of  California. 

Riverside.  CA  92521 
Shimomura,  Osamu,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 
Shipley,  Alan  M.,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 
Siegel,  Irwin  M.,  Department  of  Ophthalmology,  New  York 

University  Medical  Center.  550  First  Avenue.  New  York.  NY 

10016 
Silver.  Robert  B.,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 
Siwicki,  Kathleen  K.,  Biology  Department,  Swarthmore  College,  500 

College  Ave.,  Swarthmore.  PA  19081 
Sjodin,  Raymond  A.,  Department  of  Biophysics.  University  of 

Maryland.  Baltimore.  MD  21201 
Skinner,  Dorothy  M.,  Biology  Division,  Oak  Ridge  National 

Laboratory.  P.  O.  Box  2009.  Oak  Ridge,  TN  37831 
Sloboda,  Roger  D.,  Department  of  Biological  Sciences.  306  Oilman. 

Dartmouth  College,  Hanover,  NH  03755 


Sluder,  Greenfield,  Worcester  Foundation  for  Experimental  Biology, 

222  Maple  Ave.,  Shrewsbury,  MA  01545 
Smith,  Peter  J.  S.,  Marine  Biological  Laboratory.  National  Vibrating 

Probe  Facility.  Woods  Hole,  MA  02543 
Smith,  Stephen  J.,  Department  of  Molecular  &  Cellular  Physiology, 

Beckman  Center,  Stanford  University  School  of  Medicine,  Stanford, 

CA  94305-5426 
Smolowitz.  Roxanna  M.,  Laboratory  of  Marine  Animal  Health, 

Marine  Biological  Laboratory,  Woods  Hole,  MA  02543 
Sogin,  Mitchell,  Marine  Biological  Laboratory.  Woods  Hole.  MA 

02543 
Sorenson,  Martha  M.,  Cidade  Universitana-RFRJ,  Department  de 

Bioquimica-ICB/CCS.  Rio  de  Janeiro.  RJ  21910.  Brasil 
Speck.  William  T.,  Columbia  Presbyterian  Medical  Center.  161  Ft. 

Washington  Avenue,  New  York.  NY  10032 
Spector,  Abraham,  Department  of  Ophthalmology,  Columbia 

University.  630  West  168th  Street,  New  York,  NY  10032 
Speer,  John  \\.,  Marine  Biological  Laboratory,  Woods  Hole,  MA 

02543 
Speksnijder,  Johanna  E.,  University  of  Groningen.  Department  of 

Genetics,  Kerklaan  30.  975 1  NN  Haren.  The  Netherlands 
Sperelakis,  Nicholas,  Department  of  Physiology  &  Biophysics, 

University  of  Cincinnati,  231  Bethesda  Ave..  Cincinnati.  OH 

45267-0576 
Spiegel,  Evelyn,  Department  of  Biological  Sciences,  Dartmouth 

College.  204  Gilman.  Hanover.  NH  03755 
Spiegel,  Melvin,  Department  of  Biological  Sciences,  Dartmouth 

College,  204  Gilman,  Hanover,  NH  03755 
Spray,  David  C.,  Albert  Einstein  College  of  Medicine.  Department  of 

Neurosciences.  1300  Moms  Park  Avenue.  Bronx.  NY  10461 
Steele,  John  Hyslop,  Woods  Hole  Oceanographic  Institution,  Woods 

Hole,  MA  02543 
Steinacker,  Antoinette,  Dept.  of  Otolaryngology,  Washington 

University,  School  of  Medicine.  Box  8115.  4566  Scott  Avenue,  St. 

Louis,  MO  63 110 
Steinberg,  Malcolm,  Princeton  University,  Department  of  Molecular 

Biology,  M-18  Moffett  Laboratory,  Princeton,  NJ  08544-1014 
Stemmer,  Andreas  C.,  Marine  Biological  Laboratory,  Woods  Hole, 

MA  02543 
Stelten,  Jane  Lazarow.  4701  Willard  Ave.,  Apt.  1413.  Chevy  Chase, 

MD  208 15-4635 
Steudler,  Paul  A..  Ecosystems  Center,  Marine  Biological  Laboratory, 

Woods  Hole,  MA  02543 
Stokes,  1 1. n  nil  R.,  Emory  University.  Department  of  Biology,  1510 

Clifton  Rd.,  NE,  Atlanta.  GA  30322-1 100 
Stommel,  Elijah  VV.,  P.O  Box  31,  E.  Thetford,  VT  05043 
Stracher,  Alfred,  Department  of  Biochemistry,  SUNY  Health  Science 

Center.  450  Clarkson  Ave..  Brooklyn.  NY  I  1203 
Strehler,  Bernard  L.,  2310  North  Laguna  Circle  Dr.,  Agoura,  CA 

91301-2884 
Strumwasser,  Felix,  USUHS.  Department  of  Psychiatry,  F.  E.  Herbert 

School  of  Medicine.  4301  Jones  Bridge  Rd..  Bethesda.  MD  20814- 

4799 
Stuart,  Ann  E.,  Department  of  Physiology,  Medical  Sciences  Research 

Bldg.  206H.  University  of  North  Carolina.  Chapel  Hill.  NC  27599- 

7545 
Sugden,  Donata  O.,  University  of  Wisconsin.  Department  of 

Neurophysiology.  281  Medical  Science  Building,  Madison,  WI 

53706 
Sugimori,  Mutsuyuki,  Department  of  Physiology  and  Neuroscience, 

Room  442,  New  York  University  Medical  Center.  550  First 

Avenue,  New  York.  NY  10016 
Summers,  William  C.,  Huxley  College  of  Environmental  Studies. 

Western  Washington  University.  Bellingham,  WA  98225 


Members  of  the  Corporation  R69 


Suprenanl,  Katln  A.,  Department  of  Physiology  and  Cell  Biology. 

4010  Haworth  Hall,  University  of  Kansas.  Lawrence,  KS  66045 
Sussman,  Maurice,  72  Carey  Lane.  Falmouth,  MA  02540 
Sussman,  Raquel  B.,  Marine  Biological  Laboratory.  Woods  Hole,  MA 

02543 
SHeet.  Frederick,  Department  of  Obstetrics  and  Gynecology,  Box 

8064,  Washington  University  School  of  Medicine.  499  South 

Euclid,  St.  Louis.  MO  631 10 
Swenson,  Katherine  I.,  Duke  University  Medical  Center.  Department 

of  Molecular  Cancer,  P.O.  Box  3686,  Durham.  NC  27710 
Sydlik,  Mary  Anne,  Department  of  Biology,  Westfield  State  College, 

Westtield,  MA  01086 
Szent-Gybrgyi,  Andrew,  Department  of  Biology,  Brandeis  University, 

Bassine  244.  415  South  Street.  Waltham,  MA  02254 
Szent-Gyorgyi,  Gwen  P.,  45  Nobska  Road.  Woods  Hole,  MA  02543 
Szuts,  F.te  /..,  1  Elm  Street,  Byfield,  MA  01922-2728 

Tabares,  Lucia,  Department  of  Physiology.  University  of  Seville 

School  of  Medicine,  Avda.  Sanchez  Pizjuan.  4.  Seville  41009,  Spain 
Tamm.  Sidney  L.,  Boston  University  Marine  Program.  Marine 

Biological  Laboratory.  Woods  Hole,  MA  02543 
Tanzer,  Marvin  L.,  University  of  Connecticut  School  of  Dental 
Medicine.  Department  of  Biostructure  and  Function.  Farmington, 
CT  06030-3705 
Tasaki,  Ichiji,  Laboratory  of  Neurobiology,  NIMH/NIH,  Bldg.  36. 

Rm.  2B-16,  Bethesda.  MD  20892 
Taylor,  Douglass  L.,  Center  for  Fluorescence  Research,  Carnegie 

Mellon  University,  4400  Fifth  Avenue,  Pittsburgh,  PA  15213 
Teal,  John  M.,  Department  of  Biology.  Woods  Hole  Oceanographic 

Institution.  Woods  Hole,  MA  02543 
Telfer,  William  II.,  Department  of  Biology,  University  of 

Pennsylvania,  Philadelphia,  PA  19104 
Telzer,  Bruce,  Pomona  College,  Department  of  Biology.  Thille 

Building.  175  W.  6th  Street,  Claremont,  CA  9171 1 
Thorndikc,  \V.  Nicholas,  Wellington  Management  Company,  200 

StateSt..  Boston.  MA  02 109 
Townsel,  James  G.,  Department  of  Physiology,  Meharry  Medical 

College.  Nashville,  TN  37208 
Travis.  David  M.,  300  River  Road.  #311.  Manchester,  NH  03104- 

2483 

Treistman,  Steven  N.,  University  of  Massachusetts  Medical  Center, 
Department  of  Pharmacology,  55  Lake  Avenue  North.  Worcester. 
MA  01655 

Trigg.  D.  Thomas,  One  Federal  Street,  9th  Floor.  Boston.  MA  0221 1 
Trinkaus,  J.  P.,  Department  of  Biology,  Yale  University.  New  Haven, 

CT  065 1  1 
Troll,  Walter,  Department  of  Environmental  Medicine,  College  of 

Medicine.  New  York  University.  New  York.  NY  10016 
Troxler,  Robert  F.,  Department  of  Biochemistry.  School  of  Medicine, 

Boston  University,  80  East  Concord  St.,  Boston.  MA  02  1  1 8 
Tucker,  Fdward  B.,  Department  of  Natural  Sciences.  Baruch  College, 

CUNY.  17  Lexington  Ave..  New  York,  NY  10010 
Turner,  Ruth  D.,  Mollusk  Department.  Museum  of  Comparative 

Zoology.  Harvard  University.  Cambridge,  MA  02138 
Tweedell,  Kenynn  S.,  Department  of  Biological  Sciences,  University  of 

Notre  Dame.  Notre  Dame,  IN  46656 
Tykocinski,  Mark  L..  Institute  of  Pathology.  Case  Western  Reserve 

University.  2085  Adelbert  Rd..  Cleveland.  OH  44106 
Tytell,  Michael.  Department  of  Anatomy  &  Neurobiology.  Bowman 
Gray  School  of  Medicine,  Wake  Forest  University,  Winston-Salem. 
NC  27103 

I'eno,  Hiroshi.  Kyoto  University.  Faculty  of  Agriculture,  Department 
of  Agricultural  Chemistry,  Sakyo,  Kyoto  606.  Japan 


Yaliela,  Ivan,  Boston  Llniversity  Marine  Program.  Marine  Biological 

Laboratory,  Woods  Hole,  MA  02543 
Vallee,  Richard,  Cell  Biology  Group,  Worcester  Foundation  for 

Experimental  Biology,  Shrewsbury.  MA  01545 
Valois,  John,  420  Woods  Hole  Road.  Woods  Hole.  MA  02543 
Van  Holde,  Kensal,  Department  of  Biochemistry  and  Biophysics. 

Oregon  State  University.  Corvallis.  OR  97331-7503 
Vogel,  Steven  S.,  LTPB/NICHD.  Bldg.  10,  Rm.  6C205,  Bethesda, 

MD  20892 


Waksman,  Byron,  Foundation  for  Microbiology,  300  East  54th  St., 

#5K,  New  York,  NY  10022 

Wall,  Betty,  9  George  St.,  Woods  Hole,  MA  02543 
Wallace,  Robin  A.,  Whitney  Laboratory.  9505  Ocean  Shore  Blvd..  St. 

Augustine,  FL  32086  (resigned) 
Wang,  Ching  Chung,  Department  of  Pharmaceutical  Chemistry. 

University  of  California.  San  Francisco,  CA  94143 
Wang,  Hsien-yu,  Department  of  Physiology  &  Biophysics,  HSC. 
University  Medical  Center.  SUNY-Stony  Brook.  Stony  Brook.  NY 
11794-8633 
\Vangh,  Lawrence  J..  Department  of  Biology,  Brandeis  Llniversity, 

415  South  St.,  Waltham,  MA  02254 
Warner,  Robert  C,  Department  of  Molecular  Biology  and 
Biochemistry.  University  of  California,  Irvine.  CA  92717 
Warren,  Kenneth  S.,  The  Picower  Institute  for  Medical  Research,  350 

Community  Drive,  Manhasset.  NY 
Warren,  Leonard,  Wistar  Institute,  36th  and  Spruce  Streets. 

Philadelphia,  PA  19104 
Waterbury,  John  B.,  Department  of  Biology,  Woods  Hole 

Oceanographic  Institution,  Woods  Hole.  MA  02543 
Watson,  Stanley,  Associates  of  Cape  Cod.  Inc.,  P.  O.  Box  224,  Woods 

Hole,  MA  02543  (deceased) 
Waxman,  Stephen  G.,  Department  of  Neurology.  P.O.  Box  208018, 

Yale  School  of  Medicine,  333  Cedar  Street,  New  Haven.  CT  06510 
Webb,  H.  Marguerite,  Marine  Biological  Laboratory.  Woods  Hole. 

MA  02543 

Weber,  Annemarie,  Department  of  Biochemistry  and  Biophysics. 
School  of  Medicine.  University  of  Pennsylvania,  Philadelphia,  PA 
19066 
Weidner,  Earl,  Department  of  Zoology  and  Physiology.  Louisiana 

State  University.  Baton  Rouge.  LA  70803 
Weiss,  Dieter  G.,  Fachbereich  Biologic,  Institute  Tierphysiologie, 

University  of  Rostock,  D- 18051  Rostock  Germany 
Weiss,  Leon  P.,  Department  of  Animal  Biology,  School  of  Veterinary 

Medicine,  University  of  Pennsylvania,  Philadelphia,  PA  19104 
Weissmann,  Gerald,  New  York  University  School  of  Medicine,  550 

First  Avenue,  New  York,  NY  10016 
\\erman,  Robert,  Neurobiology  LInit,  The  Hebrew  Llniversity, 

Jerusalem,  Israel 
Westerfield,  R.  Monte,  The  Institute  of  Neuroscience,  University  of 

Oregon,  Eugene,  OR  97403 

\\  hittaker,  J.  Richard,  Department  of  Biology.  Bag  Service  #451 1 1. 
University  of  New  Brunswick.  Fredericton.  NB  E3B  6E1,  Canada 
Wichterman,  Ralph,  3 1  Buzzards  Bay  Avenue,  Woods  Hole,  MA 

02543 
Wilson,  Darcy  B.,  San  Diego  Regional  Cancer  Center,  3099  Science 

Park  Road.  San  Diego.  CA  92121 
Wilson,  T.  Hastings,  Department  of  Physiology,  Harvard  Medical 

School,  25  Shattuck  Street.  Boston.  MA  021 15 
Witkovsky,  Paul,  Department  of  Ophthalmology,  New  York 

University  Medical  Center.  550  First  Ave.,  New  York,  NY  10016 


R70  Annual  Report 


\\  ittenberg,  Beatrice,  Department  of  Physiology  &  Biophysics,  Albert 

Einstein  College  of  Medicine,  1300  Morris  Park  Ave.,  Bronx,  NY 

10461 
Wittenberg,  Jonathan  B.,  Department  of  Physiology  and  Biophysics, 

Albert  Einstein  College,  1300  Morris  Park  Ave.,  Bronx,  NY  01461 
Wolken,  Jerome  J.,  Department  of  Biological  Sciences,  Carnegie 

Mellon  University.  440  Fifth  Ave.,  Pittsburgh.  PA  15213 
\Vonderlin,  \\  illiam  F..  Department  of  Pharmacology  &  Toxicology, 

West  Virginia  University.  Morgantown.  WV  26506 
Worden,  Mary  Kate,  Department  of  Neurobiology,  Harvard  Medical 

School,  220  Longwood  Ave.,  Boston,  MA  021  15 
\\orgul,  Basil  V.,  Department  of  Ophthalmology,  Columbia 

University,  630  West  168th  St.,  New  York,  NY  10032 
\V'u,  Chau  Hsiung,  Department  of  Pharmacology,  S2 1 5.  Northwestern 

University  Medical  School.  Chicago.  IL  6061 1 
VVyttenbach,  Charles  R.,  Department  of  Physiology  and  Cell  Biology. 

University  of  Kansas,  Lawrence,  K.S  66045 


Yen,  Jay  Z.,  Department  of  Pharmacology,  Northwestern  University 
Medical  School,  Chicago,  IL  606 1 1 

Zigman,  Seymour,  Ophthalmology  Research,  LIniversity  of  Rochester 

Medical  School,  Box  314,  601  Elmwood  Avenue,  Rochester,  NY 

14642 
Zigmond,  Michael  J.,  University  of  Pittsburgh,  570  Crawford  Hall, 

Pittsburgh.  PA  15260 
Zimmerberg,  Joshua  J.,  NIH.  Bldg.  12A,  Room  2007,  Bethesda.  MD 

20892 
Zottoli,  Steven  J.,  Department  of  Biology,  Williams  College. 

Williamstown,  MA  01267 
Zucker,  Robert  S.,  Neurobiology  Division,  Department  of  Molecular 

and  Cellular  Biology,  University  of  California,  Berkeley.  CA  94720 
Zukin,  Ruth  Suzanne,  Department  of  Neuroscience,  Albert  Einstein 

College  of  Medicine,  1410  Pelham  Parkway  South.  Bronx,  NY 

10461 


MBL  Associates 


Executive  Board 

Megan  Jones,  President 

Mary  Ulbrich,  Vice  President 

Deborah  G.  Sen  ft.  Treasurer 

Priscilla  Roslansky.  Secretary 

Alfred  F.  Borg 

Jennie  P.  Brown 

Julia  S.  Child 

Elaine  Pear  Cohen.  Arts  Chair  (deceased) 

Hanna  Hastings 

Doris  B.  Hiatt 

Ruth  Ann  Laster.  Membership  Chair 

Evelyn  Laufer 

Barbara  Little 

Robert  Livingstone,  Jr. 

Luigi  Mastroianm.  Jr. 

Jack  Pearce 

Robert  M.  Reynolds 

Ted  Rowan 

John  Valois 

Barbara  Wheeler 

Sustaining  Associate 
Dr.  and  Mrs.  Neal  W.  Cornell 
Mr.  and  Mrs.  Jonathan  O'Herron 
Plymouth  Savings  Bank 

Supporting  Associate 

Dr.  and  Mrs.  Richard  Armstrong 

Benthos 

Mr.  James  M.  Clark 

Mr.  and  Mrs.  LeRoy  Clark.  Jr. 

Dr.  and  Mrs.  Alexander  W.  Clowes 

Mrs.  Margaret  Clowes 

Mr.  and  Mrs.  Ian  D.  W.  Cramer 

Dr.  and  Mrs.  James  D.  Ebert 

Mr.  and  Mrs.  David  Fausch 

Drs.  Gerald  and  Ruth  Fischbach 

Mr.  and  Mrs.  David  Gaiser 

Dr.  and  Mrs.  Prosser  GifTord 

Dr.  and  Mrs.  Howard  H.  Hiatt 

Dr.  and  Mrs.  Leonard  Laster 


Mr.  and  Mrs.  William  K.  Mackey 

Drs.  Luigi  and  Elaine  Mastroianni 

Dr.  and  Mrs.  J.  Wister  Meigs 

Mr.  and  Mrs.  David  Palmer 

Ms.  Linda  Sallop  and  Mr.  Michael  Fenlon 

Mr.  and  Mrs.  John  E.  Sawyer 

Drs.  Christina  and  John  Tochko 


Dual  Membership 

Mr.  and  Mrs.  David  C.  Aheam 

Mr.  and  Mrs.  Douglas  F.  Allison 

Dr.  and  Mrs.  Samuel  C.  Armstrong 

Mr.  and  Mrs.  Henry  Ashworth 

Mr.  and  Mrs.  Duncan  P.  Aspinwall 

Mr.  and  Mrs.  Donald  R.  Aukamp 

Dr.  and  Mrs.  H.  Thomas  Ballantine,  Jr. 

Mr.  and  Mrs.  William  L.  Banks 

Mr.  and  Mrs.  R.  Channing  Barlow 

Dr.  and  Mrs.  Robert  B.  Barlow.  Jr. 

Mr.  and  Mrs.  John  E.  Barnes 

Mr.  and  Mrs.  Richard  T.  Baum 

Dr.  and  Mrs.  Robert  M.  Berne 

Drs.  Alan  and  Harriet  Bernheimer 

Mr.  and  Mrs.  Robert  O.  Bigelow 

Dr.  and  Mrs.  Edward  G.  Boettiger 

Dr.  and  Mrs.  Alfred  F.  Borg 

Dr.  and  Mrs.  Thomas  A.  Borgese 

Dr.  and  Mrs.  Francis  P.  Bowles 

Dr.  and  Mrs.  Thornton  Brown 

Dr.  and  Mrs.  John  B.  Buck 

Dr.  and  Mrs.  John  E.  Burris 

Dr.  and  Mrs.  Francis  D.  Carlson 

Mr.  and  Mrs.  Winslow  G.  Carlton 

Dr.  and  Mrs.  Frank  M.  Child.  Ill 

Dr.  and  Mrs.  Arnold  M.  Clark 

Mr.  and  Mrs.  David  L.  Crabb 

Mr.  and  Mrs.  Melvin  C.  Cram 

Mr.  and  Mrs.  Thomas  S.  Crane 

Dr.  and  Mrs.  John  M.  Cummings 

Mr.  and  Mrs.  Bruce  G.  Daniels 

Dr.  and  Mrs.  Clyde  J.  Dawe 

Drs.  Charles  and  Mollv  DiCecca 


Mr.  and  Mrs.  William  P.  Dugan 

Dr.  and  Mrs.  James  J.  Ferguson.  Jr. 

Mr.  and  Mrs.  Frederick  S.  Fisher,  III 

Mr.  and  Mrs.  Howard  G.  Freeman 

Mr.  and  Mrs.  Leonard  D.  Friedman 

Dr.  and  Mrs.  Robert  A.  Frosch 

Mr.  and  Mrs.  Robert  S.  Gilette 

Mr.  and  Mrs.  Charles  Goodwin 

Dr.  and  Mrs.  Harlyn  O.  Halvorson 

Drs.  Alexander  and  Carol  Hannenberg 

Mrs.  Janet  M.  Harvey 

Dr.  and  Mrs.  Richard  B.  Harvey 

Dr.  and  Mrs.  J.  Woodland  Hastings 

Mr.  and  Mrs.  Gray  G.  Hayward 

Dr.  and  Mrs.  John  E.  Hobbie 

Dr.  and  Mrs.  Stuart  Hodge 

Drs.  Francis  C.  G.  Hoskin  and  Elizabeth  M. 

Farnham 

Dr.  and  Mrs.  Robert  J.  Huettner 
Mrs.  Mary  D.  Janney 
Mr.  and  Mrs.  DeWitt  C.  Jones,  III 
Dr.  and  Mrs.  Benjamin  Kaminer 
Dr.  Peter  N.  Kivy 
Dr.  and  Mrs.  S.  Andrew  Kulin 
Dr.  and  Mrs.  Hans  Laufer 
Mr.  William  Lawrence 
Dr.  and  Mrs.  Berton  J.  Leach 
Dr.  and  Mrs.  Rachmiel  Levine 
Mr.  and  Mrs.  James  E.  Lloyd 
Mrs.  Ermine  W.  Lovell 
Mrs.  Anne  Camille  Maher 
Mr.  and  Mrs.  Bernard  Manuel 
Mr.  and  Mrs.  Joseph  C.  Martyna 
Mr.  and  Mrs.  Frank  J.  Mather,  III 
Mr.  and  Mrs.  James  W.  Mavor,  Jr. 
Mr.  John  J.  McMahon 
Mr.  and  Mrs.  Arthur  V.  Meigs 
Dr.  and  Mrs.  Jerry  M.  Melillo 
Dr.  and  Mrs.  Charles  B.  Metz 
Mr.  and  Mrs.  Richard  Meyers 
Mr.  and  Mrs.  Charles  A.  Mitchell 
Dr.  and  Mrs.  Charles  H.  Montgomery 
Mr.  and  Mrs.  Basset!  K..  Morse 


Members  of  the  Corporation   R71 


Mr.  and  Mrs.  Frank  L.  Nickerson 

Mr.  and  Mrs.  Clifford  T.  O'Connell 

Dr.  and  Mrs.  George  D.  Pappas 

Mr.  and  Mrs.  Robert  Parkinson 

Dr.  and  Mrs.  John  B.  Pearce 

Mr.  and  Mrs.  John  B.  Peri 

Dr.  and  Mrs.  Philip  Person 

Mr.  and  Mrs.  Frederick  S.  Peters 

Mr.  and  Mrs.  E.  Joel  Peterson 

Dr.  and  Mrs.  Anthony  Pires 

Mr.  and  Mrs.  George  H.  Plough 

Dr.  and  Mrs.  Aubrey  Pothier.  Jr. 

Dr.  and  Mrs.  C.  Ladd  Prosser 

Mr.  Allan  Ray  Putnam 

Mr.  and  Mrs.  Robert  M.  Reynolds 

Dr.  and  Mrs.  Renato  A.  Ricca 

Mr.  and  Mrs.  Harold  Righter 

Mr.  and  Mrs.  John  Ripple 

Ms.  Jean  Roberts 

Drs.  John  and  Priscilla  F.  Roslansky 

Dr.  and  Mrs.  John  D.  Rummel 

Dr.  and  Mrs.  John  W.  Saunders,  Jr. 

Dr.  and  Mrs.  R.  Walter  Schlesinger 

Mr.  John  Seder  and  Ms.  Frances  Plough 

Dr.  and  Mrs.  Sheldon  J.  Segal 

Drs.  Cecily  C.  Selby  and  James  S.  Coles 

Dr.  and  Mrs.  Douglas  R.  Shanklin 

Dr.  and  Mrs.  David  Shepro 

Mr.  and  Mrs.  Bertram  R.  Silver 

Mr.  and  Mrs.  Jonathan  O.  Simonds 

Mr.  and  Mrs.  Daniel  M.  Singer 

Drs.  Frederick  and  Marguerite  Smith 

Mr.  and  Mrs.  Homer  P.  Smith 

Mr.  and  Mrs.  Heinz  Specht 

Dr.  and  Mrs.  William  K.  Stephenson 

Mr.  and  Mrs.  Gerard  L.  Swope 

Mr.  and  Mrs.  Gordon  F.  Todd 

Mr.  and  Mrs.  D.  Thomas  Trigg 

Dr.  and  Mrs.  Walter  Troll.  Ph.D. 

Mr.  and  Mrs.  Volker  Ulbrich 

Mr.  and  Mrs.  John  Valois 

Mr.  and  Mrs.  Ronald  Veeder 

Ms.  Susan  Veeder 

Mr.  and  Mrs.  Samuel  Vincent 

Mr.  and  Mrs.  Henry  Walter 

Dr.  and  Mrs.  Henry  B.  Warren 

Mr.  and  Mrs.  John  T.  Weeks 

Mr.  and  Mrs.  Alfred  Weisberg 

Dr.  and  Mrs.  Gerald  Weissman 

Dr.  and  Mrs.  Paul  S.  Wheeler 

Mr.  and  Mrs.  Leslie  J.  Wilson 

Mr.  and  Mrs.  Bruce  Zimmerh 

Dr.  and  Mrs.  Donald  J.  Zinn 

Individual  Associates 

Dr.  Frederick  W.  Ackroyd 

Mrs.  Marion  S.  Adelberg 

Mr.  Henry  Albers 

Dr.  Nina  S.  Allen 

Drs.  James  and  Helene  Anderson 

Mrs.  Kamball  C.  Atwood,  III 

Mr.  Everett  E.  Bagley 

Mr.  and  Mrs.  C.  John  Berg 


Ms.  Carol  L.  Bissonnette 

Dr.  Thomas  P.  Bleck 

Mr.  Robert  D.  Boche 

Mrs.  Julie  Boettiger 

Mr.  Theodore  A.  Bonn 

Mrs.  Frank  A.  Brown,  Jr. 

Mrs.  Thomas  A.  Brown 

Dr.  Robert  H.  Broyles 

Dr.  Alan  H.  Burghauser 

Mrs.  Beatrice  F.  Buxton 

Mr.  Bruce  E.  Buxton 

Mrs.  David  Campbell 

Mr.  Frank  C.  Carotenuto 

Dr.  Robert  H.  Camer 

Mrs.  Patricia  A.  Case 

Mrs.  Shirley  R.  Chaet 

Mrs.  Christie  L.  Chapman 

Dr.  Sallie  Chisholm 

Dr.  Peter  L.  Clark 

Ms.  Ann  P.  Cleary 

Dr.  Laurence  P.  Cloud 

Mr.  Allen  W.  Clowes 

Dr.  Jewel  Plummer  Cobb 

Mrs.  Elaine  Pear  Cohen  (deceased) 

Prof.  Donald  Eugene  Copeland 

Dr.  Helen  M.  Costello 

Dr.  Vincent  Cowling 

Dr.  Sylvia  E.  Crane 

Ms.  Charlotte  E.  Cross 

Ms.  Dorothy  Crossley 

Miss  Helen  Crossley 

Mrs.  Villa  B.  Crowell 

Dr.  Morton  Davidson 

Mr.  David  L.  Donovan 

Ms.  Suzanne  Droban 

Mr.  and  Mrs.  Charles  E.  Eastman 

Mr.  Raymond  Eliott 

Mr.  Gordon  C.  Estabrooks 

Mr.  William  M.  Ferry 

Mr.  John  W.  Folino,  Jr. 

Mr.  Paul  J.  Freyheit 

Dr.  John  J.  Funkhouser 

Mrs.  Paul  M.  Fye 

Miss  Eleanor  Garfield 

Dr.  James  L.  German.  Ill 

Mr.  Charles  Gifford 

Mrs.  Rebeckah  D.  Glazebrook 

Mr.  Michael  P.  Goldring 

Mrs.  Phyllis  Goldstein 

Mr.  and  Mrs.  John  Grassle 

Mrs.  Edith  T.  Grosch 

Mrs.  Mona  Gross 

Mrs.  Barbara  Grossman 

Dr.  Harry  O.  Haakonsen 

Mrs.  Valerie  A.  Hall 

Ms.  Mary  Elizabeth  Hamstrom 

Dr.  Robert  R.  Haubnch 

Dr.  David  S.  Hays 

Mrs.  H.  D.  Hibbitt 

Mrs.  Bertha  V.  Hill 

Mrs.  Eleanor  M.  Hirschfteld 

Mrs.  Helen  Hodosh  (deceased) 

Mrs.  Man  Jean  Howard 


Ms.  Susan  A.  Huettner 

Miss  Elizabeth  B.  Jackson 

Mrs.  Margaret  Jenkins 

Mrs.  Barbara  W.  Jones 

Mr.  Fred  Karush 

Mrs.  Jessie  Keosian 

Mrs.  Patricia  E.  Keoughan 

Dr.  Ben  Korgen 

Mr.  Ezra  Laderman 

Mrs.  Rodney  C.  Larcom 

Ms.  Rebecca  Lash 

Mr.  and  Mrs.  F.  Arthur  Le  Blond 

Dr.  Marian  E.  LeFevre 

Dr.  Mortimer  Levitz 

Mr.  Lennart  Lindberg 

Mr.  Timothy  Lindner 

Mrs.  Barbara  C.  Little 

Mr.  Robert  Livingstone,  Jr. 

Mrs.  Sarah  Loessel 

Mr.  Richard  C.  Lovering 

Miss  Doris  L.  Low 

Dr.  and  Mrs.  Philip  B.  Maples 

Dr.  Julian  B.  Marsh 

Mrs.  Jane  C.  McCormack 

Mrs.  Nella  W.  McElroy 

Mr.  Paul  McGonigle 

Ms.  Mary  W.  McKoan 

Ms.  Cornelia  McMurtrie 

Mr.  Mentor  Metaxas 

Dr.  Daniel  G.  Miller 

Mrs.  Florence  E.  Mixer 

Mrs.  Anna  Monroy 

Mrs.  Mary  E.  Montgomery 

Dr.  Isabel  Mountain 

Mrs.  Eleanor  M.  Nace 

Mr.  Paul  F.  Nace,  Jr. 

Mr.  John  E.  Naugle 

Dr.  Pamela  Nelson 

Ms.  Catherine  N.  Norton 

Mr.  Thomas  O'Neil 

Dr.  Renee  Bennett  O'Sullivan 

Dr.  Janice  S.  Olszowka 

Mrs.  Malcolm  S.  Park 

Ms.  Joan  Pearlman 

Dr.  Judith  Pederson 

Mr.  Raymond  W.  Peterson 

Mrs.  F.  Carol  Price 

Mr.  John  S.  Price 

Mrs.  Cynthia  Rankin 

Mrs.  Julia  S.  Rankin 

Dr.  Samuel  O.  Raymond 

Ms.  A.  Kathy  Regis 

Mr.  John  Riina 

Dr.  Monica  Riley 

Mr.  Alexander  Meigs  Rives 

Mrs.  Lola  E.  Robertson 

Ms.  Hilde  Rosenthal 

Mrs.  Atholie  K.  Rosett 

Ms.  Virginia  F.  Ross 

Mr.  Edward  Rowan 

Mr.  Francis  C.  Ryder 

Mrs.  Ruth  L.  Saz 

Dr.  Edward  K.  Scheer 


R72  Annual  Report 


Mr.  Peter  J.  Schwamb 

MBL  Associates  Gift  Shop 

Lorraine  Mizell 

Mrs.  Elsie  M.  Scott 

Volunteers 

Eleanor  Nace 

Mrs.  Deborah  G.  Senft 

Bertha  Person 

Mrs.  Harriet  S.  Shapiro 

Margaret  Armstrong 

Liz  Price 

Dr.  Charlotte  Shemin 

Barbara  Atwood 

Linda  Rakowski 

Dr.  James  Sidie 

Harriet  Bernheimer 

Julie  Rankin 

Mrs.  Virginia  B.  Sinnott 

Gloria  Borgese 

Jean  Ripps 

Mrs.  Diana  M.  Smith 

Jennie  Brown 

Lilyan  Sauders 

Mrs.  Perle  Sonnenblick 

Kitty  Brown 

Marilyn  Shepro 

Dr.  William  T.  Speck 

Elizabeth  Buck 

Cynthia  Smith 

Dr.  Evelyn  Spiegel 

Julia  Child 

Peggy  Smith 

Mrs.  H.  Burr  Steinbach 

Vera  Clark 

Louise  Specht 

Ms.  Gail  Stetten 

Peggy  Clowes 

Susie  Steinbach 

Mrs.  Jane  Lazarow  Stetten 

Jewel  Cobb 

Jane  Stetton 

Mr.  Robert  Stump 

Janet  Daniels 

Peg  Talcot 

Mr.  Albert  H.  Swain 

Fran  Eastman 

Eleanor  Troll 

Mr.  James  K..  Taylor 

Alma  Ebert 

Natalie  Trousof 

Mrs.  Linda  L.  Timmins 

Margaret  German 

Mary  Ulbrich 

Mrs.  Ida  Trager 

Violet  Gilford 

Barbara  Van  Holde 

Miss  Natalie  Trousof 

Rose  Grant 

Alice  Veeder 

Ms.  Ciona  Ulbrich 

Edie  Grosch 

Joan  Wheeler 

Mrs.  Barbara  Van  Holde 

Barbara  Grossman 

Clare  Wilber 

Dr.  Claude  A.  Villee,  Jr. 

Jean  Halvorson 

Mrs.  Dorothy  Villee 

Pat  Hancock 

MBL  Summer  Tour  Guides 

Mrs.  Eve  Warren 

Hanna  Hastings 

Betsy  Bang 

Dr.  Gary  Wessel 

Helen  Hodosh 

John  Buck 

Dr.  William  M.  Wheeler 

Sally  Karush 

Sears  Crowell 

Mrs.  Barbara  Whitehead 

Barbara  Little 

Barbara  Little 

Mr.  Geoffrey  G.  Whitney,  Jr. 

Sally  Loessel 

Julie  Rankin 

Mrs.  A.A.T.  Wickersham 

Winnie  Mackey 

Lola  Robertson 

Mrs.  Clare  M.  Wilber 

Miriam  Mauzerall 

Priscilla  Roslansky 

Dr.  William  M.  Winn 

Mary  Mavor 

Man,  Ulbrich 

Ms.  Nancy  Woitkoski 

Polly  Miles 

Donald  Zinn 

Dr.  Sumner  Zacks 

Florence  Mixer 

Margery  Zinn 

Certificate  of  Organization 
Articles  of  Amendment 

Bylaws 


Certificate  of  Organization 

(On  File  in  the  Office  of  the  Secretary  of  the  Commonwealth) 
No.  3170 

We.  Alpheus  Hyatt.  President.  William  Stanford  Stevens.  Treasurer,  and  W'llliam 
T.  Sedgwick,  Edward  G.  Gardiner.  Susan  Minis  and  Charles  Sedgwick  Minot  being 
a  majority  of  the  Trustees  of  the  Marine  Biological  Laboratory  in  compliance  with 
the  requirements  of  the  fourth  section  of  chapter  one  hundred  and  fifteen  of  the 
Public  Statutes  do  hereby  certify  that  the  following  is  a  true  copy  of  the  agreement 
of  association  to  constitute  said  Corporation,  with  the  names  of  the  subscribers 
thereto: 


We,  whose  names  are  hereto  subscnbed.  do.  by  this  agreement,  associate  ourselves 
with  the  intention  to  constitute  a  Corporation  according  to  the  provisions  of  the 
one  hundred  and  fifteenth  chapter  of  the  Public  Statutes  of  the  Commonwealth  of 
Massachusetts,  and  the  Acts  in  amendment  thereof  and  in  addition  thereto. 

The  name  b\  which  the  Corporation  shall  be  known  is 
THE  MARINE  BIOLOGICAL  LABORATORY 


Articles  of  Amendment 


(On  File  in  the  Office  of  the  Secretary  of  the  Commonwealth) 

We.  James  D.  Ebert,  President,  and  David  Shepro,  Clerk  of  the  Marine  Biological 
Laboratory,  located  at  Woods  Hole.  Massachusetts  02543,  do  hereby  certify  that 
the  following  amendment  to  the  Articles  of  Organization  of  the  Corporation  was 
duly  adopted  at  a  meeting  held  on  August  15,  1975.  as  adjourned  to  August  29. 
1975,  by  vote  of  444  members,  being  at  least  two-thirds  of  its  members  legally 
qualified  to  vote  in  the  meeting  of  the  corporation: 

Voted:  That  the  Certificate  of  Organization  of  this  corporation  be  and  it  hereby  is 

amended  by  the  addition  of  the  following  provisions: 
"No  Officer,  Trustee  or  Corporate  Member  of  the  corporation  shall  be  personally 

liable  for  the  payment  or  satisfaction  of  any  obligation  or  liabilities  incurred 

as  a  result  of.  or  otherwise  in  connection  with,  any  commitments,  agreements. 

activities  or  affairs  of  the  corporation. 
"Except  as  otherwise  specifically  provided  by  the  Bylaws  of  the  corporation,  meetings 

of  the  Corporate  Members  of  the  corporation  may  be  held  anywhere  in  the 

United  States. 
"The  Trustees  of  the  corporation  may  make,  amend  or  repeal  the  Bylaws  of  the 

corporation  in  whole  or  in  part,  except  with  respect  to  any  provisions  thereof 

which  shall  by  law.  this  Certificate  or  the  bylaws  of  the  corporation,  require 

action  by  the  Corporate  Members." 


The  purpose  for  which  the  Corporation  is  constituted  is  to  establish  and  maintain 
a  laboratory  or  station  for  scientific  study  and  investigations,  and  a  school  for 
instruction  in  biology  and  natural  history. 

The  place  within  which  the  Corporation  is  established  or  located  is  the  city  of 
Boston  within  said  Commonwealth. 
The  amount  of  its  capital  stock  is  none. 

In  Witness  Whereof,  we  have  hereunto  set  our  hands,  this  twenty  seventh  day  of 
February  in  the  year  eighteen  hundred  and  eighty-eight,  Alpheus  Hyatt.  Samuel 
Mills.  William  T.  Sedgwick.  Edward  G.  Gardiner.  Charles  Sedgwick  Minot.  William 
G.  Farlow,  William  Stanford  Stevens.  Anna  D.  Phillips,  Susan  Mims,  B.  H.  Van 
VIeck. 

That  the  first  meeting  of  the  subscribers  to  said  agreement  was  held  on  the  thirteenth 
day  of  March  in  the  year  eighteen  hundred  and  eighty-eight. 
In  Witness  Whereof,  we  have  hereunto  signed  our  names,  this  thirteenth  day  of 
March  in  the  year  eighteen  hundred  and  eighty-eight.  Alpheus  Hyatt.  President, 
William  Stanford  Stevens.  Treasurer.  Edward  G.  Gardiner,  William  T.  Sedgwick. 
Susan  Mims.  Charles  Sedgwick  Minot. 
(Approved  on  March  20.  1988  as  follows: 

I  hereby  certify  that  it  appears  upon  an  examination  of  the  within  written  certificate 
and  the  records  of  the  corporation  duly  submitted  to  my  inspection,  that  the  re- 
quirements of  sections  one.  two  and  three  of  chapter  one  hundred  and  fifteen,  and 
sections  eighteen,  twenty  and  twenty-one  of  chapter  one  hundred  and  six.  of  the 
Public  Statutes,  have  been  complied  with  and  I  hereby  approve  said  certificate  this 
twentieth  dav  of  March  A.D,  eighteen  hundred  and  eighty-eight. 


Charles  Endicott 
Commissioner  of  Corporations) 


The  foregoing  amendment  will  become  effective  when  these  articles  of  amendment 
are  filed  in  accordance  with  Chapter  180,  Section  7  of  the  General  Laws  unless 
these  articles  specify,  in  accordance  with  the  vote  adopting  the  amendment,  a  later 
effective  date  not  more  than  thirty  days  after  such  filing,  in  which  event  the  amend- 
ment will  become  effective  on  such  later  date. 

In  Witness  whereof  and  L'nder  the  Penalties  of  Perjury,  we  have  hereto  signed  our 

names  this  2nd  day  of  September,  in  the  year  1975,  James  D.  Ebert,  President: 

David  Shepro,  Clerk. 

(Approved  on  October  24.  1975.  as  follows: 

1  hereby  approve  the  within  articles  of  amendment  and.  the  filing  fee  in  the  amount 

of  $10  having  been  paid,  said  articles  are  deemed  to  have  been  filed  with  me  this 

24th  day  of  October,  1975. 

Paul  Guzzi 

Secretary1  of  the  Commonwealth) 


Bylaws 


(Revised  August  7,  1992  and  December  10.  1992) 
ARTICLE  I— THE  CORPORATION 

A.  Name  ami  Purpose  The  name  of  the  Corporation  shall  be  The  Marine  Bio- 
logical Laboratory.  The  Corporation's  purpose  shall  be  to  establish  and  maintain 


R73 


R74   Annual  Report 


a  laboratory  or  station  tor  scientific  study  and  investigation  and  a  school  for  in- 
struction in  biology  .ind  natural  history. 

B.  Nondiscrimination  The  Corporation  shall  not  discriminate  on  the  basis  of 
age,  religion,  color,  race,  national  or  ethnic  origin,  sex  or  sexual  preference  in  its 
policies  on  employ  ment  and  administration  or  in  its  educational  and  other  programs. 

ARTICLE  II— MEMBERSHIP 

A  Members.  The  Members  of  the  Corporation  (•'Members")  shall  consist  of 
persons  elected  by  the  Board  of  Trustees  (the  "Board"),  upon  such  terms  and 
conditions  and  in  accordance  with  such  procedures,  not  inconsistent  with  law  or 
these  Bylaws,  as  may  be  determined  by  the  Board.  At  any  regular  or  special  meeting 
ot  the  Board,  the  Board  may  elect  new  Members.  Members  shall  have  no  voting 
or  other  rights  with  respect  to  the  Corporation  or  its  activities  except  as  specified 
in  these  Bylaws,  and  any  Member  may  vote  at  any  meeting  of  the  Members  in 
person  only  and  not  by  proxy.  Members  shall  serve  until  their  death  or  resignation 
unless  earlier  removed  with  or  without  cause  by  the  affirmative  vote  of  two-thirds 
of  the  Trustees  then  in  office.  Any  Member  who  has  retired  from  his  or  her  home 
institution  may,  upon  written  request  to  the  Corporation,  be  designated  a  Life 
Member.  Life  Members  shall  not  have  the  right  to  vote  and  shall  not  be  assessed 
for  dues. 

B.  Meetings.  The  annual  meeting  of  the  Members  shall  be  held  on  the  Friday 
following  the  first  Tuesday  in  August  of  each  year,  at  the  Laboratory  of  the  Cor- 
poration in  Woods  Hole,  Massachusetts,  at  9:30  a.m.  The  Chairperson  of  the  Board 
shall  preside  at  meetings  of  the  Corporation.  If  no  annual  meeting  is  held  in  ac- 
cordance with  the  foregoing  provision,  a  special  meeting  may  be  held  in  lieu  thereof 
with  the  same  effect  as  the  annual  meeting,  and  in  such  case  all  references  in  these 
Bylaws,  except  in  this  Article  II. B.,  to  the  annual  meeting  of  the  Members  shall  be 
deemed  to  refer  to  such  special  meeting.  Members  shall  transact  business  as  may 
properly  come  before  the  meeting.  Special  meetings  of  the  Members  may  be  called 
by  the  Chairperson  or  the  Trustees,  and  shall  be  called  by  the  Clerk,  or  in  the  case 
of  the  death,  absence,  incapacity  or  refusal  by  the  Clerk,  by  any  other  officer,  upon 
written  application  of  Members  representing  at  least  ten  percent  of  the  smallest 
quorum  of  Members  required  for  a  vote  upon  any  matter  at  the  annual  meeting 
of  the  Members,  to  be  held  at  such  time  and  place  as  may  be  designated. 

C.  Quorum   One  hundred  (100)  Members  shall  constitute  a  quorum  at  any 
meeting.  Except  as  otherwise  required  by  law  or  these  Bylaws,  the  affirmative  vote 
of  a  majority  of  the  Members  voting  in  person  at  a  meeting  attended  by  a  quorum 
shall  constitute  action  on  behalf  of  the  Members. 

D.  Nonce  ul  .Meeting    Notice  of  any  annual  meeting  or  special  meeting  of 
Members,  if  necessary,  shall  be  given  by  the  Clerk  by  mailing  notice  of  the  time 
and  place  and  purpose  of  such  meeting  at  least  15  days  before  such  meeting  to 
each  Member  at  his  or  her  address  as  shown  on  the  records  of  the  Corporation. 

E.  tt'awr  ol  Nonce  Whenever  notice  of  a  meeting  is  required  to  be  given  a 
Member,  under  any  provision  of  the  Articles  or  Organization  or  Bylaws  of  the 
Corporation,  a  written  waiver  thereof,  executed  before  or  after  the  Meeting  by  such 
Member,  or  his  or  her  duly  authorized  attorney,  shall  be  deemed  equivalent  to 
such  notice. 

F.  Adjournments.  Any  meeting  of  the  Members  may  be  adjourned  to  any  other 
time  and  place  by  the  vote  of  a  majority  of  those  Members  present  at  the  meeting, 
whether  or  not  such  Members  constitute  a  quorum,  or  by  any  officer  entitled  to 
preside  at  or  to  act  as  Clerk  of  such  meeting,  if  no  Member  is  present  or  represented. 
It  shall  not  be  necessary  to  notify  any  Members  of  any  adjournment  unless  no 
Member  is  present  or  represented  at  the  meeting  which  is  adjourned,  in  which  case, 
notice  of  the  adjournment  shall  be  given  in  accordance  with  Article  II. D.  Any 
business  which  could  have  been  transacted  at  any  meeting  of  the  Members  as 
originally  called  may  be  transacted  at  an  adjournment  thereof. 

ARTICLE  III— ASSOCIATES  OF  THE  CORPORATION 

I\MI  iiHo  of  the  Corporation  The  Associates  of  the  Marine  Biological  Laboratory 
shall  be  an  unincorporated  group  of  persons  (including  associations  and  corporations) 
interested  in  the  Laboratory  and  shall  be  organized  and  operated  under  the  general 
supervision  and  authority  of  the  Trustees.  The  Associates  of  the  Marine  Biological 
Laboratory  shall  have  no  voting  rights. 

ARTICLE  IV— BOARD  OF  TRUSTEES 

A.  Powers.  The  Board  of  Trustees  shall  have  the  control  and  management  of 
the  affairs  of  the  Corporation.  The  Trustees  shall  elect  a  Chairperson  of  the  Board 
who  shall  serve  un,til  his  or  her  successor  is  elected  and  qualified.  They  shall  annually 
elect  a  President  of  the  Corporation.  They  shall  annually  elect  a  Vice  Chairperson 
of  the  Board  who  shall  be  Vice  Chairperson  of  the  meetings  of  the  Corporation. 


They  shall  annually  elect  a  Treasurer.  They  shall  annually  elect  a  Clerk,  who  shall 
be  a  resident  of  Massachusetts.  They  shall  elect  Trustees-at-Large  as  specified  in 
this  Article  IV.  They  shall  appoint  a  Director  of  the  Laboratory  for  a  term  not  to 
exceed  five  years,  provided  the  term  shall  not  exceed  one  year  if  the  candidate  has 
attained  the  age  of  f>5  years  prior  to  the  date  of  the  appointment.  They  shall  choose 
such  other  officers  and  agents  as  they  shall  think  best.  They  may  fix  the  compensation 
of  all  officers  and  agents  of  the  Corporation  and  may  remove  them  at  any  time. 
They  may  fill  vacancies  occurring  in  any  of  the  offices.  The  Board  shall  have  the 
power  to  choose  an  Executive  Committee  from  their  own  number  as  provided  in 
Article  V,  and  to  delegate  to  such  Committee  such  of  their  own  powers  as  they 
may  deem  expedient  in  addition  to  those  powers  conferred  by  Article  V.  They 
shall,  from  time  to  time,  elect  Members  to  the  Corporation  upon  such  terms  and 
conditions  as  they  shall  have  determined,  not  inconsistent  with  law  or  these  Bylaws. 

B.  Composition  and  Eleeium 

( 1 )  The  Board  shall  include  24  Trustees  elected  by  the  Board  as  provided 
below: 

(a)  At  least  six  Trustees  ("Corporate  Trustees")  shall  be  Members  who  are 
scientists,  and  the  other  Trustees  ("Trustees-at-Large")  shall  be  individuals  who 
need  not  be  Members  or  otherwise  affiliated  with  the  Corporation. 

(b)  The  24  elected  Trustees  shall  be  divided  into  four  classes  of  six  Trustees 
each,  with  one  class  to  be  elected  each  year  to  serve  for  a  term  of  four  years,  and 
with  each  such  class  to  include  at  least  one  Corporate  Trustee.  Such  classes  of 
Trustees  shall  be  designated  by  the  year  of  expiration  of  their  respective  terms. 

(2)  The  Board  shall  also  include  the  Chief  Executive  Officer.  Treasurer  and 
the  Chairperson  of  the  Science  Council,  who  shall  be  ex  officio  voting  members  of 
the  Board. 

(3)  Although  Members  or  Trustees  may  recommend  individuals  for  nomi- 
nation as  Trustees,  nominations  for  Trustee  elections  shall  be  made  by  the  Nom- 
inating Committee  in  its  sole  discretion.  The  Board  may  also  elect  Trustees  who 
have  not  been  nominated  by  the  Nominating  Committee. 

C.  Eligibility  A  Corporate  Trustee  or  a  Trustee-at-Large  who  has  been  elected 
to  an  initial  four-year  term  or  remaining  portion  thereof,  of  which  he/she  has 
served  at  least  two  years,  shall  be  eligible  for  re-election  to  a  second  four-year  term, 
but  shall  be  ineligible  for  re-election  to  any  subsequent  term  until  one  year  has 
elapsed  after  he/she  has  last  served  as  a  Trustee. 

D.  Removal   Any  Trustee  may  be  removed  from  office  at  any  time  with  or 
without  cause,  by  vote  of  a  majority  of  the  Members  entitled  to  vote  in  the  election 
of  Trustees;  or  for  cause,  by  vote  of  two-thirds  of  the  Trustees  then  in  office.  A 
Trustee  may  be  removed  for  cause  only  if  notice  of  such  action  shall  have  been 
given  to  all  of  the  Trustees  or  Members  entitled  to  vote,  as  the  case  may  be,  prior 
to  the  meeting  at  which  such  action  is  to  be  taken  and  if  the  Trustee  to  be  so 
removed  shall  have  been  given  reasonable  notice  and  opportunity  to  be  heard 
before  the  body  proposing  to  remove  him  or  her. 

E.  I'aeancics.  Any  vacancy  in  the  Board  may  be  filled  by  vote  of  a  majority  of 
the  remaining  Trustees  present  at  a  meeting  of  Trustees  at  which  a  quorum  is 
present.  Any  vacancy  in  the  Board  resulting  from  the  resignation  or  removal  of  a 
Corporate  Trustee  shall  be  filled  by  a  Member  who  is  a  scientist. 

F.  Meetings   Meetings  of  the  Board  shall  be  held  from  time  to  time,  not  less 
frequently  than  twice  annually,  as  determined  by  the  Board.  Special  meetings  of 
Trustees  may  be  called  by  the  Chairperson,  or  by  any  seven  Trustees,  to  be  held 
at  such  time  and  place  as  may  be  designated.  The  Chairperson  of  the  Board,  when 
present,  shall  preside  over  all  meetings  of  the  Trustees.  Written  notice  shall  be  sent 
to  a  Trustee's  usual  or  last  known  place  of  residence  at  least  two  weeks  before  the 
meeting.  Notice  of  a  meeting  need  not  be  given  to  any  Trustee  if  a  written  waiver 
ol  notice  executed  by  such  Trustee  before  or  after  the  meeting  is  filed  with  the 
records  of  the  meeting,  or  if  such  Trustee  shall  attend  the  meeting  without  protesting 
prior  thereto  or  at  its  commencement  the  lack  of  notice  given  to  him  or  her. 

G.  Quorum  and  Action  by  Trustees.  A  majority  of  all  Trustees  then  in  office 
shall  constitute  a  quorum.  Any  meeting  of  Trustees  may  be  adjourned  by  vote  of 
a  majority  of  Trustees  present,  whether  or  not  a  quorum  is  present,  and  the  meeting 
may  be  held  as  adjourned  without  further  notice.  When  a  quorum  is  present  at 
any  meeting  of  the  Trustees,  a  majority  of  the  Trustees  present  and  voting  (excluding 
abstentions)  shall  decide  any  question,  including  the  election  of  officers,  unless 
otherwise  required  by  law.  the  Articles  of  Organization  or  these  Bylaws. 

H.  r™«\frn  (i/  InlcrcM*  in  Land  There  shall  be  no  transfer  of  title  nor  long- 
term  lease  of  real  property  held  by  the  Corporation  without  prior  approval  of  not 
less  than  two-thirds  of  the  Trustees.  Such  real  property  transactions  shall  be  finally 
acted  upon  at  a  meeting  of  the  Board  only  if  presented  and  discussed  at  a  prior 
meeting  of  the  Board.  Either  meeting  may  be  a  special  meeting  and  no  less  than 
four  weeks  shall  elapse  between  the  two  meetings.  Any  property  acquired  by  the 
Corporation  after  December  I.  1989  may  be  sold,  any  mortgage  or  pledge  of  real 


Bylaws  of  the  Corporation  R75 


property  (regardless  of  when  acquired)  lo  seeure  borrowings  by  the  Corporation 
may  be  granted,  and  any  transfer  of  title  or  interest  in  real  property  pursuant  to 
the  foreclosure  or  endorsement  of  any  such  mortgage  or  pledge  of  real  property 
may  be  effected  by  any  holder  of  a  mortgage  or  pledge  of  real  property  of  the 
Corporation,  with  the  prior  approval  of  not  less  than  two-thirds  of  the  Trustees 
(other  than  any  Trustee  or  Trustees  with  a  direct  or  indirect  financial  interest  in 
the  transaction  being  considered  for  approval)  w-ho  are  present  at  a  regular  or 
special  meeting  of  the  Board  at  which  there  is  a  quorum. 

ARTICLE  V— COMMITTEES 

A.  Executive  Committee  There  shall  be  an  Executive  Committee  of  the  Board 
of  Trustees  which  shall  consist  of  not  more  than  eleven  (11)  Trustees,  including  i'.v 
othi'io  Trustees,  elected  by  the  Board. 

The  Chairperson  of  the  Board  shall  act  as  Chairperson  of  the  Executive  Committee 
and  the  Vice  Chairperson  as  Vice  Chairperson.  The  Executive  Committee  shall 
meet  at  such  times  and  places  and  upon  such  notice  and  appoint  such  subcommittees 
as  the  Committee  shall  determine. 

The  Executive  Committee  shall  have  and  may  exercise  all  the  powers  of  the 
Board  dunng  the  intervals  between  meetings  of  the  Board  except  those  powers 
specifically  withheld,  from  time  to  time,  by  \ote  of  the  Board  or  by  law.  The  Executive 
Committee  may  also  appoint  such  committees,  including  persons  who  are  not 
Trustees,  as  it  may.  from  time  to  time,  approve  to  make  recommendations  with 
respect  to  matters  to  be  acted  upon  by  the  Executive  Committee  or  the  Board. 

The  Executive  Committee  shall  keep  appropnate  minutes  of  its  meetings,  which 
shall  be  reported  to  the  Board.  Any  actions  taken  by  the  Executive  Committee 
shall  also  be  reported  to  the  Board. 

B  .Vi  'iiiiihiniii:  ( \  unmitlee  There  shall  be  a  Nominating  Committee  which  shall 
consist  of  not  fewer  than  four  nor  more  than  six  Trustees  appointed  by  the  Board 
in  a  manner  which  shall  reflect  the  balance  between  Corporate  Trustees  and  Trustees- 
at-Large  on  the  Board.  The  Nominating  Committee  shall  nominate  persons  for 
election  as  Corporate  Trustees  and  Trustees-at-Large.  Chairperson  of  the  Board. 
Vice  Chairperson  of  the  Board,  President.  Treasurer,  Clerk.  Director  of  the  Lab- 
oratory and  such  other  officers,  if  any,  as  needed,  in  accordance  with  the  requirements 
of  these  Bylaws.  The  Nominating  Committee  shall  also  be  responsible  for  overseeing 
the  training  of  new  Trustees.  The  Chairperson  of  the  Board  of  Trustees  shall  appoint 
the  Chairperson  of  the  Nominating  Committee.  The  Chairperson  of  the  Science 
Council  shall  be  an  t'.v  officio  voting  member  of  the  Nominating  Committee. 

C.  Sciem-e  Council  There  shall  be  a  Science  Council  (the  "Council")  which  shall 
consist  of  Members  of  the  Corporation  elected  to  the  Council  by  vote  of  the  Members 
of  the  Corporation,  and  which  shall  ad\ise  the  Board  with  respect  to  matters  con- 
cerning the  Corporation's  mission,  its  scientific  and  instructional  endeavors,  and 
the  appointment  and  promotions  of  persons  or  committees  with  responsibility  for 
matters  requiring  scientific  expertise.  L'nless  otherwise  approved  by  a  majority  of 
the  members  of  the  Council,  the  Chairperson  of  the  Council  shall  be  elected  annually 
by  the  Council.  The  chief  executive  officer  of  the  Corporation  shall  be  an  ex  officio 
voting  member  of  the  Council. 

D.  Board  o)  Overseers-  There  shall  be  a  Board  of  Overseers  which  shall  consist 
of  not  fewer  than  five  nor  more  than  eight  scientists  who  have  expertise  concerning 
matters  with  which  the  Corporation  is  involved.  Members  of  the  Board  of  Overseers 
may  or  may  not  be  Members  of  the  Corporation  and  may  be  appointed  by  the 
Board  of  Trustees  on  the  basis  of  recommendations  submitted  from  scientists  and 
scientific  organizations  or  societies.  The  Board  of  Overseers  shall  be  available  to 
review  and  offer  recommendations  to  the  officers.  Trustees  and  Science  Council 
regarding  scientific  activities  conducted  or  proposed  by  the  Corporation  and  shall 
meet  from  time  to  time,  not  less  frequently  than  annually,  as  determined  by  the 
Board  of  Trustees. 

E.  Board  Committees  Generally.  The  Trustees  may  elect  or  appoint  one  or  more 
other  committees  (including,  but  not  limited  to.  an  Investment  Committee,  a  De- 
velopment Committee,  an  Audit  Committee,  a  Facilities  and  Capital  Equipment 
Committee  and  a  Long-Range  Planning  Committee)  and  may  delegate  to  any  such 
committee  or  committees  any  or  all  of  their  powers,  except  those  which  by  law. 
the  Articles  of  Organization  or  these  Bylaws  the  Trustees  are  prohibited  from  del- 
egating, provided  that  any  committee  to  which  the  powers  of  the  Trustees  are 
delegated  shall  consist  solely  of  Trustees.  The  members  of  any  such  committee 
shall  have  such  tenure  and  duties  as  the  Trustees  shall  determine.  The  Investment 
Committee,  which  shall  oversee  the  management  of  the  Corporation's  endowment 
funds  and  marketable  securities  shall  include  as  ex  officio  members,  the  Chairperson 
of  the  Board,  the  Treasurer  and  the  Chairperson  of  the  Audit  Committee,  together 
with  such  Trustees  as  may  be  required  for  not  less  than  two-thirds  of  the  Investment 
Committee  to  consist  of  Trustees.  Except  as  otherwise  provided  by  these  Bylaws 
or  determined  by  the  Trustees,  any  such  committee  may  make  rules  for  the  conduct 


of  its  business,  but,  unless  otherwise  provided  by  the  Trustees  or  in  such  rules,  its 
business  shall  be  conducted  as  nearly  as  possible  in  the  same  manner  as  is  provided 
by  these  Bylaws  for  the  Trustees. 

F.  Action*  Without  a  Meeting  Any  action  required  or  permitted  to  be  taken  at 
any  meeting  of  the  Executive  Committee  or  any  other  committee  elected  by  the 
Trustees  may  be  taken  without  a  meeting  if  all  members  of  such  committees  consent 
to  the  action  in  writing  and  such  written  consents  are  filed  with  the  records  of 
meetings.  Members  of  the  Executive  Committee  or  any  other  committee  elected 
by  the  Trustees  may  also  participate  in  any  meeting  by  means  of  a  telephone  con- 
ference call,  or  otherwise  lake  action  in  such  a  manner  as  may.  from  time  to  time, 
be  permitted  by  law. 

G.  Manual  of  /Vnuv/(/ri'\   The  Board  of  Trustees,  on  the  recommendation  of 
the  Executive  Committee,  shall  establish  guidelines  and  modifications  thereof  to 
be  recorded  in  a  Manual  of  Procedures.  Guidelines  shall  establish  procedures  for: 
( I )  Nomination  and  election  of  members  of  the  Corporation,  Board  of  Trustees 
and  Executive  Committee:  (2)  Election  of  Officers:  (3)  Formation  and  Function  of 
Standing  Committees. 

ARTICLE  VI— OFFICERS 

A.  Enumeration  The  officers  of  the  Corporation  shall  consist  of  a  President,  a 
Treasurer  and  a  Clerk,  and  such  other  officers  having  the  powers  of  President. 
Treasurer  and  Clerk  as  the  Board  may  determine,  and  a  Director  of  the  Laboratory. 
The  Corporation  may  have  such  other  officers  and  assistant  officers  as  the  Board 
may  determine,  including  (without  limitation)  a  Chairperson  of  the  Board,  Vice 
Chairperson  and  one  or  more  Vice  Presidents,  Assistant  Treasurers  or  Assistant 
Clerks.  Any  two  or  more  offices  may  be  held  by  the  same  person.  The  Chairperson 
and  Vice  Chairperson  of  the  Board  shall  be  elected  by  and  from  the  Trustees,  but 
other  officers  of  the  Corporation  need  not  be  Trustees  or  Members.  If  required  by 
the  Trustees,  any  otficer  shall  give  the  Corporation  a  bond  for  the  faithful  performance 
of  his  or  her  duties  in  such  amount  and  with  such  surety  or  sureties  as  shall  be 
satisfactory  to  the  Trustees. 

B.  Tenure.  Except  as  otherwise  provided  by  law.  by  the  Articles  of  Organization 
or  by  these  Bylaws,  the  President,  Treasurer,  and  all  other  officers  shall  hold  office 
until  the  first  meeting  of  the  Board  following  the  annual  meeting  of  Members  and 
thereafter,  until  his  or  her  successor  is  chosen  and  qualified. 

C.  Resignation  Any  officer  may  resign  by  delivering  his  or  her  written  resignation 
to  the  Corporation  at  its  principal  office  or  to  the  President  or  Clerk  and  such 
resignation  shall  be  effective  upon  receipt  unless  it  is  specified  to  be  effective  at 
some  other  time  or  upon  the  happening  of  some  other  event. 

D.  Removal  The  Board  may  remove  any  officer  with  or  without  cause  by  a  vote 
of  a  majority  of  the  entire  number  of  Trustees  then  in  office,  at  a  meeting  of  the 
Board  called  for  that  purpose  and  for  which  notice  of  the  purpose  thereof  has  been 
given,  provided  that  an  officer  may  be  removed  for  cause  only  after  having  an 
opportunity  to  be  heard  by  the  Board  at  a  meeting  of  the  Board  at  which  a  quorum 
is  personally  present  and  voting. 

E.  I  'acancy  A  vacancy  in  any  office  may  be  filled  for  the  unexpired  balance  of 
the  term  by  vote  of  a  majority  of  the  Trustees  present  at  any  meeting  of  Trustees 
at  which  a  quorum  is  present  or  by  written  consent  of  all  of  the  Trustees,  if  less 
than  a  quorum  of  Trustees  shall  remain  in  office. 

F.  Chairperson  The  Chairperson  shall  have  such  powers  and  duties  as  may  be 
determined  by  the  Board  and.  unless  otherwise  determined  by  the  Board,  shall 
serve  in  that  capacity  for  a  term  coterminous  with  his  or  her  term  as  Trustee. 

G.  I  'tee  Chairperson.  The  Vice  Chairperson  shall  perform  the  duties  and  exercise 
the  powers  of  the  Chairperson  in  the  absence  or  disability  of  the  Chairperson,  and 
shall  perform  such  other  duties  and  possess  such  other  powers  as  may  be  determined 
by  the  Board.  LInless  otherwise  determined  by  the  Board,  the  Vice  Chairperson 
shall  serve  for  a  one-year  term. 

H.  Director  The  Director  shall  be  the  chief  operating  officer  and.  unless  otherwise 
voted  by  the  Trustees,  the  chief  executive  officer  of  the  Corporation.  The  Director 
shall,  subject  to  the  direction  of  the  Trustees,  have  general  supervision  of  the  Lab- 
oratory and  control  of  the  business  of  the  Corporation.  At  the  annual  meeting,  the 
Director  shall  submit  a  report  of  the  operations  of  the  Corporation  for  such  vear 
and  a  statement  of  its  affairs,  and  shall,  from  time  to  time,  report  to  the  Board  all 
matters  within  his  or  her  knowledge  which  the  interests  of  the  Corporation  may 
require  to  be  brought  to  its  notice. 

1.  Deputy  Director  The  Deputy  Director,  if  any.  or  if  there  shall  be  more  than 
one.  the  Deputy  Directors  in  the  order  determined  by  the  Trustees,  shall,  in  the 
absence  or  disability  of  the  Director,  perform  the  duties  and  exercise  the  powers 
of  the  Director  and  shall  perform  such  other  duties  and  shall  have  such  other 
powers  as  the  Trustees  may.  from  time  to  time,  prescribe. 


R76   Annual  Report 


J.  President  The  President  shall  have  the  powers  and  duties  as  may  be  vested 
in  him  or  her  In  the  Board. 

K.  TreuHiici  and  Assistant  Treasurer.  The  Treasurer  shall,  subject  to  the  direction 
of  the  Trustees,  have  general  charge  of  the  financial  affairs  of  the  Corporation, 
including  its  long-range  financial  planning,  and  shall  cause  to  be  kept  accurate 
books  of  account.  The  Treasurer  shall  prepare  a  yearly  report  on  the  financial  status 
of  the  Corporation  to  be  delivered  at  the  annual  meeting.  The  Treasurer  shall  also 
prepare  or  oversee  all  filings  required  by  the  Commonwealth  of  Massachusetts,  the 
Internal  Revenue  Service,  or  other  Federal  and  State  Agencies.  The  account  of  the 
Treasurer  shall  be  audited  annually  by  a  certified  public  accountant. 

The  Assistant  Treasurer,  if  any,  or  if  there  shall  be  more  than  one,  the  Assistant 
Treasurers  in  the  order  determined  by  the  Trustees,  shall,  in  the  absence  or  disability 
of  the  Treasurer,  perform  the  duties  and  exercise  the  powers  of  the  Treasurer,  shall 
perform  such  other  duties  and  shall  have  such  other  powers  as  the  Trustees  may. 
from  time  to  time,  prescribe 

L.  Clerk  and  Assistant  Clerk  The  Clerk  shall  be  a  resident  of  the  Commonwealth 
of  Massachusetts,  unless  the  Corporation  has  designated  a  resident  agent  in  the 
manner  provided  by  law.  The  minutes  or  records  of  all  meetings  of  the  Trustees 
and  Members  shall  be  kept  by  the  Clerk  who  shall  record,  upon  the  record  books 
of  the  Corporation,  minutes  of  the  proceedings  at  such  meetings.  He  or  she  shall 
have  custody  of  the  record  books  of  the  Corporation  and  shall  have  such  other 
powers  and  shall  perform  such  other  duties  as  the  Trustees  may.  from  time  to  time, 
prescribe. 

The  Assistant  Clerk,  if  any,  or  if  there  shall  be  more  than  one,  the  Assistant 
Clerks  in  the  order  determined  by  the  Trustees,  shall,  in  the  absence  or  disability 
of  the  Clerk,  perform  the  duties  and  exercise  the  powers  of  the  Clerk  and  shall 
perform  such  other  duties  and  shall  have  such  other  powers  as  the  Trustees  may, 
from  time  to  time,  prescribe. 

In  the  absence  of  the  Clerk  and  an  Assistant  Clerk  from  any  meeting,  a  temporary 
Clerk  shall  be  appointed  at  the  meeting. 

M.  Oilier  Powers  and  Duties  Each  officer  shall  have  in  addition  to  the  duties 
and  powers  specifically  set  forth  in  these  Bylaws,  such  duties  and  powers  as  are 
customarily  incident  to  his  or  her  office,  and  such  duties  and  powers  as  the  Trustees 
may,  from  time  to  time,  designate. 

ARTICLE  VII— AMENDMENTS 

These  Bylaws  may  be  amended  by  the  affirmative  vote  of  the  Members  at  any 
meeting,  provided  that  notice  of  the  substance  of  the  proposed  amendment  is  stated 
in  the  notice  of  such  meeting.  As  authorized  by  the  Articles  of  Organization,  the 
Trustees,  by  a  majority  of  their  number  then  in  office,  may  also  make,  amend  or 
repeal  these  Bylaws,  in  whole  or  in  part,  except  with  respect  to  (a)  the  provisions 
of  these  Bylaws  governing  (i)  the  removal  of  Trustees  and  (ii)  the  amendment  ol 
these  Bylaws  and  (b)  any  provisions  of  these  Bylaws  which  by  law,  the  Articles  of 
Organization  or  these  Bylaws,  requires  action  by  the  Members. 

No  later  than  the  time  of  giving  notice  of  meeting  of  Members  next  following 
the  making,  amending  or  repealing  by  the  Trustees  of  any  Bylaw,  notice  thereof 
stating  the  substance  of  such  change  shall  be  given  to  all  Members  entitled  to  vote 
on  amending  the  Bylaws. 

Any  Bylaw  adopted  by  the  Trustees  may  be  amended  or  repealed  by  the  Members 
entitled  to  vote  on  amending  the  Bylaws. 

ARTICLE  VIII— INDEMNITY 

Except  as  otherwise  provided  below,  the  Corporation  shall,  to  the  extent  legally 
permissible,  indemnify  each  person  who  is,  or  shall  have  been,  a  Trustee,  director 
or  officer  of  the  Corporation  or  who  is  serving,  or  shall  have  served  at  the  request 
of  the  Corporation  as  a  Trustee,  director  or  officer  of  another  organization  in  which 
the  Corporation  directly  or  indirectly  has  any  interest  as  a  shareholder,  creditor  or 
otherwise,  against  all  liabilities  and  expenses  (includingjudgments,  fines,  penalties, 
and  reasonable  attorneys'  fees  and  all  amounts  paid,  other  than  to  the  Corporation 
or  such  other  organization,  in  compromise  or  settlement)  imposed  upon  or  incurred 
by  any  such  person  in  connection  with,  or  arising  out  of.  the  defense  or  disposition 
of  any  action,  suit  or  other  proceeding,  whether  civil  or  criminal,  in  which  he  or 
she  may  be  a  defendant  or  with  which  he  or  she  may  be  threatened  or  otherwise 
involved.  directK  or  indirectly,  by  reason  of  his  or  her  being  or  having  been  such 
a  Trustee,  director  or  officer. 

The  Corporation  shall  provide  no  indemnification  with  respect  to  any  matter  as 
to  which  any  such  Trustee,  director  or  officer  shall  be  finally  adjudicated  in  such 
action,  suit  or  proceeding  not  to  have  acted  in  good  faith  in  the  reasonable  belief 
that  his  or  her  action  was  in  the  best  interests  of  the  Corporation.  The  Corporation 
shall  provide  no  indemnification  with  respect  to  any  matter  settled  or  comprised 
unless  such  matter  shall  have  been  approved  as  in  the  best  interests  of  the  Cor- 


poration, after  notice  that  indemnification  is  involved,  by  (i)  a  disinterested  majority 
of  the  Board  of  the  Executive  Committee,  or  (n)  a  majority  of  the  Members. 

Indemnification  may  include  payment  by  the  Corporation  of  expenses  in  de- 
fending a  civil  or  criminal  action  or  proceeding  in  advance  of  the  final  disposition 
of  such  action  or  proceeding  upon  receipt  of  an  undertaking  by  the  person  indem- 
nified to  repay  such  payment  if  it  is  ultimately  determined  that  such  person  is  not 
entitled  to  indemnification  under  the  provisions  of  this  Article  VIII,  or  under  any 
applicable  law. 

As  used  in  the  Article  VIII,  the  terms  "Trustee,"  "director,"  and  "officer"  include 
their  respective  heirs,  executors,  administrators  and  legal  representatives,  and  an 
"interested"  Trustee,  director  or  officer  is  one  against  whom  in  such  capacity  the 
proceeding  in  question  or  another  proceeding  on  the  same  or  similar  grounds  is 
then  pending. 

To  assure  indemnification  under  this  Article  VIII  of  all  persons  who  are  determined 
by  the  Corporation  or  otherwise  to  be  or  to  have  been  "fiduciaries"  of  any  employee 
benefits  plan  of  the  Corporation  which  may  exist,  from  time  to  time,  this  Article 
VIII  shall  be  interpreted  as  follows:  (i)  "another  organization"  shall  be  deemed  to 
include  such  an  employee  benefit  plan,  including  without  limitation,  any  plan  of 
the  Corporation  which  is  governed  by  the  Act  of  Congress  entitled  "Employee 
Retirement  Income  Security  Act  of  1974,"  as  amended,  from  time  to  time,  ("ER- 
ISA");  (ii|  "Trustee"  shall  be  deemed  to  include  any  person  requested  by  the  Cor- 
poration to  serve  as  such  for  an  employee  benefit  plan  where  the  performance  by 
such  person  of  his  or  her  duties  to  the  Corporation  also  imposes  duties  on,  or 
otherwise  involves  services  by,  such  person  to  the  plan  or  participants  or  beneficiaries 
of  the  plan:  (iii)  "fines"  shall  be  deemed  to  include  any  excise  tax  plan  pursuant 
to  ERISA:  and  (iv)  actions  taken  or  omitted  by  a  person  with  respect  to  an  employee 
benefit  plan  in  the  performance  of  such  person's  duties  for  a  purpose  reasonably 
believed  by  such  person  to  be  in  the  interest  of  the  participants  and  beneficiaries 
of  the  plan  shall  be  deemed  to  be  for  a  purpose  which  is  in  the  best  interests  of  the 
Corporation. 

The  nght  of  indemnification  provided  in  this  Article  VIII  shall  not  be  exclusive 
of  or  affect  any  other  rights  to  which  any  Trustee,  director  or  officer  may  be  entitled 
under  any  agreement,  statute,  vote  of  Members  or  otherwise.  The  Corporation's 
obligation  to  provide  indemnification  under  this  Article  VIII  shall  be  offset  to  the 
extent  of  any  other  source  of  indemnification  of  any  otherwise  applicable  insurance 
coverage  under  a  policy  maintained  by  the  Corporation  or  any  other  person.  Nothing 
contained  in  the  Article  shall  affect  any  rights  to  which  employees  and  corporate 
personnel  other  than  Trustees,  directors  or  officers  may  be  entitled  by  contract,  by 
vote  of  the  Board  or  of  the  Executive  Committee  or  otherwise. 


ARTICLE  IX— DISSOLUTION 

The  consent  of  every  Trustee  shall  be  necessary  to  effect  a  dissolution  of  the 
Marine  Biological  Laboratory.  In  case  of  dissolution,  the  property  shall  be  disposed 
of  in  such  a  manner  and  upon  such  terms  as  shall  be  determined  by  the  affirmative 
vote  of  two-thirds  of  the  Trustees  then  in  office  in  accordance  with  the  laws  of  the 
Commonwealth  of  Massachusetts. 

ARTICLE  X— MISCELLANEOUS  PROVISIONS 

A.  fi\uil  Year  Except  as  otherwise  determined  by  the  Trustees,  the  fiscal  year 
of  the  Corporation  shall  end  on  December  31st  of  each  year. 

B.  Seal   Unless  otherwise  determined  by  the  Trustees,  the  Corporation  may 
have  a  seal  in  such  form  as  the  Trustees  may  determine,  from  time  to  time. 

C.  Execution  of  Instruments  All  checks,  deeds,  leases,  transfers,  contracts,  bonds, 
notes  and  other  obligations  authorized  to  be  executed  by  an  officer  of  the  Corporation 
in  its  behalf  shall  be  signed  by  the  Director  or  the  Treasurer  except  as  the  Trustees 
may  generally  or  in  particular  cases  otherwise  determine.  A  certificate  by  the  Clerk 
or  an  Assistant  Clerk,  or  a  temporary  Clerk,  as  to  any  action  taken  by  the  Members, 
Board  of  Trustees  or  any  officer  or  representative  of  the  Corporation  shall  as  to  all 
persons  who  rely  thereon  in  good  faith  be  conclusive  evidence  of  such  action. 

D.  Corporate  Records  The  original,  or  attested  copies,  of  the  Articles  of  Orga- 
nization, Bylaws  and  records  of  all  meetings  of  the  Members  shall  be  kept  in  Mas- 
sachusetts at  the  principal  office  of  the  Corporation,  or  at  an  office  of  the  Corpo- 
ration's Clerk  or  resident  agent.  Said  copies  and  records  need  not  all  be  kept  in  the 
same  office.  They  shall  be  available  at  all  reasonable  times  for  inspection  by  any 
Member  for  any  proper  purpose,  but  not  to  secure  a  list  of  Members  for  a  purpose 
other  than  in  the  interest  of  the  applicant,  as  a  Member,  relative  to  the  affairs  of 
the  Corporation. 

E.  Articles  of  Organization   All  references  in  these  Bylaws  to  the  Articles  of 
Organization  shall  be  deemed  to  refer  to  the  Articles  of  Organization  of  the  Cor- 
poration, as  amended  and  in  effect,  from  time  to  time. 


Bylaws  of  the  Corporation  R77 


F.  Transactions  milt  Interested  Portia  In  the  absence  of  fraud,  no  contract  or 
other  transaction  between  this  Corporation  and  an>  other  corporation  or  an>  firm, 
association,  partnership  or  person  shall  he  affected  or  invalidated  by  the  fact  that 
an\  Trustee  or  officer  of  this  Corporation  is  pecuniarily  or  otherwise  interested  in 
or  is  a  director,  member  or  officer  of  such  other  corporation  or  of  such  firm,  as- 
sociation or  partnership  or  in  a  party  to  or  is  pecuniarily  or  otherwise  interested 
in  such  contract  or  other  transaction  or  is  in  any  way  connected  with  any  person 
or  person,  firm,  association,  partnership,  or  corporation  pecuniarily  or  otherwise 
interested  therein;  provided  that  the  fact  that  he  or  she  individually  or  as  a  director, 
member  or  officer  of  such  corporation,  firm,  association  or  partnership  in  such  a 


party  or  is  so  interested  shall  be  disclosed  to  or  shall  have  been  known  by  the  Board 
of  Trustees  or  a  majority  of  such  Members  thereof  as  shall  be  present  at  a  meeting 
of  the  Board  of  Trustees  at  which  action  upon  any  such  contract  or  transaction 
shall  be  taken;  any  Trustee  may  be  counted  in  determining  the  existence  of  a 
quorum  and  may  vote  at  any  meeting  of  the  Board  of  Trustees  for  the  purpose  of 
authorizing  any  such  contract  or  transaction  with  like  force  and  effect  as  if  he/she 
were  not  so  interested,  or  were  not  a  director,  member  or  officer  of  such  other 
corporation,  firm,  association  or  partnership,  provided  that  any  vote  with  respect 
to  such  contract  or  transaction  must  be  adopted  by  a  majority  of  the  Trustees  then 
in  office  who  have  no  interest  in  such  contract  or  transaction. 


CONTENTS 


HISTORICAL  REVIEW 


ECOLOGY  AND  EVOLUTION 


Shimomura,  Osamu 

A  short  story  of  aequorin 


DEVELOPMENT  AND  REPRODUCTION 

Morisawa,  Sachiko 

Fine  structure  of  spermatozoa  of  the  hagfish  Epta- 

tretus  burger'i  (Agnatha)     6 

Glas,  Patricia  S.,  Jeffrey  D.  Green,  and  John  W.  Lynn 
Oxidase  activity  associated  with  the  elevation  of  the 
penaeoid  shrimp  hatching  envelope  13 

PHYSIOLOGY 


Chadwick-Furman,    Nanette    E.,    and    Irving    L. 
Weissman 

Life  histories  and  senescence  of  Botr\llu.\  vWmwd 
(Chordata,  Ascidiacea)  in  Monterey  Bay    36 

Hairston,  Nelson  G.,  Jr.,  and  Colleen  M.  Kearns 

The  interaction  of  photoperiodand  temperature  in 
diapause  timing:  a  copepod  example 42 

Woodin,  Sarah  A.,  Sara  M.  Lindsay,  and  David  S. 
Wethey 

Process-specific  recruitment  cues  in  marine  sedi- 
mentary systems     49 


Scholnick,  David  A. 

Sensitivity  of  metabolic  rate,  growth,  and  fecundity 
of  tadpole  shrimp  Tri<>/>\  longicaudatus  to  environ- 
mental variation  22 


IMMUNOLOGY 

Hirose,  Euichi,  and  Teruhisa  Ishii 

Microfilament  contraction  promotes  rounding  of 
tunic  slides:  an  integumentary  defense  system  in  the 
colonial  ascidian  A/iI/iliinn  \nnifnu  29 


FUNCTIONAL  MORPHOLOGY 


Carefoot,  Thomas  H.,  and  Deborah  A.  Donovan 

Functional  significance  of  varices  in  the  muricul 
gastropod  (.ruiii^lniiiii  foliatuin    ............... 


59 


Annual  Report  of  the  Marine  Biological  Laboratory       R 1 


Volume  189 


THE 


Number  2 


BIOLOGICAL 
BULLETIN 


• 


OCTOBER/NOVEMBER,  1995 


Published  by  the  Marine  Biological  Laboratory 


THE 


Marine  Biological  Laboratory/ 
Wood*  Hole  Omonogniphle  Institution 


JAN  0  5  1996 


Woods  Hole,  MA  0250 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 
THE  MARINE  BIOLOGICAL  LABORATORY 

Associate  Editors 

PETER  A.  V.  ANDERSON,  The  Whitney  Laboratory,  University  of  Florida 

WILLIAM  D.  COHEN.  Hunter  College,  City  University  of  New  York 

DAVID  EPEL,  Hopkins  Marine  Station,  Stanford  University 

J.  MALCOLM  SHICK,  University  of  Maine.  Orono 

Editorial  Board 


PETER  B.  ARMSTRONG,  University  of  California,  Davis 
THOMAS  H.  DIETZ,  Louisiana  State  University 
DAPHNE  GAIL  FAUTIN,  University  of  Kansas 

WILLIAM  F.  GILLY.  Hopkins  Marine  Station,  Stanford 

University 

ROGER   T.    HANLON,    Marine   Biomedical    Instituie, 
University  of  Texas  Medical  Branch 

MICHAEL  LABARBERA.  University  of  Chicago 
CHARLES  B.  METZ,  University  of  Miami 


K.  RANGA  RAO,  University  of  West  Florida 

BARLICH     RINKEVICH,     Israel    Oceanographic    & 
Limnoiogical  Research  Ltd. 

RICHARD  STRATHMANN,  Friday  Harbor  Laboratories, 
University  of  Washington 

STEVEN  VOGEL,  Duke  University 

J.  HERBERT  WAITE,  University  of  Delaware 

SARAH  ANN  WOODIN,  University  of  South  Carolina 

RICHARD   K.   ZIMMER-FALIST,   University  of  South 

Carolina 


Editor:  MICHAEL  J.  GREENBERG.  The  Whitney  Laboratory.  University  of  Florida 
Managing  Editor:  PAMELA  L.  CLAPP.  Marine  Biological  Laboratory 

OCTOBER/NOVEMBER,  1995 


Printed  and  Issued  by 
LANCASTER  PRESS,  Inc. 

3575  HEMPI.AND  ROAD 
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THE  BIOLOGICAL  BULLETIN 


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Reference:  Biul.  Bull  189:  69-76.  (October/November. 


Sulfide  as  a  Chemical  Stimulus  for  Deep-Sea 
Hydrothermal  Vent  Shrimp 


G.  H.  RENNINGER1*,   L.  KASS2,  R.  A.  GLEESON3,  C.  L.  VAN  DOVER4t, 
B.-A.   BATTELLE1,  R.  N.  JINKS5,  E.  D.  HERZOG6.  AND  S.  C.  CHAMBERLAIN5 

^Biophysics  Group,  Department  of  Physics.  University  ofGuelph.  Giie/ph.  Ontario  NIG  211']. 
Canada;2  Department  of  Zoology.  University  of  Maine,  Orono.  Maine  04469: 3C.  I'.  Whitney 

Laboratory,  University  of  Florida,  St.  Augustine.  Florida  32086;  ^Diike  University  Marine 

Laboratory,  Beaufort,  NC  28516;  -Institute  for  Sensory  Research  and  Department  of  Bioengineering 

&  Neuroscience,  Syracuse  University,  Syracuse,  New  York  13244-5290;  and ''Department  of  Biology. 

University  of  I  'irginia,  Charlottesville.  I  'irginia  22903 


Organisms  dependent  on  deep-sea  hydrothermal  vents 
for  their  existence  face  extinction  when  their  vents  expire, 
unless  they  can  establish  populations  on  neighboring  vents 
or  on  new  vent  sites.  Propagules.  including  larvae  and  mo- 
tile adults,  are  readily  dispersed  broadly  by  seajloor  cur- 
rents, but  how  they  recognise  active  hydrothermal  sites  is 
problematical.  Compelling  evidence  that  vent  organisms 
can  find  and  colonize  hydrothermal  sites  has  been  provided 
by  a  series  of  observations  on  the  East  Pacific  Rise  (I). 
New  hydrothermal  vents  created  there  following  a  volcanic 
eruption  on  the  seajloor  in  March  1991  were  colonized  by 
sessile  invertebrates  in  less  than  one  year.  On  the  Af id- 
Atlantic  Ridge,  shrimp  that  normally  cluster  on  sulfide 
surfaces  have  been  observed  to  swim  directly  back  to  the 
surfaces  when  displaced  from  them.  How  do  vent  animals 
locate  new  or  existing  vents?  Passive  transport  by  currents 
(2)  or  active  swimming  without  guidance  by  some  physical 
cue  is  not  likely  to  result  in  success  (3).  Chemicals  present 
in  hydrothermal  fluids  have  been  proposed  as  attractants. 
H  'e  provide  the  first  evidence  of  a  chemosensory  response 
in  a  vent  invertebrate  to  stilfides.  which  are  prevalent  in 
vent  fluids  and  provide  the  energy  for  chemosynthelic  pri- 
mary production  at  vents. 

During  recent  field  work  at  hydrothermal  vents  on  the 
Mid-Atlantic  Ridge  where  extremely  motile  shrimp 
(Rimicaris  cxoculata  and  a  smaller,  possibly  new,  Rinu- 


Received  3  May  1995:  accepted  28  July  1995. 
*  To  whom  correspondence  should  be  addressed. 
1  Present  address:  West  Coast  National  Undersea  Research  Center. 
PO  Box  757220,  University  of  Alaska,  Fairbanks,  AK  99775. 


caris  sp.)  dominate  the  surfaces  of  sulfide  chimneys,  we 
documented  a  strong  orientation  behavior  of  shrimp,  per- 
haps guided  by  chemical  cues,  to  a  piece  of  sulfide  re- 
moved from  a  chimney  (Fig.  1 ).  Specimens  of  Rimicaris 
sp.  and  R.  exoculala  collected  by  the  deep  submergence 
vehicle  (DSV)  Alvin  survived  sufficiently  long  aboard  ship 
to  allow  us  to  examine  physiological  responses  of  antennal 
nerves  to  various  chemical  stimulants,  as  well  as  to  pre- 
serve antennal  filaments  for  later  electron  microscopic 
examination.  The  shrimp  possess  first  and  second  anten- 
nae (Fig.  2A)  similar  to  those  of  other  decapod  crustaceans 
whose  antennae  are  known  to  respond  to  chemical  as  well 
as  to  tactile  stimuli  (4).  The  first  and  second  antennae 
both  bearsensilla  (Fig.  2B;  1  sensillum/66  ^m  of  filament 
circumference  on  average  for  Rimicaris  sp.  and  1/48  ^m 
for  R.  exoculata)  with  an  open  pore  at  their  tips  (Fig.  2C, 
D).  Transmission  electron  microscopy  (TEM)  reveals  that 
each  sensillum  is  innervated  by  1 0  to  14  sensory  dendrites 
(Fig.  2E,  F).  Light  microscopic  and  TEM  studies  indicate 
that  the  channel  containing  the  sensory  dendritic  segments 
extends  to  the  pore  at  the  tip  of  the  sensillum,  and  that 
at  least  some  of  the  dendritic  processes  in  the  channel 
reach  the  pore. 

Nerves  in  excised  filaments  of  both  antennae  frequently 
showed  spontaneous  activity  and  responded  to  tactile 
stimuli.  Antennal  filaments  also  responded  to  a  variety 
of  chemical  stimuli,  including  an  homogenate  of  bacterial 
cells  isolated  from  the  vents  and  mixtures  of  amino  acids. 
We  tested  7  filaments  of  the  second  antenna,  and  4  medial 
filaments  and  5  lateral  filaments  of  the  first  antenna.  The 
largest  and  most  robust  response  was  evoked  in  filaments 


69 


70 


G.  H.  RENNINGER  ET  AL. 


Figure  1.  (A)  A  startled  hydrothermal  vent  shrimp  swims  off  a  sulride- 
bcaring  rock  sample  taken  from  a  vent  chimney.  (B)  reorients  itself  to- 
wards the  rock,  and  then  (C)  swims  back  to  the  rock.  Similar  behavior 
by  other  shrimp  occurred  several  times  during  collection  of  the  rock 
sample  by  DSV  Alvin.  From  videotape  of  DSV  A/rui  dive  number  2613 
mi  14  June  1993  at  the  Snake  Pit  site  on  the  Mid-Atlantic  Ridge. 


of  the  second  antenna  on  exposure  to  solutions  of  Na:S 
(Fig.  3A).  All  seven  filaments  tested  responded  to  Na2S; 
the  response  could  be  eliminated  by  removing  the  stim- 
ulant and  could  be  evoked  repeatedly.  The  steady-state 
response  increased  with  the  concentration  of  Na2S.  In  the 
four  filaments  of  the  second  antenna  used  to  study  the 
response  to  Na2S  at  different  concentrations,  the  relative 
responses  (Fig.  3B)  were  (mean  ±  standard  error  of  the 
mean):  0. 1 1 3  ±  0.054  (/;  =  3)  at  [Na2S]  =  1 .3  mM;  0.567 
±0.137  (n  =  4)  at  13  mA/;  0.654  ±  0.148  (n  =  4)  at 
130  mA/;  and  1.014  ±  0.140  (n  =  4)  at  1300  mA/.  Two 
control  solutions  were  used  on  two  of  these  four  filaments. 
The  first  was  artificial  seawater  (ASW)  with  pH  =  8, 
matching  the  pH  of  the  lowest  Na2S  concentration  used, 
which  gave  a  relative  response  of  -0.078  ±  0.010.  The 
second  control  solution  (pH  13)  was  ASW  with  pH  ad- 
justed to  1 3.  equal  to  that  of  the  1 300  mA/  Na2S  solution, 
which  gave  a  relative  response  of  -0.030  ±  0.050.  Three 
each  of  the  medial  and  lateral  filaments  of  the  first  antenna 
were  tested  for  sensitivity  to  Na2S.  The  medial  filament 
of  the  first  antenna  responded  less  consistently  than  fila- 
ments of  the  second  antenna  in  what  appeared  to  be  a 
transient  manner.  The  lateral  filament  responded  little,  if 
at  all,  to  Na2S. 

Of  the  three  antennal  filaments,  only  the  responses  of 
the  second  antenna  showed  a  significant  concentration 
dependence.  A  linear  regression  analysis  of  the  mean  val- 
ues of  the  second  antenna's  responses  as  a  function  of 
log[Na:S]  gave  a  slope  significantly  different  from  zero  (P 
<  0.05).  Based  on  general  experience  with  chemical  senses, 
we  expect  that  the  actual  dependence  of  the  second  an- 
tenna's response  on  sulfide  concentration  is  not  this  linear 
one,  but  rather  a  sigmoidal  dependence  (10).  The  trend 
of  the  responses  suggests  that  the  threshold  concentration 
for  the  sensory  cells  in  the  second  antenna  lies  in  the  mi- 
cromolar  range  of  sulfide  concentrations. 

The  responses  of  excised  filaments  of  the  second  an- 
tenna of  Rimicaris  sp.  and  R.  exoculata  to  stimulation 
by  dissolved  sulfide  suggest  that  vent  shrimp  may  be  able 


Figure  2.  (A)  Lateral  view  of  Rimicaris  sp.,  a  small  orange-colored 
hydrothermal  vent  shrimp  collected  at  the  Snake  Pit  site,  together  with 
dorsal  view  of  cephalothorax.  showing  the  two  first  antennae  (each  with 
a  short  medial  and  lateral  filament  close  to  the  midline)  and  the  two 
second  antennae  (each  having  one  long  filament  shown  deployed  later- 
ally); scale  bar  =  5  mm.  (B)  The  second  antenna  of  Rimicuris  sp.  showing 
the  distribution  of  sensilla  near  the  distal  border  of  each  segment.  Bacteria! 
populations  encrust  the  surface,  giving  it  a  mottled  appearance  (scale 
=  25  jim).  The  second  antenna  of  Rimicaris  exoculata  is  similar  in 
structure.  For  SEM  examination,  antennae  were  removed  from  animals 
which  had  been  fixed  in  5%  paraformaldehyde  in  0. 1  A/Sorensen's  phos- 
phate buffer  (pH  7.2)  immediately  after  arrival  at  the  ocean's  surface. 
The  tissue  was  dehydrated  in  a  graded  ethanol  series,  immersed  in  hexa- 
methyldisilazane  for  5-10  min  and  air  dried  (5).  Pieces  of  dried  anten- 
nae were  mounted  on  stubs,  coated  with  gold,  and  examined  using  an 


CHEMOSENSORY  RESPONSE  IN  VENT  SHRIMP 


71 


Hitachi  4000  scanning  electron  microscope  at  6.0  kV.  (C)  A  sensillum  on  the  second  antenna  of  Rimicaris 
sp.  At  the  tip  of  the  sensillum.  hngerlike  projections  extend  above  the  laterally  facing  terminal  pore  (arrowhead). 
Proximal  to  the  pore  are  regularly  arranged  button-like  microstructures  (arrow)  which  are  associated  with 
the  distal  third  of  the  sensrllum  (scale  =  1  jim).  (D)  The  sensilla  on  the  second  antenna  of  R.  exuculaui  are 
similar  in  structure,  with  irregular  crenulations  replacing  the  button-like  microstructures  (scale  =  1  ^m). 


72 


G.  H.  RENNINGER  ET  AL. 


ca 


, 


1  pm 


Figure  2.  (E)  Cross  section  in  the  proximal  region  of  a  sensillum  from  the  second  antenna  of  R  exoadala. 
The  sensillum  is  composed  of  three  major  layers:  a  cuticle  (c):  an  inner  core  of  amorphous  material  (am) 
penetrated  by  canals  (ca):  and  a  central  channel  containing  the  processes  of  auxiliary  cells  (in  •).  together  with 
the  outer  dendritic  segments  (ods)  of  sensory  neurons.  In  this  region  the  outer  dendritic  segments  are  sur- 
rounded by  a  well-developed  dendritic  sheath  (ds).  For  TEM  examination,  antennae  were  removed  from 
animals  immediately  after  they  were  brought  to  the  ocean's  surface.  The  antennae  were  fixed  overnight  in 
0. 1  M Sorensen's  phosphate  buffer  (PB;  pH  7.2)  containing  5%  paraformaldehyde,  0.8%  glutaraldehyde,  3% 
NaCl  and  4.5%  sucrose.  Subsequently,  antennae  were  (1)  washed  in  PB  containing  8%.  sucrose,  3  times  for 
5  mm  each;  (2)  post-fixed  in  PB  containing  8%  sucrose  and  1%  osmium  tetroxide  for  1  h;  (3)  washed  in 
distilled  water,  3  times  for  5  min  each;  (4)  dehydrated  in  a  graded  ethanol  series  followed  by  transfer  to 
propylene  oxide;  and  (5)  embedded  in  Epon-araldite.  Thin  cross  sections  of  the  antennular  sensilla  were  cut 
on  an  RMC  MT-6000  XL  ultramicrotome,  stained  with  5%  aqueous  uranyl  acetate  and  lead  citrate,  and 
viewed  on  a  Zeiss  IOC  transmission  electron  microscope. 


to  sense  sulfides  at  concentrations  occurring  naturally  in 
their  environment,  e.g..  2-15  mMin  vent  fluids  from  the 
Menez  Gwen  and  Lucky  Strike  segments,  0.01-1.0/uM 
in  the  vicinity  of  known  venting  sites  2-5  m  above  the 
bottom  (7),  1-300  nM  at  diffuse,  low  temperature  vents 
(8),  and  6  mM  at  the  orifice  of  a  black  smoker  at  the 


Snake  Pit  site  (9).  Sulfide  may  be  useful  principally  as  a 
short-range  stimulus  over  distances  of  tens  of  meters  from 
its  source.  Sulfide  emitted  from  vents  is  oxidized  in  the 
seawater  surrounding  the  vents,  with  a  half-life  of  380  h 
(11,  12).  This  half-life,  however,  is  significantly  shortened 
by  the  presence  of  sulfide-utilizing  bacteria  associated  with 


CHEMOSl  NSORV   RESPONSE  IN  VENT  SHRIMP 


73 


E 


Figure  2.  (F)  Cross  section  in  the  distal  portion  of  a  sensillum  from  the  second  antenna  of  R 
In  this  region  the  outer  dendritic  segments  are  more  dispersed  and  the  dendritic  sheath  is  fragmented. 


the  vents  (13).  Other  compounds  associated  with  hydro- 
thermal  plumes,  such  as  methane  which  can  be  detected 
analytically  tens  of  kilometers  from  the  plume  source  ( 14), 
should  be  investigated  for  their  ability  to  stimulate  vent 
invertebrate  chemoreceptors  and  thus  possibly  serve  as 
long-distance  cues. 

Our  observations  that  the  antennae  of  vent  shrimp  are 
sensitive  to  sulfides  led  us  to  ask  whether  the  antennae  of 
other  shrimp  are  sensitive  to  sulfides.  The  suction  elec- 
trodes used  on  hydrothermal  vent  shrimp  axons  failed  to 
reveal  any  response  to  sulfides  or  other  chemical  stimuli 
from  excised  antennal  filaments  of  two  species  of  shallow- 
water  shrimp,  Penaeus  a:tecus  and  Palaemonetes  pugio, 
although  the  filaments  responded  to  tactile  stimuli  and 


were  spontaneously  active.  We  then  used  suction  elec- 
trodes with  finer  tips  especially  developed  to  study  che- 
mosensory  responses  from  antennal  filaments  of  decapod 
crustaceans  (15,  16)  which  allowed  better  discrimination 
between  axon  bundles  in  the  second  antenna  of  P.  aztecus. 
We  found  that  chemosensory  axons  which  responded  to 
broad-spectrum  odorants  also  responded  to  sulfide  stim- 
ulation in  a  concentration-dependent  way  (Fig.  4).  The 
relative  responses  were  0.010  ±  0.012  (n  =  3)  for  [Na2S] 
=  1.3  mM:  0.083  ±  0.009  (n  =  3)  for  13  mM;  0.138 
±0.019  (n  =  4)  for  130mA/;  and  0.52  (/;  ==  1)  for 
1300  mM.  We  used  three  control  solutions  in  these  ex- 
periments: ASW  [-0.030  ±  0.020  (n  =  2)];  pH  13  [0.26 
(/;  =  1 )];  and  ASW  with  the  pH  adjusted  to  10  to  match 


74 


A  Second  Antenna 

1.3 


G.  H.  RENNINGER  ET  AL. 
Medial  Filament 


Lateral  Filament 


13 


130 


1300 
[Na2S] 

(mM) 


ASW 


Control        • 


T 


B 


1.5 


0) 

w 

c 


0) 

OC 

«  0.5 

15 

C  0.0 


1.5 


1.0 


0.5 


0.0 


1.5 


1.0 


0.5- 


0.0 


101     10°     101     102     103 


101     10°     101     102     103 

[Na.S]  (mM) 


10-'     10°     101     102     103 


Figure  3.  (A)  Examples  of  multiunit  nerve  responses  to  solutions  of  Na2S  in  the  second  antenna,  and  in  the  medial  and  lateral  filaments  of  the  first 
antenna  excised  from  Rimicarix  sp.  The  bold  vertical  arrow  0.5  s  after  the  beginning  of  each  2  s  record  indicates  when  the  stimulus,  whether  one  of  the  Na>S 
or  control  solutions,  was  applied.  Transient  responses  following  the  exposure  may  have  been  due  to  tactile  receptors  activated  by  fluid  motion.  The  activity 
following  the  transient  response  increased  significantly  with  [Na2S]  in  the  second  antenna.  Exposure  of  filaments  of  either  antenna  to  control  solutions,  either 
artificial  seawater  (ASW)  or  pH  1 3,  evoked  little  or  no  response.  These  electrophysiological  recordings  from  antennal  filaments  of  hydrothermal  vent  shrimp 
collected  from  the  Snake  Pit  site  on  the  Mid-Atlantic  Ridge  (depth  cti.  3600  m)  were  made  aboard  R/V  Atlantis  II  Filaments  were  excised  from  active 
shrimp  and  maintained  in  Linuihis  physiological  solution  (6).  Several  segments  of  the  exoskeleton  were  removed  to  expose  a  short  length  of  antennal  nerve, 
which  was  either  wholly  or  partially  drawn  into  a  suction  electrode  filled  with  the  same  solution.  Filaments  with  spontaneous  nerve  activity  and  sensitivity 
to  tactile  stimuli  were  judged  to  be  in  good  physiological  condition  and  were  chosen  for  further  experimentation.  A  single  drop  of  the  test  solution  was  gently 
merged  with  the  fluid  surface  next  to  the  antenna  away  from  the  tip  of  the  suction  electrode.  Because  of  the  differential  behavior  of  the  axonal  responses 
from  the  various  filaments,  it  is  unlikely  that  the  responses  of  the  second  antenna  are  due  to  some  general  injury  response  of  the  exposed  ends  of  the  axons 
to  sulfide.  Test  solutions  were  as  follows:  Na2S  was  dissolved  in  distilled  water  at  a  concentration  of  1. 3  M  (pH  13),  which  was  then  diluted  with  ASW  to 
produce  lower  concentrations  ( 1 30  mM.  pH  10.5;  1 3  mM.  pH  9.5;  1 .3  mM.  pH  8).  The  stimulating  solutions  contained  several  chemical  species  of  sulfide, 
namely,  HS~.  H,S,  Sr,  and  NuHS.  with  concentrations  dependent  on  the  pH  of  the  solution.  In  the  pH  range  of  the  experiments,  sulfide  was  present 
predominantly  (>  90%)  in  the  form  HS".  (B)  Steady-state  relative  responses  to  concentrations  of  Na:S  and  controls.  Individual  responses  have  been  occasionally 
offset  in  the  plot  for  the  sake  of  clarity.  The  relative  responses  to  Na:S  calculated  for  the  sample  recordings  shown  in  (A)  are  represented  here  by  the  symbols 
shown  in  (A):  •  represents  the  second  antennal  responses  in  (A);  •,  the  medial  filament  of  the  first  antenna;  and  A,  the  lateral  filament.  The  other  solid 
symbols  represent  responses  to  sulfides  of  the  other  filaments  tested,  whose  responses  are  not  shown  in  (A).  Open  symbols  represent  responses  to  the  ASW 
control  (shown  near  O.I  on  the  abscissa)  or  to  the  pH  13  control  (shown  at  or  near  1300)  for  the  filaments  whose  responses  to  sulfide  are  indicated  by  the 
corresponding  solid  symbols.  Because  individual  nerve  impulses  could  not  be  distinguished  in  these  recordings,  we  determined  the  relative  response  for  each 
filament  in  the  following  way:  we  calculated  the  root-mean-square  (rms)  deviation  of  the  signal  about  its  mean  over  an  interval  of  duration  0.25  s  beginning 
0.5  s  before  exposure  (B),  together  with  the  rms  deviation  over  a  similar  0.25  s  interval  beginning  1  s  after  exposure  (R):  then  we  took  the  relative  response 
r  to  equal  (R/B)  -  1.  These  intervals  are  indicated  by  heavy  horizontal  bars  in  (A). 


CHEMOSENSORY  RESPONSE  IN  VENT  SHRIMP 


75 


Second  Antenna 


B 


13 


130 

1300 

[Na2S] 

(mM) 


o> 

(0 

c 
o 

Q. 
(/> 
O 
DC 

0 


0.5- 


v 


jo  o.o 

o>          

DC      -|  o -V  10°101102103 

[Na2S]  (mM) 


ASW 


pH10 


Control 


Figure  4.  (A)  Examples  of  multiunit  nerve  response  from  the  second  antenna  of  the  shallow-water 
shrimp  Panaeus  a:lecus  to  solutions  of  Na:S  and  to  control  solutions.  The  experimental  procedures  used 
for  P.  a:lecus  are  described  in  ( 1 5.  16).  See  Figure  3A  for  other  details.  (B)  Steady-state  relative  responses 
to  concentrations  of  Na2S  and  controls.  Note  the  expanded  response  scale  in  comparison  with  Figure  3B. 
The  solid  s\  mbol  •  represents  the  relative  responses  to  Na:S  shown  in  (A):  the  other  solid  symbols  represent 
sulfide  responses  of  the  other  second  antennae  tested.  The  open  symbols  represent  responses  to  the  three 
control  solutions  used  on  the  antennal  filaments  whose  sulfide  responses  are  indicated  by  the  corresponding 
solid  symbols,  namely,  ASW  (shown  near  0.1).  pH  10  (shown  at  or  near  130).  and  pH  13  (shown  at  or  near 
1300).  See  Figure  3B  for  other  details. 


the  pH  of  the  130  mM  Na2S  solution  [pH  10,  giving  a 
response  =  0.040  ±0.018  (n  =  4)]. 

While  the  second  antennae  of  both  vent  and  shallow 
water  shrimp  responded  to  Na:S.  there  are  major  differ- 
ences between  them.  First,  the  axons  of  P.  aitccus  re- 
sponded vigorously  to  the  pH  13  control  solution,  unlike 
those  of  Rimicaris  sp.  or  R  c.xociiluici.  Thus,  the  response 
to  Na:S  in  P.  azlecits  was  confounded  by  a  sensitivity  to 
high  pH.  Second,  the  axons  of  P.  a~teciis  responded  only 
for  about  40  min  after  excision,  while  axons  of  Rimicaris 
sp.  and  R.  exociilalu  responded  repeatedly  to  sulfide  stim- 
ulation for  several  hours,  and.  in  one  experiment  on  R. 
exoculata,  for  24  h.  Furthermore,  sensilla  on  the  second 


antenna  of  Rimicaris  sp.  and  R.  e.\ociiluta  are  more  nu- 
merous than  those  in  P.  aztcais  (1  sensillum/231  /urn  of 
filament  circumference)  and  P.  pugio  ( 1  sensillum/ 
1 10  fim).  Although  we  do  not  know  how  many  chemo- 
sensory  neurons  there  are  in  each  sensillum  or  what  their 
individual  sensitivities  are,  the  larger  density  of  sensilla 
in  the  vent  species  suggests  that  they  may  have  an  en- 
hanced chemosensory  capacity. 

To  our  knowledge,  this  is  the  first  physiological  dem- 
onstration of  a  sensory  response  to  a  geochemical  stimulus 
in  a  vent  invertebrate.  Sulfides  have  been  reported  to  serve 
as  physical  cues  for  other  invertebrates:  a  shallow-water 
polychaete  may  use  sulfides  as  a  settlement  cue  (17),  but 


76 


G    H    RENNINGER  ET  AL. 


this  is  controversial  (18);  and  a  tropical  tick  is  attracted 
to  sulfides  in  mammalian  breath  (19).  In  behavioral  ex- 
periments on  the  shrimp  Palaemonetex  vulgaris.  concen- 
trations of  H2S  <  0.08  mM  evoked  an  avoidance  response 
(N.  Sofranko  and  C.  L.  Van  Dover,  unpub.  data).  The 
sulfide  response  which  we  have  found  in  hydrothermal 
vent  shrimp  may  thus  be  an  adaptation  of  a  widespread 
sulfide  sensitivity  in  shrimp  to  the  particular  environment 
created  by  the  hydrothermal  vents.  Behavioral  studies  on 
sulfide  sensitivity  of  vent  shrimp  will  be  required  to  de- 
termine whether  the  responses  we  have  observed  are  in 
fact  used  by  the  shrimp  in  orientation  or  other  behaviors. 
If  the  antennal  responses  initiate  behavior  in  vent  shrimp, 
the  behavioral  threshold  may  well  lie  below  the  neural 
threshold.  Such  differences  between  thresholds  for  sensory 
cells  and  for  behavior,  which  have  been  attributed  to  the 
convergence  of  the  sensory  arTerents  onto  a  relatively 
smaller  population  of  cells  in  the  central  nervous  system, 
have  been  discussed  for  the  spiny  lobster,  Panulirus  in- 
lerniplus,  in  which  the  behavioral  sensitivity  to  ATP  is 
30  times  greater  than  the  sensory  cellular  sensitivity  (20), 
and  for  the  American  cockroach  (2 1 )  and  moths  (22,  23). 

Acknowledgments 

We  thank  the  scientists  aboard  R/V  Atlantis  //on  cruise 
129-7  for  helpful  advice  and  discussions.  We  are  indebted 
to  the  Alvin  Group  and  the  crew  of  R/V  Atlantis  II  for 
the  success  of  the  cruise.  We  thank  Lorraine  McDowell 
and  Richard  Mitchell  for  technical  assistance.  This  work 
was  supported  in  part  by  NSF  USA  and  NSERC  Canada. 

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Cephalopods  Occupy  the  Ecological  Niche  of 
Epipelagic  Fish  in  the  Antarctic  Polar  Frontal  Zone 

PAUL  G.  RODHOUSE  AND  MARTIN  G.  WHITE 

British  Antarctic  Survey,  Natural  Environment  Research  Council,  High  Cross,  Madinglev  Road, 

Cambridge  CB3  GET,  UK 


Recent  data  from  research  cruises  and  exploratory  fish- 
ing in  the  Antarctic  Polar  Frontal  Zone  (APFZ)  of  the 
Scotia  Sea,  together  with  data  from  dietary  studies  of  Ant- 
arctic vertebrate  predators,  have  revealed  a  large,  previ- 
ously overlooked  trophic  .system  in  the  Southern  Ocean 
(Fig.  1).  The  upper  trophic  levels  of  this  open-ocean  epi- 
pe/agic  community  are  exceptional  in  that  they  contain 
no  fish  species.  Fishes  are  replaced  by  cephalopods,  in- 
cluding the  ommastrephid  squid,  Martialia  hyadesi.  This 
squid  preys  on  mesopelagic  myctophids  (lanternfi.sh).  which 
feed  largely  on  copcpods.  H  'c  identify  here  a  geographically 
distinct.  Antarctic,  open-ocean  food  chain  which  is  of  im- 
portance to  air  breathing  predator  species  but  where  Ant- 
arctic krill,  Euphausia  superba,  is  absent.  This  system  is 
probably  prevalent  in  areas  of  higher  primary  productivity, 
especially  the  Scotia  Sea  and  near  the  peri-Antarctic  is- 
lands. Squid  slocks  in  the  APFZ  may  have  potential  for 
commercial  exploitation,  but  they,  and  the  predators  they 
support,  are  likely  to  be  sensitive  to  overftshing.  Squid  have 
a  short,  semelparous  lifecycle,  so  overfishing  in  a  single 
year  can  cause  a  stock  to  collapse. 

The  presence  of  this  trophic  system  was  already  evident 
among  the  results  of  the  Discovery  expeditions.  Large 
quantities  of  squid  remains,  especially  their  indigestible 
beaks,  were  found  in  the  gut  contents  of  several  albatross 
and  seal  species  ( 1 ).  But  because  E.  superba  is  so  con- 
spicuous and  plays  such  an  important  role  in  the  diet  of 
vertebrate  predators  (especially  commercially  exploited 
baleen  whales),  and  as  these  crustaceans  were  amenable 
to  marine  biological  research  methods  of  the  day,  the 
cephalopod  trophic  system  in  the  vast  but  remote  region 
of  the  APFZ  was  largely  ignored. 

Received  18  May  1995:  accepted  27  July  1995. 
Abbreviations:  APFZ  =  Antarctic  Polar  Frontal  Zone;  BAS  =  British 
Antarctic  Survey. 


Following  a  review  of  the  resources  of  the  Southern 
Ocean  (2)  that  noted  the  possible  presence  of  large  ceph- 
alopod stocks,  studies  of  the  cephalopod  prey  of  vertebrate 
predators  breeding  at  South  Georgia  (Fig.  2)  in  the  1970s 
(3)  revealed  that  an  unidentified  ommastrephid.  Toda- 
rodes?sp.  dominated  the  diet  of  some  species.  Collections 
of  cephalopod  remains  in  the  predator  regurgitations  at 
South  Georgia  (4.5)  made  in  the  1980s  by  scientists  of  the 
British  Antarctic  Survey  (BAS),  coincided  with  a  large 
"by-catch"  of  the  little-known  ommastrephid  M.  hyadesi 
in  the  new  fishery  for  the  squid  I/lex  argentinus  on  the 
Patagonian  Shelf  during  1986  (6).  A  comparison  of  ma- 
terial from  both  sources  revealed  that  the  species  being 
taken  by  predators,  previously  identified  as  Todarodes? 
sp.,  was  M.  hyadesi.  Exploratory  fishing  by  Japanese  squid 
jiggers  in  the  APFZ  west  of  South  Georgia  subsequently 
caught  commercial  quantities  of  M.  hyadesi  near  the  sur- 
face (<50  m),  confirming  the  presence  of  this  species  in 
the  Scotia  Sea  (6).  Although  stock  size  cannot  be  assessed 
at  present,  annual  predator  consumption  of  M.  hyadesi 
in  the  region  is  estimated  at  >  3  30,000  tonnes  (7).  Stomach 
contents  of  jigged  specimens  showed  they  had  fed  on  a 
community  of  myctophid  fish  dominated  by  Kreffiichthys 
anderssoni  (8).  Myctophids  feed  largely  on  copepods.  so 
this  food  chain  may  be  partially  or  fully  independent  of 
the  Euphausia  superba  trophic  system.  Elsewhere  in  the 
APFZ,  myctophids  have  recently  been  identified  as  the 
major  food  resource  for  other  higher  predators  such  as 
the  king  penguin  (9). 

During  the  1994  research  cruise  of  RRS  James  Clark 
Ross  (BAS)  satellite-tagged  albatross  predators  of  M.  hy- 
adesi and  other  squid  were  tracked  to  the  APFZ,  north 
of  South  Georgia;  there  the  pelagic  community  exploited 
by  the  birds  was  sampled  with  a  commercial  trawl.  The 
samples  included  the  squids  M.  hyadesi,  Aforoteuthis  kni- 
povitchi  (the  major  prey  of  southern  elephant  seals),  Go- 


77 


78 


P.  G.  RODHOUSE  AND  M.  G.  WHITE 

ANTARCTIC  POLAR  FRONTAL  ZONE  SOUTH  GEORGIA  SHELF/SLOPE 


Albatrosses 


Albatrosses 


500m  -i 


1000  m  -j 


Ommastrephid  squid      ••  Toothed  whales 
Mesopelagic  fish 


Figure  1.  Spatial  relationship  between  the  myctophid/cephalopod  trophic  system  at  the  Antarctic  Polar 
Frontal  Zone  and  the  Euphaiisia  supcrba  based  system  on  the  South  Georgia  shelf  and  slope  (positions  of 
organisms  in  the  water  column  do  not  represent  their  bathymetnc  distribution). 


minis  anlarcticus,  Galiieiitliis  glacialis.  and  Brachioteuthis 
sp.  (Rodhouse,  P.  G.  ct  al.,  BAS,  in  prep).  The  data  con- 
firmed that,  in  the  Southern  Ocean,  in  the  vicinity  of  the 
APFZ,  M.  hyailt'si  occurs  near  the  surface  (<  100  m).  Al- 
batross predators  breeding  at  South  Georgia  and  large 
male  sperm  whales  feeding  in  the  vicinity  of  the  islands 
(10)  exploit  different,  geographically  separated,  cephalo- 
pod  communities.  The  whales'  diet  does  not  include  sig- 
nificant numbers  of  M.  hyadcsi:  rather  it  is  dominated, 
in  terms  of  biomass,  by  the  gigantic  Antarctic  cranchiid 
squid.  Mesonychoteuthis  humilioni. 

The  biological  oceanography  of  the  APFZ  is  poorly  un- 
derstood. Primary  productivity  in  the  ice-free  zone  of  the 
Southern  Ocean  is  generally  low  (11).  Strong  westerly 
winds  generally  maintain  a  deep  mixed  layer,  but  com- 
posite Coastal  Zone  Color  Scanner  images  reveal  regions 
of  high  concentrations  of  phytoplankton  pigment,  indi- 
cating enhanced  productivity  in  the  Scotia  Sea.  in  the 
vicinity  of  the  Scotia  Arc.  and  near  the  peri- Antarctic  is- 
lands ( 1 2).  In  the  ice- free  zone  of  the  Scotia  Sea,  the  pelagic 
community  is  dominated  by  copepods,  small  euphausiids, 
gelatinous  zooplankton,  and  myctophids  (13.14).  Else- 
where in  the  APFZ,  M.  hytu/csi  has  been  recorded  from 
the  Kerguelen  (15)  and  Macquarie  (16)  islands,  suggesting 
that  this  squid  occurs  in  areas  of  enhanced  productivity 
in  the  pelagic  community. 

The  composition  of  the  Antarctic  pelagic  fish  com- 
munity is  unusual  by  comparison  with  such  communities 


in  other  oceans,  because  epipelagic  fish  families  are  absent. 
Key  components  of  the  pelagic  food-web  in  temperate 
and  tropical  seas — clupeids.  carangids,  scombrids  and 
their  predators,  the  oceanic  sharks — are  absent  or  rare 
vagrants  on  the  periphery  of  the  Southern  Ocean.  At  shal- 
low depths  around  the  Antarctic  Continent  and  the  peri- 
Antarctic  islands,  demersal  fish,  mostly  members  of  the 
endemic  suborder  Notothenioidei,  dominate  the  fish 
fauna.  Most  species  are  demersal  as  adults,  but  a  small 
number,  notably  Pleuragramma  antarctiann  near  the 
continent  and  Champsocephalus  gunnari  in  the  vicinity 
of  islands,  have  become  secondarily  adapted  to  inhabit 
pelagic  habitats.  Others  are  temporarily  pelagic  during 
early  ontogeny.  By  contrast,  the  fish  fauna  of  the  open 
ocean  is  limited  to  deepwater  bathypelagic  and  mesope- 
lagic  species.  Of  these,  the  myctophids  predominate,  and 
are  sufficiently  abundant  to  support  a  fishery  (17).  In  the 
north  Scotia  Sea,  mesopelagic  fish,  mainly  myctophids. 
constitute  up  to  18%  of  the  total  nekton  biomass  and  are 
the  main  component  of  the  biomass  available  to  higher 
predators  (13,14).  But  epipelagic  fish  are  absent,  and  we 
now  conclude  these  are  replaced  by  a  cephalopod  com- 
munity dominated  by  ommastrephid  squid. 

An  epipelagic  system  dominated  by  cephalopods  is 
possibly  the  consequence  of  physiological  constraints  on 
fish  in  cold  sub-Antarctic  or  Antarctic  waters,  that  do  not 
apply  to  the  cephalopods;  or  may  have  arisen  because  the 
life  cycle  traits  of  cephalopods  may  be  better  adapted  to 


ECOLOGICAL  NICHE  IN  POLAR  FRONTAL  ZONE 


79 


50"- 


ANTARCJIC,  POLAR.FR 
S      (Approximate  position) 


SOUTH 
GEORGIA 


60 


50° 


SCOTIA  SEA 
40' 


Figure  2.     Atlantic  sector  of  the  Southern  Ocean  showing  the  position  of  South  Georgia  and  the  Antarctic 
Polar  Front. 


the  physical  environment  of  the  APFZ.  The  relatively 
short  life  span  of  cephalopods  might  allow  them  to  com- 
plete the  life  cycle,  at  least  from  the  planktonic  to  the 
nektonic  phase,  before  they  are  flushed  out  of  productive 
regions  by  the  fast-flowing  Antarctic  Circumpolar  Current. 
If  epipelagic  communities  in  other  remote  oceanic  regions 
are  similarly  dominated  by  cephalopods,  then  compara- 
tive studies  may  shed  light  on  these  questions. 

As  finfish  stocks  have  declined  globally,  cephalopod 
catches  have  grown  and,  in  terms  of  dollar  value  of  the 
catch,  are  currently  rated  third  in  world  importance  after 
shrimp  and  tuna  (18).  Pressures  on  fish  stocks  continue 
to  increase,  and  conflict  at  sea  between  fishing  nations  is 
likely  to  escalate  in  the  absence  of  political  will  to  reduce 
fishing  effort.  Under  these  circumstances,  new  cephalopod 
stocks  are  likely  to  be  sought  as  an  alternative,  high-value 
resource.  Stocks  of  M.  /n-flflts/  straddle  the  region  ad- 
ministered by  the  Commission  for  the  Conservation  of 
Antarctic  Marine  Living  Resources,  and  the  high  seas  to 
the  north.  This  will  complicate  management  of  any  future 
fishery  in  the  region.  Given  the  important  role  of  ceph- 
alopods in  the  diet  of  several  species  of  higher  predator 
in  the  Antarctic  ( 19)  and  the  vulnerability  of  short-lived, 
semelparous  species  to  overexploitation,  the  ecological 
consequences  of  an  unmanaged  fishery  for  cephalopods 
in  the  Southern  Ocean  are  potentially  severe. 

Acknowledgments 

Cephalopod  research  at  the  British  Antarctic  Survey 
(BAS)  owes  much  to  collaboration  with  John  Croxall  and 
Peter  Prince  in  the  Higher  Predators  Programme. 


10 


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lite  Conservation  o/  Antarctic  Marine  Living  Resources.  Hobarl. 
Australia  SC-CAMLR-SSP/7:  433-457. 

18.  FAO  Fisheries  Department.  1993.     Fisheries  and  the  law  of  the 
sea:  a  decade  of  change.  FAO  Fisheries  Circular.  No.  853:  66  pp. 
Rome. 

19.  Laws,  R.  M.  1985.     Ecology  of  the  Southern  Ocean.  Am.  Sci.  73: 
26-40. 


Reference:  Bio/.  Bull  189:  81-90.  (October/November,  1995) 


Phylogenetic  Position  of  the  Dicyemid  Mesozoa 
Inferred  from  18S  rDNA  Sequences 


TOMOE  KATAYAMA1,  HIROSHI  WADA2,  HIDETAKA  FURUYA3,  NORIYUKI  SATOH2 

AND  MASAMICHI  YAMAMOTO1 

^Ushimado  Marine  Laboratory,  Okayama  University,  Okayama  701-43,  2 Department  oj  Zoology, 

Kyoto  University,  Kyoto  606-01,  and*  Department  oj  Biology, 

Osaka  University,  Toyonaka  560,  Japan 


Abstract.  The  dicyemid  mesozoa,  obligate  symhionts  in 
the  cephalopod  kidney,  are  simply  organized  multicellular 
animals.  They  have  long  been  the  subject  of  phylogenetic 
debates.  Some  authors  have  suggested  that  dicyemids  rep- 
resent an  offshoot  from  an  early  metazoan  ancestor.  Other 
workers  considered  them  to  be  degenerated  progeny  of 
higher  metazoa.  possibly  parasitic  trematodes.  We  deter- 
mined the  almost  complete  nucleotide  sequences  of  18S 
rDNA  in  two  species  of  dicyemid,  Dicyema  orientate  and 
Dicyema  acuticephalwn.  isolated  purely  from  cephalopod 
urine.  We  compared  these  sequences  with  sequences  de- 
termined in  the  present  study  from  three  flatworm  species, 
as  well  as  with  a  variety  of  eukaryote  sequences  obtained 
from  databases.  The  phylogenetic  trees  reconstructed  with 
the  use  of  the  neighbor-joining,  maximum-parsimony, 
and  maximum-likelihood  methods  indicated  that  the  di- 
cyemids belong  among  the  triploblastic  animals  (Bila- 
teria).  However,  we  cannot  firmly  establish  the  position 
of  the  dicyemids  within  the  Bilateria  because  we  cannot 
ignore  the  problem  of  long  branch  attraction  between  the 
myxozoans,  dicyemids,  nematodes.  and  acoel  flatworms. 
The  present  results  favor  the  hypothesis  that  the  dicyemids 
do  not  represent  an  early  divergent  metazoan  group,  but 
rather  a  group  degenerated  from  a  triploblastic  ancestor. 

Introduction 

The  dicyemids  are  simply  organized  multicellular  an- 
imals consisting  of  an  outer  layer  of  20-40  ciliated  somatic 
cells  and  an  inner  core  of  one  long  axial  cell.  They  are 

Received  7  July  1994;  accepted  5  July  1995. 

Correspondence:  Dr.  Masamichi  Yamamoto.  Ushimado  Marine  Lab- 
oratory. Okayama  University,  Ushimado.  Okayama.  701-43  Japan. 


obligate  symbionts  in  the  kidney  of  cephalopods.  Their 
life  cycle  is  complex;  the  asexually  produced  vermiform 
embryos  increase  the  population  in  the  host,  while  the 
infusoriform  embryos  arising  from  fertilized  eggs  pass  out 
of  the  host  body  with  the  urine  and  are  thought  to  infect 
another  cephalopod  host  (Brusca  and  Brusca,  1990). 

The  dicyemid  mesozoans  have  long  been  the  subject 
of  a  phylogenetic  controversy  (Brusca  and  Brusca.  1990; 
Willmer,  1990).  They  were  at  first  considered  to  be  an 
extant  link  between  the  Protozoa  and  the  Metazoa  (Hy- 
man,  1959).  However,  the  resemblance  of  their  complex 
life  cycles  to  those  of  parasitic  trematodes  has  led  some 
authors  to  propose  that  the  dicyemids  are  descended  from 
an  established  metazoan  group  and  that  their  simple  body 
organization  results  from  degeneration  attributable  to 
parasitism  (Nouvel.  1948;  McConnaughey,  1951:  Stun- 
kard,  1954;  Ginetsinskaya,  1988).  Others  still  view  the 
simple  body  construction  of  dicyemids  as  truly  primitive 
and  hold  that  the  group  represents  an  offshoot  from  early 
divergent  metazoa  (Dodson,  1956;  Hyman,  1959;  Lapan 
and  Morowitz,  1974). 

The  phylogenetic  relationships  of  eukaryotes  have  re- 
cently come  under  intense  scrutiny  in  the  light  of  new 
molecular  data.  Phylogenetic  analyses  using  nucleotide 
sequences  of  5S  rRNA  suggested  that  a  dicyemid  (Di- 
cyema misakiense]  diverged  early  among  such  lower  me- 
tazoa as  sponges,  cnidarians.  and  flatworms  (Ohama  et 
a/..  1984;  Hori  and  Osawa,  1987).  But  phylogenetic  trees 
based  upon  comparisons  of  about  120  sites  in  the  nu- 
cleotide sequences  of  5S  rRNA  were  different  from  those 
inferred  from  longer  nucleotide  sequences  of  18S  or  28S 
ribosomal  RNA  (Field  el  ai.  1988;  Christen  et  ai,  1991; 
Katayama  el  a/.,  1993:  Wainright  et  al.,  1993:  Kobayashi 
el  u/..  1993).  We  have  sequenced  18S  ribosomal  RNA 


81 


82 


T.   KATAYAMA  ET  AL 


genes  ( 1 8S  rDN  A)  in  two  species  of  the  dicyemid  mesozoa. 
Our  comparison  of  the  nucleotide  sequences  of  small- 
subunit  rDNA  for  a  variety  of  organisms  indicates  that 
the  dicyemids  belong  among  the  triploblastic  animals. 

Materials  and  Methods 

Biological  materials 

We  determined  almost  the  entire  sequence  of  18S 
rDNA  in  two  species  of  dicyemid  mesozoans  and  three 
species  of  turbellarians  (Platyhelminthes).  Pure  samples 
of  the  dicyemids  Dicyema  aculiccphalum  and  Dicyema 
orientate  were  collected  from  the  urine  of  Octopus  vu/garis 
and  Sepioteuthis  lessoniana,  respectively  (Furuya  el  al., 
1992a).  Specimens  ofConvoluta  naikaiensis  (Acoela)  and 
Planocera  multitentaculata  (Polycladida)  were  collected 
on  the  shore  near  the  Ushimado  Marine  Laboratory. 
Specimens  of  Dngesia  japonica  (Tricladida)  were  obtained 
from  the  brook  near  the  Ushimado  Marine  Laboratory. 
All  were  frozen  quickly  and  kept  at  — 80°C  until  use. 

In  addition  to  the  sequences  of  the  above  five  species, 
we  used  the  sequences  of  23  eukaryotes — including  ani- 
mals, protists,  plants,  and  fungi — for  which  almost  com- 
plete 18S  rDNA  sequences  were  available  in  databases. 
The  species  used  and  their  accession  numbers  are  as  fol- 
lows: Parameditm  ictraiirelia  (Ciliophora),  X03772; 
Oxytricha  nova  (Ciliophora),  X03948;  Crypthecodinium 
coluiii :(Dinozoa),  M64245:  Theileria  annulata  (Apicom- 
plexa),  M64243;  Sarcocystis  nniris  (Apicomplexa), 
M64244;  Hartmanella  vermiformis  (Rhizopoda), 
M95168;  Saccharomyces  cerevisiae  (Fungi),  JO  1353;  Fi- 
lobasidii'lla  neoformans  (Fungi).  X60183;  Arahidopsis 
thaliana  (Plantae),  X16077;  J'o/vav  carter/  (Plantae), 
X53904;  Berne  atcwnis  (Ctenophora),  D15068;  Tricho- 
plax  adhaerens  (Placozoa),  LI 0828:  Anemonia  sulcata 
(Cnidaria),  X53498;  Scyplia  ciliata  (Porifera),  L 10827: 
Henneguya  sp.  (Myxozoa),  U 13826;  Caenorhabditis  ele- 
gans  (Nematoda),  X03680;  Moliniformis  moliniformis 
(Acanthocephala),  Z 19562;  Schistosoma  mansoni  (Tre- 
matoda),  X53047;  Crassostrea  gigas  (Bivalvia),  X60315; 
Artemia  salina  (Crustacea),  X01723;  Sugitta  crassa 
(Chaetognatha),  D 1 4363;  Aster/as  annirensis  (\s\em\dea). 
D14358;  and  Xenopus  laevis  (Vertebrata),  X04025. 

DNA  isolation 

Genomic  DNA  was  extracted  by  the  method  described 
previously  (Wada  et  al,  1992).  In  brief,  the  frozen  samples 
were  lysed  in  TE  buffer  (10  mAf  Tris-HCl,  0. 1  AI  EDTA, 
pH  8.0)  containing  0.5%  sodium  dodecyl  sulfate.  After 
digestion  with  proteinase  K.  (100  Mg/ml)  at  50°C  for  3  h. 
DNA  was  extracted  with  phenol  and  precipitated  in 
ethanol  and  an  equal  volume  of  5.0  M  ammonium  ace- 
tate. Samples  resuspended  in  TE  buffer  were  further  pu- 


rified by  RNase  A  digestion  (20  fig/ml)  at  37 °C  for  1  h 
followed  by  ethanol  precipitation. 

Amplification  of  ISS  rDNA 

The  1 8S  rDNA  was  amplified  by  the  polymerase  chain 
reaction  (PCR;  Saiki  et  al..  1988)  in  an  Air  Thermo-cycler 
1645  (Idaho  Technology).  Almost  the  entire  length  of  18S 
rDNA  was  amplified  using  synthetic  oligonucleotides, 
5'-CTGGTTGATCCTGCCAG-3'  (primer  0)  and  5'- 
CCTTGTTACGACTT-3'  (primer  10)  as  the  terminal 
primers.  Amplifications  were  performed  in  50  n\  of 
50  mM  Tris-HCl  (pH  8.5),  250  /ug/ml  BSA,  2  mAf  Mg2+, 
with  0.2  mA/each  dNTP,  50  pM  primers,  template  DNA 
(5-10  ng).  and  2  U  Taq  DNA  polymerase  (TOYOBO). 
The  temperature  regimen  for  35  cycles  was  20  s  at  94°C, 
30  s  at  50°C,  and  90  s  at  74°C. 

Determination  of  DNA  sequences 

After  purification  of  the  amplified  DNA  by  electro- 
phoresis  in  a  0.8%  agarose  gel,  the  nucleotide  sequence 
was  directly  determined  by  dideoxy  chain-termination 
(Sanger  et  al..  1977)  using  Sequenase  ver  2.0  (USB)  and 
[1?S]-dATP  (Amersham).  All  DNA  samples  were  se- 
quenced  in  both  directions  and  from  several  separate  am- 
plifications with  terminal  primers  (0  and  10)  and  internal 
primers.  The  internal  primers  used  were  primer- 1  (5- 
CCGGAGAGGGAGCCTGA-3'),  primer-2  (antisense  of 
primer- 1),  primer-3  (5'-CAGCAGCCGCGGTAATT-3'), 
primer-4  (antisense  of  primer-3),  primer-5  (5'-GCGAA- 
AGCATTTGCCAA-3').  primer-6  (antisense  of  primer  5), 
primer-7  (5'-GAAACT(TC)AAAGGAAT-3'),  primer-8 
(antisense  of  primer-7),  and  primer-9  (5'-ACGGGC- 
GGTGTGT(AG)C-3').  The  positions  corresponding  to 
these  primers  in  18S  rDNA  sequences  are  shown  in  Figure 
1.  The  continuity  of  the  DNA  fragments  was  confirmed 
by  overlapping  of  the  sequences. 

Phylogenetic  analyses 

Sequences  were  aligned  manually  on  the  basis  of  max- 
imum nucleotide  similarity  (Fig.  1 ).  Alignment  gaps  were 
inserted  to  account  for  putative  length  differences  between 
sequences.  Some  regions  could  be  confidently  aligned  and 
were  presumed  to  be  homologous.  However,  we  could 
not  unequivocally  determine  the  optimal  alignment  for 
the  regions  containing  deletions,  insertions,  or  highly 
variable  sequences.  We  excluded  positions  from  the  anal- 
ysis according  to  the  following  rule:  Positions  where  a  gap 
was  present  for  any  taxon  were  not  used  in  analyses.  In 
the  present  study,  we  found  this  simple  rule  alone  ade- 
quate for  excluding  the  regions  of  ambiguous  homology 
(the  regions  where  two  or  more  equally  optimal  align- 
ments were  present)  from  the  analysis  because  in  those 


MOLECULAR  PHYt.OGENY  OF  DICYEMIDS 


83 


regions  alignment  gaps  were  always  serially  inserted  in 
many  sequences.  The  phylogenetic  trees  were  recon- 
structed using  the  PHYLIP  package  version  3.5c  (Felsen- 
stein,  1989)  and  fastDNAML  (Olsen  ct  at..  1993).  Tree- 
building  procedures  used  were  the  neighbor-joining  (Sai- 
tou  and  Nei,  1987),  the  maximum-parsimony  (Fitch, 
1971),  and  the  maximum-likelihood  (Felsenstein,  1981). 
For  the  neighbor-joining  analysis,  evolutionary  distance 
values  were  calculated  by  the  formula  of  Jukes  and  Cantor 
(1969).  The  degree  of  support  for  internal  branches  of  the 
trees  in  the  neighbor-joining  and  the  maximum-parsi- 
mony trees  was  assessed  by  bootstrap  levels  of  support 
(Felsenstein,  1985)  determined  by  500  bootstrap  repeti- 
tions. 

Results 

In  D.  acitticcphaliim,  D.  orientate.  Convoluta  naikaien- 
sis,  Dugesiajaponica,  and  Planocera  multitentaculata,  al- 
most the  entire  length  of  18S  rDNA  was  amplified  by 
PCR  from  the  genomic  DNA.  The  sequence  ( 1 500- 
1700bp)  was  determined  directly  from  PCR  products. 
The  sequences  have  been  deposited  in  databases  (GSDB, 
DDBJ,  EMBL,  and  NCBI)  under  the  following  accession 
numbers:  D26529  for  D.  aaiticepluilnm;  D26530,  D.  ori- 
entate; Dl  7558.  Convoluta  naikaiensis;  Dl  7560,  Ditgesia 
japonica;  D 17562,  Planocera  multitentaculata.  To  infer 
the  phylogenetic  position  of  the  dicyemids  within  the  eu- 
karyotes,  we  aligned  the  almost  complete  nucleotide  se- 
quences of  18S  rDNA  of  the  above  five  species  with  the 
23  eukaryote  sequences  we  obtained  from  databases.  Me- 
tazoan  taxa  were  chosen  to  represent  phyla  broadly;  pro- 
tozoan taxa  were  chosen  to  represent  the  more  recently 
derived  groups.  Figure  1  shows  a  sample  of  the  alignment 
for  9  out  of  28  species  included  in  the  present  analysis. 
This  alignment  reveals  that  throughout  the  eukaryotes 
the  sequences  are  highly  conserved  in  some  regions  and 
highly  variable  in  others.  After  exclusion  of  the  regions 
of  ambiguous  homology,  1070  sites  (Fig.  1 )  remained  for 
phylogenetic  inference.  Phylogenetic  trees  shown  were  re- 
constructed by  the  neighbor-joining  (Fig.  2),  the  maxi- 
mum-parsimony (Fig.  3),  and  the  maximum-likelihood 
(Fig.  4)  analyses. 

Among  the  phylogenetic  trees  reconstructed  by  the 
three  methods,  the  topologies  were  largely  congruent  with 
one  another,  though  branching  with  low  bootstrap  support 
within  the  metazoan  lineage  showed  somewhat  conflicting 
arrangements.  The  metazoans — including  triploblasts 
(Bilateria).  diploblasts,  dicyemids,  and  a  myxozoan — 
formed  a  monophyletic  assemblage  in  the  three  trees. 
Within  the  metazoan  assemblage,  triploblasts  formed  a 
discrete  monophyletic  unit  together  with  the  mesozoa  and 
the  Myxozoa.  The  branches  of  triploblasts  were  in  general 
longer  than  those  of  other  taxa. 


The  grouping  of  the  dicyemids  with  the  triploblastic 
animals  was  supported  by  a  bootstrap  value  of  100%  in 
both  the  neighbor-joining  (Fig.  2)  and  the  maximum-par- 
simony (Fig.  3)  analyses  (because  of  the  enormous  com- 
putation time  required,  bootstrapping  was  not  performed 
in  the  maximum-likelihood  analysis).  In  the  trees  recon- 
structed by  the  three  methods,  dicyemids  were  grouped 
with  Caenorhabdilis  elegans  (a  nematode),  Henneguya 
sp.  (a  myxozoan),  and  Convoluta  naikaiensis  (an  acoel 
flatworm),  though  bootstrap  confidence  level  for  this 
grouping  was  low. 

To  corroborate  the  inclusion  of  the  dicyemids  in  the 
triploblastic  lineage,  we  analyzed  subsets  of  taxa  shown 
in  the  present  paper  as  well  as  several  different  sets  of  taxa 
including  some  of  the  following  species  (the  accession 
numbers  for  18S  rDNA  data  are  shown  in  parentheses): 
Cryptomonas  phi  (X57162),  Bahesia  bovis  (M87566), 
Telillci  japonica  (D15067),  Sycon  calcaravis  (D15066), 
Mnemiopsis  leidyi  (LI 0826),  Tripedalia  cystophora 
(L10829).  Paraspadella  gotoi  (D14362),  Antedon  serrata 
(D14357),  Strongylocentrotus  intermedius  (D14365), 
Balanoglossus  carnosus  (D14359),  Oikopleura  sp. 
(D14360),  Branchiostoma  floridae  (M19571),  Homo  sa- 
piens (X03205).  In  all  sets  of  taxa  analyzed,  triploblasts 
formed  a  monophyletic  unit  and  the  dicyemids  were 
placed  within  the  triploblastic  clade  with  high  bootstrap 
confidence  level.  Grouping  of  Caenorhabdilis,  Convoluta. 
Henneguya.  and  Dicyema  were  consistently  observed. 
Triclioplax.  another  enigmatic  animal  whose  phylogenetic 
position  is  controversial  (Brusca  and  Brusca,  1990;  Will- 
mer,  1990),  was  always  positioned  outside  the  triploblastic 
assemblage,  confirming  analyses  by  Wainwright  ct  a/. 
(1993). 

Discussion 

The  present  molecular  phylogenetic  study  based  upon 
comparisons  of  nucleotide  sequences  of  1 8S  rDNA  shows 
that  triploblastic  animals  form  a  monophyletic  assemblage 
within  the  metazoan  subtree  and  that  the  dicyemid  me- 
sozoa are  an  ingroup  of  the  monophyletic  unit  of  triplo- 
blastic animals.  Monophyly  of  triploblastic  animals  has 
repeatedly  been  shown  in  previous  molecular  phylogenetic 
analyses  with  18S  or  28S  rDNA  sequences  (Field  ct  a/.. 
1988;  Christen  el  a/..  1991;  Wainright  et  at..  1993;  Ko- 
bayashi  ct  til..  1993;  Smother  et  ai.  1994).  The  present 
analysis  confirms  the  recent  claim  by  Smother  et  al.  ( \  994) 
based  upon  1 8S  rDNA  sequences  that  the  Myxozoa  are 
closely  related  to  the  triploblastic  animals. 

As  in  phylogenetic  trees  previously  constructed  on  the 
basis  of  rDNA  sequences  (Christen  et  at..  1991;  Wainright 
et  ai.  1993;  Smother  ct  al..  1994),  Trichoplax.  which  had 
once  tentatively  been  grouped  in  the  phylum  Mesozoa 
(see  Brusca  and  Brusca,  1990),  was  positioned  within  the 


84  T.  KATAYAMA  ET  AL 

5  0  100 

D.acuticephaJum    ATGCATGCGTAAGCTCATGCTCT-CTAATGAGCGAAACCGCAGACGGCTCATTAAATCGGACATAACTTACTTGATAATCTC-ACACGACGGTGGATAAC 

D.  orient  ale       nnnnnn.TA.  ...T - G - 

C.naikaiensis       T TAT.  .CT TG  .  .AA.  .T GA  T A.TT.CT.TC.  .  AAG  .  C  .  G  .GTGA-CATTCTACAA.  A  .  .  .G. 

D. japonic a        nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn. .G-.T T.A.A.C. . .G.T. . . AGA . . . .TAA. .T.TTT .C .ACAA 

p.muJtitentacuJata  nnnnnnnnn.C.  .TA.  .CATCA — TATTACGAT GA.T A.CT.  .GGT.CCT.G. .  .GC.G.  . .ATTACA 

A.sulcata  TC.  .  .  .TAT. A.  .A.--T.GTACT.T T.  .GA.T A.TT.  .CGT.  .  .T TG  .  ACG-TTTACTACT 

P. tetraureJia       TC  .  .  .  .TAT.AATAG .  .TAG.  .T T.  .GA.T A.A.TT.  .  .GT.  .  .T G  .  GCA .ATTA.A 

A.  thaliana          T TATG  AA  .  GAATTC  .  GACT  .  T T.  .GA.T A.TT.  .  .GT.  .GT GG.AA. TACTACTC 

S.cerevisiae        TC  .  .  .  .TAT.  A.  .AA.-T.  .TAG.  .T T.  .GA.T A.TT.  .CGT.  .  .T G.TC.TTT.  .T.  .ATG.T 

150  200 


D.acuticepnaJum    TGTTGTAAATCTAGAGCTAATACATGCGTACAGCTTCTCG AAGCGCAATTATTAGA 

D.  orient  a  le        AG.A 

C.naikaiensis  . .CGC. . .C-.GC.T.T. . . .G.T. . .AG.TT. . . .GGGA GACTGAGA.TT. . .CG. . .T ATCCACCGGGGAGCTCTGAAACACCGAG 

D. japonica  .  .  .G.  .  .  .T TT.TAAAGA.TGCCG.GACT — AACGAAGCG  .T.G  .T T-CAAAACTAAC-CTGTCGCAAGAACCA 

P.muJtitentacuJata    .  .  .G.G.  .T ACCA. AGCCA.GACCGCAAGGGAATG T T-CAAAACCAAC-CGGCTTC 

A.sulcata  C.  .G.  .  .  .T A.G.  .TCC  .  GACTTCT--GGAAGGGAT.T  .T TTCAAAACCAATGCGGGTTCT 

P.tetraure-Ha  C.  .G.  .  .  .T C  .A.TACCGGACGC AAGAATGGTA.  .  .T TTTAA--CCAT 

A.  thaliana  C.  .A.  .  .  .T G.  .  .AACA  .  A.CC  .GACTTAT--GGAAGGGA  .  .  .  .T TAAAAGGTCGACGCGGGCTCTG 

S.cerevisiae  C.  .G.  .  .  .T T.  .A.ATC.  .GACCCTTT-GGAAG  .GAT.T.T TAAAAAATCAATG-T--CTTCG 

250  300 

D.acuticepnalum     ATTAGTTGACTCTGGATAAACTTGCTGATCACA GnAGTGCCTGCGACGTATTAATT-AGATGTCCGCCCCATCAATTT 

D. orient a Je        T G G.CT.  ......T C....- G 

C.naikaiensis  ACCCAGGCGATGATG. A.C .CGT.G . .GT.TG GAAA . . . .GT .CAAAGGCCGT. AAGGCTG .TGACG . AGTC .GC . . .TG.T.AG.T CC. 

D.  japonica  C.  .T.A CT.  .A.GT.  .  .  GT  .  C — GACCATTA.  .T.GA.  .  .  .AA.  .  CTC  .  .G.AG..G.T.A..T 

P.muJtitentacuJata GGC--CTGTTTG  .T.A AGC  .GAT.GCATGG-CCAT.  A.  .  .G CTT.C-  .A T....T 

A.suJcata  GCC--CGGTGC. .T.G. . .T. .ATAG. . .CTGAT.GA. . .G. .T GGCCTT.C. .TG....T..T.C.T.C-.A..T..T....T C.. 

P.  tetraureJia       C.CAT.G.  .  .A.  .  .TAG.  .  .CT.A C.G.GT .  .  ATAC  .  T  .  GTAA .  .C.T.C-.AG.T..T....T....GC.. 

A.  t  hall  an  a         GC--T — TGC.CT.A.  .  .T.  .AT CTCGA.G.  .  .  .G.  .T--GGCCTCT.  ...TG C..C.T.C-.A..T..T....T C.. 

S.cerevisiae       GA-CTC  .  .T.A.  .  .T.  .ATA.  .  .  .CT.  .T.GA.  .  .G.  .T GGCCTT.  ...TG....T.GT.C.T.C-.A..T..T....T C.. 

350  400 

D.acuticepnaJim    TCGACGGTGAGGTAGTGGCTTACCGTGGTTA-CAACGGGTAACGG-GAATCAGGGTTCGATTCCGGAGAGGGAGCATGAGAAATGGCTGCCAGATCTATG 

D. orient a Je        .T - - 

C.naiJcaiensis  AT ACT.  .  .-A.CAC G-TG.  ...C G A C C....A.T.C...C.A. 

D.  japonica  .  .  .  .T.  .  .A.  .A.CAAA A G-T G T C C....A...C..  .ACG. 

P.muJtitentacuJata  A.  .TT.  .  .AG  .T.  .CCT.  .C....A....G.T G C....A A...C. 

A.sulcata  ...AT...A....GT A - G....A....T C C....A...C...C.A. 

P.  tetraureJia       .  .  .  .T.  .  .ACT.  .  .T.  .  .AC  .  .  .  .A.  .  .CAG-TC A....T C C....A A. 

A.  thaliana          ....T...AG.A C....A....GG-T G....A....T C C C...C... 

S.cerevisiae       ...  .T.  .  .AG.A C  .  .  .  .A T- G.  .  .  .A ...C....A...C...C.A. 

Primer  1,(2) 

450  500 

D.acuticepnaJum    GATTGCAGCAGGTGCGT-AAATTATCCACTTTTGGCAT AAAGAGATAGTGATCATAAATAACGGATCCGGGCAT TTGCCAGGAGATCGTAA 

D.orientaJe        A- T ...TTCA 

C.naikaiensis       ..A CA..C- C...A...-.  .A.C GGG.  .  .G CG.G TCAT.TAA.C  .T.  A A.  .GTTTATA.  .GAG.  . 

D.  japonica  .  .AG C  .  .  .AT C.  .  .AC.A-.  .  .CGTAGCCCT.  .  .G CA AATATG  .  .  .  .C. AG.G  .TTT  .ATA.  .  T.C.  . 

P.muJtitentacuJata  .T C...C- C C — ...  .C GGG.  .  .G CG.A AATA.G.  .T.T.TTTATAG.  .  .  CC  .  TA  .  .  T  .  G  .  . 

A.suJcata  ..AG C...C- C...A.CC..A.TC .GG...G CA.G..C...  .AATA.A.  .  .  .  T.  -TTGTAA  .T  .TT.TA.  .T.G.  . 

P.  tetraureJia       .  .AG C.  .  .  .- C.  .  .A.CCC.ATTC GGG.  .  .G CA.G G.AAC.  .GT.CGGG-TTTCC-.TT.C.G.  .  .T.C.  . 

A.  thaliana          .  .AG C.  .  .C- C.  .  .A.CC  .  .A.  .C GGG.  .  .G CA AATA TC-TTTCGA.T.T  .  .TA.  .T.G.  . 

S.cerevisiae        .  .AG C.  .  .C- C.  .  .A.CC.AATTC .GG.  .  .G CA ATA.  A.  .  .  .  CC-ATTCGG  .T  .TT.TA.  .T.G.  . 


Figure  1.  Nucleotide  base  sequences  of  18S  rDNA  from  two  dicyemids.  Dicyema  acuticephalum  and 
Dicvema  orientate,  and  three  flatworms,  Convoluta  naikaiensis,  Dugesia  japimica.  and  Pltinocera  multiten- 
taculata.  The  sequences  are  aligned  to  that  of  small-subunit  rDNA  of  a  diploblast.  Anemonia  Mileiitu;  a 
protist,  ParaimriiiiH  Iclnuiir/m:  a  plant.  Arabidopsis  thaliana.  and  a  fungus.  Sacchromyces  cm'visiae  A 
period  indicates  that  the  base  at  that  position  is  identical  to  that  in  D  acuticephalum,  a  h\phen  indicates  a 
gap.  and  an  "n"  indicates  an  undetermined  site.  The  1070  positions  used  for  phylogenetic  inference  are 
shown  by  single  lines  above  the  alignment.  The  positions  corresponding  to  the  internal  primers  are  shown 
by  double  lines  below  the  alignment.  The  pnmers  whose  numbers  are  in  parentheses  are  antisense  to  the 
sequence  shown  here. 


MOLECULAR  PHYLOGENY  OF  DICYEMIDS 


85 


5  5  0 


D 
D 
C 
D 
P 
A 
P. 
A 
S 

.acuticephalum          TGAAATAACT-GTAAAAGCTTTAATGAATG-CAATTGGAGGGCAAGTCTGGTGCCAG-CAGCCGCG-CAATTCCAGT-TCCAATAGTGTATACTAAAGTT 

.naikaiensis 
.  japonica 
.multitentaculata    . 
.sulcata 
.tetraurelia 
.thaliana 
.cerevisiae 

GGA.-CG. 
..G.ACTA.TT.  . 
..GT.C.  .C-T.  . 
..GT.C.AC-T.  . 
.  .G.  .  .  .G.-CC. 
.  .GT.C.A.-C.  . 
.  .GT.C.  A.-.  .  . 

CC 

,  .TA.  .  .  . 
.  .CC.A.  . 
.  .  TC  .  .  .  . 
.  .TC.G.A 
.  .  TC  .  C  .  . 
.  .TA.C.  . 

,TCA 
.  .C. 
.  .C. 
.  .C. 

.  .C. 
.  .C. 

.TGAC.G.G.A  
.G.AT  
.  G  .  AC  
.GGAT.C  
.T.AA  
.GGAT.C  
.GGAA  

-.  .C.  .  .  T.  .  .A.  . 
-  -T  
G  -T  
-  GT  
-  GT  
-  GT  
-  GT  

.  .CC.  .T.CA. 
.  .C-  
.  .C-  
.  .C-  
.  .C-  
.  .C-  
.  .C-  

.C.  . 
.C.  . 
.C.  . 
.C.  . 
.C.  . 
.C.  . 
.C.  . 

.  .CGATG.  .  .  . 
.  .  .T  
.  .  .T  
.  .  .T  
T  
.  .  .T.T  
.  .  .T  

Primer   3, (4 ) 


650 


700 


D.acuticepnalu/n 
D.orientale 
C .naikaiensis 
D . japonica 


GCTGCAGTTGAAAAGCTCGTAGTTGGATCTCGGTGTGCTAGTACGTAATCGCG-TGCTAGTAGGCCTTTG--CTATAGTTAG—  ACTATG  ---------- 

.................................  CT  ......  CG.  .C.  .A-.  .--  .  .AA  ............  —  CTGTAGT.G.  .T  ---------------- 

.....  GT.  .A  ....................  G  —  .T.  .G.  .T.TGA.  .CATC.  .  C  .  AT—  AC  .CATTGCGCGTA  .  .G.  .GCCTCG.  .GCCCATCCTT  —  CG 

.T.CG.  .  .  .A  ...............  A.AT.GA.GAAATG.T.TATAT.A--A.T.TA.GACT.ATTA.C.AGA.CCT  —  .CCTTCTCCGTCGTGATATTC 

P.multitentaculata  .T  .......  A  ....................  GA.GA.TAG.  .CCA-C.GGTTG.CC.  .AT.GCTA-.AC.TGTGGC.CAGCCTGC.TAG.CGGTGAT  —  TG 

A.  sulcata  .T  .......  A  .................  CT.  .  .  .G.  .  .G  —  CACG.CCGGTC.  .CC.  .A.  .  .GT.T.AC.  .G  ---  CCG  .GCC  .CTCT.C  .TCGCAAAGAC- 

.T  .......  A  ...............  A.  AT.  .  .  AGTCAG  .TACTA.  .TGG.T.TTC.TC.  .  .  TA  .  TTAA  .  TGAT  .C  .CCG  .CTAC  .  A.CCCTTTTGCGCT- 

.1  .......  A  ...............  A.C.  .T.  .GA.  .G  —  .  .CG.CCGGTC.  .CCTT.G.  .GT.-.A.  .  .G  ---  TCG.C.T.TCC.  .TC  .GTCGGCGATA 

.T  .......  A  ...............  A.CT.T.  .GCCCG  —  .  .TG.CCGGTC.  .A.TT.TTCGT.T-AC.  .GAT  .TCCAACG  .GGC  .  .T  .CCTTCTGGCTA 


P.  tetraurelia 
A.  thaliana 
S.  cerevisiae 


750 


800 


CATCGATGATATTAGTTGCACTTTGTTGTGACGACTAGTGGAAACGGTGTT. .C. .T. . 
GTTAAGTGCACTTTATTGGGATCTTTTACAA--TAACCGACAA G 


D.acuticephalum 
D. orient ale 
C. naikaiensis 
D. japonica 

P.multitentaculata  TTTTGGTGCTCTTAATTGAGTGCCTTAATTGCCCG GCCA CG. . 

A. sulcata 
P. tetraurelia 
A. thaliana 
S. cerevisiae 


GC--TTTACCTTGAACAAA-ATAGAGTGCTTAAGGC-AAGCATTCTGCTTG-A 


CGCGTGTGCTCTTGACTGAGTGTGCGCGGGAGTTGCGA CG.  . 

TTAGGGTTGCAGCTGGGCGAGTAG ACAA .  . 

CGCTCCTGGTCTTAATTGGCCGGGT-CGTGCCTCCGGCG CTG. 

ACCTTGAGTCCTTG — TGGCTCT-TG-GCGAACCAGGA CT.  . 


T 

GTT.  .C. 

.T.  . 

.  .GT. 

.  .-T.  .  .  . 

.  .  .T 

,C. 

,A.  .- 

.  .C 

.T.  .GG.A 

,C. 

- 

—  G.  .  .  . 

.T.  . 

.  .  .A. 

.  .-T.  .  .  . 

.A.  .G 

,G. 

.T.ATGCT 

.GCAT 

- 

-CG.  .  .  . 

.T.. 

.  .  .A. 

.  .AT.G.  . 

.C. 

.AT.- 

.G. 

.CCAA.  .  . 

C.  . 

.A. 

--CG.  .  .  . 

.T.  . 

.  .  .A. 

.  .-T.  .  .  . 

.  .  .T 

.C. 

.A.  .- 

.G. 

.-CAGC.  . 

,A. 

- 

.  .  .A. 

.  .-T.  .  .  . 

.  .  .T 

.CC 

.G. 

T-C.TC.  . 

CG 

.A. 

.- 

-CTG.  .  . 

.T.  . 

.  .  .G. 

.  .-T.  .  .  . 

.C. 

.A.  .- 

.  .  .  .C.A.GCTC. 

.G. 

_ 

-CT  

.T.  . 

.  .  .A. 

.  .-T.  .  .  . 

.  .  .T 

.C. 

.A.  .- 

.G. 

.G.AT.  .  . 

.C 

.A. 

850 


900 


D.  acuticephal  usn 

TATCTAAGCATGGAATAATAGAATAAGAC-TTTTC' 

_.c  -    A  -  

PA  T 

rGGTT-ACG-A-TAGTAAAAGTAATG-TTAACAGAGACAGCCGGGGGCATCCGT 
.  .T.  .r-  .G.  .                            .  .A.  .                          .  .A.  .               .  .T.  . 

G         —  —  —  — 

C.  naikaiensis 

.  .ATAA  

.  .  .G.  .GA.T. 

.  .  -C  .  GAG 

.  C  AACGTTTTGAACGCGGT  .  GCTGTTGCTCG  .  .  G 

.  .  .A.  . 

G.A. 

.GA. 

.  .  .G  

.  .  .  .  TT  .  . 

D.  japonica 

AT.  .GTT  

.  .  .GA  

.  .-.  .CGG 

.TTTATTTTG. 

TT.  .-.AAC--TG  

.  .  .A.  . 

.  .A. 



.  .  .  .AT.  . 

P.multitentaculata 

C.GA 

.CTCG.  .CC.  .  . 

.  .  .CA.  .A  —  . 

.  .-.  .CnG 

.  TCTATTTTG  . 

.-.  .  .TT.  .G.AC.--TG  

.  .  .A.  . 

.  .G. 

.G. 

.  .  .A  

.  .  .  .T.  .  . 

A.  sulcat  a 

.  .CA 



.  .  .G  G. 

.  .-.  .GGG 

.TCTATTTTG. 

TCT.G.ACC--TG  

.  .  .A.  . 

.  .G. 

.G. 

.  .  .TT.  .  .  . 

.  .  .  .T.  .  . 

P  .  tetraurelia 

.  .CA 

.T  

.  .  .G  G. 

.  .-.  .GGG 

.C  TTTTG. 

TTA.G.C.  TG.  .  .  . 

.  .  .A.  . 

.  .T. 

.G. 

,  .  .  .AT.  .  .  . 

.  .  .  .TA.  . 

A.  thaliana 

.  .CA 

.T  G.  . 

.  .C.TC.  .  .G. 

.T-.  .CGA 

.CCTATTGTG. 

.  .  .C.TCG.G.TC  GG.  .  .  . 

.  .  .A.  . 

.G. 

.  .  .  .T  

.  .  .  .T.  .  . 

S  _  rfrfv  i  <;  j  af> 

.  .A 

.T.  . 

.  .G. 

.  .G.  .  .GG 

.TCTATTTTG. 

.  .TCTAGGACC  .TC.  . 

.  .A.  . 

.  .T. 

.G. 

.  .G.T.  . 

.  .G.  . 

950 


1000 


D.acuticephalum 

D.orientale 

c.naifcaiensis 

D. japonica 

P.multitentaculata 

A. sulcata 

P. tetraurelia 

A. thaliana 

S. cerevisiae 


D. acuticephalum 

D.orientale 

C. naikaiensis 

D. japonica 

P.multitentaculata 

A. sulcata 

P.  tetraurelia 

A. thaliana 

S. cerevisiae 


ATTGCTCCGTTATAGGTGAAATTCGTAGATCGGTGCAGGACGTA-CTACAGCGAA-GCATTTGCCAAC-ATGTTTTCATTAATCAAGAACGACAGTTGGA 


A  .  . 
A. 

.... 

.u  
,G  

T. 

G. 

.  .  .  .TG.  . 

,A. 

.  .AA.- 

.  .  .  .T.  . 

.  .  .A  

.  GA  

A 

.  .GCTGGT 

,C. 

.G  

TG 

.  .  .C.ATCAGCA. 

.  .  .A.  A 

.  .  .  .T.  . 

.  .  .A  

.  GA  

CA 

GGT 

.GG 

.G  

T. 

G. 

.  ..ATC.  . 

,A. 

.GCC  — 

.  .  .A  

.  GA  

CA 

.  .  .-.GTT 

.  .C 

.G  

T. 

G. 

.  .TTAC.A 

.A. 

.  .  .A.- 

.  .  .  .T. 

.  .  .A  

.  GA  

A 

.  .  .TAATT 

.  .C 

.G  

T. 

G. 

.  .TTA.TA 

.A. 

.  .  TA  .  - 

.  .  TAT  . 

.  .  .A  

.  GG  

A 

.G 

.  .  .T.ATA 

.  .C 

.G  

T. 

G. 

.  .TTA.  .A 

.A. 

.  .  .A.- 

.A.  .T. 

.  .  .  .A  

.GG  

.G 

.  .  .CAATT 

.  .c-c  

T. 

G. 

.  .TTA.TG 

.A. 

..TA.- 

.  .  .  .T. 

..  .  .A  

.  .GG.C  

A 

.G 

Primer  5,  (  6) 

1100 


A.  .  .  .G.  .--TTTG  T.  .  .  . 

.  .  .  .  TA  .  . 

.AG. 

.  .AC.C. 

.A..  .T.  . 

.C.  .  . 

.  .  .TT.A. 

TA.GA. 

.TT.TCCC.GTCCC-.CTCG.  .  TGGGC  AA  .  AATTTAA 

.G  

.A.  . 

.  .C.  . 

.  .AC.  .  . 

.C. 

TG  . 

.C.  .  . 

.  .  .T.  .  .  . 

.  .T.  .GA.GAGTTAGC.  .AGGATA.  .TA.  .CATC.T.  .TA.AA.TCA  

.  GT  

.A.  . 

.  .C.  . 

.  .  AC  .  .  . 

.C. 

TG  . 

.C.  .  . 

G.  .  .A 

,  .  .  .T.  .CG.T.GCGA.TTCGATCC.A.  .  .  .CA.  .C. 

.  GC  .  .  .  . 

.  .A.  . 

.  .C.  . 

.  .AC.  .  . 

.C. 

.  .  .  .AT.  . 

.C.  .  . 

.G  GGA 

,  .A.AGAG.GT.  .  .-  GACC.CTTT.  .CAC.  .  . 

AG 

.G.  .  .A. 

.  .A.  . 

.  .C.  . 

.  .AC.  .  . 

.  .  .CTT.  . 

.  .  .T.  .A. 

.G  AG. 

,  .G.AA.GGT.ATA-  .ATTAGTCCCTTTC  .  .CAT.G. 

AA 

.A. 

.  GC  .  .  .  . 

.  .A.  . 

.  .C.  . 

.  .AC.  .  . 

.C. 

.  .  .CT.  .  . 

.C.  .  , 

.G.  .C.  .GGA 

,  .  A  .  .  G  .  .  .  GT  .  GC  .  .  AT  .  GGAC  .  CC  .  .  T  .  .  CAC  .  .  . 

AT 

,A. 

AT.  .  .C.  .  .  .AC CTT.  ..C T G...  .  —  GA.  .G.G  .  .G  .GT  .T.  .  .TA  .  .OACCC.CTC.  .  .AC.  .  .A.  .A. 


86 


T.  K.ATAYAMA  ET  AL 


D.acuticephalum 

D.orientale 

C.naikaiensis 

D. japonica 

p.multitentacula 

A.sulcata 

p . tetraurelia 

A . thaliana 

S . cerevisiae 


D.acuticephalum 

D.orientale 

C.naikaiensis 

D . japonica 

P  .multitentaculata 

A . sulcata 

P. tetraurelia 

A . thai iana 

S. cerevisiae 


D.acuticephalum 

D.orientale 

C.naikaiensis 

D. japonica 

P  .multitentaculata  -C 

A . sulcata 

P. tetraurelia 

A. thaliana 

S. cerevisiae 


1150 1100 

AAACCAAAGTCTTTGGGTTCCGGGGGAAGT-ATGGTTGCAAAGCTGAAACTTAAAGGAATTGAC-GGAAGGGCACCACCAGGAGTGGAGCTTGCG-CTCA 


TAT                            ATC      

C        T 

A                  G                    

C        T 

T 

A          A             C     G   T 

T  . 

T  G...-  C  ...G  -.. 

C     G   T 

.  .  .T.  . 

T  G...-  C....G  -.. 

C.  .  .  .G.  .-. 

Primer  7(8) 

1250 

1300 

ATTTGAi 

:TCAACGCAGAAAAACTCACCCGGGCCGAACACAGTGAGGATTGACAGACTGATAGCTTTT' 
A 

rCTTG  ATACTGTGGGTAGTGGTGC  ATGGCCGTT 

A.G.GG  A.  .T.  .AG.  .  .TCT.A  TA.TATA.-C.C 

.  .A.  A.  .CAGA.  .  .  .CG  

A.G.G...TT..C  T...G....T  T  C.. 

T.G  G  

AGG     C.  -G....T  T...A....C.. 

T.G  G  

A.G.GG  A.  .T.  .AG.  .  .T.  .GA  T.  .  .G.  .  .  .C.  . 

T.  .A  G  

A.G.GG  T...A..T.AA...  .TG.ATG  T...A....C.. 

T.  .A  G  

....   A.G.GG  T...A..T..AG...T...A  G....C.. 

T..A  G  

A.G.GG  A..T..AG  A.  A  T...G....C.. 

TT  G  

1350 

1400 

.  .  .  .  -  C  A  AG  .  C  

.  .T.  .TT.  .  .  .G.T.G.CG  AT-.  A.  . 

-C.  .A. 

..A.-  C  A  TCTT  .  G  .  CA  .  T  .  A  .  A  .  . 

.  GAAAG  .  AG  .  A  .  CGAGGTA  TTGCAA  .  -  AG  . 

-C.  .A. 

-  C  A.  ...A  TA....G..A.A.. 

.  .  GTATTT  .  .  .  T  .  .  AC  .  CGTC  AAATAAT-  .  AC  . 

C   A 

C               A                         TAG  C     A  A 

ACACTC   C  ATT  GTG   TGAGTGC 

-C.  .A. 

....-  A  T  CTTA  ..C.G..A.A.. 

.  .TACG.CCA.  .GC.A.  .G  GCAAC-TA.  . 

C   A 

A  CTTA.  C  G  .  A 

TTGCTTG  GAACAACAG-  GTA-TA 

-C.  .A. 

C  A  T  CTCAG.C.G.  -A.  ... 

.CTACGT.GAGGC.TCCC.  —  TCACGGC-.  .G. 

TC.CA. 

..-..               ..C..A...G..               .  .CTTA.  .C.  .  ..A.A.. 

.  .G.  .G.T.GCA.TTGCTG--  -GTTAT-.C.  . 

1450 


1500 


D. acuticephal  um 

D.orientale 

C .naikaiensis 

D . japonica 

P. multitentaculata 

A.sulcata 

P. tetraurelia 

A. thaliana 

S . cerevisiae 


D . acuticephal  um 

D.orientale 

C.naikaiensis 

D. japonica 

P. multitentaculata 

A.sulcata 

P. tetraurelia 

A.  thaliana 

S .cerevisiae 


D.acuticephalum 

D.orientale 

C.naikaiensis 

D. japonica 

P. multitentaculata 

A. sulcata 

P . tetraurelia 

A. thaliana 

S. cerevisiae 


TACTAAGAAGGATCAGTG TGAAAACACTTGAAAATGAGCAATAACAGGTCTGTGATTGCCCTTAGA-CGTTCGGGGC-GCACGCGTGCTAC 

.  .  .-T.  .  .G- .  TT.  .  .ACAC.T - - T.A C 

.T.  .T.  .  .G.C.CTGT.CCGAATTTAAAT.CGGA.AGC.  .  .GTGAG - -T....T..A.T C 

.T..T A.  .A.  A.  A GCG.CT.CGT.AA A - A.  .  .  .-T.  .C GC C 

.T.  .T.  .GG.  .  .CA.  .  .  . GCAT.C.  .GC.  .TAC - -T.  .C C C 

.T. .T. . .G. . .CTGT. . GTG.TT. .C. . AAGTC . GGAAG . .  ..-..       . . -T. . . .T . . . .C . .    ..C.. 


.T. 
T 

.T. 
.T. 

.  .G. 

,  .G. 

.  .CT 
.  .CT 

.TG. 
.TC, 

,  CCGTT.AGGCCA. 
,  GTTTCAAGCCGA-] 

GGAAGTTTGAG  

T 

T 

c 

c 

'GGAAGTTTGAG  

-  A  .  .  . 

.  .T  .  . 

.  .C  .  . 

...     c  

1550 

1600 

AATGAAGAAAGCAGAGGG GGTTTTGCTTGGAAAAGCGAACTAAGCCT-TAAAATTTCTTCGTGGCAGGAATCGAGGCTTGTAATTATTCCTCG 


.C. 

.T.CTCTA. 

T 

-TGAAAAACC  . 

AAT  .  .  A  . 

ftfi 

GGATTGGGA. 

T 

.  ACAG  .  .  CGGAGAAATT  .  T  .  AG  . 

n 

.  .  .T.CA. 

C.T.  .G.A.  . 

.  GCAGTTC. 

AC.  A 

,  —  TAATACC.  . 

AAC.  .A. 

A.  . 

T.  .TTGGGC. 

.T 

.T.G.G.  .  .CACTG.  .  .  .AA.  .  . 

.G. 

.T.T.AA. 

T.A.  A 

.c. 

.  .  .T.GC.T 

AC.  A 

,  --TTATTCTCC 

.GATCC. 

A.  . 

GG.TCGGGC. 

.C 

.TGT.G.  .  .CC.A  A.  .  . 

.G. 

.  .  .G.  .  .  . 

C.  . 

.c. 

.  .C.  .TGT. 

AC.  A 

.  TCTCTCC 

.  .  .GCC. 

A.  . 

GGT.TGGG.  . 

.T 

.T.C.C.  .  .CA.  .G  CTG. 

.G. 

.A.  .TCA. 

.  .C.  .  .  .T.  .GA.  .T 

.c. 

.  .CACGTT. 

.C.A 

.CTTATTTACC. 

G  .  C  .  CGAA  .  GGGCACGGGA  . 

.T 

.T.G.T.GGACGTG  CTG. 

.G. 

.A.  .TCT. 

.  .C  AGA.  .T 

.c. 

.  .  T  .  T  .  TT  .  . 

.AC.  A 

.  TTCACACC  .TGCC  . 

AC. 

GGC.CGGG.  . 

.T 

.T.-.G  A  ATG. 

.G. 

.A.  .TCA. 

.  .C.  .  .  .G.  .GG.  .T 

.c. 

.  .C  .G.GC. 

.C.A 

,  TCTAACC 

.  .  .GCC. 

AG. 

GGT.TTGG.  . 

.T 

.T.G.G.  .  .C.C.G  CTG. 

.G. 

.  A  .  .  .  CA  . 

G.  .  .T 

1650 

1684 

TGCACAAGGAATTCCTAGTAATCGC-AGGTCATTAGCCTGCAATGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTAC 

.  .A 

.  .CC  

,  .  .  .C 

.  .  .  .AT.G-.A. 

...  AC  .  A 

.T. 

CT.T  

-  .  .  . 

A 

G 

G    A  A 

.  .A 

.  .G  

.  .  .  .G.  .  .-.A. 

.  .  .  .A.  A 

.T. 

.  .GT  

.  .A 

.  .G  

.  .  .  .G    -GA 

.C  .  . 

Tr 

GT 

.  .A 

.  .G  

.  .  .  .T 

.  .  .  .G.A.-.  .  . 

.  .  .  .C.  . 

.TGC.  .  .A.  . 

C. 

.CA 

.CA 

.  .G  
.  .G  

.  .  .  .G.  .  .-GA. 
.  .  .  .G.  .  .-.A. 

.  .  .  .c.  . 

.  .  .  .c.  . 

.TC 
.T. 

.  .  GT  .  .  .  C  .  . 
.  .GT  

C. 
G 

Primer ( 9 ) 


MOLECULAR  PHYLOGENV  OF  DICVEMIDS 


87 


Xennpus  laevis 
Anemia  salina 


Sagitta  crassa 


•  P/anncera  multitentaculata 

Moliniformis  moliniformis 

•  Caenorhabditis  elegans 

-Convoluta  naikaiensis 

Henneguva  sp. 
Dicyema  acuticephalum 
Dicvema  orientate 


Asteruii  amurensu 


Crassostrea  glgas 

Shistosoma  mansoni 

Dugesiajaponica 


Anemonia  sulcata 

Trichoplax  adhaerens 
Beroe  cucumis 


Diploblasts 
Plants 


Scypha  ciliata 
Arabidopsis  lhaliana 
Volvox  carteri 

Hartmanella  vermijormis 
Oxytricha  nova 

Paramecium  tetraurelia 


Sacrcocvstis  mitris 


Theuena  annulata 

Crypthecodinitan  cohnn 


Triploblasts 


Saccharomyces  cerevisiae 
Firobasidiella  neoformans 


Protists 


Fungi 


0.05 


Figure  2.  Neighbor-joining  trees  showing  the  phylogenetic  position  of  the  dicyemids  among  28  repre- 
sentative eukaryotic  taxa.  The  tree  was  reconstructed  on  the  basis  of  the  pairwise  distances  of  Jukes  and 
Cantor  (1969)  using  DNADIST  and  NEIGHBOR  programs  (PHYLIP  package,  version  3.5c).  The  tree  was 
rooted  by  using  Saccharomyces  cerevisiae  as  an  outgroup.  Branch  lengths  are  proportional  to  the  scale  given 
in  substitutions  per  sequence  position.  The  percentage  of  500  neighbor-joining  bootstrap  replicates  is  shown 
at  the  node  the  value  is  supporting. 


diploblastic  assemblage.  This  study  does  not  support  a 
close  relationship  between  Trichoplax  and  mesozoans. 

These  sequence  data  do  not  firmly  establish  the  position 
of  the  dicyemids  within  the  triploblastic  assemblage.  In 
the  phylogenetic  trees  obtained  by  three  different  methods, 
the  dicyemids  formed  a  monophyletic  unit  with  the 
myxozoans,  nematodes,  and  acoel  flatworms.  They  are 
all  considered  to  be  early  divergent  groups  in  one  widely 
accepted  phylogeny.  The  early  divergence  of  acoel  flat- 
worms  in  triploblastic  evolution  has  been  suggested  by 
Katayama  et  al.  (1993)  from  comparisons  of  partial  18S 
rDNA  sequences.  However,  the  myxozoans,  dicyemids, 
nematodes.  and  acoel  flatworms  were  all  represented  by 
a  long  branch  in  the  phylogenetic  trees  (a  high  nucleotide 
substitution  rate).  Hence  we  cannot  ignore  the  possibility 
that  these  long  branches  produce  artifactual  groupings 
within  the  triploblastic  assemblage  (Van  de  Peer  et  at.. 
1993). 

With  regard  to  the  topology  of  triploblast  phyla,  the 
present  phylogenetic  trees  contradict  those  of  some  pre- 
vious analyses  of  18S  rDNA  sequences  in  some  points. 


The  Platyhelminthes  did  not  form  a  monophyletic  unit 
as  previously  shown  by  Katayama  et  al.  (1993).  Mono- 
phyly  of  the  deuterostomes  and  the  protostomes  has  re- 
peatedly been  shown  in  the  molecular  phylogeny  of  1 8S 
rDNA  (Wada  and  Satoh,  1994;  Raff  et  at..  1994;Halanych 
et  u/..  1995),  but  neither  group  was  monophyletic  in  all 
trees  of  the  present  analysis.  Triploblastic  phyla  are  poorly 
resolved  in  the  molecular  phylogenetic  trees  of  18S  rDNA; 
i.e.,  nodes  defining  phyla  are  not  supported  by  high 
bootstrap  values.  Therefore,  the  topology  of  the  trees  de- 
pends largely  on  the  choice  of  taxa.  Philippe  et  al.  (1994) 
have  showed  that  by  eliminating  rapidly  evolving  species 
from  the  analysis,  discrepancies  between  molecular  and 
traditional  phylogeny  partly  disappear  and  bootstrap  val- 
ues rise  at  some  nodes.  Since  we  only  intended  to  show 
the  placement  of  the  dicyemids  firmly  within  the  triplo- 
blasts,  we  chose  taxa  representing  a  broad  spectrum  of 
eukaryote  phyla  without  regard  to  consistency  with  the 
traditional  view  of  triploblast  phylogeny. 

There  have  been  opposing  views  on  the  role  of  the  di- 
cvemid  mesozoa  in  the  storv  of  metazoan  evolution.  Some 


88 


T.  KATAYAMA  ET  AL 


27 


10 


24 


1  Xenopus  laevis 


-Sagina  crassa 


13 


—  Anemia  sa/ina 
Asteriax  amurensis 


40 


54 


100 

1                Crassostrea  giga 

,,I-^ 

17          _^J        '  
L.                   PI 

->il                               ., 

791 

,„               ~1                 100  i- 

loor 

1 

1           Anemonia  sulcata 

Shistosoma  mansoni 
^^— ^^—  Dugesiajaponica 
Planocera  multitentaculata 

'oliniformis  mo/ini/ormis 

^— ^—  Caenorhabditis  elegans 


—  Dicyema  acuticephalum 
•  Dic\etna  oriental? 


Convoluta  naikaiensis 

Henneguva  sp. 


Triploblasts 


'  Beroe  cucumis 
451  Scvpha  ciliata 

100  i  Arabidopsis  thaliana 

Volvox  carter! 

Hartmanella  vermiformis 

•  Ox\triclia  nova 

631         ;!  I  Paramecium  letraurelia 

'Sacrcocvstis  muris 
Theileria  annulata 
—  Crypthecodinium  colmii 


•  Saccliaromvces  cerevisiae 
'  FirobasiJiella  neoformans 


Diploblasts 
Plants 

Protists 


Fungi 


40 


Figure  3.  The  consensus  tree  obtained  using  the  maximum-parsimony  algorithm  with  bootstrap  resam- 
pling (DNAPARS,  SEQBOOT,  and  CONSENSE  programs  of  PHYLIP  package,  version  3.5c).  showing  the 
phylogenetic  position  of  the  dicyemids  among  28  representative  eukaryotic  taxa.  The  percentage  of  500 
parsimony  replicates  is  shown  at  the  node  the  value  is  supporting.  The  tree  was  rooted  by  using  Saccharomyces 
cerevisiue  as  an  outgroup.  Branch  lengths  are  proportional  to  the  scale  given  in  number  of  substitutions  (a 
total  of  2807).  This  tree  is  different  from  the  three  most  parsimonious  trees  in  the  positions  of  Crassostrea 
gigax  and  Molinitormis  mulinil'ormis  within  the  assemblage  of  the  coelomate  triplohlasts. 


authors  have  proposed  that  the  dicyemids  are  a  missing 
link  between  unicellular  organisms  and  multicellular  an- 
imals (Dodson,  1956;  Hyman,  1959;  Lapan  and  Morow- 
itz,  1974;  Ohama  ct  a/..  1984).  while  others  have  claimed 
that  they  are  an  animal  group  degenerated  as  a  result  of 
parasitism  (Nouvel,  1948;  McConnaughey.  1951;  Stun- 
kard,  1954;  Ginetsinskaya,  1988).  The  phylogenetic  trees 
inferred  from  comparisons  of  nucleotide  sequences  of  5S 
rRNA  suggested  that  the  dicyemids  emerged  first  among 
the  metazoa  examined  and  that  triclad  flatworms,  nem- 
atodes,  cnidarians,  and  sponges  followed,  in  that  order 
(Ohama  el  al.  1984,  Hori  and  Osawa,  1987).  This  sug- 
gestion does  not,  however,  accord  with  the  present  result 
and  the  previous  inferences  about  metazoan  phylogeny 
based  upon  18S  and  28S  rDNA  sequences  (Field  el  al, 
1988;  Christen  el  al.  1991;  Wainright  el  al.,  1993;  Ko- 
bayashi  el  al..  1993).  Discrepancies  are  partly  ascribable 
to  differences  in  the  methods  used  to  infer  phylogenetic 
relationships.  In  contrast  to  the  18S  and  28S  rDNA  trees 
reconstructed  by  the  neighbor-joining,  maximum-parsi- 
mony, and  maximum-likelihood  methods,  the  above  5S 


rRNA  trees  have  been  reconstructed  by  unweighted  and 
weighted  pair  group  methods  using  arithmetic  averages 
(UPGMA  and  WPGMA,  respectively),  which  are  valid 
under  the  assumption  that  rates  of  nucleotide  substitution 
are  constant  among  taxa  analyzed  (Sokal  and  Mitchener, 
1958).  However,  the  essential  point  is  that  the  5S  rRNA 
is  too  small  to  contain  signal  sufficient  to  allow  precise 
inference  of  phylogenetic  relationships.  Because  of  large 
standard  errors,  sequential  orders  of  branching  of  the  di- 
cyemids. flatworms,  nematodes,  cnidarians,  and  sponges 
shown  in  the  above  5S  rRNA  trees  appear  to  be  statistically 
insignificant.  Recently  Halanych  (1991)  analyzed  the  se- 
quence data  of  5S  rRNA  with  the  maximum-parsimony 
method.  The  phylogenetic  tree  obtained  was  inconsistent 
with  phylogenies  based  on  18S  and  28S  rDNA  data,  and 
few  nodes  in  the  tree  were  supported  by  bootstrap  value 
at  a  significant  level. 

The  present  results  do  not  appear  to  support  the  prop- 
osition that  the  dicyemids  are  a  truly  primitive  group 
linking  unicellular  organisms  with  multicellular  metazoa. 
Instead,  our  results  favor  the  view  that  the  dicvemids  are 


MOLECULAR  PHYLOGENY  OF  DICYEMIDS 


89 


•  \eni>pus  laevis 

—  Artemia  salina 

—  Asterta*  amurenxix 


j~  Crasso 

ITI= 

i PI 


•  Sagitta  crassa 


Crassostrea  gigux 

Shistosoma  mansoni 

Dugesia  japonica 


•  Planncera  mullitentaculata 
'Molini/nrmix  molimformis 
—^—^^—  Caenorhabditis  elegans 

l^""""/"'"  naika 
^_____^_^_^—  Henneguya  sp. 


•          Dicvema  acuticephalum 
"I—    Dic\ema  oriental? 


Triploblasts 


.\nemonia  xulcata 

Trichoplax  adhaerens 
Beroe  cucumis 
Scvpha  ciliata 
Arabidapsis  thaliana 

Vnlvox  carteri 

Hartmanella  vermiformis 
Oxytriclia  nova 

Paramecium  tetraurelia 
^—^—  Crypthecodinium  cohmi 


Diploblasts 
I     Plants 


Protists 


Sacrcocystis  murix 

'  Theilena  annulala 
•  Saccharomyces  cerevisiae 
Firobasidiella  neoformans 


Fungi 


0.05 


Figure  -4.  Maximum-likelihood  tree  showing  the  phylogenetic  position  of  the  dicyemids  among  28  rep- 
resentative eukaryotic  groups.  The  tree  was  obtained  using  the  fastDNAML  algorithm  with  a  transition/ 
transversion  ratio  of  1.48.  which  gave  the  best  maximum-likelihood  score  (In  likelihood  =  -  1 1909.45548). 
Branch  lengths  are  proportional  to  the  scale  given  in  substitutions  per  sequence  position.  Because  of  the 
long  computation  time  (more  than  12  h  per  replication),  bootstrapping  was  not  performed. 


degeneratively  simple  animals  descended  from  a  more 
complex  triploblastic  ancestor.  Recent  close  observations 
of  dicyemid  development  (Furuya  t'/fl/.,  1992b.  1994)  do 
not  contradict  the  present  inference;  spiral  cleavage,  a  de- 
terminative mode  of  cell  division,  and  the  formation  of 
stereoblastula-like  structure  through  epiboly.  as  seen  in 
the  development  of  vermiform  and  infusoriform  embryos, 
are  reminiscent  of  flatworms.  Myxozoans  are  also  re- 
garded as  an  extreme  example  of  the  degeneration  char- 
acteristic of  parasitic  evolution  (Smother  el  a/..  1994).  At 
present  we  can  say  little  about  the  ancestor  from  which 
the  dicyemids  were  derived.  Molecular  analyses  that  in- 
clude other  lower  turbellarian  groups  (for  example,  the 
Catenulida  and  the  Nemertodermatida)  and  the  Ortho- 
nectida.  a  group  tentatively  included  in  the  Mesozoa,  will 
provide  further  information  for  understanding  the  phy- 
logenetic position  of  the  dicyemids. 

Acknowledgments 

We  thank  Dr.  T.  Miyata  and  Mr.  N.  Nikoh  of  Kyoto 
University  for  their  kind  help  in  reconstructing  the  phy- 
logenetic trees.  We  also  thank  Dr.  K.  Tsuneki  and  Dr.  Y. 


Koshida  of  Osaka  University  for  their  valuable  sugges- 
tions. T.  Katayama  and  H.  Wada  are  supported  by  a  JSPS 
(Japan  Society  for  the  Promotion  of  Science)  Predoctoral 
Fellowship  for  Japanese  Junior  Scientists  with  Research 
Grant  0360  and  2447.  respectively.  H.  Furuya  is  partly 
supported  by  a  grant  from  Fujiwara  Natural  History 
Foundation. 


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Reference:  fl/r >/  Bull  189:  9 1  -105.  (October/November.  1995) 


The  Incidence  and  Morphology  of  Subcuticular 
Bacteria  in  the  Echinoderm  Fauna  of  New  Zealand 

MAEVE  S.  KELLY1,  M.  F.  BARKER2,  J.  DOUGLAS  McKENZIE1  •*.   AND  JAN  POWELL3* 

{ The  Scottish  Association  for  Marine  Science,  PO  Box  3,  Ohan,  Argyll.  Scotland. 

Departments  of  Marine  Science  and  Zoology  and  ^Department  of 

Microbiology.  University  ofOtago,  Dunedin,  New  Zealand 


Abstract.  New  Zealand  echinoderms  (33  species  drawn 
from  all  five  extant  classes)  were  examined  for  the  presence 
of  symbiotic  bacteria  by  fluorescence  and  electron  mi- 
croscopy. Gram-negative,  subcuticular  bacteria  (SCB) 
were  found  in  1 7  species  from  four  classes.  The  SCB  could 
be  classified  into  two  major  morphological  types.  Some 
species  had  both  types  of  SCB.  The  distribution  of  SCB 
was  not  obviously  linked  to  host  ecology  but  did  appear 
to  be  related  to  host  phylogeny.  Related  species  usually 
all  have  SCB  or  all  lack  them.  The  number  of  SCB  in  five 
species  was  estimated  to  be  between  8.41  X  108  and  4.96 
X  109g~'  ash-free  dry  weight  of  host  tissue.  Significant 
differences  in  bacterial  load  and  relative  proportions  of 
the  different  types  of  bacteria  were  found  among  three 
congeneric  ech'mo\ds(Pseudec/iinns  hutloni.  P.  albocinctus 
and  P.  novaezealandia).  Ophiocoma  bollonsi  was  peculiar 
in  having  groups  of  bacteria  enclosed  in  host  cells  (bac- 
teriocytes)  within  the  connective  tissue  of  the  tube  feet. 

Introduction 

Symbiotic  associations  are  increasingly  seen  as  path- 
ways for  evolutionary  innovation,  allowing  organisms  to 
transcend  the  biochemical  limitations  of  their  own  ge- 
nome by  harnessing  the  different  biochemical  capabilities 
ofasymbiont(Margulis,  1981;  Douglas,  1994).  Examples 
of  marine  organisms  utilizing  symbionts  to  exploit  oth- 
erwise closed  energy  sources  include  corals  that  use  di- 
noflagellates  to  provide  them  with  carbon  fixed  via  pho- 
tos) nthesis(Muscatine  ct  ai.  1984)  and  a  variety  of  marine 
invertebrates  that  exploit  the  energy  potential  of  free  sul- 


Received  15  August  1994:  accepted  25  July  1995. 
*  Author  to  whom  correspondence  should  be  addressed. 
'  Current  address:   Division  of  Infectious  Diseases,   University  of 
Maryland  School  of  Medicine.  Baltimore.  Maryland  21201. 


fide  through  the  activities  of  sulfide-oxidizing  bacteria 
(e.g.,  Cavanaugh  et  a!..  1981).  Symbioses  between  inver- 
tebrates and  bacteria  occur  in  protozoans  (Fenchel  et  ai, 
1977;Saffo,  1990);  sponges  (Vacelet  and  Donadey,  1977); 
cnidarians  (Palincsar  et  al.,  1989);  nematodes  and  tur- 
bellarians  (Ott  et  al..  1982);  annelids  (Giere,  1981;  Haus- 
mann,  1982);  pogonophorans  (Cavanaugh  et  al.,  1981); 
echiurans  (Bosch,  1976);  bivalve  molluscs  (Southward, 
1986);  cephalopods  (McFall-Ngai,  1994);  bryozoans  (Lu- 
taud,  1969);  echinoderms  (Holland  and  Nealson,  1978); 
tunicates  (Mackie  and  Bone,  1978);  and  pterobranchs 
(Welsch,  1984).  New  symbioses  are  frequently  reported 
(e.g.,  Menon  and  Arp.  1993;  Haszprunar  et  al..  in  press). 
In  some  cases  the  biological  role  of  the  symbionts  is  ob- 
vious (e.g..  chemoautotrophic  associations  where  the  host 
lacks  a  gut),  but  in  most  it  is  enigmatic. 

Echinoderms  from  all  five  extant  classes  are  known  to 
harbor  symbiotic  bacteria  between  the  epidermal  cells  and 
the  overlying  layers  of  the  cuticle  (Holland  and  Nealson. 
1978;  Feral.  1980;  McKenzie,  1987).  These  so-called  sub- 
cuticular bacteria,  or  SCB  (Holland  and  Nealson,  1978), 
have  been  recorded  from  Atlantic  species  of  echinoderms 
(Holland  and  Nealson,  1978;  Feral,  1980;  McKenzie, 
1987;  Walker  and  Lesser,  1989;  McKenzie  and  Kelly, 
1994).  from  Australian  crinoids  (McKenzie,  1992),  and 
from  northeast  Pacific  ophiuroids  (McKenzie  and  Kelly, 
1994).  The  symbiosis  is,  therefore,  geographically  and 
phylogenetically  widespread.  Information  on  the  exact 
distribution  of  SCB  amongst  echinoderms  is,  however, 
very  vague.  Some  species  are  known  to  lack  SCB 
(McKenzie  and  Kelly,  1994),  and  it  may  be  possible  to 
correlate  the  presence  or  absence  of  SCB  with  some  com- 
mon aspect  of  their  hosts'  biology  if  there  is  a  large  enough 
data  set  upon  which  to  make  such  a  comparison.  The 
morphology  of  bacteria  can  also  be  useful  in  determining 
the  probable  trophic  role  of  the  symbionts.  Likewise,  a 


91 


92 


M.  S.  KELLY  ET  AL. 


high  ratio  of  symbiont-to-host  biomass  may  help  identify 
particularly  important  associations.  To  this  end  we  have 
been  surveying  a  large  number  of  echinoderm  species  from 
around  the  world  for  the  presence,  abundance,  and  mor- 
phology of  SCB.  This  paper  details  the  results  of  a  study 
of  33  species  of  echinoderms  that  are  found  around  New 
Zealand.  Some  preliminary  results  of  this  study  were  re- 
ported in  Kelly  el  a/,  (in  press). 

Materials  and  Methods 

Echinoderms  were  collected  between  October  1992  and 
January  1993,  by  beam  trawl  on  the  mid-shelf  region  off 
Otago  harbor  at  a  depth  of  64  m  (45°46.632'  170° 52.881') 
and  at  a  deeper  water  site  of  120m  (45°44.539' 
1 7 1  °0 1 . 197'),  and  by  shore  collections  from  the  intertidal 
zone  adjacent  to  Portobello  Marine  Laboratory.  Some 
species  were  also  collected  from  the  intertidal  zone  at 
Matheson  Rocks,  north  of  Auckland,  North  Island.  An- 
imals were  examined  with  epi fluorescence  light  micros- 
copy and  tissues  promptly  fixed  for  transmission  electron 
microscopy  (TEM). 

The  epifluorescent  microscopy  was  according  to  Hobbie 
ct  al.  (1977)  as  adapted  by  Kelly  and  McKenzie  (1992). 
A  tube  foot  was  removed,  placed  on  a  glass  slide,  stained 
with  acridine  orange  at  a  concentration  of  0.003%  and 
gently  squashed  with  the  coverslip.  This  action  frees  many 
of  the  SCB  from  the  tissue.  Usually,  3-10  individuals  of 
each  species  were  examined  for  the  presence  of  SCB.  The 
bacterial  load  was  quantified  in  three  species  of  Pseud- 
echinus:  in  Asterodon  miliaris,  Ophiocoma  hollon.si,  and 
Amphipholis  squamata:  and  in  juvenile  Pseudechinus 
huttoni  (diameters  of  14-18  mm).  Rods  and  spirals  were 
counted  separately  for  the  three  species  of  Pseudechinus 
and  for  the  juvenile  P.  huttoni 

For  direct  counts  of  bacterial  numbers,  larger  pieces  of 
tissue  were  removed  from  the  seven  species  mentioned 
above  and  homogenized  with  either  a  hand-held  glass  tis- 
sue grinder  or  an  Ultraturrex  mechanical  tissue  homog- 
enizer.  For  ophiuroids,  a  piece  of  arm  was  used;  for  echi- 
noids,  a  portion  of  test  was  taken  from  the  ambulacral 
groove  area.  Care  was  taken  not  to  rupture  the  gut  as  the 
test  was  removed.  The  tissue  was  homogenized  at  a  ratio 
of  1  g  tissue:  2  ml  of  filtered  (0.1  /urn)  seawater  and  then 
mixed  with  an  equal  volume  of  acridine  orange.  An  exact 
volume  of  the  homogenate  (5  ^1)  was  slide-mounted  using 
No.  1  22  X  22  mm  coverslips.  In  each  homogenate,  all 
the  bacteria  observed  within  an  eyepiece-mounted  Whip- 
pie  grid  (at  1000X  magnification)  were  counted  from  20 
randomly  selected  areas.  The  whole  process  was  repeated 
twice  for  each  of  10  animals.  Because  individual  Amphi- 
pfioli.s  squamata  are  small,  homogenates  were  made  from 
pooled  individuals  (one  of  8  and  one  of  1 1  individuals). 
To  check  the  accuracy  of  the  counting  technique,  10 
counts  were  made  from  one  homogenate  of  P.  huttoni 


test.  External  or  contaminant  bacteria,  which  were  only 
rarely  seen,  differed  obviously  in  size  and  appearance  from 
the  SCB.  The  number  of  SCB  was  expressed  per  gram  of 
tissue  wet  weight  and  per  gram  of  ash-free  dry  weight,  to 
allow  a  comparison  between  species  with  different  ratios 
of  soft  tissue  to  skeletal  calcite.  For  wet  weights,  tissue 
samples  were  rinsed  in  filtered  (0.1  pm)  seawater,  shaken 
to  remove  excess  surface  water,  and  then  weighed.  For 
dry  weights,  tissues  were  dried  at  40°C  until  they  reached 
a  constant  weight.  Ash  weights  were  obtained  after  tissues 
were  incinerated  in  a  muffle  furnace  at  400°C.  Ash-free 
dry  weights  were  obtained  by  subtracting  the  ash  weight 
from  the  dry  weight  for  each  sample.  Microscopical  ob- 
servations and  counts  of  the  bacteria  were  made  with  an 
Olympus  Vanox  epi  fluorescence  microscope. 

In  preparation  for  TEM,  tissues  from  three  individuals 
of  each  species  (where  available)  were  fixed  in  4%  glutar- 
aldehyde  in  0. 1  A/cacodylate  buffer,  rinsed  in  fresh  buffer 
and  decalcified,  if  necessary,  in  saturated  EDTA.  Post- 
fixation  was  with  1%  osmium  tetroxide  in  filtered  sea- 
water,  followed  by  dehydration  in  ethanol  and  embedding 
in  Agar  100  resin.  Silver  sections  were  cut  on  an  LKB  III 
microtome,  stained  with  ethanoic  uranyl  acetate  and 
aqueous  Reynold's  lead  citrate,  and  examined  on  a  JEOL 
100S  at  60  Kv. 

Attempts  were  made  to  culture  symbionts  from  the 
ophiuroids  A.  squamata  and  Ophiocoma  bollonsi  and  the 
echinoid  Pseudechinus  huttoni.  The  experimental  pro- 
cedures for  A.  squamata  were  (a)  that  of  Walker  and  Lesser 
(1989) — surface  sterilization  of  whole  animals  in  70% 
isopropyl  alcohol  for  30  s  followed  by  two  rinses  in  75% 
sterile  artificial  seawater  (ASW)  prior  to  homogenization 
in  sterile  glassware  and  plating  on  agar;  (b)  a  modified 
procedure  (Lesser,  pers.  comm)  as  above  but  with  surface 
sterilization  time  reduced  to  5  s;  and  (c)  an  alternative 
method  using  protocol  b  but  separating  the  arms  from 
the  disk  of  the  animals  before  homogenization.  For  O. 
bollonsi,  only  tissue  from  the  arm  was  used;  a  portion  of 
the  arm  was  surface  sterilized  and  then  the  tube  feet  were 
removed  and  homogenized.  The  homogenate  was  then 
spread  on  plates  or  used  to  inoculate  liquid  culture  me- 
dium. For  P.  huttoni,  small  squares  of  test  from  the  am- 
bulacral groove  were  surface  sterilized  and  treated  as 
above.  Animals  that  had  not  been  subjected  to  the  surface 
sterilization  procedures  were  used  as  controls.  The  brit- 
tlestar  Ophionereis  fasciata,  which  does  not  have  SCB, 
was  used  as  a  further  control.  The  tube  feet  or  portions 
of  tissue  were  placed  in  a  watch  glass  and  ground  with  a 
glass  grinder.  The  glassware  was  surface  sterilized  with 
70%  ethanol.  Marine  broth  (Difco,  300  ^1)  was  used  as  a 
diluent. 

Ultraviolet  (UV)  exposure  and  repeated  washing  in  au- 
toclaved  filtered  seawater  were  investigated  as  alternative 
methods  for  surface  sterilization.  Tube  feet  from  O.  bol- 
lonsi and  arms  from  A.  squamata  were  exposed  to  UV 


SYMBIOTIC  BACTERIA  IN  ECHINODERMS 


93 


for  2.  5.  10.  and  15  min  prior  to  homogenization  in  300  ^1 
diluent.  Whole  .1.  siiuanuita  and  squares  of  the  test  of  P. 
hutloni  were  washed  up  to  five  times  in  autoclaved  filtered 
seawater.  Homogenates  were  made  of  the  A.  sciuamaia 
arms  and  of  the  squares  of  urchin  test.  The  homogenates 
were  prepared  in  300  1  diluent,  then  plated  on  agar  and 
inoculated  in  broth  as  outlined  below.  Equivalent  un- 
treated tissues  were  used  for  controls. 

The  medium  used  was  Zobell's  modified  2216E  pre- 
pared with  75%  (w/v)  ASW.  Salt  solutions  ( 1  Af)  for  the 
ASW  were  prepared  and  autoclaved.  The  agar  was  then 
prepared,  the  yeast  and  peptone  were  added,  and  the  agar 
was  autoclaved  and  held  at  56°C.  To  prevent  precipitation 
of  the  sterile  salt  solutions,  they  were  warmed  and  added 
to  the  agar  before  the  plates  were  poured.  To  determine 
the  number  of  colony-forming  units  (CPU)  per  animal, 
logarithmic  dilutions  were  prepared  in  marine  broth  and 
spread  plated.  The  dilution  broths  were  also  cultured  as 
enrichment  for  slow-growing  or  stressed  organisms  unable 
to  grow  initially  on  solid  medium.  The  plates  were  in- 
cubated at  room  temperature  (18-21°C)  and  examined 
daily  for  bacterial  growth.  After  48  h,  the  resulting  bac- 
terial colonies  were  counted  and  CPU  per  sample  were 
determined.  The  plates  were  then  maintained  for  up  to 
21  days  to  observe  the  appearance  of  any  slower  growing 
colonies. 


Table  1 

Number  of  species  examined  and  number  and  percentage  with  SCB 


Results 


General  observations 


Table  I  shows  the  number  of  species  within  each  class 
that  were  examined  and  the  number  and  percentage  that 
were  found  to  harbor  SCB.  Of  the  33  species  of  New  Zea- 
land echinoderms  examined.  17  had  SCB  (Table  II).  When 
the  SCB  load  was  substantial,  the  bacteria  were  easy  to 
find  with  epifluorescence.  However,  in  some  cases.  TEM 
examination  demonstrated  SCB  that  had  escaped  detec- 
tion by  epifluorescence. 

Bacterial  morphology 

In  all  of  the  echinoderms  examined,  the  SCB  appeared 
to  be  gram-negative  as  they  had  two  membranes  (Figs.  4. 
11)  surrounding  the  cytosol  (Neidhart  el  a/..  1990).  The 
penplasmic  layer  between  the  two  membranes  was  usually 
thin  and  homogeneous.  None  of  the  bacteria  appeared 
flagellated,  nor  were  pili  observed.  The  cytosol  usually 
evenly  surrounded  the  nucleoid  area  and  was  usually 
rather  homogeneous,  with  no  evidence  of  either  internal 
membranes  (other  than  membrane-bound  vacuoles)  or 
granular  inclusions.  Round  spaces  in  the  cytosol,  indi- 
cating where  material  had  been  lost  during  processing, 
were  not  infrequent  and  may  be  poly-/3-hydroxybutyrate 
storage  areas  (Berkeley,  1979).  The  DNA  in  the  nucleoid 
was  often  condensed  into  an  electron-dense  lobulated 


Class 


Number  of  species 


Number  (%)  with  SCB 


Echinoidea 

7 

5(71) 

Ophiuroidea 

9 

5(56) 

Asteroidea 

14 

5(36) 

Holothuroidea 

2 

2(100) 

Crinoidea 

1 

0(0) 

Total 

33 

17(51.5) 

structure  (Fig.  3),  but  in  some  SCB  it  was  much  more 
diffuse  (Fig.  5). 

The  SCB  from  the  New  Zealand  species  of  echinoderms 
were  more  variable  in  their  morphology  than  those  pre- 
viously encountered.  McKenzie  and  Kelly  (1994)  de- 
scribed three  morphological  types  of  bacteria  from  ophiu- 
roids  (Types  1-3).  Type  1  SCB  are  short  rods  that  char- 
acteristically occur  as  paired  bacteria  within  a  single, 
complex  capsule.  This  type  has  been  found  only  in  species 
of  Ophiothnx.  although  another  brittlestar,  Ophiopholis 
aculeata,  has  SCB  that  are  intermediate  between  Type  1 
and  Type  3  SCB.  SCB  of  Type  2  were  the  most  common. 
They  are  long,  thin  (0.1-0.2 /jm).  often  electron-dense 
rods  with  little  evident  ultrastructure.  They  lack  capsules 
and  rarely  have  vacuoles.  They  are  usually  spirals,  but 
vary  from  perfectly  straight  rods  through  spirals  with  long 
wave-lengths  (Figs.  5.  7A)  to  tightly  kinked  spirals  with 
short  wave-lengths  (Fig.  9).  Type  3  SCB  are  straight,  broad 
(0.2-0.5  /j.m)  rods,  often  with  capsules  and  characteristi- 
cally with  vacuoles,  though  these  are  not  always  present. 
The  New  Zealand  species  could  be  further  categorized 
into  three  subdivisions  of  Type  3  SCB.  The  first  subtype 
is  the  "classic"  Type  3  as  described  by  McKenzie  and 
Kelly  (1994).  The  second  subtype  differs  in  having  far 
more  vacuoles  than  normally  encountered  in  Type  3  SCB. 
This  subtype  was  found  in  Ophiomyxa  brevimira  (Fig. 
10)  and  Stichopus  mol/is  (Fig.  2).  The  third  subtype  was 
found  in  the  three  species  of  Pseitdechinus.  It  was  a 
straight,  baton-shaped  rod  with  a  well-defined,  granular 
penplasmic  layer  and  no  obvious  vacuoles  (Fig.  3). 

In  some  species,  more  than  one  type  of  SCB  was  found 
within  a  single  host.  Fixation  artifact  or  pathology  can. 
however,  result  in  Type  2  SCB  resembling  Type  3  SCB, 
and  care  has  to  be  taken  not  to  confuse  such  artifacts  with 
genuine  Type  3  SCB.  Bacteria  appear  to  swell  in  fixation, 
causing  the  membranes  to  become  more  distinct  and  space 
to  appear  around  the  chromatin  (Fig.  7B).  The  shape  of 
the  bacterium  can  become  more  rounded  and  eventually 
irregular.  In  severe  cases,  the  chromatin  is  isolated  in  the 
center  of  the  bacterium  and  the  cell  membranes  are  greatly- 
disrupted.  Such  bacteria  can  be  mistaken  for  poorly  fixed 
host  microvilli  or  blebbed  pieces  of  epidermal  tissue. 


94  M.  S.  KELLY  ET  AL 

Table  II 

Observation  unit  description  of  SCB  as  seen  under  epifluorescenl  (EF)  and  transmission  electron  microscopy  (TEM) 


CLASS 
Order 

Family 

Species  and  authority* 

SCB 

EF/TEM' 

Descnption* 

CRINO1DEA 

Comatulida 

Comastendae 

Comanthus  inn'ti-t'altiiuliiit.'2  Clark 

n/n 

Some  bacteria  found  on  surface  not  SCB,  TEM  only. 

HOLOTHUROIDEA 

Aspidnchirotida 

Stichopodidae 

Stichopus  /HC///S  (Hutton) 

n/y 

Short  rods,  T3. 

\  |>Ull  1(1.1 

Chmdotidae 

Tniiin'thiiu  dunedinensis  (Parker) 

n/y 

Short,  electron  dense  rods.  T? 

ECHINOIDEA 

Cidaroida 

Cidaroidae 

Goniocidaris  unihruciiliini*  (Hutton) 

y/y 

Short  rods,  \-2nm.  T3. 

Echinacea 

Temnopleundae 

Pscuticcltmus  Inillimi  Benham 

y/y 

Rods  2-3  jjm  and  spirals,  spirals  can  be  >  10  /im.  T2  &  T3. 

Pscuitecliini(\  novaezealandlae  (Mortensen) 

y/y 

Rods  and  spirals,  spirals  can  be  >  10  ^m,  T2  &  T3. 

Pseudechinus  albocinctus  (Hutton) 

y/y 

Rods  and  spirals,  spirals  can  be  >  10  /jm.  T2  &  T3. 

Echinometndae 

/-.ivt/j/'mv  i  /?/r"c//(  nv  (Valenciennes) 

n/n 

Clypeasleroida 

Arachnoidae 

Fellaxii'r  zelandiaem  (Gray) 

y/y 

Spirals,  difficult  to  find  T2. 

Spatangoida 

Loveniidae 

Echinocardium  cordatum**  (Pennant) 

n/- 

TEM  specimens  poorly  fixed. 

ASTEROIDEA 

Haxillosida 

Asteropectimdae 

Astropecten  pn>nit;enius  (Mortensen) 

y/y 

Short  rods.  2-3  (*m  and  spirals.  T2. 

A.\iKipccU'n  polyacanthus  '  Muller  &  Troschel 

n/n 

Cuticle  not  preserved. 

Valtatida 

Astennidae 

fatint'llii  i'c\;uliin\  V'emll 

y/y 

Spirals  5-8  ^m.  T2. 

Gomastendae 

Pentagonaster  pulchetlus  Gray 

n/- 

Odontastendae 

Asteniiton  iniluim  (Gray) 

y/y 

Rods  3  ><m,  sometimes  in  short  chains  of  2-3.  Spirals  seen  only  with  TEM.  T2  &  T3. 

OihtnuiMei  hi'iihumi  {Mortensen) 

y/y 

Straight  rods.  10  ^m  and  spirals  10  ^m.  T2  &  T3. 

Ophidiastendae 

Ophitliasler  kermadecensis  m 

n/y 

Rods  with  vacuoles  and  coats,  T3? 

Spinulnsida 

Echmasteridae 

Hctintui  I'ci/pluu'  Fell 

n/n 

Forcipulatida 

Astenidae 

Sclerasterias  mi>lli\  (Hutton) 

n/n 

t'i>vc  niaMcriiH  calamaria  (Gray) 

n/n 

Allit.'ilii-lni.-.lcr  /in/cw/.v  (Farquhar) 

n/n 

Allostichaster  p»tvpla\  (Muller  &  Troschel) 

n/n 

islrostole  scabra'  (Hutton) 

n/n 

Calvaslciui-'  \uicn  (de  Lono!) 

n/- 

OPHIUROIDEA 

Asteroschematidae 

Astrobrachion  conslrictum2  (Farquhar) 

n/y 

Spirals,  T2. 

Ophiomyxidac 

Opliiumvxa  hrcrimira  Clark 

y/y 

Short  rods,  occasional  spirals.  Only  T3  with  TEM. 

Ophiurida 

Amphiundae 

Amplnpholi!,  squamata  Delle  Chiaje 

y/y 

Long  rods  3-4  urn,  shorter  fat  rods  (2  ^m)  often  in  chains  of  2.  T3. 

Amphiura  abemethyi1  Fell 

y/- 

Rods  4-5  fim  EF  only. 

Amphiura  amokurae  Mortensen 

y/v 

Spirals  6-8  f/m.  T2.  Unusually  broad:  0.25-0.30  /im. 

Ophiocomidae 

Optunctinni  ^('//('/n/  Farquhar 

y/B 

5  tim  thin  rods  seen  with  EF.  sometimes  faint,  bacteriocytes  with  TEM. 

Ophinplern  antipodum  Smith 

y/n 

5  //m  rods  seen  with  EF.  sometimes  faint. 

Ophiodermalidae 

Peclinura  maculala  (Venll) 

n/n 

Ophionereidac 

Ophionereis  /a.scuiiti  (Hutton) 

n/n 

*  1.  2.  or  3  -  number  of  specimens  available  if  3  or  less:  N  -  collected  from  North  Island. 
'  y  =  seen;  n  =  not  seen;  -  =  no  sample:  B  =  bactenocyte. 

*  T2  =  Type  2  SCB;  T3  =  Type  3  SCB.  Lengths  are  from  epifluorescence  measurements. 


Occurrence  within  class 

Crinoidea:  Examination  of  the  pinnules  of  Coman- 
thus  novaezealandiae  by  epifluorescent  microscopy  re- 
vealed no  evidence  of  SCB.  A  few  large  bacteria  were 
found  in  TEM  sections,  but  these  were  outside  the  cu- 


ticle and  were  not  considered  to  be  invasive  or  to  be 
any  type  of  SCB.  The  same  type  of  bacteria  were  found 
in  both  specimens  examined,  collected  at  the  same  time 
and  location. 

Holothuroidea:  The  apodous  holothurian  Troclioclota 
dnncdinensix  appeared  to  have  an  unusual  form  of  SCB 


SYMBIOTIC  BACTERIA  IN  ECHINODERMS 


95 


that  could  not  be  easily  classified  as  belonging  to  any  of 
the  major  types  of  SCB  (Fig.  1 ).  These  were  uncommon 
and  had  an  electron-dense,  filamentous  appearance.  SCB 
of  a  similar  morphology  have  been  found  in  Labidoplax 
digitata,  another  apodous  holothurian  (Kelly  and  Mc- 
Kenzie,  1995).  SCB  were  commonly  present  in  TEM  sec- 
tions of  the  aspidochirote  holothurian  Stichopus  mollis. 
although  none  had  been  found  previously  with  epifluo- 
rescence.  These  short  rods  had  numerous  vacuoles 
(Fig.  2). 

Echinoidt'ti:  SCB,  in  the  form  of  short  rods,  were  found 
in  Goniocidaris  itinbraculwn,  a  representative  of  the 
primitive  subclass  Perischoechinoidea.  SCB  were  easily 
found  by  epi fluorescence  in  all  the  specimens  that  were 
examined.  TEM  fixation  of  both  the  host  cytoplasm  and 
bacteria  was  poor,  but  the  symbionts  appeared  to  be  Type 
3  SCB.  with  internal  vacuoles.  The  material  in  these  vac- 
uoles was  often  lost,  forming  a  hole  in  the  section. 

SCB  were  found  in  all  three  species  of  the  genus  Pseud- 
echinus.  Each  had  two  distinct  morphological  types  of 
bacteria:  straight,  baton-shaped  rods  (Fig.  3)  and  a  typical 
Type  2  spiral  form  (Fig.  4).  Both  forms  could  be  seen  with 
epifluorescence  and  TEM.  The  SCB  were  found  lying  both 
below  and  within  the  fibrous  layer  of  the  cuticle.  In  both 
types  of  SCB  the  chromatin  was  usually  condensed  into 
thick,  electron-dense  fibers  running  up  the  center  of  the 
bacteria.  The  baton-shaped  rods  had  the  appearance  of 
being  rigid  and  had  rounded  caps  at  either  end  of  the 
baton  (Fig.  3),  rather  than  tapering  at  their  ends.  The 
most  striking  feature  of  this  type  of  SCB  was  the  well- 
defined,  granular  periplasmic  space.  The  three  species  of 
Pseudec/iinus  were  collected  throughout  the  austral  sum- 
mer months,  and  SCB  were  always  present  regardless  of 
the  reproductive  condition  or  size  of  the  host.  The  smallest 
P.  Inittoni  collected  had  a  diameter  of  14  mm  and  was 
probably  less  than  2  years  old. 

Detection  of  SCB  by  epifluorescence  microscopy  of  tube 
feet  from  the  sand  dollar  Fellaster  zelandiae  was  difficult. 
A  few  irregular  rods  were  seen,  but  the  fluorescence  faded 
unusually  quickly.  The  same  dampening  effect  has  been 
noted  when  examining  some  other  echinoids  (for  example, 
Evechinus  chloroticus)  and  some  darkly  pigmented 
ophiuroids.  TEM  showed  that  the  symbionts  in  F.  :elan- 
diae  were  spiral  Type  2  SCB,  though  few  were  seen. 

No  SCB  were  found  in  specimens  of  Echinocardium 
cordantm  with  either  technique.  None  of  the  specimens 
of  E.  corclatiim  fixed  for  TEM  had  retained  their  cuticles, 
and  their  morphology  was  also  poorly  preserved. 

Asteroidea:  With  epifluorescence,  SCB  were  difficult  to 
find  in  Astropecten  primigenius.  In  only  one  specimen 
were  a  few  rods  and  spirals  observed.  In  contrast,  TEM 
of  this  species  revealed  that  typical  Type  2  SCB  were  com- 
mon in  the  fibrous  part  of  the  cuticle  (Fig.  5),  where  they 
sometimes  appeared  to  be  aligned  parallel  to  the  fibers  of 
the  lower  cuticle.  Larger,  misshapen  bacteria,  which  were 


probably  distorted  Type  2  SCB,  were  also  seen.  No  SCB 
were  found  in  the  single  specimen  of  A.  polyacantluts  from 
Auckland.  The  cuticle  was  not  intact  in  this  specimen. 

In  the  cushion  star  Patiriclla  rcgularis,  abundant  spiral- 
shaped  SCB  with  little  internal  specialization  were  found 
with  epifluorescence,  and  TEM  revealed  Type  2  SCB  in 
the  lower,  fibrous  part  of  the  cuticle.  Asterodon  miliaris 
and  Odontaster  henluimi  are  biscuit  stars  that  are  ex- 
tremely difficult  to  tell  apart,  being  identifiable  by  the 
number  of  projections  on  the  oral  plates  (Fell,  1962).  Even 
so,  the  two  species  were  readily  distinguished  on  the  basis 
of  their  SCB.  With  epifluorescent  microscopy  (Fig.  6A), 
A.  miliaris  had  mostly  straight,  brightly  fluorescing  rods, 
forming  short  chains  of  2-3  cells;  whereas  O.  benhami 
had  a  mixture  of  two  morphological  types.  TEM  showed, 
however,  that  A.  miliaris  did  have  both  Type  3  and  Type 
2  SCB,  though  the  latter  were  much  less  common  than 
they  were  in  O.  benhami.  The  Type  3  SCB  in  O.  benhami 
were  often  dumb-bell  shaped  (Fig.  6B).  In  the  one  spec- 
imen of  Ophidiaster  kemuidecensis  that  was  examined, 
no  SCB  were  found  with  epifluorescence  microscopy.  A 
few  Type  3  rods  with  vacuoles  and  well-developed  capsules 
were  found  amongst  the  microvilli  in  TEM  sections.  These 
were  probably  SCB  but,  as  the  specimen  lacked  its  cuticle, 
this  requires  confirmation.  The  remaining  seastar  of  the 
Order  Valvatida,  Pentagonaster  pulchellns.  was  examined 
only  by  epifluorescent  microscopy  and  no  SCB  were 
noted. 

No  SCB  were  found  in  Henricia  or  in  any  of  the  seastars 
belonging  to  the  order  Forcipulatida  with  either  epifluo- 
rescence or  TEM.  Rod-shaped  bacteria  were  commonly 
found  in  TEM  sections  of  a  single  specimen  of  Calvas- 
lerias  suteri;  however,  the  tissues  were  considerably  dis- 
rupted and  bacteria  were  found  throughout  the  sections, 
including  in  connective  tissue.  These  bacteria  are  probably 
not  SCB  and  may  be  pathogenic,  but  further  study  will 
be  necessary  to  confirm  this.  A  single  bacterium  was  found 
embedded  in  an  indentation  of  the  outermost  layer  of  the 
cuticle  of  a  specimen  of  Sclerasterias  mollis.  This  was  not 
thought  to  be  an  SCB. 

Ophiwoidea:  Many  SCB  were  found  in  TEM  exami- 
nation of  decalcified  arm  tips  from  Astrobrachion  con- 
strictum  (Fig.  7A).  They  were  spirals  but  were  occasionally 
seen  to  vary  in  cross-sectional  area  (Fig.  7B).  The  larger 
types  are  probably  an  artifact  resulting  from  the  swelling 
of  the  more  commonly  seen  form.  However,  no  bacteria 
were  found  when  the  tube  feet  were  examined  with  epi- 
fluorescence. The  small  amount  of  soft  tissue  on  the  arms 
and  the  small  tube  feet  make  epifluorescent  examination 
of  tissue  squashes  difficult,  whereas  the  SCB  are  found 
more  easily  with  TEM  after  decalcification. 

Numerous  thin,  rod-shaped  bacteria  were  found  in 
tube-foot  squash  preparations  from  Ophiocoma  bollonsi 
and  Ophiopteris  antipodum.  The  fluorescence  faded  very 
rapidly  in  homogenates  of  the  tissue  of  these  species, 


96 


M.  S.  KELLY  ET  AL 


| 


Figures  1-5.     Scale  bars  represent  I 


SMV1BIOIIC  BACTERIA  IN  ECHINODERMS 


97 


making  counting  difficult.  Despite  intensive  searching 
with  TEM,  no  SCB  were  ever  found  King  in  the  suhcu- 
ticular  space  of  either  species,  although  the  bacteria  were 
always  found  in  tube  foot  squash  preparations  with  epi- 
fluorescent  microscopy.  This  initially  led  to  the  assump- 
tion that  the  bacteria  were  in  discrete  areas  of  the  sub- 
cuticular  space  and  might  have  been  missed  during  TEM 
examination.  However,  large  bacteriocytes  packed  with 
long,  spiral-shaped  bacteria  were  later  found  in  the  con- 
nective tissue  of  O  bollonxi  (Fig.  8).  The  bacteriocytes 
were  about  10  /urn  diameter  in  TEM  micrographs.  Oc- 
casionally the  spiral  bacteria  were  seen  free  in  the  tissue. 
Ophiomyxa  brevimira  had  a  mixture  of  morphological 
types  of  SCB:  short  rods,  often  forming  chains  of  two; 
longer  rods;  and  a  few  spiral  forms  were  seen  with  epiflu- 
orescence.  With  TEM,  short,  heavily  vacuolated  Type  3 
rods  were  found  to  be  the  predominant  type.  Holes  often 
formed  in  the  sections  where  there  were  vacuoles 
(Fig.  10). 

Two  species  ofAmphhira  were  examined.  A.  amokwae 
was  very  common  under  stones  along  the  beach  adjacent 
to  the  Portobello  laboratory.  One  specimen  of  A.  aber- 
nethyi  was  obtained  by  trawl  from  a  depth  of  1 20  m  out- 
side Otago  Harbor.  A.  amokwae  had  long,  tightly  bound, 
spiral  SCB  that  were  obvious  in  both  epifluorescence  and 
electron  microscopy  (Fig.  9).  These  had  an  unusually  large 
diameter  for  Type  2  SCB.  In  A.  abernethyi.  straight  rods, 
4-5  Mm  long,  were  seen  with  epifluorescence.  but  the  only 
available  specimen  was  in  poor  condition  when  fixed  for 
TEM.  and  no  cuticle  or  bacteria  could  be  found  around 
the  surface.  Amphipholis  smtamata  had  long,  vacuolated, 
rod-shaped  bacteria  (Fig.  1 1).  These  were  very  common 
and  were  easily  seen  with  both  electron  and  light  micros- 
copy. 

Quantification 

The  results  of  the  quantification  of  bacterial  load  for 
all  three  species  of  Pscmk-chinits.  Astcrodon  miliaris. 
Ophiocoma  bollonsi.  Amphipholis  sqitamata.  and  juvenile 
Pseitdechinits  hiittoni  (test  diameters  of  14-18  mm)  are 


given  in  Table  III.  No  significant  differences  were  found 
between  arms  or  ambulacra!  areas  from  the  same  indi- 
vidual animal  for  any  of  the  species  (Student's  /  and  Wil- 
coxon  signed  rank  test).  To  measure  the  variability  of  the 
counting  technique,  10  counts  were  made  from  one  ho- 
mogenate  of  P.  lutitoiii  test.  The  mean  count  per  20  fields 
of  view  was  1 7.9,  with  a  standard  deviation  of  4.63.  Rods 
and  spirals  were  also  counted  separately  for  the  three  spe- 
cies of  Psendechinus  and  for  juvenile  P.  huttoni.  A  one- 
way analysis  of  variance  (ANOVA)  and  a  Fisher's  least 
significant  difference  test  (LSD)  was  then  applied  to  the 
data  (Table  IV).  P.  huttoni  adults  and  juveniles  both  had 
greater  numbers  of  rods  than  spirals  (P  <  0.05).  The  other 
two  species  had  more  spirals  than  rods,  though  these  dif- 
ferences were  not  significant.  As  can  be  seen  from  Table 
IV,  there  are  numerous  significant  differences  between 
the  species  in  their  relative  numbers  of  rods  and  spirals. 

Culturing 

The  methods  described  in  Walker  and  Lesser  (1989) 
and  Lesser  and  Blakemore  (1990)  (i.e..  plating  the  ho- 
mogenates  of  A.  sqitamata  following  a  surface  sterilization 
in  70%  isopropyl  alcohol  for  30  s),  failed  to  produce  bac- 
terial colonies  on  agar  plates.  However,  when  whole  an- 
imals were  treated  for  only  5  s,  prior  to  separate  homog- 
enization  of  arms  and  disks,  bacterial  colonies  grew  on 
plates  inoculated  from  homogenates  of  the  disks  (1.5  X 
10:  mixed  CFU/disk)  but  not  from  the  arms.  This  com- 
pares with  2.6  X  104  and  2.6  X  103  CFU  for  control  (not 
surface  sterilized)  arms  and  disks  respectively.  Although 
the  plates  were  examined  daily,  no  fresh  colonies  appeared 
in  a  10-14  day  period  as  described  by  Lesser  and  Blake- 
more  (1990)  or  after  14-21  days  as  described  by  Lesser 
(pers.  comm.  to  MSK). 

The  other  species  produced  results  similar  to  those  ob- 
tained from  A.  smtamata.  The  surface  sterilization  for  5  s 
usually  resulted  in  no  colony  formation  on  plates,  but 
nonsterilized  animals  produced  colonies  regardless  of 
whether  they  contained  SCB  (P.  huttoni)  or  not  (Ophio- 
coma bollonsi  and  Ophionereisfasciata).  In  the  few  cases 


Figure  1.  Transverse  section  of  SCB  (arrow)  from  Trochodota  dunedinensis  body  wall,  lying  beneath 
the  cuticle  (C)  of  the  body  wall.  SCB  coat  has  fibrous  appearance. 

Figure  2.  Transverse  sections  through  Type  3  SCB  (arrows)  beneath  the  cuticle  (C)  of  Slichopux  mollis 
tube  foot.  SCB  have  numerous  vacuoles  (v).  E,  epidermal  support  cell. 

Figure  3.  Baton-shaped  SCB  from  Pseudechinus  Imltoni  tube  foot,  longitudinal  section  and  transverse 
section.  The  chromatin  is  condensed  into  electron-dense  fibers  running  along  the  center  of  the  bacterium 
(arrow).  Note  granular  appearance  of  periplasmic  space  (curved  arrow)  and  rounded  cap-like  ends  (open 
arrow). 

Figure  4.  Type  2  spiral  form  SCB  from  Pseudechinus  hitltoni  tube  foot,  showing  condensed  chromatin 
fibers  (arrows)  and  double  membranes  characteristic  of  Gram  negative  bacterium  (curved  arrow).  E.  epidermal 
support  cell. 

Figure  5.  Typical  Type  2  SCB  (arrows)  situated  in  the  lower  fibrous  part  of  the  cuticle  (F)  in  Astropecten 
primigeniux  tube  foot,  SCB  sometimes  appear  aligned  with  the  fibers  of  the  lower  cuticle.  C,  outer  layer  of 
cuticle:  E.  epidermal  support  cells. 


98 


M.  S.  KELLY  ET  AL 


*•  •  1 


•V'. 


; 


Figures  6-7.     Scale  bars  represent  1 


SYMBIOTIC  BACTERIA  IN  ECHINODERMS 


99 


in  which  colonies  did  form,  these  were  always  of  more 
than  one  type  and  of  similar  morphology  to  the  colonies 
growing  on  control  plates. 

The  UV  treatments  reduced  the  number  of  bacterial 
colonies  from  the  A.  siiitannihi  arms  but  did  not  totally 
eliminate  the  bacteria,  even  at  longer  exposure  times.  The 
resulting  bacterial  colonies  were  of  a  variety  of  morpho- 
logical types  and  were  similar  to  those  on  the  control  agar 
plates.  Few  colonies  were  isolated  from  the  nonsterilized 
tube-foot  homogenates  of  O.  bollonsi  or  from  those  given 
a  short  U  V  exposure;  no  bacteria  were  cultured  from  tube 
feet  exposed  to  UV  for  longer  time  periods.  Repeated 
washing  with  sterile  ASW  had  no  apparent  effect  in  re- 
ducing the  number  of  colony-forming  bacteria  associated 
with  the  echinoderms  tested.  Whenever  bacteria  were  cul- 
tured, the  variety  of  colony  morphologies  was  similar  to 
that  of  control  animals. 

Discussion 

About  half  of  the  echinoderm  species  we  examined 
from  New  Zealand  contained  SCB.  In  a  survey  of  63  spe- 
cies of  echinoderms  from  the  shelf  seas  around  the  British 
Isles,  more  than  60%  had  SCB  (Kelly  and  McKenzie,  in 
press).  These  surveys  and  most  of  the  other  information 
on  SCB  distribution  have  been  from  temperate,  shallow- 
water  echinoderm  faunas.  The  distribution  of  SCB  in 
tropical  and  polar  echinoderms  is  unknown,  though 
McKenzie  (1992)  described  SCB  in  crinoids  from  the 
Great  Barrier  Reef,  Australia.  SCB  have  also  been  found 
in  deep-sea  holothurians  (Roberts  et  ai.  1991)  and  deep- 
sea  ophiuroids  (unpub.  obs.).  There  is  no  obvious  corre- 
lation between  the  occurrence  of  SCB  and  the  habitat, 
feeding  strategy,  or  other  ecological  aspects  of  the  host. 
The  New  Zealand  echinoderms  were  collected  from  a 
range  of  habitats,  including  sandy  beaches,  rocky  shores, 
and  depths  of  60  m  and  120  m.  SCB  were  found  in  ani- 
mals of  different  reproductive  condition  and  size  or  age. 
Again,  the  number  of  SCB  within  a  species  was  apparently 
not  linked  to  host  ecology.  Similar  conclusions  were 
reached  in  other  studies  (McKenzie  and  Kelly,  1994;  Kelly 
and  McKenzie,  in  press).  SCB  are  thus  a  general,  though 
not  universal,  phenomenon  of  echinoderms  rather  than 
being  found  only  in  particular  ecological  groupings  of 
species. 

One  factor  that  does  relate  to  SCB  distribution  is  host 
phytogeny.  The  following  generalities  are  supported  by 


the  present  observations  and  those  of  earlier  studies.  ( 1 ) 
When  SCB  are  recorded  from  a  species,  then  all  individ- 
uals of  that  species  will  have  symbionts;  (2)  species  con- 
generic with  a  symbiont-containing  species  will  all  have 
SCB;  and  (3)  co-familial  species  will  probably  all  have  or 
all  lack  SCB  (McKenzie  and  Kelly,  1994;  Kelly  and 
McKenzie,  in  press).  Every  individual  of  well-studied  spe- 
cies, such  as  the  three  Pseudechinus  species,  had  SCB. 
Ampliipholis  squamata  from  New  Zealand  (this  study). 
North  America  (Walker  and  Lesser,  1989)  and  North  Eu- 
rope (McKenzie  and  Kelly,  1994)  all  appear  to  have  mor- 
phologically identical  SCB.  It  would  be  interesting  to  check 
the  molecular  similarities  between  the  hosts  and  between 
the  symbionts  from  these  separate  regions.  The  presence 
of  SCB  in  all  three  species  of  Pseudechinus  supports  the 
second  generality;  this  is  also  true  for  the  brittlestar  genus 
Ampliiwa.  SCB  were  present  in  both  New  Zealand  species 
and  have  also  been  found  in  three  European  species  of 
Amphiura  (Kelly  and  McKenzie,  in  press).  SCB  have  been 
recorded  from  Asiropcctcn  irregularis  and  three  species 
of Echinocardium  (Holland  and  Nealson.  1978;  Kelly  and 
McKenzie  in  press).  The  failure  to  demonstrate  SCB  in 
the  New  Zealand  species  Astropecten  polyacanthus  and 
Echinocardium  cordatum  was  probably  due  to  the  poor 
cuticular  fixation  in  all  the  specimens  examined;  further 
investigations  of  these  species  may  reveal  their  presence. 
Three  Australian  species  of  the  feather  star  genus  Com- 
anlhus  (C.  tiinorensis,  C.  parvicirnis,  and  C.  altemans) 
were  examined  by  McKenzie  (1992).  None  had  SCB,  as 
was  the  case  for  the  New  Zealand  C.  novozealandiae.  Ex- 
amples of  co-familial  species  either  having  or  lacking  SCB 
are  best  seen  in  the  asteroids.  Asterodoti  and  Odontaster 
both  have  SCB.  Patiriella  belongs  to  the  Asterinidae  and 
SCB  have  also  been  reported  from  other  species  in  this 
family  (Cameron  and  Holland,  1983;  Souza  Santos  and 
Sasso,  1970;  Kelly  and  McKenzie,  in  press).  None  of  the 
species  belonging  to  the  family  Asteriidae  (Sclerasterias, 
Coscinasterias,  Allostichaster,  Astrostole  and  Calvasterias) 
had  SCB.  The  earlier  report  of  Calvasterias  having  SCB 
(Kelly  L'l  ai.  1994)  was  erroneous. 

McKenzie  and  Kelly  ( 1 994)  noted  a  correlation  between 
SCB  morphology  and  host  phylogeny  in  ophiuroids.  Spe- 
cies within  a  genus  usually  all  have  similar  SCB,  and  this 
is  sometimes  also  true  of  co-familial  species.  This  corre- 
lation is  partially  supported  by  the  results  from  the  New 
Zealand  species,  but  there  are  exceptions.  In  specimens 


Figure  6.  (A)  Mixed  morphological  types  of  SCB  in  Odimiasti'r  benlianii  tube  foot.  Type  2  SCB  (white 
arrows)  in  the  lower  fibrous  layer  (F)  of  the  cuticle  (C)  and  Type  3  SCB  mostly  in  transverse  section  (arrows). 
L.  lamellae  of  support  cells;  E.  epidermal  support  cell.  (B)  Dumbbell-shaped  Type  3  SCB  (arrows)  lying 
below  the  cuticle  (C).  L.  lamellae  of  support  cells;  E,  epidermal  support  cell. 

Figure  7.  (A)  Numerous  Type  2  SCB  (arrows)  lying  beneath  the  cuticle  (C)  ofAstrobrachion  consiricluni 
tube  foot.  BL.  basal  lamima.  (B)  Two  opposing  surfaces  of  tube  foot  from  A\n«hraclut>n  cini\tncnii»  (C). 
On  the  upper  left  the  SCB  in  transverse  section  appear  enlarged  and  distorted  (arrows).  On  the  lower  right 
the  SCB  appear  as  typical  Type  2  SCB  (curved  arrows).  E.  epidermal  support  cell. 


100 


M.  S.  KELLY  ET  AL 

• 


S^'^finBwi^P 

%>  %«£&**  *W      *  % 


Figures  8-11.     Scale  bars  represent  I 


SYMBIOTIC  BACTERIA  IN  ECH1NODERMS 


101 


of  Ainpliinra  tiliformis  and  A.  chiajci  collected  from  the 
British  Isles  (McKenzie  and  Kelly.  1994).  the  SCB  are 
Type  3  rods,  classically  with  membrane-bound  vacuoles 
tearing  to  form  holes  in  the  sections.  Spiral  forms  have 
occasionally  been  seen  with  the  epifluorescent  microscope 
but  not  so  far  with  TEM,  the  straight  rods  being  much 
more  numerous.  McKenzie  and  Kelly  ( 1994)  found  a  sin- 
gle Type  2  SCB  in  another  amphiurid  (Microphiopholis 
atra).  A.  amokiirac  is  apparently  unusual  amongst  am- 
phiurids  in  having  a  predominance  of  Type  2  SCB  instead 
of  the  Type  3  that  might  have  been  predicted.  The  fact 
that  some  species  have  two  types  of  SCB,  combined  with 
the  low  overall  level  of  variation  in  SCB  morphology, 
limits  the  usefulness  of  bacterial  morphology  as  a  character 
in  investigating  possible  co-evolution  between  the  sym- 
bionts  and  their  hosts. 

It  is  not  known  if  the  different  morphological  types  of 
SCB  represent  separate  genotypes.  Other  symbioses  show 
evidence  that  bacteria  with  different  morphologies  have 
different  genetic  identities.  Among  homopteran  insects. 
55%  of  the  species  are  thought  to  contain  more  than  one 
type  of  symbiont  (Buchner,  1966).  and  both  sulnde-oxi- 
dizing  and  methylotrophic  symbionts  have  been  found 
within  the  same  bivalve  host  (Fisher  el  ai,  1993).  The 
fact  that  the  three  Pscudcchinus  species  (Table  IV)  and 
Astcrodon  miliaris  and  Odontaster  benhami  consistently 
differ  in  the  ratio  of  the  SCB  types  within  them  may  in- 
dicate that  the  morphotypes  reflect  different  bacterial  ge- 
notypes. Alternatively,  this  difference  could  result  from  a 
single  SCB  genotype  having  morphological  plasticity 
within  its  echinoderm  host.  The  ability  of  a  single  genotype 
to  exhibit  considerable  pleomorpnism  in  response  to  sub- 
tle environmental  variations  is  known  in  many  bacteria 
(Berkeley,  1979)  and  has  been  recorded  in  symbioses  such 
as  some  mycetocyte-insect  associations  (Houk  and  Grif- 
fiths, 1980;  Smith  and  Douglas,  1987).  The  presence  of 
both  major  types  of  SCB  is  phylogenetically  widespread, 
and  it  may  be  that  they  are  both  potentially  present  in  all 
species.  This  would  strengthen  the  argument  that  the  types 
are  phenotypic  variants  rather  than  different  genotypes. 
Molecular  investigations  of  16S  rRNA  variation  within 
the  symbionts  of  single  species  could  be  the  best  way  of 
resolving  question. 

The  estimates  of  bacterial  loading  in  New  Zealand's 
echinoderms  were  similar  to  those  recorded  for  ophiuroids 
from  the  British  Isles.  McKenzie  and  Kelly  (1994)  esti- 


mated a  bacterial  load  of  4.60  X  10"  g~"  AFDW  for  Am- 
pliipholis  squamata  collected  from  the  west  coast  of  Scot- 
land. This  compares  to  4.96  X  109  g~ '  AFDW  for  A  squa- 
mata  from  New  Zealand.  The  figure  for  A.  squamata  given 
by  Lesser  and  Blakemore  (1990)  is  not  directly  compa- 
rable: it  relates  to  bacterial  colonies  per  animal  rather  than 
to  SCB  g~'  AFDW.  Pscudcclunus  Inittoni  had  a  high  bac- 
terial loading,  only  slightly  lower  than  that  recorded  from 
A.  squamata  and  considerably  higher  than  that  of  the  other 
two  species  of  Pscudcchinus.  The  three  species  of  Pseu- 
dechiniis  are  broadly  sympatric  subtidally  on  the  Otago 
coast  and  maintain  genetic  isolation  from  each  other,  al- 
though they  can  be  readily  hybridized  in  the  laboratory. 
Color  is  the  most  obvious  character  distinguishing  the 
species,  although  there  are  other  morphological  differences 
such  as  test  thickness  and  length  of  spines.  The  differences 
in  overall  bacterial  load  and  in  the  ratios  of  the  types  of 
SCB  present  therefore  warrant  further  investigation. 

Few  comparative  figures  are  available  from  other  sym- 
bioses. The  density  of  SCB  in  all  the  species  is  similar  to 
the  bacterial  loading  in  the  trophosome  of  vestimentifer- 
ans  ( up  to  1 09  bacteria  g~ '  wwt — Cavanaugh  el  al.  1981; 
Powell  and  Somero,  1983).  The  echinoderm  symbionts 
clearly  have  the  potential  to  be  metabolically  important 
to  their  hosts.  However,  the  trophosome  is  a  much  higher 
proportion  of  the  total  body  mass  of  pogonophorans  than 
the  integumental  tissues  are  in  echinoderms,  so  any  con- 
tribution of  SCB  to  their  hosts'  holistic  energy  budget  is 
likely  to  be  proportionately  lower. 

Walker  and  Lesser  (1989)  claimed  to  have  cultured  SCB 
from  Ampliipholis  squamata,  which  they  identified  as  an 
undescribed  species  of  I  'ibrio.  Attempts  to  reproduce  this 
result  from  New  Zealand  A.  squamata  and  from  other 
symbiont-containing  echinoderms  (this  study  and  un- 
published observations  from  Scottish  echinoderms)  did 
not  produce  any  evidence  of  a  symbiont  in  culture.  It  is 
difficult  to  see  how  any  SCB  could  have  survived  the  ster- 
ilization described  in  Lesser  and  Blakemore  (1990).  Even 
a  5-s  exposure  to  isopropyl  alcohol  killed  the  host,  so  30  s 
of  exposure  would  have  penetrated  the  cuticle  and  pre- 
sumably killed  the  SCB.  In  our  experiments,  bacteria 
never  grew  when  the  animals  had  more  than  a  5-s  exposure 
to  isopropyl  alcohol.  Even  those  that  occasionally  grew 
at  an  exposure  of  5  s  were  probably  associated  with  the 
surface  or  gut  rather  than  being  symbionts,  because  they 
always  formed  mixed  colonies  morphologically  identical 


Figure  8.  Spiral-shaped  bacteria  (arrows)  in  a  bacteriocyle  from  the  connective  tissue  of  an  Ophiocoma 
htillonsi  tube  foot.  HC.  host  cell;  open  arrows,  host  cell  membrane. 

Figure  9.  Tightly  kinked  spiral  form  Type  2  SCB  (arrow)  from  a  tube  foot  of  Amphiwa  umnkiirac.  C, 
cuticle. 

Figure  10.  Type  3  SCB  (arrows)  from  a  tube  foot  of  Ophiomyxa  brevimira.  SCB  have  numerous  vacuoles 
(v).  C.  cuticle;  HV  vacuoles  of  host  origin  in  sub-cuticular  space. 

Figure  11.  Vacuolated  Type  2  rods  from  a  tube  foot  of.4niphiph<>ln<  \tiiiamata  Curved  arrow  indicates 
double  membranes  around  SCB.  C.  cuticle;  v.  vacuoles. 


102 


M.  S.  KELLY  ET  AL 


to  those  from  controls.  No  bacterial  colonies  of  the  type 
reported  by  \Valker  and  Lesser  (1989)  were  observed,  even 
after  2 1  days.  These  results  do  not  necessarily  mean  that 
the  bacterium  isolated  by  Walker  and  Lesser  (1989)  was 
not  the  symbiont.  The  SCB  of  A.  sqitamata  from  the  east 
coast  of  the  United  States  could  be  different  from  the  New 
Zealand  or  European  symbionts  and  could  either  be  re- 
sistant to  isopropyl  alcohol  or  have  simpler  culturing  re- 
quirements. Successful  isolation  into  culture  of  a  bacterial 
symbiont  from  marine  organisms  is,  however,  very  rare. 
Only  the  luminescent  symbionts  of  some  fish  and  squid 
(Hastings  and  Nealson,  1981;  McFall-Ngai.  1994)  have 
definitely  been  cultured,  and  these  bacteria  are  commonly 
occurring,  free-living  forms.  The  problems  of  symbiont 
isolation  are  illustrated  by  a  meticulous  attempt  by  Wood 
and  Kelly  (1989)  to  culture  chemoautotrophic  bacteria 
from  the  bivalve  Thyasira  flexuasa.  They  isolated  a  sul- 
fide-oxidizer  of  the  genus  Thiobarillm  and  proposed  it  as 
the  symbiont  of  T.  Jk'xitosa;  however,  when  genetic  se- 
quences of  the  cultured  bacterium  were  compared  to  the 
symbionts,  it  was  found  that  they  were  not  the  same 
(Marine  Biological  Association  of  the  United  Kingdom 
Annual  Report  1991-1992). 

As  evidence  that  the  bacteria  they  cultured  were  indeed 
SCB,  Walker  and  Lesser  ( 1989)  produced  polyclonal  an- 
tibodies to  whole  bacterial  cells,  then  used  these  antibodies 
to  label  sections  of  the  host  material.  The  SCB  reacted 
with  the  polyclonal  antibodies.  Unfortunately,  these  au- 
thors did  not  try  to  determine  the  specificity  of  their  an- 
tibodies to  the  cultured  bacterial  isolate  by  testing  them 
against  either  a  range  of  bacteria  isolated  from  the  gut 
and  outer  surfaces  of  A.  sijucunata,  or  against  other  marine 
bacteria.  It  is  therefore  possible  that  these  antibodies  were 
not  1 7/v/o-specific.  Nor  did  these  authors  cross-react  the 
antibodies  with  other  }'ihri<>  strains.  It  would  not  be  sur- 
prising if  SCB  were  related  to  the  genus  Mhrio,  because 
unculturable  symbionts  from  flashlight  and  angler  fishes 
(Haygood  and  Distel,  1993)  were  shown  to  be  related  to 
vibrios.  The  polyclonal  antibody  results  do  show  that  the 


Table  IV 

Fisher's  least-square  difference  table  for  comparisons  of  rods  (Type  3 
SCB)  and  spirals  (Type  2  SCB)  between  Pseudechinus  huttoni  (PH); 
P.  huttoni  juveniles  (PHJ);  P.  novaezealandiae  (PN),  and  P. 
albocinctus  (PA);  significance  is  at  P  <  0.05 


Comparison 


Significance 


PA  rods  >  PN  rods 
PH  rods  >  PA  rods 
PHJ  rods  >  PA  rods 
PA  spirals  >  PA  rods 
PA  rods  >  PN  spirals 
PA  rods  >  PH  spirals 
PA  rods  >  PHJ  spirals 
PH  rods  >  PN  rods 
PHJ  rods  >  PN  rods 
PA  spirals  >  PN  rods 
PN  spirals  >  PN  rods 
PH  spirals  >  PN  rods 
PHJ  spirals  >  PN  rods 
PH  rods  >  PHJ  rods 
PH  rods  >  PA  spirals 
PH  rods  >  PN  spirals 
PH  rods  >  PH  spirals 
PH  rods  >  PHJ  spirals 
PHJ  rods  >  PA  spirals 
PHJ  rods  >  PN  spirals 
PHJ  rods  >  PH  spirals 
PHJ  rods  >  PHJ  spirals 
PA  spirals  >  PN  spirals 
PA  spirals  >  PH  spirals 
PA  spirals  >  PHJ  spirals 
PN  spirals  >  PH  spirals 
PN  spirals  >  PHJ  spirals 
PHJ  spirals  >  PH  spirals 


0.0260 
<0.0001 
0.0017 
NS 
NS 
NS 
NS 

<0.0001 

<0.0001 

0.002 

NS 

NS 

NS 

<0.0001 
<0.0001 
<0.0001 
<0.0001 
<0.0001 
NS 

0.0005 
<0.0001 
<0.0001 
0.0486 
0.0015 
0.0066 
NS 
NS 
NS 


cultured  bacterium  shares  an  antigen  with  the  SCB,  but 
further  investigations — preferably  with  monoclonal  an- 
tibodies and  //(  situ  hybridization  with  species-specific 
rRNA  probes  (Distel  el  at.,  1991) — are  needed  to  show 
that  the  cultured  bacteria  are  genuinely  the  same  as 
the  SCB. 


Table  III 

Bacteria/  counts  per  grain  oj  tissue  wet  weight  (\V\VT)  and  ash-free  dry  weight  (AFDH'i 


Species 

Weight  of  tissue 

(g> 

Count  from  homogenate 
(standard  deviation) 

SCBg-' 
WWT 

SCBg-1 
AFDW 

Pseudechinus  huttoni 

0.958 

37.0    (13.0) 

2.19  x  108 

4.48  X  10" 

I1  iill'ncinctiis 

0.986 

16.8    (10.5) 

9.96  x  107 

2.17  x  10° 

P  novaezealandiae 

0.608 

6.37(5.2) 

3.77  x  10' 

8.41  X  108 

P.  Intttont  juveniles 

0.314 

22.1    (12.4) 

1.31  x  108 

2.67  X  109 

Asterodon  miliaris 

0.577 

34.3    (19.6) 

2.03  x  108 

1.99  X  109 

Amplupholis  suuamatu* 

0.030 

83.8    (14.0) 

4.96  X  10* 

4.96  X  109 

Oplnocoma  hollonsi 

0.849 

6.9    (6.0) 

4.09  x  107 

4.41  X  10s 

The  wet  weight  is  an  average  per  homogenate;  the  counts  are  an  average  of  20  fields  of  view  and  a  total  count  of  rods  and  spirals. 
*  AFDW  conversion  factor  taken  from  Scottish  west  coast  specimens  (McKenzie  and  Kelly.  1994). 


SYMBIOTIC  BACTERIA  IN  ECHINODERMS 


103 


It'SCB  are  unculturable.  identifying  the  nature  of  their 
interaction  with  their  hosts  is  more  difficult.  There  has 
been  a  great  deal  of  interest  in  associations  between  che- 
moautotrophic  bacteria  and  various  marine  invertebrates. 
There  is.  however,  no  positive  evidence  suggesting  that 
SCB  are  chemoautotrophs.  Bacterial  morphology  some- 
times provides  clues  to  trophic  biology  (Berkeley,  1979), 
but  the  morphology  of  the  SCB  from  the  New  Zealand 
echinoderms  was  not  particularly  informative.  Type  2 
SCB  showed  little  internal  specialization;  even  vacuoles 
were  rare.  The  Type  3  SCB  from  the  New  Zealand  species 
were  more  diverse.  The  profusion  of  vacuoles  seen  in  the 
brittlestar  Ophiomyxa  and  the  holothurian  Stichopus 
inollis  may  indicate  that  these  bacteria  are  more  unusual 
than  other  SCB  so  far  observed.  There  was,  however,  no 
evidence  of  the  specialized  membrane  stacks  known  from 
symbiotic  methylotrophs  (Fisher  et  al..  1993)  and  nitrifiers 
(Stanier  et  al..  1977).  Nor  was  there  any  evidence  of  the 
sulfur  storage  observed  in  some  sulfide-oxidizing  sym- 
bionts  (Southward.  1986).  The  lack  of  obvious  morpho- 
logical clues  need  not  exclude  chemoautotrophy.  Some 
other  sulnde-oxidizers,  for  example,  do  not  store  sulfur 
and  they  resemble  SCB  (Southward,  1986).  Given  the  large 
number  and  diversity  of  host  species,  some  SCB  may  yet 
prove  to  be  chemoautotrophic. 

Many  investigators  favor  the  suggestion  that  SCB  me- 
tabolize dissolved  organic  material  (DOM)  (Holland  and 
Nealson.  1978:  Walker  and  Lesser,  1989),  and  some  ev- 
idence supports  this  hypothesis.  In  Amphipholis  sqnamata. 
SCB  can  take  up  dissolved  amino  acids,  and  this  uptake 
precedes  any  translocation  of  synthesized  proteins  to  the 
host  (Walker  and  Lesser.  1989:  Lesser  and  Walker.  1992). 
Host  epidermal  cells  frequently  phagocytose  SCB  (Walker 
and  Lesser,  1989;  Roberts  et  al..  1991;  McKenzie  and 
Kelly,  1994).  SCB  could  have  a  nutritive  role  if  they  used 
DOM  as  an  energy  source,  then  were  "cropped"  by  the 
host  through  phagocytosis.  It  is  more  usual,  however,  for 
a  host  to  benefit  from  the  products  of  its  symbionts  than 
to  ingest  the  bacteria  themselves  (Douglas.  1994).  and  it 
may  be  that  only  moribund  SCB  are  phagocytosed.  The 
division  rate  of  the  SCB  is  unknown — there  are  only  es- 
timates for  standing  crop — but  given  the  frequency  of  ob- 
servations of  phagocytosis,  it  may  be  high.  Lesser  and 
Walker  (1992)  concluded,  however,  that  the  rate  of  DOM 
uptake  was  too  low  to  provide  a  significant  energy  source 
to  the  host.  They  based  this  conclusion  on  a  comparison 
between  a  symbiont-containing  ophiuroid.  Amphipholis 
sqnamata,  and  a  species  that  lacked  SCB.  Unfortunately, 
the  latter  was  Ophiopholis  aculeata,  a  species  that  defi- 
nitely possesses  SCB.  albeit  in  lower  numbers  than  are 
found  in  A.  sqnamata  (McKenzie  and  Kelly,  1994).  The 
role  of  SCB  in  DOM  uptake  has,  therefore,  still  to  be 
denned.  Because  of  the  density  of  symbionts  observed  in 
some  species,  the  possible  contribution  of  SCB  to  DOM 


uptake  in  echinoderms  must  be  considered  in  investiga- 
tions of  growth  and  regeneration. 

Although  the  bacteria  seen  in  the  bacteriocytes  of  O. 
hollonsi  had  a  regular,  "crimped"  appearance  when 
viewed  with  TEM,  they  are  probably  the  same  as  the 
faintly  fluorescing  rods  seen  in  the  tube-foot  squash  prep- 
arations, the  "crimped"  shape  being  too  small  to  be  re- 
solved with  epifluorescence  microscopy.  These  bacteria 
are  not  SCB,  and  both  this  species  and  Ophiopteris  anti- 
podum  have  been  discounted  from  previous  estimates  of 
the  numbers  of  species  that,  on  the  basis  of  epifluorescent 
evidence,  are  thought  to  harbor  SCB  (Kelly  et  al..  1994). 
The  bacteria  counted  in  the  homogenate  of  arm  tissue 
from  Ophiocoma  bollonsi  were  probably  also  released 
from  the  bacteriocytes.  The  counts  made  from  homoge- 
nates  ofOphiocoma  bollonsi  (Table  III)  suggest  that  these 
bacteria  are  almost  as  abundant  as  SCB  in  other  species. 
Ophiopteris  papillosa  from  the  northwestern  coast  of 
America  is  similar  to  Ophiopteris  antipodum  in  that  the 
rod-shaped  bacteria  seen  with  epifluorescence  could  not 
be  found  under  the  cuticle  with  TEM  (McKenzie  and 
Kelly,  1994).  Further  investigations  may  reveal  bacter- 
iocytes in  these  species  also.  Nothing  is  known  of  the  bi- 
ology of  the  bacteriocytes,  but  the  host  animals  appear 
healthy.  Large  cells,  filled  with  rod-shaped  bacteria,  have 
also  been  found  in  the  crinoid  Calamocrinus  diomedae 
(Holland  et  al..  199 1 ).  Similar  structures  have  been  found 
in  other  stalked  crinoids  (U.  Welsh,  pers.  comm.)  and  in 
the  comatulids  Antedon  bifuia  (Kelly  and  McKenzie,  in 
press)  and  A.  pelasus  (Heinzeller  and  Welsch,  1994).  These 
bacteria  may  be  a  second  type  of  symbiont,  but  they  are 
also  reminiscent  of  rickettsial  infections  of  marine  inver- 
tebrates (Sparks.  1985),  which  often  have  no  obvious 
pathological  effects  on  their  hosts. 

The  SCB  in  New  Zealand  echinoderms  are  very  similar 
to  those  in  echinoderms  from  Europe  and  North  America. 
Although  some  of  the  SCB  have  interesting  morphologies 
that  have  not  previously  been  encountered,  almost  all  of 
the  SCB  from  New  Zealand  species  can  be  classified  within 
two  major  types.  The  overall  pattern  of  SCB  distribution 
is  similar  to  that  in  other  echinoderm  faunas  (Kelly  and 
McKenzie,  in  press)  and  no  ecological  trends  in  their  dis- 
tribution are  obvious.  The  New  Zealand  study  highlights 
both  the  restricted  degree  of  variation  in  SCB  morphology 
and  the  possibility  that  more  than  one  type  of  SCB  can 
occur  within  a  single  host  species.  It  also  strengthens  the 
observation  that  closely  related  species  are  all  likely  to 
either  have  SCB  or  lack  them  (McKenzie  and  Kelly,  1994). 
The  recorded  densities  of  SCB  indicate  that  they  are  po- 
tentially important  to  their  hosts,  although  their  exact 
functions  are  as  yet  unknown.  None  appear  to  be  che- 
moautotrophs. Molecular  techniques  will  be  the  best  way 
to  investigate  the  links  between  host  phylogeny  and  SCB 
distribution.  In  the  absence  of  isolated  symbionts,  exper- 
iments comparing  nonsymbiotic  species  with  ones  con- 


104 


M.  S.  KELLY  ET  AL. 


taining  SCB  p  -.he  most  productive  means  to  explore 

trophic  inte      .;ons  between  SCB  and  their  hosts. 

Acknowledgments 

This  work  was  partly  funded  by  the  Waitangi  Fellow- 
ship to  MSK  from  The  Bank  of  Scotland  and  by  Mobil 
North  Sea  Ltd.  We  thank  the  Directors  of  the  Portobello 
and  Dunstaffnage  Marine  Laboratories  for  the  use  of  the 
facilities;  and  Mr.  Alan  Mitchell  and  the  staff  at  the  South 
Campus  EM  Unit,  University  of  Otago;  the  crew  of  the 
R.  V.  Munida;  and  Dr.  Mark  James  for  their  technical 
support. 

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Coexistence  and  Possible  Parasitism  of  Somatic  and 

Germ  Cell  Lines  in  Chimeras  of  the  Colonial 

Urochordate  Botryllus  schlosseri 

ZEEV  PANCER' 2.  HARRIET  GERSHON2,  AND  BARUCH  RINKEVICH1  * 

^Israel  Oceanographic  &  Limnological  Research,  National  Institute  of  Oceanography,  Tel-Shikmona, 

P.O.B.  8030.  Haifa  31080,  Israel,  and2 Department  of  Immunology.  The  Bruce  Rappaport  Faculty  of 

Medicine.  Teclmion- Israel  Institute  of  Technology.  P.O.B.  9649.  Haifa  31096.  Israel 


Abstract.  Fusion  between  conspecifics  (chimerism)  is  a 
well-documented  phenomenon  in  a  variety  of  taxa.  Chi- 
merism and  the  subsequent  mixing  of  genetically  different 
stem  cell  lines  may  lead  to  competition  between  cell  lin- 
eages for  positions  in  the  germ  line  and  to  somatic  and 
germ  cell  parasitism.  It  is  suggested  that  somatic  com- 
patibility systems  evolved  to  alleviate  the  costs  and  the 
threat  of  such  cell  lineage  competition.  Allogeneic  colonies 
of  the  ascidian  Botryllus  schlosseri  form  vascular  chimeras 
based  on  matching  in  one  or  both  alleles  on  one  highly 
polymorphic  fusibility  haplotype.  Thereafter,  one  of  the 
partners  is  completely  or  partially  resorbed.  Here  we  used 
a  polymorphic  molecular  marker  (PCR  typing  at  a  mi- 
crosatellite  locus)  to  follow  somatic  and  gametic  conse- 
quences of  chimera  formation.  Twenty-two  chimeras  and 
subclone  samples  were  established  from  1 2  different  ge- 
notype combinations,  in  which  blood  cells,  zooids,  and 
gonads  were  typed  45- 130  days  thereafter.  Somatic  co- 
existence of  both  partners  was  recorded  in  73%  of  the 
subcloned  chimeras  (83%  of  chimeric  entities)  up  to  100 
days  after  disconnection  between  genotypes  and  in  all 
chimeras  where  colony-resorption  was  completed.  Both 
genotypes  were  present  in  23%-  of  the  sampled  gonads  (in 
33%  of  the  chimeras),  and  in  22%  of  the  cases,  germ  cells 
of  the  second  partner  only  were  detected.  Injection  of  al- 
logeneic  but  compatible  blood  cells  into  three  recipient 
colonies  revealed  proliferation  of  the  donor  cells  in  one 
case,  100  days  after  injection.  To  further  evaluate  somatic 
and  germ  cell  parasitism  in  chimeric  organisms,  we  pro- 
pose four  key  features  that  characterize  cell  lineage  corn- 


Received  16  March  1995;  accepted  27  July  1995. 
*  To  whom  correspondence  should  be  addressed. 


petition  processes.  These  include  the  somatic  embryogen- 
esis  mode  of  development,  the  capability  for  independent 
existence  of  stem  cells,  the  disproportionate  share  of  ga- 
metic output  within  chimeras,  and  the  existence  of  hier- 
archial  responses. 

Introduction 

Genetically  non-homogeneous  organisms  may  be  es- 
tablished through  somatic  mutations  or  via  chimerism, 
the  fusion  between  genetically  distinct  conspecifics.  While 
most  somatic  mutation  variants  are  evolutionarily  irrel- 
evant (Van  Valen,  1988),  chimeric  entities  have  been 
documented  in  nature  from  a  variety  of  protists,  plants, 
and  animals,  belonging  to  at  least  nine  phyla  (Buss.  1982). 
Several  studies  (Buss,  1982;  Grosberg  and  Quinn,  1986; 
Rinkevich  and  Weissman,  1987a,  1992a)  have  discussed 
the  evolutionary  significance  of  these  natural  chimeras  by 
evaluating  the  fitness  costs  and  benefits  of  chimerism  as 
compared  to  the  state  of  genetically  homogeneous  entities. 
While  several  sets  of  benefits  were  attributed  to  natural 
chimerism  (Buss,  1982;  Grosberg  and  Quinn,  1986),  par- 
ticular attention  has  been  paid  to  the  potential  costs  re- 
sulting from  the  mixing  of  genetically  distinct  cell  lines 
within  chimeras.  This  is  relevant  especially  in  those  cases 
where  germ  line  sequestration  remains  undetermined  until 
late  in  ontogeny,  or  is  never  accomplished  during  the  life- 
span  of  an  organism  (Buss,  1982,  1983;  Grosberg  and 
Quinn.  1986;  Rinkevich  and  Weissman,  1987a,  1992a). 
Within  the  shared  morphological  and  physiological  en- 
vironments of  chimeric  entities,  one  genotype  could  gain 
a  disproportionate  share  of  germ  cells  at  the  expense  of 
the  other  partner  (germ  cell  parasitism),  or  one  genotype 
may  use  the  tissues  and  energy  reservoirs  of  the  other 


106 


CELL  PARASITISM   IN  AN  ASCIDIAN 


107 


member  in  the  chimera  for  general  maintenance  (somatic 
cell  parasitism).  Examples  on  the  morphological,  cellular, 
and  biochemical  levels  of  the  displacement  of  one  cell 
lineage  in  the  chimera  at  the  expense  of  another  were 
documented  in  several  groups  of  organisms  (reviewed  in 
Rinkevich  and  Weissman,  1987a;Grosberg,  1988).  How- 
ever, there  is  still  no  direct  documentation  for  such  a  pro- 
cess in  which  molecular  markers  were  used. 

A  typical  colony  of  the  urochordate  Botryllus  schlosseri, 
a  subtidal  species  that  can  be  found  worldwide,  is  com- 
posed of  a  few  to  several  hundreds  of  modular  units  called 
zooids,  which  are  arranged  in  star-shaped  structures  (sys- 
tems) and  are  connected  to  each  other  via  a  ramified  blood 
system.  In  this  group  of  organisms,  a  line  of  stem  cells 
retains  the  ability  to  differentiate  into  either  germ  cells  or 
somatic  tissue  throughout  the  lifespan  of  the  colony  (Ber- 
rill  and  Liu.  1948),  continuously  replacing  aging  differ- 
entiated cells.  Consequently,  all  zooids  within  a  single 
colony  are  genetically  identical,  having  been  derived  by 
blastogenesis  from  a  single  founder  zooid,  by  a  complex 
but  highly  synchronized  weekly  developmental  cycle. 
Zooids  and  blood  vessels  are  embedded  within  the  tunic, 
a  translucent  organic  matrix,  which  bears  sausage-like 
termini  of  blood  vessels,  called  ampullae,  in  the  colony's 
periphery.  When  a  colony  is  split  in  nature  or  experi- 
mentally into  two  or  more  fragments,  each  subclone  usu- 
ally continues  to  grow  independently  to  form  a  larger 
colony. 

Recent  interest  in  botryllid  ascidians  has  centered  on 
allogeneic  recognition  and  its  consequences.  Pairs  of  col- 
onies that  meet  naturally  in  the  wild  or  are  placed  in  con- 
tact under  laboratory  conditions  either  fuse  their  con- 
tacting peripheral  ampullae  to  form  a  vascular  parabiont 
(cytomictical  chimera;  Rinkevich  and  Weissman.  1987a). 
or  develop  cytotoxic  lesions  in  the  contact  zone  (reviewed 
in  Taneda  el  al..  1985;  Weissman  el  ai,  1990:  Rinkevich, 
1992).  This  allorecognition  is  genetically  controlled  by  a 
single,  highly  polymorphic,  fusibility/histocompatibility 
(Fu/HC;  Weissman  et  al..  1990)  haplotype  with  multiple 
codominantly  expressed  alleles.  Fusion  may  be  established 
between  genotypes  which  match  in  one  or  both  Fu/HC 
alleles.  Rejecting  colonies  share  no  Fu/HC  alleles  (Scofield 
et  al.,  1982).  Controlled  laboratory  experiments  on  Bo- 
tryllus chimeras  revealed  that  the  zooids  from  one  ge- 
notype in  each  specific  chimera  are  all  morphologically 
eliminated  within  a  few  days  to  several  months  by  massive 
phagocytosis,  leaving  the  zooids  of  the  other  partner  intact 
(Rinkevich  and  Weissman,  1987a.  b.  1992a.  b:  Weissman 
rf  al..  1990;  Rinkevich  ct  al..  1993).  This  phenomenon, 
called  "colony  resorption"  (Rinkevich  and  Weissman. 
1987b),  typically  occurs  at  the  end  of  a  blastogenic  cycle 
and  appears  to  be  controlled  genetically  by  a  multilevel 
hierarchial  organization  of  histocompatibility  alleles 
(Rinkevich,  1993;  Rinkevich  el  a!..  1993).  In  addition  to 


this  resorption  phenomenon,  a  few  studies  have  docu- 
mented, within  chimeras,  germ  cell  transfer  and  estab- 
lishment between  different  partners  (Sabbadin  and  Zan- 
iolo,  1979;  Rinkevich  and  Weissman.  1987a).  These  pre- 
liminary results  further  indicate  that  chimerism  may 
present  substantial  fitness  costs  over  the  long  term  through 
cell  lineage  competition,  parasitism,  or  a  combination  of 
both  processes  (Buss,  1982;  Grosberg  and  Quinn,  1986; 
Rinkevich  and  Weissman,  1987a,  1992a;  Sabbadin  and 
Astorri,  1988). 


Materials  and  Methods 


Animals 


Botryllus  schlosseri  colonies  were  cultured  in  the  lab- 
oratory as  previously  described  (Boyd  et  at..  1986;  Rink- 
evich and  Weissman.  1987b.  1992a;  Rinkevich  et  al., 
1993).  Experimental  colonies  were  isolated  from  among 
the  progeny  of  our  laboratory  stock,  which  originated 
about  a  year  earlier  from  Monterey  marina,  CA,  USA. 
Newly  metamorphosed  colonies  were  raised  individually, 
each  attached  to  glass  slides  (50  X  75  mm). 

Colony  allorecognition  assays 

The  technique  of  colony  allorecognition  assay  (CAA) 
is  the  most  common  assay  used  for  revealing  self-nonself 
discrimination  potential  in  botryllid  ascidians  (Rinkevich, 
1992.  1995;  Saitoh  ai,  1994).  In  this  assay,  small  or  large 
groups  of  zooids  at  the  growing  edges  of  the  colonies  are 
isolated  by  dissecting  them  from  each  colony  without  in- 
juring their  surrounding  ampullae.  Subclones  from  two 
colonies  are  put  on  glass  slides  in  pairs  and  placed  so  that 
they  contact  one  another  with  their  extending  ampullae. 
They  are  usually  allowed  to  fasten  themselves  to  the  slides 
by  placing  them  in  a  moisture  chamber  for  30-45  min 
before  transferring  to  the  tanks  or  to  the  sea.  The  CAA 
was  performed  on  all  botryllid  ascidians  where  specificity 
was  analyzed.  Observations  on  the  contact  sites  and  col- 
onies were  made  under  the  dissecting  microscope  at  least 
once  a  week  to  confirm  the  location  of  "each  partner"  in 
the  chimera  in  accordance  with  the  developing  buds  dur- 
ing successive  blastogenic  cycles.  A  complete  mixture  of 
blood-borne  pigment  cells  was  recorded  <  14  days  after 
fusion  (Rinkevich  and  Weissman,  1987b). 

Experimental  procedures 

Polymorphic  molecular  markers,  such  as  microsatellite 
loci,  may  be  most  suitable  for  individual  identification  of 
genotypes  within  B.  schlosseri  chimeras.  Microsatellites 
are  tandem  repeats  (usually  >100  bp  long)  of  very  short 
nucleotide  motifs  ( 1-6  bp  long)  that  are  dispersed  abun- 
dantly and  randomly  through  eukaryotic  genomes.  Since 
each  of  the  microsatellites  is  flanked  within  the  DNA  ma- 


108 


Z.  PANCER  ET  AL 


terial  by  unique  sequences,  they  can  be  amplified  in  vitro 
using  the  pol>  merase  chain  reaction  (PCR:  Queller  el  ai. 
1983).  We  used  the  protocol  developed  for  PCR  typing 
at  B  scklosseri  microsatellite  locus  811.  which  has  been 
found  to  be  highly  polymorphic  in  two  Botryllus  popu- 
lations (Pancer,  1994;  Pancer  et  ai,  1994).  Twenty-three 
laboratory-raised  colonies  were  typed  at  microsatellite  8 1 1 
and  were  assigned  to  their  fusibility  status  by  employing 
colony  allorecognition  assays  (Rinkevich,  1992,  1995; 
Saito  et  ai,  1994)  on  different  pair  combinations. 

Samples  for  typing  were  either  a  small  tissue  fragment 
(T);  or  hemolymph  (H);  or  a  single  zooid  (Z),  and  the 
gonad  (G),  if  present  in  that  zooid.  Gonads  are  situated 
on  both  sides  of  the  zooids,  composed  of  lobulated  testes 
and  ovaries.  In  this  study  the  sperm  and  the  small  oocytes 
from  each  gonad  were  sampled  as  a  whole  in  the  PCR 
reaction.  Whenever  the  state  of  the  chimeras  allowed,  si- 
multaneous sampling  of  several  zooids  was  performed. 
Tissue  samples  were  transferred  into  1.5-ml  test  tubes. 
rinsed  with  0.5  ml  filtered  (0.2  ^M.  Schleichter  &  Schuell) 
seawater.  and  were  then  boiled  for  5  min  in  100  ^1  TE, 
dispersed  by  pipetting  through  an  aerosol-free  tip  (EL- 
KAY,  Labsystems)  and  centrifuged  at  12000  X  g  for 
5  min.  The  supernatant  was  discarded,  the  pellet  resus- 
pended  in  10-20  ^1  of  PCR-lysis  buffer  (20  mAl  DTT, 
10~3%  SDS  and  0.5  mg/ml  Proteinase  K)  and  incubated 
for  1  h  at  55°C  with  occasional  vortexing.  At  the  end  the 
sample  was  boiled  again  for  5  min.  Amplifications  were 
performed  on  a  PTC- 100  thermal  cycler  (MJ  Research) 
in  10  jul  reaction  mixtures  containing  1  ^1  of  the  sample, 

4  pmoles  each  of  the  forward  and  reverse  primers,  200  pM 
of  each  dNTP,  0.1  MCi  [«--2P]dCTP,  5%  DMSO,  0.5  U 
Taq  DNA  polymerase  and  buffer  (Boehnnger.  Mann- 
heim). The  cycling  parameters  were  3  min  at  96°C  fol- 
lowed by  30  cycles  of  45  s  at  95°C,  75  s  at  60°C,  and  1 5  s 
at  74°C.  The  reactions  were  stopped  by  the  addition  of 

5  n\  Sequenase  stop  solution  (USB),  denatured  for  5  min 
at  95°C,  then  2  jul  of  each  sample  were  electrophoresed 
on  a  6%  denaturing  polyacrylamide  gel.  Detection  thresh- 
old was  determined  experimentally  in  cell  mixtures  of 
1000  cells  total,  containing  different  proportions  of  two 
homozygous  colonies.  Cells  of  a  genotype  mixed  1:10-1: 
20  with  cells  of  a  second  genotype  could  be  detected  fol- 
lowing three  exposure  days  of  the  film  (Pancer,  1994). 

For  microinjections.  a  siliconized  micropipette 
mounted  on  a  micromanipulator  was  inserted  into  a 
large  blood  vessel.  Five  to  seven  microliters  of  hemo- 
lymph were  drawn.  The  micropipette's  contents  were 
then  injected  into  the  recipient  colony's  blood  vessels. 
Several  cell  counts  revealed  approximately  2.5-5  X  104 
blood  cells  in  that  volume. 

Results 

We  studied  22  whole  chimeras  and  chimeric  subclones 
that  were  established  from  12  different  genotype  combi- 


nations (Table  I,  Fig.  la-c).  Different  parts  of  the  colony's 
body,  including  blood  cells,  individual  zooids.  pieces  of 
tissue,  and  gonads  were  sampled  45- 1 30  days  after  estab- 
lishment of  the  chimeras  or  15-100  days  after  disconnec- 
tions between  the  partners  in  the  chimera  as  a  result  of 
subcloning,  spontaneous  disconnection,  or  after  immu- 
nological  resorption  (Table  I). 

In  four  genotype  combinations  (chimeras  A,  B,  C,  G, 
Table  I,  Fig.  la-c),  we  sampled  2-5  subclones  from  each 
original  chimera  at  different  times  following  chimera  for- 
mation or  disconnection  (experimentally  or  sponta- 
neously). In  one  of  these  chimeras  (G)  a  hierarchial  re- 
sponse was  documented.  Seventy  days  following  chimera 
formation,  genotype  BE  was  recorded  in  all  DD  regions 
sampled  (cases  13.  15,  16),  while  genotype  DD  was  not 
detected  in  the  BE  regions  (cases  12,  14).  In  pair  combi- 
nation C  (cases  8.  9)  the  two  subclones  varied,  since  one 
contained  both  genotypes  in  the  zooid  soma  55  days  fol- 
lowing fusion,  while  the  second  was  not  detected  in  the 
other  partner's  soma  70  days  after  chimera  formation. 
Although  subclones  of  pair  combinations  A  and  B  were 
not  assigned  to  their  pretyped  genotypes,  it  is  clear  that 
six  out  of  the  seven  subclones  possessed  soma  of  both 
genotypes  up  to  125  days  after  chimera  formation  (Table 
I,  Fig.  la-c).  In  six  (75%)  of  the  remaining  eight  chimeras, 
the  other  partner's  soma  was  detected,  together  with  the 
resident  partner's  soma,  up  to  60  days  after  fusion.  Most 
interestingly,  in  all  four  chimeras  where  colony  resorption 
was  completed  (cases  17,  19-21.  Table  I,  Fig.  Ic).  the 
resorbed  genotype  was  clearly  detected  together  with  the 
"winner's"  genotype,  even  35  days  after  resorption.  In 
summary,  both  genotypes  were  recorded  in  16  (73%)  of 
the  subclones  and  10  (83%)  of  the  chimeras  (Table  I). 

In  13  of  the  cases  (9  chimeras)  studied,  gonads  (ex- 
cluding mature  oocytes)  were  sampled  from  the  same 
zooids  where  the  soma  was  typed.  In  three  cases  (33%  of 
chimeras,  nos.  H,  J.  L.  Table  I,  Fig.  Ic)  both  genotypes 
were  present  simultaneously  in  the  gonads.  These  chi- 
meras were  sampled  20-30  days  after  a  complete  im- 
munological  resorption  of  one  partner  in  the  chimera  was 
recorded  (75%  of  the  studied  resorptions).  In  all  the  nine 
cases  (five  chimeras)  where  the  partners  were  disconnected 
experimentally  or  spontaneously,  we  did  not  record  any 
chimeric  situation  in  the  gonads  up  to  100  days  after  sep- 
aration. However,  in  two  of  these  cases  (nos.  3.  12;  Table 
I,  Fig.  la,c),  the  gonads  were  typed  as  belonging  to  the 
other  genotype  in  the  chimera  while  the  corresponding 
zooid's  soma  was  only  of  the  original  genotype. 

In  an  additional  set  of  experiments,  blood  cells  were 
injected  from  pretyped  Fu/HC-compatible  allogeneic  do- 
nors into  the  blood  system  of  three  different  recipient  col- 
onies (colonies  C1-C3.  Fig.  Id).  The  recipient  colonies 
were  sampled  20  days  after  injection  and  then  30-80  days 
thereafter  (50-100  days  after  injection).  In  one  case  (C3, 


CELL  PARASITISM  IN  AN  ASCIDIAN 


109 


Table  I 

Typing  at  /cun  nil  «l  \onui  and  gonads  within  B.  schlosscri  chimeras,  45-130  days  following  fusion 

Typing  chimeras,  following  disconnections  or  resorptions 


Case 
no. 

Microsatellite 
typing  of 
chimera  partners 
1:11 

Typing  times  (days) 

Type  of 

Chimera  and              Following  chimera 
subclone  code"                    formation 

Following  disconnection 
or  rcsorption*1 

Soma                    Gonads 
1:11                          1:11 

1 

BE:DD 

Al 

125 

95.  E 

+  ++:-  +  +                   nd 

2 

A2 

45 

15.  E 

+  ++:-  +  +                   nd* 

3 

A3 

130 

100,  E 

—  :+  +  +            +++:  — 

4 

AA:DD 

61 

45 

15.  E 

+  ++:-  +  +                   nd* 

5 

B2 

45 

15,  E 

+  ++:  —  +                   nd* 

6 

B3 

125 

95,  E 

+  ++:+++            +++:  — 

7 

B4 

125 

95,  E 

+  +  +  :  —  +             +++:  — 

8 

AA:DD 

Cl  [AA] 

55 

20,  E;  40,  S 

+  ++:+  +  +                   nd* 

9 

C2  [AA] 

70 

35.  E:  55.  S 

+++:—                       nd* 

10 

AA:BE 

D  [AA] 

45 

30.  S 

+  ++:+  +  +                   nd* 

11 

AD:DD 

E[DD] 

45 

35.  S 

—  :+  +  +                   nd* 

12 

BE:DD 

Gl  [BE] 

70 

20.  S 

+  ++:  —                     —  :+++ 

13 

Gl  [DD] 

70 

20,  S 

+  ++:  +  +  +                   nd 

14 

G2  [BE] 

70 

50,  E 

+  ++:  —                 +  +  +  :  — 

15 

G3  [DD] 

70 

50.  E 

+  +  +  :  +  +  +                 —  :+++ 

16 

G4  [DD] 

70 

50.  E 

+  ++:+  +  +                 —  :+  +  + 

17 

AD:BE 

H[BE] 

60 

30,  R 

+  +  +  :  +  +  +             +  ++:+  +  + 

18 

BE:DD 

I  [BE] 

65 

40,  S 

+++:  —                +++:  — 

19 

AA:CC 

J[CC] 

55 

25,  R 

+  +  +  :  +  +  +                -+:  +  +  + 

20 

AA:BE 

K[BE] 

55 

35.  R 

+  ++:+  +  +                 —  :  +  +  + 

21 

AA:CC 

L[AA] 

50 

20,  R 

+  ++:+++             +++:  +  +  + 

"I  ") 

CC:DD 

N  [DD] 

40 

30.  S 

+  ++:+++                 —  :+  +  + 

Both 

genot>  pes  recorded  in: 

subclones 

16(73%)                  3(23%) 

chimeric  entities 

10(83%)                  3(33%) 

A  single  genotype  recorded  in: 

subclones 

6(27%)                 10(77%) 

chimeric  entities 

2(17%)                   6(67%) 

Only 

the  other  partner  recorded  in: 

subclones 

0(0%)                     2(15%) 

chimenc  entities 

0  (0%)                     2  (22%) 

'  Twelve  chimeras  are  designated  by  capital  letters  ( A-N).  When  applicable,  their  corresponding  subclones  are  marked  with  numbers.  In  brackets: 
The  original  resident's  typed  alleles  at  locus  81 1  (not  recorded  for  chimeras  A  and  B).  Chimeras  A,  B  refer  to  Figure  la,  chimera  G  to  Figure  Ib,  c 
and  chimeras  H,  J,  K.  L  to  Figure  Ic. 

b  Time  elapsed  since  disconnections  in  the  chimeras  due  to  subcloning.  an  experimental  manipulating  (E);  spontaneous  disconnection  (Rinkevich 
and  Weissman,  1989)  between  the  partners  in  the  chimera  (S).  or  resorption  (R)of  one  partner  (Rinkevich  and  Weissman,  1987b.  I992a.  b;  Weissman 
a  til..  1990;  Rinkevich  el  al..  1993). 

c  Soma  typing  was  performed  from  either  a  tissue  fragment  or  a  single  zooid.  The  gonad  was  from  the  same  zooid  sampled  for  soma.  nd  =  not 
determined  due  to  non-reproductive  state.  'Asterisks  designate  dying  colonies,  sampled  from  the  last  remains.  Autoradiographic  patterns  were 
digitized  into  one  of  four  categories:  a  strong  signal  on  the  film  after  an  overnight  exposure  (  +  +  +  )  or  a  weak  signal  (-  +  +  );  detection  only  after  three 
exposure  days  ( h)  or  below  the  detection  threshold  ( ). 


Fig.  Id),  the  hemolymph  of  "ie  recipient  unequivocally 
showed  also  the  injected  part)  er's  genotype,  which  may 
indicate  a  proliferative  process  for  the  injected  blood  cells. 
However,  in  the  sampled  zooid  and  gonads  of  this  recip- 
ient, the  injected  genotype  was  below  the  detection 
threshold. 

Discussion 

In  five  of  the  nine  ovigerous  chimeras  (56%)  we  doc- 
umented situations  where  vascular  fusion  between  allo- 


genic  B.  schlosseri  colonies  led  to  free  exchange  of  stem 
cells  (that  eventually  become  germ  cells)  across  the  former 
boundary  between  the  two  genotypes  and  to  incubation 
of  foreign  germ  lines  within  the  gonads.  In  two  of  the 
cases  only  the  other  partner's  germ  cells  were  detected. 
The  immunological  resorption  phenomenon  (Rinkevich 
and  Weissman,  1987b,  1992a,  b:  Weissman  el  al.,  1990; 
Rinkevich  et  al.,  1993),  in  which  one  partner  in  a  Botryllns 
chimera  is  morphologically  eliminated  by  phagocytosis, 
does  not  exclude  the  possibility  that  the  "resorbed  ge- 
nome" will  be  reestablished  and  coexist  within  the  "win- 


10 


Z.  PANCER  ET  AL. 


ID 

CSI 

45(151 

_     0 
IT      O 

en    i- 
5?   5 
r*    n    V 

f     r-      M 

45(151 

125(951 

125(951 

i 

5 

a 

+DD1  T 

N  N  ID  I 

0      0     0 
DOG 

O 

a 

N  IS  N  (D 

O      O 
G      O 

<  << 

S  S       S 


G4IDDJ         lil  [Mill 


I  .  Ill"   ] 


G3IUD] 


8     S 


•    •  •*•    •    .» 
.*4..t*VM.. 


-  .  • 


a     <   a     a 
0500 

A          A       A          A 

5   g  5   5 


Figure  1.  (A)  Typing  at  microsatellite  locus  8 1 1  of  genotype  combinations  A  and  B  (divided  into  subclones 
AI-A3  and  BI-B4),  Allelic  typing  (21)  of  the  partners  is  shown  in  brackets.  Compartments  sampled  are 
the  hcmolymph  (H);  a  tissue  fragment  (T);  a  single  zooid  (Z)  and  if  present,  from  the  gonads  (G)  of  the 
same  zooid.  Chimeras  were  first  sampled  25  days  after  vascular  anastomosis,  after  subcloning  (numbers  in 
parentheses  denote  days  after  subcloning).  and  upon  sexual  maturation.  Predetermined  alleles  in  the  population 
(A  =  2 16  bp,  B  =  232.  C  =  236.  D  =  240.  E  =  268)  are  depicted  at  the  left  side.  Horizontal  bar-lines  group 
simultaneous  sampling  of  subclones,  or  samples  from  several  compartments  of  a  single  subclone.  An  overnight 
exposure  of  the  film  is  shown.  (B)  A  pictorial  outline  of  genotype  combination  G  depicting  the  corresponding 
subclones.  The  chimera  was  cut  into  4  subclones.  20  days  after  fusion  (marked  lines  in  the  figure):  Gl.  the 
original  chimera  with  three  systems;  G2.  a  single  system,  an  outgrowth  from  genotype  BE;  G3  and  G4,  each 
a  single  system  originating  from  genotype  DD.  Thirty  days  later  (50  days  after  chimera  formation)  subclone 
Gl  disconnected  naturally  along  the  region  of  fusion,  into  subclone  Gl  [BE]  and  subclone  Gl  [DD].  The 
bar  line  represents  I  mm.  (O  Typing  at  microsatellite  locus  81  1  of  genotype  combinations  G,  H.  J.  K.  L. 
Allelic  typing  of  the  partners  in  each  chimera  is  shown  in  brackets.  Sampling  days  and  compartments  as  in 
Figure  la.  Subclone  sampling  from  genotype  combination  G  is  as  depicted  in  Figure  Ib.  Genotype  com- 
binations H.  J,  K,  L  were  sampled  after  the  resorption  of  one  partner  in  each  (numbers  are  for  day  after 
fusion;  numbers  in  parentheses  denote  days  after  resorption).  An  overnight  exposure  of  the  film  is  shown. 
(D)  Typing  of  three  recipient  colonies  which  were  microinjected  with  allogeneic-compatible  hemolymph 
(C1-C3).  Only  C3  was  in  good  condition,  which  allowed  full  screening  of:  hemolymph  (H).  a  zooid  (Z)  and 
the  gonad  it  contained.  Sampling  of  Cl  and  C2  was  from  the  last  remaining  tissue  fragment  (T).  Horizontal 
bars  group  chronological  sampling,  or  from  various  sources  (C3).  A  three-day  exposure  of  the  film  is  shown. 


ner"  partner's  body  in  both  the  soma  (100%)  and  the  go- 
nads (75%).  Under  these  conditions,  one  genotype  in  the 
chimera  could  parasitize  the  other  (Buss.  1982,  1983; 
Grosberg  and  Quinn.  1986;  Rinkevich  and  Weissman, 
1987a,  1992a;  Grosberg,  1988)  by  somatic  or  germ  cell 
parasitism  through  competitive  processes  among  the  ge- 
notypes over  somatic  and  gametic  positions.  The  potential 
for  cell  lineage  competition  and  parasitism  in  botryllid 
ascidians  stems  from  the  documentation  that  these  ani- 
mals maintain  self-perpetuating  stem  cell  lineages 


throughout  their  lifespan  (Berrill  and  Liu,  1948)  as  do 
many  other  organisms  (Buss,  1982,  1983).  The  displace- 
ment of  one  cell  lineage  by  another  in  some  other  groups 
has  been  suggested  to  occur  in  chimeras  of  cellular  slime 
molds  (Buss,  1982),  myxomycetes  (Clark  and  Collins, 
1973),  fungi  (Davis,  1959),  sponges  (Van  de  Vyver,  1988; 
Mukai,  1992),  and  hydractiniid  hydroids  (Hauenschild, 
1956;  Muller,  1964). 

The  results  of  the  present  study  do  not  unequivocally 
prove  somatic  and  germ  cell  parasitism.  Parasites  by  def- 


CELL  PARASITISM   IN  AN  ASC1DIAN 


11 


inition  live  at  the  expense  of  a  host  organism  (Michalakis 
el  a/..  1992).  However,  parasite/host  relationships  may 
reflect  a  continuum  flanked  by  two  extremes.  At  the  one, 
the  parasite  may  cause  the  host's  death.  At  the  other,  the 
parasite  may  evolve  as  "benign."  This  occurs  when  par- 
asite and  host  have  a  relatively  large  "shared  desiderata 
list"  (Dawkins,  1990).  Unfortunately,  the  literature  deals 
primarily  with  parasitism  by  species  of  distant  taxi',  from 
the  hosts,  and  there  is  almost  no  theoretical  treatment  to 
intraspecific  parasitism  on  the  cellular  level.  Therefore, 
we  define  germ/somatic  cell  parasitism  in  the  Botryllux 
chimeric  system  as  follows:  1.  Incubation  of  any  number 
of  alien  male/female  germ  cells  within  gonads.  This  is 
especially  marked  where  one  genotype's  oocytes  acquire 
nutrients  from  follicle  cells  (Manni  ct  a/..  1994)  of  another 
genotype,  or  compete  for  space  and  nutrition  (Sabbadin 
and  Zaniolo,  1979)  with  other  developed  germ  cells.  2. 
Penetration  and  establishment  of  alien  somatic  cells  as 
an  integral  part  of  the  resident's  soma  (zooids),  where 
they  can  support  the  development  of  germ  cells.  However, 
in  the  absence  of  cell  lineage  competition,  chimeras  should 
show  stable  sexual  expression,  with  the  original  germ  cells 
in  the  appropriate  region  of  the  chimera  (Sabbadin  and 
Astorri.  1988;  Shenk,  1991).  When  cell  lineage  competi- 
tion occurs,  four  key  features  may  be  assigned  to  char- 
acterize the  processes  involved  within  chimeric  organisms: 
1.  Cell  lineage  competition  is  evolutionarily  relevant  to 
taxa  developing  through  the  somatic  embryogenesis  or 
epigenetic  developmental  types,  where  totipotent  stem 
cells  are  competent  to  produce  germ  cells  at  any  point  in 
ontogeny  or  during  most  of  the  lifespan  of  the  organism, 
respectively  (Buss,  1982,  1983;  Tuomi  and  Vuorisalo, 
1989).  2.  These  cell  lineages  should  be  capable  of  increas- 
ing in  frequency,  eventually  establishing  an  independent 
existence  (Buss,  1982).  3.  Competing  cell  lineages  in  chi- 
meric entities  should  gain  a  disproportionate  share  of  ga- 
metic  output  by,  for  example,  restricting  the  differentia- 
tion of  the  totipotent  cells  to  gametic  descendants,  while 
using  the  somatic  constituents  of  the  other  partner  for 
maintenance  and  space  provisions  for  gametes  (Buss, 
1982.  1983;  Buss  and  Green,  1985;  Grosberg.  1988;  Sab- 
badin and  Astorri,  1988).  4.  Either  transitive  or  nontran- 
sitive  hierarchial  responses  for  somatic/germ  cell  parasit- 
ism should  be  established  in  chimeric  combinations  from 
each  studied  group  of  compatible  genotypes.  This  also 
provides  further  insights  into  the  genetic  rules  operating 
for  the  cell  lineage  competition  processes.  Somatic  em- 
bryogenesis (Berrill  and  Liu,  1948;  Buss.  1982.  1983; 
Rinkevich  and  Weissman.  1987a,  1 992a:  Grosberg.  1988). 
proliferation  of  foreign  cells  in  the  blood  (Fig.  Id:  Sab- 
badin and  Zaniolo.  1979).  and  foreign  gametic  output 
(Sabbadin  and  Zaniolo.  1979;  Sabbadin  and  Astorri.  1988; 
Fig.  la-c.  Table  I)  have  already  been  attributed  to  B. 
schlosxcri  chimeras.  There  is  also  evidence  for  a  hierarchial 


relationship  for  somatic  positions  in  Botryllus  chimeras 
(cases  12-16,  Table  I,  Fig.  lb,c). 

In  the  vast  majority  of  the  chimeras  (83%)  including 
all  chimeras  where  colony  resorption  was  recorded,  and 
in  73%  of  all  subclones  studied,  both  genotypes  were  re- 
corded in  the  soma  (Table  I,  Fig.  la-c).  One  may  therefore 
postulate  for  the  opposite  conclusion  that  these  outcomes 
are  characteristic  to  synergistic  relationships  between  the 
genotypes  within  the  chimeras  in  which,  for  example,  each 
partner  in  the  chimera  expresses  proportionally  its  ca- 
pacities for  somatic  growth  or  gametic-product  differen- 
tiation. In  such  a  case,  the  chimeric  growth/reproductive 
output  should  be  greater  than  that  of  each  genotype  alone. 
Previous  results,  however,  showed  that  neither  growth 
rates  nor  reproductive  activities  were  improved  in  Bo- 
tryllus chimeras  as  compared  to  single  colonies  or  rejecting 
partners  (Rinkevich  and  Weissman.  1992a).  It  should  also 
be  taken  into  consideration  that  the  above  results  may  be 
the  outcome  of  a  chaotic  situation  within  the  chimeras 
in  which  free  circulating  germ  cell  primordia  are  posi- 
tioned haphazardly  within  the  developing  gonads.  This 
possibility  may  be  critically  evaluated  by  establishing  pos- 
sible hierarchial  relationships  for  gametic  positions  in  Bo- 
tryllus chimeras.  The  surprising  result  that  even  after  a 
complete  resorption  of  one  partner  in  the  chimera,  the 
resorbed  genotype  may  continue  to  thrive,  not  only  in 
the  form  of  germ  cells  but  also  as  an  integral  part  of  the 
"winner's"  soma,  may  further  suggest  that  the  Fu/HC 
(Weissman  el  a/..  1990)  and  the  resorption-histocompat- 
ibility  (Re/HC;  Rinkevich,  1993)  loci  that  are  very  effec- 
tive in  self-nonself  discrimination  of  many  already  deter- 
mined cell  lineages  do  not  distinguish  (or  do  not  activate 
the  effector  arm  against)  the  most  important  group  of  the 
stem  cells.  In  such  a  scenario,  the  freely  circulating  stem 
cells  may  uninterruptedly  differentiate  into  both  gametes 
and  somatic  tissues  throughout  the  life  span  of  the  original 
chimera  and  its  corresponding  subclones,  which  are  cre- 
ated by  splitting  the  chimera  into  fragments.  Different 
subclones  probably  possess  unlike  proportions  of  the 
mixed  genotype's  stem  cells,  which  may  result  in  the  vari- 
ations observed  for  the  chimeric  constituents  of  both  soma 
and  gametes.  Therefore,  more  attention  should  be  paid 
to  possible  interactions  between  different  lines  of  stem 
cells  (such  as  the  displacement  of  one  at  the  expense  of 
another)  rather  than  to  the  idea  of  somatic  germ  cell  par- 
asitism in  the  context  of  the  evolution  of  somatic  tissue 
compatibility  (Buss.  1982;  Buss  and  Green,  1985;  Rin- 
kevich and  Weissman.  1987a:  Grosberg.  1988). 

Within  B.  stiilosseri  chimeras,  transferred  cells  between 
genotypes  may  persist  for  many  blastogeneic  generations 
as  circulating  elements  before  maturing  as  sperm  or  eggs 
(Sabbadin  and  Zaniolo,  1979;  Sabbadin  and  Astorri, 
1988).  In  B.  sclilosscri,  therefore,  stem  cells  are  probably 
the  level  at  which  natural  selection  could  act,  rather  than. 


112 


Z.  PANCER  ET  AL. 


as  believed,  at  the  colony  level  (Tuomi  and  Vuorisalo, 
1989).  By  using  molecular  markers,  such  as  microsatel- 
lites, new  discoveries  can  shed  more  light  on  the  evolu- 
tionary processes  that  shaped  chimerism  and  the  detailed 
consequences  of  fusion  between  compatible  allogeneic 
organisms. 

Acknowledgments 

We  are  grateful  to  R.  K.  Grosberg  and  D.  Stoner  for 
critically  reading  the  manuscript,  to  R.  Pancer  for  editing 
a  first  draft  of  the  manuscript,  and  to  Elul  productions 
for  graphic  assistance.  This  study  was  supported  by  a  Ca- 
reer Development  Award  to  B.R.  from  the  Israel  Cancer 
Research  Foundation — U.S.,  a  grant  from  the  U.S.-Israel 
Binational  Science  Foundation,  and  a  grant  from  S.  Price, 
NAF/IOLR. 

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Sexual  Dimorphism  and  Niche  Divergence  in 
a  Mid-Water  Octopod  (Cephalopoda:  Bolitaenidae) 


JANET  R.   VOIGHT 

Department  of  Zoology,  The  Field  Museum  of  Natural  History,  Roosevelt  Rd.  at  Lake  Shore  Drive, 

Chicago,  Illinois  60605 


Abstract.  In  the  translucent  mid-water  octopod  Ele- 
donella  pygmaea.  the  posterior  salivary  glands  that  release 
proteolytic  enzymes  into  the  esophageal  crop  grow  five 
times  faster  in  males  than  in  females.  I  suggest  that  the 
sexes  vertically  partition  the  water  column  and  that  large 
glands  have  evolved  in  males  as  a  result  of  their  deep- 
water  habitat.  Members  of  the  species  undergo  ontogenetic 
vertical  descent  and  are  suggested  to  mate  at  the  lower 
end  of  the  adult  depth  range  where  receptive  females  signal 
males  with  light  organs.  Selection  for  increased  fitness  is 
inferred  to  result  in  females  increasing  their  fecundity  by 
feeding  at  the  upper  limit  of  the  adult  range  and  in  mature 
males  increasing  their  encounters  with  mates  by  living  at 
depths  where  mating  occurs.  To  further  increase  their  fit- 
ness, mature  males — despite  occurring  in  a  prey-limited 
habitat — must  expend  energy  to  visually  detect  potential 
mates,  to  travel  over  wide  areas,  and  to  attempt  to  copu- 
late. To  increase  the  energy  available  to  them,  males  at 
depth  may  exploit  bioluminescent  prey.  The  large  glands 
protect  the  translucent  males  from  increased  predation 
by  physically  blocking  light  emitted  by  bioluminescent 
prey  in  their  crops,  and  by  speeding  digestion. 

Introduction 

Because  it  acts  directly  on  sexually  dimorphic  traits, 
sexual  selection,  produced  by  interaction  between  the 
sexes,  has  been  assigned  a  primary  role  in  the  evolution 
and  maintenance  of  sexual  dimorphism.  Ecological  factors 
contribute  to.  and  theoretically  drive,  the  evolution  of 
sexual  dimorphism  but  are  rarely  considered  to  be  major 
factors  in  its  evolution  (Slatkin.  1984;Shine,  1989).  Slatkin 
( 1984)  noted  three  ways  in  which  ecological  factors,  pro- 
duced by  the  interaction  of  members  of  each  sex  with  the 

Received  2  September  1994:  accepted  7  July  1995. 


environment,  could  result  in  sexual  dimorphism.  Sexual 
dimorphism  could  evolve  when  a  species  has  a  dimorphic 
niche,  due  to  sex-linked  differences  in  ecological  or  social 
roles,  when  two  or  more  optima  exist  for  both  sexes,  or 
when  very  high  competitive  pressure  results  in  divergence 
of  the  niches  the  sexes  occupy,  allowing  resource  parti- 
tioning. Selander  ( 1972)  argued  that  ecological  factors  are 
most  likely  to  result  in  sexual  dimorphism  of  the  trophic 
organs,  although  this  need  not  always  be  the  case. 

This  paper  describes  sexual  dimorphism  in  posterior 
salivary  gland  size  in  the  mid-water  octopod  Eledonella 
pygmaea  Verrill.  Selection  on  males  to  find  mates  and 
on  females  to  increase  their  fecundity  is  hypothesized  to 
have  led  to  sexually  dimorphic  niches.  Dimorphism  in 
the  glands,  which  are  thought  to  produce  and  release  pro- 
teolytic enzymes  (Boucaud-Camou  and  Boucher-Rodoni, 
1983),  is  hypothesized  to  be  due  to  the  divergence  of  pos- 
terior salivary  gland  growth  in  males,  as  an  ecological  ad- 
aptation to  their  deep-water  distribution. 

Biology  of  Eledonella  pygmaea  I  'errill 

Members  of  the  species  Eledonella  pygmaea,  typical  of 
the  little-known  bolitaenid  octopods,  occur  at  depths 
greater  than  100  m  in  mid-latitudes  (Thore,  1949).  Mem- 
bers of  the  family  Bolitaenidae  descend  in  the  water  col- 
umn as  they  mature.  Although  juveniles  occur  near  the 
upper  limits  of  the  species  range,  larger  individuals  occur 
variably  between  depths  of  500  and  3250  m  (Thore.  1949: 
Young,  1978).  Gravid  and  nearly  gravid  females  are  col- 
lected only  from  the  deepest  part  of  the  species  range, 
although  brooding  females  are  collected  from  shallow 
depths  of  the  adult  range.  This  distributional  pattern  led 
Young  ( 1978)  to  conclude  that  mating  occurs  at  the  lower 
limit  of  the  species  depth  range  and  that  hatchlings  are 
released  near  the  upper  limit  of  the  adult  distribution. 


113 


14 


J.  R.  VOIGHT 


Fully  mature  males,  defined  as  those  carrying  spermato- 
phores  (Mangold,  1987),  have  not  to  my  knowledge  been 
reported  in  the  literature.  Low-density  salts  contained  in 
fluid-tilled  vacuoles  in  the  arm  and  mantle  musculature 
may  allow  the  animals  to  approach  neutral  buoyancy 
(Denton  and  Shaw,  1961).  The  fluid  in  the  muscles  may 
also  increase  the  translucence  of  the  animals  and  their 
susceptibility  to  severe  damage  during  trawl  collection,  a 
feature  which  precludes  direct  behavioral  observations  of 
the  animals. 

The  general  anatomy  of  the  anterior  digestive  system 
is  typical  of  incirrate  octopods(Thore,  1949),  all  of  which 
are  predators.  The  esophagus  and  its  diverticulum,  the 
crop,  lie  on  the  dorsal  surface  of  the  digestive  gland  within 
the  mantle  cavity  (Fig.  1).  A  pair  of  posterior  salivary 
glands  straddle  the  esophagus  at  the  level  of  the  crop  di- 
verticulum (Fig.  1);  the  anterior  salivary  glands  are  at- 
tached to  the  buccal  mass.  Two  ducts,  one  from  each 
posterior  salivary  gland,  merge  to  follow  the  esophagus 
anteriorly  to  the  buccal  mass  at  the  center  of  the  arms.  A 
second  duct  from  each  gland  enters  the  crop  diverticulum 
directly.  The  opening  of  the  crop  is  muscular,  but  its  sac- 
cular  portion,  in  preserved  specimens,  is  nearly  transpar- 


ent. As  in  all  incirrate  octopods,  the  dorsal  viscera  are 
covered  by  a  sheath  that  carries  chromatophore  organs. 
The  distribution  of  these  organs  is  distinctive  in  bolitae- 
nids;  few  chromatophore  organs  are  broadly  scattered  over 
the  dorsal  crop,  but  chromatophore  organs  are  densely 
packed  over  the  stomach,  just  dorsal  to  the  tip  of  the 
mantle. 

Females  of  E.  pygmaea  and  other  bolitaenids  develop 
a  circumoral  light  organ  at  sexual  maturity,  apparently 
to  attract  potential  mates  (Robison  and  Young,  1981). 
The  light  organ  is  probably  not  used  in  feeding,  first  be- 
cause the  octopod  could  not  see  prey  attracted  to  it,  and 
second  because  the  green  color  of  the  emitted  light  is 
thought  to  be  ineffective  in  luring  prey  (Robison  and 
Young,  1981 ).  Females  are  not  thought  to  feed  after  the 
circumoral  light  organ  develops. 

Mature  females  are  characterized  by  the  circumoral 
light  organ  and  increased  pigmentation  on  the  web  and 
arm  crown  (Rancurel,  1970,  Plate  II:  Robison  and  Young, 
1981).  Females  brooding  eggs  are  characterized  by  a  sealed 
buccal  mass,  a  deep  web,  and  deterioration  of  the  digestive 
system  (Young,  1972a:  1978).  As  females  become  senes- 
cent, their  consistency  becomes  very  gelatinous,  parasites 


f-^- 


Figure  1.  An  oblique  view  of  a  male  (A)  and  a  dorsal  view  of  a  female  (B)  speeimen  of  Elednnclla 
pyKiiiucii.  The  dorsal  mantle  has  been  removed  in  both  specimens  to  show  the  esophagus  entering  the  mantle 
cavity  and  the  posterior  salivary  glands  ( I )  on  the  dorsal  surface  of  the  digestive  gland  which,  in  the  female 
(B),  straddles  the  esophageal  crop  (.).  ( I )  the  posterior  salivary  glands:  (2)  esophageal  crop,  visible  only  in 
the  female  (B);  (3)  stomach,  visible  only  in  ( Blatter  removal  of  the  overlying  membrane  with  its  chromatophore 
organs;  and  (4)  the  gonad  and  (in  \)  the  accessory  male  ducts. 


SEXUAL  DIMORPHISM  IN  BOLITAENIDAE 


15 


become  more  prominent,  and  the  digestive  gland  is  re- 
duced and  becomes  nearly  transparent  (Young,  1978: 
Voight,  pers.  obs.).  Deterioration  of  the  digestive  organs 
supports  the  hypothesis  that  bolitaenids  produce  a  single 
clutch  of  eggs,  as  is  typical  of  octopods  (Mangold,  1987). 
In  addition,  females  probably  brood  their  eggs  in  their 
arm  crown  until  the  eggs  hatch  (Young,  1972a).  As  the 
mouth  is  at  the  center  of  the  arm  crown,  brooding  eggs 
would  be  inconsistent  with  feeding. 

In  laboratory  experiments,  bioluminescence  has  been 
elicited  from  the  digestive  glands  of  10  bolitaenid  speci- 
mens, collected  near  Oahu,  Hawaii,  and  identified  as  E. 
pygnuiea  and  Japeiella  diaphana  (Young  el  al,  1979). 
The  digestive  glands  of  "most  specimens  of  both  species" 
(Young  el  al.,  1979:  p.  74)  examined  were  reported  to 
emit  detectable  light,  but  neither  the  gills  (/;  =  3)  nor  the 
stomach  (/;  =  1)  did  so.  Unfortunately,  neither  the  sex 
nor  the  feeding  status  of  the  bioluminescent  bolitaenids 
was  reported. 

Materials  and  Methods 

Sixty  specimens  ofEledonella  pygmaea  (Table  I)  that 
share  meristic  characters  of  the  gill  lamellae  and  suckers 
are  the  basis  of  this  study.  The  sex  of  each  specimen  was 
determined  by  internal  examination:  males  were  identified 
by  the  presence  of  a  single  genital  duct  and  females  by 
the  presence  of  paired  genital  ducts:  the  sex  of  one  indi- 
vidual could  not  be  determined.  G-tests  were  used  to  de- 
termine whether  the  sex  composition  of  the  sample  dif- 
fered from  unity  and  whether  the  presence  of  senescent 
females  and  of  juveniles  significantly  differed  with  the 


month  of  the  year.  A  Wilcoxon  two-sample  test  was  used 
to  look  for  significant  size  differences  between  the  sexes. 

Because  accurate  measurement  required  that  the  spec- 
imens be  dissected,  only  40  of  the  comparatively  rare 
specimens  were  measured  for  this  analysis.  The  1 1  mea- 
surements recorded  included  dorsal  mantle  length,  from 
the  midpoint  between  the  eyes  to  the  posterior  tip  of  the 
mantle:  mantle  width,  measured  with  the  calipers  touching 
the  digestive  gland  through  the  mantle  wall;  head  width, 
the  maximum  width  of  the  head  including  the  eyes;  diges- 
tive gland  length,  the  maximum  length  of  the  organ:  pos- 
terior salivary  gland  length,  tip  to  tip  on  the  dorsal  surface 
on  the  left  gland;  pupil  length,  along  its  longest  axis;  eye 
length,  along  its  greatest  axis;  and  arm  length,  from  the 
first  sucker  to  the  arm  tip  on  the  oral  surface.  Arm  lengths 
were  averaged  within  each  of  the  four  arm  pairs.  Addi- 
tional characters,  such  as  esophageal  crop  diameter,  were 
not  measured  because  preservation  bias  caused  by  the 
presence  or  absence  of  food  in  the  crop  at  fixation  violated 
assumptions  implicit  in  the  application  of  morphometric 
analyses  to  soft-bodied  organisms  (Voight.  1991)  and  be- 
cause clearly  defined  endpoints  on  which  to  base  the  mea- 
surements are  absent.  In  this  analysis,  preserved  specimens 
of  a  wide  range  of  sizes  are  included. 

Data  were  transformed  to  natural  logarithms  (In),  a 
technique  that  preserves  allometries,  standardizes  vari- 
ances, and  produces  a  scale-invariant  covariance  matrix 
(Jolicoeur,  1963).  The  In-transformed  data  were  entered 
into  a  principal  components  analysis  (hereafter  referred 
to  as  PCA)  using  PROC  FACTOR  in  SAS  (SAS  Inst., 
1987);  and  principal  components  (hereafter  termed  PC) 


Table  I 

Summary  oj  information  for  the  lots  oj  specimens  examined  mit\ciim  catalog  number.  collection  locality,  number  of  .V/ 
anil  Jcplli 


/WITO,  and  collection  date 


Museum 
number' 

Latitude  (°N) 

Longitude  (°W)                     A 

Collection  month,  year 

Depth  (m) 

FMNH  78332" 

32°  13.  3' 

64°37' 

I                         August  1948 

730-820 

FMNH  278057 

32°  13' 

64°40.5' 

July  1948 

1953 

USNM  792006** 

32°04' 

63°58'                                  2< 

)                            August  1971 

0-1025 

FMNH  78333 

32° 

64°51.7' 

1                          July  1948 

1000-1100 

UMML  31.2564 

29=4' 

87°37' 

April  1961 

186 

UMML  31.171 

28°58' 

88°00' 

October  1953 

1544_1730 

UMML  3  1.2030* 

26°30' 

90°42' 

July  1959 

2790 

UMML  31.2031* 

23°35.25-36.3' 

76°54.25-55.1' 

April  1975 

1000 

UMML  31.2032 

23°59.7-24°1.2' 

75°46.75-47.5' 

November  1974 

1900 

UMML  31.2033* 

23°38.0-40.5' 

76°52.4-55.25' 

August  1975 

1000 

UMML  31.1701* 

23°12.6 

90°44.r 

!                          November  1975 

2000 

UMML  31.2207 

21  "56.3-51' 

65°4.0-64°57.5I 

Jul>  1971 

1000 

UMML  3  1.2565 

19°  16' 

65°51' 

!                          July  1971 

7282-7363 

1  FMNH  =  The  Field  Museum  of  Natural  History:  L'MML  =  The  University  of  Miami  Marine  Laboratory;  USNM  =  The  United  States  Museum 
of  Natural  History. 

*  Lots  with  one  senescent  female:  **  lots  with  two  senescent  females. 


116 


J.  R.  VO1GHT 


were  computed  from  the  covariance  matrix  (e.g.  Strauss, 
1985).  The  algorithm  requires  that  individuals  without 
complete  data  be  deleted;  due  to  trawl  damage,  the  num- 
ber of  specimens  contributing  to  the  multivariate  analysis 
was  limited  to  35:  1 1  males,  23  females,  and  one  unknown. 
PCA  is  a  powerful  multivariate  technique  that  examines 
patterns  of  morphological  variation  regardless  of  a  priori 
group  definitions.  Because  culturing  individuals  through 
the  life  cycle  and  analyzing  their  growth  at  regular  intervals 
is  impossible  in  this  species,  this  analysis  uses  each  pre- 
served specimen  as  a  proxy  for  the  species  at  that  size.  In 
this  manner,  analysis  of  museum  specimens  quantifies 
allometric  patterns.  Analysis  of  specimens  of  a  wide  size 
range,  as  in  this  case,  is  predicted  to  reveal  that  size  con- 
tributes most  morphological  variation  observed.  All  mea- 
surement data  from  each  specimen  entered  in  the  analysis 
are  predicted  to  reflect,  to  a  greater  or  lesser  extent,  the 
specimen's  size,  as  the  parts  are  expected  to  increase  with 
increasing  size.  PCA  identifies  this  unique  pattern  of 
strong  positive  covariance  among  the  characters  it  ana- 
lyzes as  overall  size.  This  size  variation  is  assigned  to  a 
component,  usually  PCI,  that  can  be  recognized  by  the 
uniformly  large  positive  loadings  of  each  character.  Par- 
titioning size  to  a  single  component  allows  the  analysis 
to  consider  shape  variation  without  the  confounding  ef- 
fects of  size.  The  absolute  value  of  the  loading  of  each 
character  on  each  component  identifies  how  that  character 
contributes  to  size  (on  PCI)  and  shape  variation  (on  sub- 
sequent components).  Each  specimen  is  assigned  a  score 
on  each  component;  the  score  signifies  its  position  on  that 
component  relative  to  the  others  in  the  analysis. 

When  PCA  revealed  that  a  single  measurement — pos- 
terior salivary  gland  length — contributed  most  size-free 
shape  variation,  the  natural  logarithm  of  that  measure- 
ment was  plotted  against  In  mantle  length  to  express  the 
shape  variation  of  the  gland  in  two  dimensions.  This  pro- 
cedure also  increased  the  number  of  specimens  (12  males 
and  28  females)  contributing  to  the  calculation  of  the 
equation  of  the  line  describing  the  growth  of  the  character 
in  members  of  each  sex  relative  to  mantle  length. 

The  distribution  of  chromatophore  organs  on  the  sheath 
superficial  to  the  crop  was  compared  between  males  and 
females,  as  was  the  transparency  of  the  sheath.  The 
esophageal  crops  of  nine  individuals  were  opened  and 
their  contents  examined.  To  test  whether  the  olfactory 
papillae  (the  paired,  fan-shaped  papillae  projecting  from 
the  lateral  edges  of  the  mantle  opening)  are  sexually  di- 
morphic, as  olfactory  organs  frequently  are  in  fishes  from 
depths  of  1000-4000  m  (Marshall,  1967),  the  maximum 
dimension  of  the  right  olfactory  papilla  was  plotted  against 
mantle  length  for  7  male  and  10  female  specimens.  The 
papillae  detect  water-borne  chemicals  in  squids  (Gilly  and 
Lucero,  1 992);  whether  the  organs  function  in  this  manner 
in  octopods  is  yet  to  be  demonstrated. 


Results 

As  predicted,  size  contributed  most  (82.0%)  of  the 
morphological  variation  revealed  by  PCA  (Table  II).  One 
measurement,  posterior  salivary  gland  length,  contributed 
most  of  the  size-free  shape  variation  (8.48%  of  the  total 
morphological  variation).  This  variation  is  due  to  differ- 
ences between  males  and  females,  as  is  evident  when  in- 
dividual scores  on  PC2  (size-free  shape  variation)  are 
plotted  against  PCI  (overall  size  variation)  (Fig.  2).  Diges- 
tive gland  length  contributed  most  to  shape  variation  on 
PC3.  due  largely  to  data  from  a  single  senescent  female. 

The  dimorphism  is  evident  in  individuals  with  mantle 
lengths  greater  than  18  mm  (Fig.  3).  The  growth  rates  of 
the  posterior  salivary  glands  relative  to  mantle  length  dif- 
fered strongly  between  the  sexes.  The  positively  allometric 
growth  of  male  posterior  salivary  gland  length  (PSG)  rel- 
ative to  mantle  length  (ML),  with  both  expressed  as  nat- 
ural logs  (In),  is  described  by  the  equation; 

In  PSG  =  2.3  (In  ML)  -  4.97. 

The  allometric  coefficient  of  posterior  salivary  gland  length 
in  females  is  one-fifth  of  the  gland's  coefficient  in  males; 
its  negatively  allometric  growth  is  described  by  the  equa- 
tion: 

In  PSG  =  0.46  (In  ML). 

The  dimorphism  of  posterior  salivary  gland  size  appears 
to  correlate  with  qualitative  characters.  Although  the  dis- 


Table  II 

Loadings  of  each  n/'lhc  II  characters  on  the  first  three  principal 
cuinpiincnt.f  (PC)  fnnn  the  analysis  <>]  35  specimens  of 
Eledonella  pygmaea 


Character 

PCI 

PC2 

PC3 

Mantle  length 

0.941 

-0.007 

0.178 

Mantle  width 

0.903 

-0.135 

0.151 

Head  width 

0.950 

-0.064 

0.106 

Digestive  gland  length 

0.787 

0.194 

0.533 

Posterior  salivary  gland  length 

0.531 

0.834 

-0.146 

Pupil  length 

0.716 

0.154 

0.273 

Eye  length 

0.890 

0.015 

0.255 

Arm  length  I 

0.975 

-0.120 

-0.092 

Arm  length  11 

0.968 

-0.120 

-0.132 

Arm  length  III 

0.984 

-0.001 

-0.116 

Arm  length  IV 

0.966 

-0.138 

-0.020 

Total  proportion  of  variation 

explained  by  each 

component: 

82.00 

8.48 

3.74 

PC  1  represents  overall  size  variation,  as  is  indicated  by  the  strongly 
positive  loadings  for  each  character.  PC2  represents  size-free  variation 
in  posterior  salivary  gland  length,  as  is  indicated  by  the  character's  sin- 
gularly large  loading  on  PC2  PC3  represents  size-free  variation  in  digestive 
gland  length,  as  is  indicated  by  its  high  loading. 


SEXUAL  DIMORPHISM  IN  BOLITAENIDAE 


117 


2.0 


1.0 


i  0.0 


-1.0 


-2.0 


m 
m 


jn 


f     f     f 


ft 

f       f 


-2.0 


-1.0 


0.0 


PC  1 


1.0 


2.0 


Figure  2.  Specimen  scores  on  principal  component  2  (PC2)  (rep- 
resenting size-free  shape  variation  in  posterior  salivary  gland  length)  are 
plotted  against  scores  on  principal  component  1  (PCI)  (representing 
overall  size),  m,  male  specimens;  f,  female  specimens;  j.  specimen  of 
unknown  sex. 


tribution  of  chromatophore  organs  on  the  sheath  over  the 
dorsal  viscera  appears  to  be  very  nearly  the  same  in  both 
sexes,  the  transparency  of  the  sheath  differs  with  sex.  In 
preserved  males,  the  silvery  iridescence  of  the  sheath  ef- 
fectively obscures  the  underlying  organs:  the  sheath  had 
to  be  removed  to  see  the  underlying  posterior  salivary 
glands  that  effectively  cover  the  small  crop  (Fig.  la).  In 
females,  the  crop  and  its  contents  are  readily  visible 
through  the  sheath:  the  posterior  salivary  glands  cover 
only  the  medial  portion  of  the  large  crop  (Fig.  Ib). 

The  crops  of  three  of  the  six  males  examined  were 
empty;  the  crops  of  the  three  other  males  and  all  three 
females  contained  fish  scales,  parts  of  crustacean  exo- 
skeletons,  and  an  apparently  parasitic  worm.  The  only 
prey  item  that  was  identifiable  to  species  was  a  conspecific. 
identified  by  an  arm,  in  the  crop  of  a  female. 

The  olfactory  papillae  of  males  and  females  were  similar 
in  size.  Damage  to  the  skin  overlying  the  eye  appeared  to 
be  associated  with  distortion  of  the  papillae,  regardless  of 
the  sex  of  the  specimen. 

The  sex  ratio  was  significantly  female-biased  (38  females 
to  21  males,  G  =  4.97;  p  <  0.05).  Among  the  specimens 
examined,  the  incidence  of  individuals  smaller  than 
10  mm  mantle  length  did  not  significantly  differ  among 
the  months  for  which  samples  were  available  (G  =  1.39: 
p  >  0.05 ).  Of  the  specimens  analyzed,  those  with  the  long- 
est mantles,  the  traditional  estimator  of  cephalopod  size 
(Fig.  3),  and  with  the  highest  PCI  scores  (Fig.  2)  were 


female.  The  PCI  scores  of  males  and  females  did  not, 
however,  significantly  differ  (Wilcoxon  two-sample  test. 
/  =  \A9;p>  0.40). 

Eight  of  the  38  females  examined  appeared  to  be  re- 
productively  mature  or  nearing  senescence.  The  largest 
ovarian  eggs  found  in  a  female  were  1.85  mm  long,  only 
0. 1 5  mm  less  than  the  longest  egg  definitively  reported  as 
being  from  a  specimen  of  Eledonella  (Young.  1978);  no 
hatchlings  were  present.  The  incidence  of  senescent  fe- 
males did  not  significantly  differ  among  the  months  for 
which  samples  were  available  (G  =  1.16;  p  >  0.05).  The 
collection  depths  of  the  post-brooding,  senescent  females 
are  uninformative  about  the  depth  at  which  mating  occurs. 
Among  the  male  specimens  examined,  none  were  repro- 
ductively  mature,  i.e..  none  contained  spermatophores; 
but  males  with  enlarged  reproductive  organs  were  found, 
and  these  were  probably  nearing  sexual  maturity. 

Discussion 

The  growth  rate,  relative  to  the  mantle  length,  of  the 
posterior  salivary  glands  is  five  times  faster  in  males  of 
Eledonella  pygmaea  than  it  is  in  females,  and  as  a  result, 
the  glands  of  males  are  up  to  two-and-a-half  times  larger 
than  those  of  conspecific  females  of  similar  size  (Fig.  1). 
Relying  on  distributional  data  from  Young  (1978)  and 
our  limited  biological  knowledge  of  the  species.  I  argue 


m 

2.50 

ra 

m 

g 

ra              m 

5 

m 

o 

32.00 

m 

| 

<j 

ra 

on 

P4 

f     f 

0 
|  1.50 

f     f 
f       f          f       ff          f 
f     f         f 

Cfl 

2 

f        m                                  f 
f     m    m              f 

3 

f                                                f 

ff 

f 

1.00 

m.             f 

2.50  2.75  3.00  3.25 

LN  MANTLE  LENGTH 


3.50 


Figure  3.  The  natural  logarithm  (In)  of  posterior  salivary  gland  length 
is  plotted  against  In  mantle  length  for  40  individuals  of  Eledonella  pyg- 
maea.  m.  male  specimens;  f.  female  specimens;],  specimen  of  unknown 
sex.  For  the  equations  of  the  lines  describing  the  glands  growth  in  males 
and  females,  see  text. 


118 


J.  R.  VOIGHT 


that  this  sexual  dimorphism  results  from  the  adaptation 
of  males  to  their  deep-water  habitat.  Further.  I  posit  that 
inferred  sex-specific  selective  forces  are  responsible  for 
sexually  dimorphic  depth  distributions. 

To  discuss  the  evolution  of  a  character,  its  primitive 
condition  must  be  established,  and  in  this  case,  I  consider 
small  posterior  salivary  glands  to  be  the  ancestral  condi- 
tion. The  Bolitaenidae  appears  to  be  the  basal  lineage  of 
the  suborder  Incirrata  (Voight,  unpub.  data).  Members 
of  the  sister  taxon,  the  suborder  Cirrata,  lack  posterior 
salivary  glands  in  the  mantle  cavity;  the  glands  of  the 
outgroup,  the  order  Vampyromorpha,  are  very  small 
(Young,  1964).  Sexual  dimorphism  of  the  glands  and  the 
particularly  large  size  they  reach  in  males  appear  to  be 
uniquely  shared  among  bolitaenid  species  (Voight,  unpub. 
data). 

Given  the  difficulties  in  observing  bolitaenids  in  nature, 
we  must  infer  how  selective  pressures  on  males  and  fe- 
males differ,  as  differences  are  required  for  sexual  dimor- 
phism to  evolve.  Selection  acts  to  increase  the  depth  dis- 
tribution of  males.  A  corollary  to  Young's  (1978)  hy- 
pothesis that  bolitaenids  mate  at  great  depths  predicts  that 
mature  males  occur  at  those  depths  to  increase  the  number 
of  receptive  females  they  encounter.  Selection  also  acts 
to  intensify  the  sensitivity  of  males  to  light  cues  and  to 
increase  male  mobility.  Because  responding  to  a  female's 
light  cue  increases  a  male's  chances  of  mating  (Robison 
and  Young,  1981),  males  that  are  better  able  to  detect 
bioluminescence  will  have  higher  fitness.  To  further  in- 
crease the  number  of  receptive  females  that  they  detect, 
males  should  be  highly  mobile.  Males  that  move  across 
broad  areas  are  likely  to  see  more  mates  than  are  males 
who  search  only  locally.  The  absence  of  mature  males 
from  trawl  collections  (this  study,  u  =  60;  Young,  1978, 
//  =  80),  if  due  to  net  avoidance,  supports  the  hypothesis 
of  increased  male  mobility. 

In  females,  selection  acts  to  increase  fecundity,  a  feature 
tightly  linked  to  body  size  in  cephalopods  (Mangold, 
1987).  To  grow  large  rapidly,  females  may  remain  in 
comparatively  shallow  depths  where  the  crustaceans  and 
fishes  that  females  and  juvenile  males  exploit  as  prey  are 
more  abundant.  Females  descend  to  greater  depths  only 
when  ready  to  mate.  Selection  will  not  heighten  sensitivity 
to  bioluminescence  or  increase  mobility  in  females,  except 
to  the  extent  that  the  traits  are  under  selection  in  conspe- 
cific  males. 

The  sex-specific  selective  forces  outlined  above  suggest 
that  the  variability  in  size  at  which  bolitaenids  descend 
to  adult  depths  documented  by  Young  (1978)  is  sex- 
linked.  Reproductively  mature  males  occur  at  the  lower 
end  of  the  species'  depth  range;  females  occur  at  these 
depths  only  when  ready  to  mate.  If  this  hypothesis  of  hab- 
itat-partitioning between  the  sexes  is  supported,  the  evo- 


lution of  large  posterior  salivary  glands  in  males  can  be 
argued  to  relate  to  ecological  factors. 

As  mature  males  descend  in  the  water  column,  the 
abundance  of  familiar  prey  declines,  and  their  energy  costs 
may  increase.  Males  must  pay  the  metabolic  costs  thought 
to  be  associated  with  high  visual  acuity  (Childress,  1995) 
if  they  are  to  detect  mates.  To  meet  these  metabolic  de- 
mands, maintain  their  capacity  for  high  mobility,  and 
prolong  their  survival  in  this  habitat,  I  suggest  that  males 
use  their  visual  acuity  to  exploit  bioluminescent  prey, 
which  increases  in  abundance  with  depth. 

The  shift,  with  depth,  to  the  selection  of  bioluminescent 
prey  carries  with  it  a  major  liability.  A  translucent  boli- 
taenid risks  predation  if  prey  in  its  crop  emits  light.  En- 
larged posterior  salivary  glands,  however,  reduce  the  risk 
of  predation  in  two  ways.  First,  the  large  glands,  and  the 
iridescence  of  the  sheath  overlying  the  dorsal  viscera,  ef- 
fectively cover  the  crop  of  males  and  would  physically 
block  light  emitted  from  within  it.  Second,  assuming  that 
large  glands  release  greater  volumes  of  proteolytic  enzymes 
than  do  small  glands,  large  glands  would  speed  the  catab- 
olism  of  bioluminescent  chemicals. 

One  could  argue  that,  if  gland  size  correlates  with  the 
potency  or  volume  of  the  proteolytic  enzymes  released, 
males  with  large  glands  would  digest  prey  more  quickly, 
lowering  their  energetic  cost  of  swimming,  but  the  diges- 
tive gland  is  a  primary  site  of  food  absorption  in  cepha- 
lopods (Boucaud-Camou  and  Boucher-Rodoni.  1983). 
Therefore,  if  males  were  under  selection  to  maintain  con- 
stant body  weight  by  speeding  the  digestion  and  absorption 
of  prey,  the  digestive  gland  should  also  be  dimorphic.  PCA 
falsifies  this  prediction  (Table  II). 

Alternatively,  one  could  argue  that  females  face  a  sim- 
ilar liability.  Among  octopuses,  however,  gravid  females 
are  rarely  active  predators.  As  their  eggs  enlarge,  females 
typically  reduce  their  feeding  rates  (Mangold,  1987).  If 
this  generalization  holds  true  for  bolitaenids,  gravid  fe- 
males are  not  likely  to  ingest  prey  while  near  the  lower 
limits  of  the  species  range.  This  physiological  pattern  also 
argues  against  sexual  cannibalism  as  a  routine  strategy  in 
the  species.  Although  sexual  cannibalism  explains  the 
presence  of  a  conspecific  in  the  crop  of  a  female,  so  does 
the  animal  having  fed  in  the  trawl.  Sexual  cannibalism 
also  explains  the  rarity  of  mature  males,  as  does  the  hy- 
pothesis of  increased  male  mobility. 

The  presence  of  sexually  dimorphic  posterior  salivary 
glands  was  unsuspected  in  this  taxon.  This  striking  dif- 
ference in  the  digestive  system  had  been  attributed  to  dis- 
similar nutritional  states  of  the  specimens  (Thore,  1949) 
and  to  the  existence  of  cryptic  species  (Young,  1972b). 
Indeed,  sex-linked  differences  have  not  been  noted  in  any 
of  the  taxonomically  diverse  deep-sea  predators  in  which 
dark  peritoneums  or  digestive  organs  have  been  suggested 
to  camouflage  bioluminescent  prey  (e.g.,  vampyromorph 


SEXUAL  DIMORPHISM  IN  BOLITAENIDAE 


19 


cephalopods.  Pickford,  1949;  fishes,  McAllister,  1961). 
The  rarity  with  which  deep-sea  animals  such  as  these  are 
seen  in  nature  may  limit  our  ability  to  document  a  sex- 
linked  difference,  especially  if  the  sexes  partition  habitats, 
as  suggested  here. 

If  the  posterior  salivary  glands  camouflage  ingested  lu- 
minescent prey  as  effectively  as  this  study  indicates,  direct 
observation  of  living  animals  may  not  detect  their  pres- 
ence. Museum  specimens  collected  incidentally  in  the 
previous  half  century  through  a  variety  of  research  efforts 
do,  however,  prov  ide  the  anatomical  and  allometric  data 
that  are  critical  not  only  to  documenting  the  patterns,  but 
to  generating  this  hypothesis  of  its  evolution,  including 
polarity  assessment.  Although  the  hypotheses  could  not 
have  been  generated  using  observations  of  live  animals, 
the  critical  tests  of  the  hypotheses — determining  prey 
preferences  and  energetic  costs  experienced  by  males  at 
depth — cannot  be  conducted  on  preserved  specimens. 
Expanding  the  techniques  we  apply  to  the  study  of  these 
rare  deep-sea  animals  will  increase  our  knowledge  of  one 
of  the  least-known  habitats  of  the  world,  the  mid-water 
depths,  and  provide  evolutionary  insight  into  questions 
of  broad  importance  and  biological  complexity. 

Acknowledgments 

I  thank  David  R.  Pensgard  for  providing  the  illustra- 
tions, and  N.  A.  Voss.  the  University  of  Miami  Marine 
Laboratory.  C.  F.  E.  Roper,  the  United  States  National 
Museum,  and  F.  Naggs,  the  Natural  History  Museum 
(London),  for  allowing  me  to  study  specimens  in  their 
care.  R.  E.  Young.  M.  J.  Brooks,  and  R.  E.  Strauss  made 
helpful  comments  in  the  course  of  this  research.  Anony- 
mous reviewers  made  very  helpful  comments  to  improve 
this  paper.  The  research  was  supported  by  NSF  DEB- 
9306925. 

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Reference:  Biol.  Bull  189:  120-127.  (October/November,  1995) 


Behavioral  Control  of  Swash-Riding  in  the  Clam 

Donax  variabilis 

OLAF  ELLERS* 
Department  of  Zoology,  Duke  University,  Durham,  North  Carolina  27706 


Abstract.  Clams  of  the  species  Donax  variabilis  migrate 
shoreward  during  rising  tides  and  seaward  during  falling 
tides.  These  clams  spend  most  of  the  time  in  the  sand, 
emerging  several  times  per  tidal  cycle  to  ride  waves.  Mi- 
gration is  not  merely  a  passive  result  of  waves  eroding 
clams  out  of  the  sand;  rather  clams  actively  jump  out  of 
the  sand  and  ride  specific  waves.  Such  active  migration 
is  experimentally  demonstrated  during  a  falling  tide  by 
comparing  the  motion  of  dead  and  live  clams:  live  clams 
emerge  from  the  sand  and  move  seaward  even  when  dead 
ones  do  not.  As  low  tide  approaches,  live  clams  become 
progressively  less  active.  They  cease  migrating  for  2  hours 
around  low  tide  and  resume  jumping  to  migrate  shoreward 
after  the  tide  has  turned.  During  the  rising  tide,  far  from 
being  passive,  the  clams  jump  out  to  ride  only  the  largest 
20%  of  waves.  Specifically,  they  choose  swash  that  have 
the  largest  excursion,  i.e.,  those  swash  that  move  furthest 
on  the  beach. 

Introduction 

The  coquina  clam,  Donax  variabilis,  lives  on  south- 
eastern North  American  shores  on  coarse-grained,  sandy 
beaches  with  moderate  to  high  waves.  D.  variabilis  mi- 
grates shoreward  with  the  rising  tide  and  seaward  with 
the  falling  tide,  as  do  many  other  animals  that  live  on 
wave-exposed  beaches  (e.g.,  other  Donax  spp.,  mole  crabs, 
mysids,  gastropods,  amphipods,  and  isopods:  McLachlan 
et  al.,  1979). 

Migration  by  D.  vuriahilis  is  accomplished  in  a  series 
of  steps.  Most  of  the  time,  these  clams  stay  in  the  sand. 
Several  times  per  tidal  cycle,  each  clam  emerges  from  the 
sand  and  flow  from  waves  drags  it  to  a  new  position  where 


Received  9  September  1994;  accepted  27  July  1995. 
*  Current  address:  Section  of  Evolution  and  Ecology.  Division  of  Bio- 
logical Sciences.  University  of  California.  Davis,  CA  95616. 


it  again  digs  in.  I  have  named  this  method  of  locomotion 
"swash-riding"  (Ellers,  1987,  1988),  where  swash-riding 
is  the  process  of  emerging  from  the  sand,  riding  flow  from 
a  wave,  and  digging  in  again.  Swash-riding  does  not  nec- 
essarily lead  to  migration.  For  instance,  D.  faba  swash- 
rides  without  migrating  (McLachlan  and  Hesp,  1984),  and 
a  gastropod,  Bitllia  digitalis,  swash-rides  and  uses  positive 
chemotaxis  to  locate  moving  prey  in  the  surf  and  swash 
zones  (Odendaal  et  al..  1992).  But  in  D.  variabilis,  a  series 
of  swash-rides  usually  becomes  a  migration.  By  migrating, 
a  clam  maintains  its  position  at  the  sea's  edge,  i.e.,  in  the 
region  of  the  beach  that  is  alternately  underwater  and 
exposed  to  air  every  few  seconds  as  waves  arrive  on  the 
shore. 

In  the  tumultuous  milieu  of  breaking  waves,  eroding 
sand,  and  rip  currents  at  the  edge  of  such  a  beach,  how 
much  control  do  these  clams  have  over  their  migrations? 
One  reasonable  hypothesis  is  that  the  clams  are  eroded 
forcibly  out  of  the  sand  by  waves  during  a  falling  tide  and 
that  they  then  must  swash-ride  shoreward  during  the  in- 
coming tide  to  maintain  their  intertidal  location  on  the 
beach. 

That  hypothesis  has  some  support.  Jacobson  (1955) 
suggested  that  D.Jbssorwas  migrating  as  the  passive  result 
of  wave  action.  Wade  (1967a)  suggested  that  emergence 
of  D.  denticulatus  into  the  backwash  was  usually  a  passive 
result  of  clams  being  washed  out  of  the  sand.  Mikkelsen 
(1981)  described  two  populations  of  D.  variabilis:  one 
population  lived  on  a  high-slope  beach  with  relatively  large 
waves  and  migrated;  the  other  population  lived  on  a  low- 
slope  beach  with  smaller  waves  and  did  not  migrate. 
Beaches  with  lower  slopes  and  smaller  waves  have  been 
associated  with  non-migratory  or  partially  migratory 
populations  (e.g.,  in  D.  slrialus:  Wade,  1967b;  in  D.  gonl- 
<///.  Irwin,  1973).  Edgren  (1959)  described  a  population 
of  D.  variahilis  that  migrated  only  after  a  storm.  Those 


120 


BEHAVIORAL  CONTROL  OF  SWASH-RIDING 


121 


observations  suggest  that  migration  might  be  a  passive, 
wave-driven  process. 

An  alternative  hypothesis  is  that  the  clams  choose  to 
migrate,  behaviorally  maintaining  their  position  at  the 
sea's  edge.  In  this  alternative  view,  clams  are  not  usually 
washed  out  of  the  sand;  rather  they  actively  push  them- 
selves out  using  their  feet.  In  this  view,  physical  distur- 
bance is  neither  the  proximal  nor  the  ultimate  cause  of 
migration.  Instead,  other  ultimate  causes  are  imaginable. 
Perhaps  more  filterable  food  is  available  in  water  stirred 
up  by  waves,  and  the  clams  are  merely  following  the  food. 
Many  aspects  of  migration  could  be  beneficial:  avoiding 
predators,  or  avoiding  the  overheating  that  might  occur 
if  they  remained  behind  in  the  intertidal  during  the  falling 
tide.  No  specific  ultimate  cause  for  migration  is  advocated 
in  the  present  paper;  the  main  point  is  to  distinguish  be- 
tween clams  being  forcibly  dislodged  by  waves  and  clams 
actively  emerging  to  ride  waves. 

In  marine  invertebrates  there  are  many  other  migratory 
behaviors  in  which  the  relative  contributions  of  active 
and  passive  determinants  of  net  motion  are  important. 
For  instance,  the  diurnal  vertical  migrations  of  brachyuran 
larvae  may  determine  the  direction  of  transport  by  cur- 
rents: early  larval  stages  spend  nights  in  the  neuston  when 
land  breezes  tend  to  transport  them  offshore;  later  larval 
stages  migrate  down  during  the  night  hours,  and  surface 
currents  from  sea  breezes  thus  tend  to  transport  them 
onshore  (Shanks,  1986).  The  net  movement  of  hypothet- 
ical invertebrate  larvae  has  been  modeled  in  computer 
simulations  that  evaluated  the  relative  influence  and  in- 
teraction of  light-  and  tide-cued  vertical  migrations, 
swimming  speed,  turbulent  mixing,  and  tidal  and  nontidal 
flows  (Smith  and  Stoner.  1993);  in  specific  tidal  channels 
modeled,  nontidal  flows  dominated  the  effect  of  vertical 
migration.  For  lobster  larvae,  a  combination  of  directed 
swimming  of  fourth  stage  larvae  and  ocean  currents  was 
necessary  to  account  for  onshore  recruitment  (Katz  el  ai, 
1994).  Interactions  of  behavior  and  passive  movement  by 
flow  are  thus  important  to  the  biology  of  marine  inver- 
tebrate larvae. 

Passive  movement  by  flows  is  not  only  important  in 
the  larval  phase,  but  can  also  be  significant  in  the  juvenile 
or  adult  phases.  The  adults  of  a  botryllid  ascidian  dispersed 
200  times  further  by  rafting  in  currents  than  did  swimming 
larvae  of  the  same  species  (Worcester.  1994).  Further, 
adults  of  1 7  mollusc  species  (with  or  without  a  planktonic 
larval  phase)  and  an  asteroid  have  been  observed  dis- 
persing by  rafting  (Martel  and  Chia,  1991).  In  a  tellinid 
bivalve,  Alacoma  ba/t/iica.  postlarval  juveniles  migrated 
to  new  tidal  flat  habitats  by  secreting  long  hyaline  threads 
and  being  dragged  by  currents  (Beukema  and  de  Vlas. 
1989).  or  by  becoming  positively  buoyant  and  floating  in 
currents  (Sorlin.  1988).  The  latter  is  an  inducible  response 
to  a  combined  stimulus  of  temperature  change  and  water 


movement.  In  all  these  examples,  animals  achieve  move- 
ment to  new  locations  by  a  combination  of  behavioral 
and  passive  phenomena.  In  this  and  following  papers 
(Ellers.  1995a,  b)  I  investigate  the  relative  contributions 
of  passive  and  active  factors  to  migratory  movements  of 
another  tellinacean  clam. 

That  D.  variahilis  clams  choose  to  migrate  in  the 
shoreward  direction  is  easily  observed  because,  during  ris- 
ing tides,  clams  emerge  from  sand  before  an  incoming 
wave  reaches  the  location  where  they  are  buried.  However, 
during  the  falling  tide,  clams  emerge  directly  into  the 
backwash:  the  question  of  whether  this  emergence  is  active 
or  passive  thus  cannot  be  answered  by  direct  observation, 
but  can  be  addressed  in  a  field  experiment. 

During  the  rising  tide,  when  clams  clearly  choose  to 
migrate,  they  might  choose  specific  waves  to  ride,  or  they 
might  merely  jump  in  advance  of  random  shorewardly 
moving  waves.  I  present  three  field  experiments:  the  first 
assesses  whether  clams  actively  jump  out  into  the  back- 
wash during  falling  tides  or  are  passively  eroded  by  re- 
ceding waves;  the  second  observes  clam  behavior  at  low 
tide;  the  third  demonstrates  the  degree  to  which  these 
clams  choose  waves  during  the  rising  tide. 


Materials  and  Methods 


Location 


All  observations  were  made  on  sandy  beaches  near  Pine 
Knoll  Shores  and  Atlantic  Beach.  North  Carolina.  These 
beaches  are  on  the  outer  coast  of  a  barrier  island,  expe- 
rience moderate  to  large  waves,  and  have  relatively  coarse- 
grained sand.  D  vahahilis  is  densely  but  patchily  distrib- 
uted along  the  length  of  this  island. 

Behavior  during  the  falling  tide 

To  determine  whether  clams  are  eroded  from  the  sand 
or  actively  push  themselves  out,  paired  dead  and  live  clams 
were  planted  in  the  sand  during  a  falling  tide.  Two 
hundred  and  thirty-four  clams,  killed  by  exposure  to  30% 
alcohol,  were  marked  with  pink  nail  polish  and  planted 
in  the  beach,  each  one  about  2  to  3  cm  away  from  a  live 
clam  marked  with  red  nail  polish  and  similarly  planted. 

Locations  of  all  the  pink-  and  red-marked  clams  were 
designated  with  thin  poles  inserted  into  the  beach.  Poles 
were  inserted  sufficiently  far  from  the  planted  clams  that 
wave-caused  erosion  of  sand  around  the  poles  did  not 
excavate  any  experimental  clams. 

These  clams  were  planted  in  four  groups  during  a  falling 
tide — 4.0,  3.5,  2.5,  and  1.5  h  before  low  tide.  They  were 
planted  in  the  wetted  portion  of  the  beach,  i.e..  in  the 
swash  zone,  amidst  other,  actively  migrating,  D.  variabilis. 
At  low  tide,  when  the  poles  were  high  and  dry,  the  sand 
adjacent  to  the  poles  was  dug  up  and  clams  of  each  color 


122 


O.  ELLERS 


0> 


Figure  1.  A  smaller  and  then  a  larger  wave  on  a  beach,  viewed  in 
vertical  section.  Water  directions  are  indicated  with  unidirectional  arrows. 
UJJ!  indicates  the  place  and  time  that  clams  might  jump.  The  variables 
are  ( 1 )  Bm,  the  "beachward  maximum,"  the  position  on  the  beach  where 
the  swash  becomes  backwash:  (2)  Sm,  the  "seaward  minimum,"  the  po- 
sition on  the  beach  where  the  previous  backwash  meets  the  swash;  (3) 
E,  the  "excursion  of  the  swash."  the  distance  that  the  swash  moves  in 
the  shoreward  direction  after  passing  the  previous  backwash;  and  (4)  Ep, 
the  "excursion  ol  the  previous  backwash,"  the  distance  that  the  previous 
backwash  moved  in  the  seaward  direction  before  passing  the  swash. 


were  counted.  At  the  time  they  were  planted,  each  group 
of  clams  had  been  divided  topographically  into  subgroups 
of  4,  5,  or  10  pairs  of  clams  to  facilitate  subsequent  sta- 
tistical analysis  (35  subgroups  were  created). 

To  test  whether  the  erosion  rates  of  dead  clams  in  the 
above  experiment  were  specific  to  the  day  on  which  that 
experiment  was  done,  an  additional  450  dead  clams  were 
planted  during  6  different  days  over  3  summer  months 
and  the  proportion  remaining  in  their  original  locations 
was  surveyed  at  lo\\  tide.  This  replication  was  done  only 
with  the  dead  clams  because  seaward  movement  of  live 
clams  during  falling  tides  had  been  observed  on  hundreds 
of  summer  days  during  3  years  and  was  also  documented 
in  observations  described  in  the  next  section. 

One  question  that  arises  is  whether  marking  live  clams 
with  nail  polish  itself  influences  the  behavior  of  clams. 


Live  clams  handled  and  painted  with  nail  polish,  however, 
appeared  to  behave  quite  naturally  after  marking.  A  lab- 
oratory comparison  of  painted  and  unpainted  clams 
showed  similar  burrowing  speeds,  similar  emergence  re- 
sponses to  knocking  sound  stimuli  (see  Ellers,  1995a,  for 
an  explanation  of  the  stimulus),  and  similar  siphon  re- 
traction responses  to  sudden  light  increases.  Furthermore, 
field  observations  showed  that  marked  clams  released  on 
the  sand  surface  on  the  beach  swash-rode  and  burrowed 
in  a  manner  indistinguishable  from  naturally  swash-riding 
clams.  Finally,  live  marked  clams  were  observed  swash- 
riding  on  the  beach  up  to  2  months  after  marking.  Thus, 
the  marking  procedure  itself  did  not  appear  to  adversely 
affect  the  behavior  of  clams. 

Behavior  at  low  title 

On  6  summer  days  during  1985,  transects  were  dug  at 
low  tide,  perpendicular  to  the  long  axis  of  the  beach.  The 
number  of  clams  found  in  a  30-cm  by  30-cm  area  was 
recorded  every  meter  along  the  transect.  The  average  po- 
sition of  the  water's  edge  at  low  tide  was  also  recorded. 
Behavior  of  clams  was  observed  during  the  period  from 
1 .5  h  before  to  1 .5  h  after  low  tide. 

Behavior  during  the  rising  tide 

Flags,  painted  with  numbers  1  through  35.  were  planted 
in  a  row  perpendicular  to  the  beach,  in  numerical  se- 
quence at  1-m  intervals.  Flags  bracketed  the  area  of  the 
beach  wetted  by  swash  and  backwash — the  base  of  the 
most  shoreward  flag  was  always  dry;  the  most  seaward 
flag  was  always  wet. 

The  sea's  edge  moves  seaward  and  shoreward  alter- 
nately as  waves  reach  the  shore,  wash  up  on  the  beach, 
and  run  off  the  beach  again.  The  location  of  the  edge  of 
the  water  was  monitored  by  recording  each  beachward- 
maximum,  Bm,  and  seaward-minumum,  Sm,  position  (Fig. 
1 ).  Simultaneously,  the  activity  of  the  clams  was  moni- 
tored. If  clams  jump,  they  do  so  after  the  water's  edge  has 
reached  a  seaward-minimum  position  and  before  the  edge 
of  the  water  reaches  a  new  beachward-maximum  position. 
Their  behavior  can  thus  be  associated  with  a  particular 
incoming  swash. 

Whether  clams  jumped  for  a  particular  swash  was  re- 
corded during  rising  tides  on  4  days  in  June  and  July.  The 
excursion  of  the  swash,  E,  and  the  excursion  of  the  pre- 
vious backwash,  Ep,  were  calculated  by  the  appropriate 
subtractions  of  the  relevant  values  of  Bm  and  Sm  (Fig.  1 ). 
Observations  were  started  not  less  than  1  h  after  low  tide 
and  were  halted  no  more  than  2^  h  before  high  tide.  Ob- 
servations on  any  particular  day  were  made  continuously 
for  1  to  2  h  (Table  I). 


BEHAVIORAL  CONTROL  OF  SWASH-RIDING 


123 


Table  I 


.Y»m/'('M  nl 

iihwYiirtui 

;v  l/ci/itcncv  nl  'xwashc 

v,  iin<!  tm/iic 

'XT  Hi 

lumping  till 

i-ncli  ot'lhc 

4  days  ot  observations 

#  of 

#of 

Swashes/ 

)  u  m  ps/ 

Da\ 

Swashes 

Jumps 

Min. 

Swash 

1 

294 

69 

4.2 

0.24 

•> 

379 

85 

4.4 

0.22 

3 

602 

74 

5.2 

0.12 

4 

481 

149 

5.3 

0.31 

Results 
Behavior  during  the  falling  tide 

In  trials  involving  live  and  dead  clams,  50%-80%  of 
dead  clams  were  not  eroded  from  the  sand  by  waves  (Fig. 
2).  In  experiments  involving  only  dead  clams,  an  average 
of  49%  (range  of  43%- 100%)  of  the  dead  clams  planted 
were  not  eroded  from  the  sand;  thus  the  trials  with  dead 
and  live  clams  had  typical  erosion  rates. 

When  dead  and  live  clams  were  planted  in  pairs,  a  sig- 
nificantly larger  proportion  of  live  than  dead  clams  left 
the  sand  in  each  of  the  four  groups  (Fig.  2).  Only  a  tiny 
fraction,  4.7%,  of  live  clams  that  were  planted  4  and  3  h 
before  low  tide  stayed  in  the  sand  in  their  original  positions 
and  were  recovered  at  low  tide.  Significantly  more,  30%. 
to  50%,  of  live  clams  planted  at  2  h  before  low  tide  stayed 
in  the  sand  in  their  original  positions  and  were  recovered 
at  low  tide.  Thus,  as  low  tide  approaches,  more  live  clams 
stay  in  the  sand.  In  contrast,  dead  clams  planted  close  to 
the  time  of  low  tide  washed  out  only  slightly  less  often 
than  clams  planted  closer  to  the  time  of  high  tide  (P 
=  0.05.  Mann-Whitney  t'-test). 

Behavior  al  low  tide 

At  low  tide,  the  population  was  found  at  or  just  shore- 
ward of  the  average  position  of  the  water's  edge  (Fig.  3). 
A  large  fraction  of  the  population  was  often  found  in  sand 
that  was  no  longer  being  wetted  by  even  the  most  shore- 
wardly  reaching  swash.  The  location  of  the  center  of  the 
population  varied  relative  to  the  average  position  of  the 
waves  at  low  tide.  On  14  May,  for  instance,  most  of  the 
population  was  at  the  average  position  of  the  water's  edge 
at  low  tide.  In  contrast,  on  5  June,  the  population  was 
22  m  shoreward  of  the  water's  edge. 

Many  clams  had  stopped  migrating  and  were  essentially 
stranded  during  the  1-2  h  around  the  time  when  the  tide 
turns.  From  0.5  h  before  low  tide  until  0.5  h  after  low 
tide,  no  clams  were  seen  jumping  out  of  the  sand.  During 
the  time  from  0.5  to  1.5  h  on  either  side  of  low  tide,  the 
numbers  of  clams  seen  jumping  were  relatively  low  com- 
pared to  the  numbers  of  clams  seen  jumping  during  the 


middle  of  the  rising  tides.  Although  quantitative  results 
are  given  for  just  6  days,  I  observed  the  same  pattern  of 
cessation  of  migration  around  low  tide  during  the  three 
summers.  May  through  August.  I  watched  clam  behavior 
on  North  Carolina  beaches. 

Behavior  during  the  rising  tide 

D.  variabilis  clams  evidently  chose  specific  waves. 
Swash  arrived  on  shore  at  an  average  rate  of  5.0  swashes 
per  minute  during  1 755  observed  swashes.  Clams  jumped 
for  20%  of  these  waves  (Table  I).  On  all  4  days,  clams 
jumped  for  swash  with  relatively  large  excursion.  E  (Fig. 
4A).  Specifically,  the  clams  jumped  for  a  larger  fraction 
of  large  than  of  small  waves.  The  ratio  C/T  (where  C  is 
the  number  of  swash  chosen  in  a  swash  size-class  and  T 
is  the  total  number  of  swash  in  that  size  class)  is  the  pro- 
portion of  the  swash  of  a  given  size  that  the  clams  ride. 
C/T  increases  for  increasing  E  on  all  4  days. 

The  largest  excursions  of  the  swash  are  E  =  1 4  to  1 8  m; 
the  smallest  are  E  =  1  to  4  m.  Choosing  waves  could  make 
a  large  difference  in  the  net  motion  of  clams.  Clams  can 
move  much  further  shoreward  for  each  swash-ride  by 
jumping  into  the  largest  swash  instead  of  the  smallest 
swash.  For  comparison,  the  intertidal  width  that  clams 
traverse  is  40  to  50  m. 


1.0 


01 

c 
'c 
'ra 
E 

0) 
0) 

15 


0.8- 


0.6- 


-  -       0.4  - 


c 
o 

o 
a 


0.2- 


0.0 


D   alive 
E3   dead 


4  3.5  2.5  1.5 

time  in  hours  before  low  tide 
when  the  clams  were  planted 

Figure  2.  Fraction  of  dead  and  live  D  variahili.i  remaining  in  the 
sand  after  being  planted  a  certain  number  of  hours  before  low  tide.  More 
live  than  dead  clams  leave  the  sand  (P  <  0.0 1 ).  suggesting  that  live  clams 
choose  to  jump  out.  Fewer  live  clams  leave  the  sand  close  to  the  time 
of  low  tide  than  leave  at  mid-tide  (P  <  0.01 ).  suggesting  that  clams  reduce 
or  halt  migration  just  before  the  time  of  low  tide.  (Probabilities  were 
calculated  using  Mann-Whitney  C  -tests.) 


124 


O.  ELLERS 


D. 
O 
0_ 


O 

re 


0.4 -, 


0.2- 


May  14 


Low  tide 
0.1  m 


U.IH 
( 

1                     10                    20                    30                    40 

0.4  n 

0.2- 

June  5                                Low 

r\                      °'L 

tide 
1m 

0.0- 
( 

Jiffa. 

)                     10                    20                    30                    40 

0.4- 

Low  tide 
0.1  m 


0.2- 


0.4-, 


0.2- 


0.0 


June  10 


Low  tide 
0.2m 


10 


30 


40 


0.4-] 

Inno  19     Low  tide 
JUllc   \L       n  1  m 

0.2- 

A 

n  n- 

A  ^/276^ 

v 

10 


20 


30 


40 


Shore  -"-Position 


Figure  3.  The  fraction  of  the  population  found  at  each  position  along 
transects  perpendicular  to  the  shoreline,  on  a  beach  at  low  tide  on  several 
days.  The  vertical  black  bar  marks  the  average  position  of  the  sea's  edge 
at  low  tide.  (The  meter  markers  were  not  in  the  same  location  each  day. 
the  profile  of  the  beach  changes  daily.)  The  vertical  height  of  each  low 
tide  is  given  beside  the  vertical  line.  The  total  number  of  clams  counted 
is  given  in  the  shaded  area.  Most  days,  the  population  is  found  shoreward 
of  the  sea's  edge  at  low  tide,  out  of  reach  of  any  swash.  Usually,  the 
clams  do  not  migrate  at  all  within  I  h  of  low  tide. 

Clams  chose  waves  that  moved  relatively  large  distances 
in  the  shoreward  direction.  They  jumped  for  such  large 
swash  before  the  swash  reached  them.  How  do  they  predict 
the  size  of  incoming  swash?  One  testable  hypothesis  is 


that  there  are  patterns  to  the  waves.  For  instance,  if  the 
size  of  incoming  swash  is  correlated  with  the  excursion 
of  the  previous  backwash,  then  the  excursion  of  the  pre- 
ceding backwash,  Ep,  could  be  a  cue.  I  found  that  Ep  is 
not  correlated  with  the  next  excursion  of  the  swash.  E, 
(R2  =  0.07,  0.04.  0.28,  0.4,  on  the  4  days),  so  it  would  be 
a  poor  predictor  of  E.  Indeed  the  clams  are  not  using  Ep 
since  C/T  does  not  increase  with  increasing  Ep  (Fig.  4B). 
Two  variables  determine  the  excursion  of  the  swash, 
E,  the  variables  Bm  and  Sm  (Fig.  1 ).  On  some  days  clams 
rode  swash  that  moved  significantly  farther  shoreward 
(Bm)  than  swash  they  did  not  ride  (P  <  0.0001,  F-test, 
multiple  regression  with  dummy  variables,  Weisberg, 


1.0 


CfT         0.8- 


0.6- 


0.4- 


0.2- 


0.0 


0-2  2-4  .4-.6  .6-. 8  .8-1.0 

E/Emax 


1.0 


CAT 


0.8- 


0.6- 


0.4- 


0.2- 


B 


0-.2 


2-4 


4-6 


Ep/Epmax 


6-8 


.8-1.0 


Figure  4.  The  proportion  of  swashes  in  size  classes  chosen  for  riding 
by  clams.  C/T  is  chosen  waves  divided  by  total  waves  in  a  wave  size 
category.  Different  shading  denotes  4  separate  days  of  observations.  (A) 
Half  ol  the  largest  swashes  (high  £/£„„„)  are  ridden,  whereas  small  swashes 
are  almost  never  chosen.  E/Emax  is  excursion  divided  by  maximum  ex- 
cursion observed  on  a  given  day.  (B)  The  proportion  of  chosen  swashes 
having  relatively  large  excursions  of  the  previous  backwashes  (large  Ep/ 
Epmax)  Swashes  with  large  Ep  are  not  chosen  systematically.  D  variabilis 
choose  to  ride  swashes  that  have  a  large  excursion.  E,  not  swashes  that 
have  a  large  excursion  of  the  previous  backwash,  Ep. 


BEHAVIORAL  CONTROL  OF  SWASH-RIDING 


125 


1980).  On  other  days,  clams  rode  waves  that  started  sig- 
nificantly farther  seaward  (Sm)  than  swash  they  did  not 
ride  (P  <  0.0001.  same  kind  of  test).  Although  on  some 
days  clams  appear  to  be  riding  swash  according  to  extreme 
values  of  Bm  or  Sm  relative  to  the  average  Bm  or  Sm  at 
that  time,  this  pattern  is  inconsistent  among  days.  In  con- 
trast, riding  swash  with  large  excursion  (Bm  -  Sm  is  ap- 
plicable to  all  days. 

Discussion 

Comparison  of  the  movements  of  dead  and  live  clams 
planted  in  the  sand  shows  that,  during  a  falling  tide,  Dona.x 
variabilis  clams  actively  emerge  into  the  backwash,  which 
then  moves  them  seaward.  Such  active  emergence  con- 
tradicts the  alternative  hypothesis  of  a  more  passive  role 
for  Donax  xpp.  during  migration  (see  Introduction).  For 
instance,  it  was  suggested  that  D.  denticiilatus  emerges 
from  the  sand  passively  to  move  seaward  during  the  falling 
tide  (Wade,  1967a).  Wade  suggested  that  a  D.  denticiilatus 
clam  would  detect  the  increased  drying  of  the  sand  as  the 
tide  receded  and  respond  by  retracting  its  foot  and  siphons, 
thus  decreasing  its  purchase  in  the  sand  and  enabling 
backwash  to  scour  it  from  the  sand  and  wash  it  seaward. 
In  contrast,  the  current  experiment  shows  that,  for  D. 
variahilis  in  the  studied  beach,  mere  retraction  of  the  foot 
would  not  usually  be  sufficient  to  cause  dislodgment  by 
erosion  (dead  clams  were  dislodged  only  50%  of  the  time). 
In  fact,  live  clams  moved  seaward  much  more  often  than 
dead  clams,  which  suggests  that  live  clams  actively 
emerged  from,  or  jumped  out  of.  the  sand.  Indeed,  I  have 
observed  jumping  responses  during  times  corresponding 
to  falling  tides  in  laboratory  experiments  on  endogenous 
rhythms  (Ellers,  1995a).  Thus.  I  conclude  that  live  clams 
of  this  species  often  actively  push  themselves  out  of  the 
sand  and  into  the  backwash  to  be  moved  seaward  by  flow. 

A  series  of  such  rides  in  the  backwash  becomes  a  sea- 
ward migration.  If  live  clams  relied  on  erosion  to  initiate 
their  seaward  rides  in  the  backwash,  only  20%  to  50%  of 
the  population  at  a  given  location  on  the  beach  could 
move  seaward  (as  did  the  dead  clams.  Fig.  2).  Considering 
that  a  minimum  of  three  swash-rides  is  required  to  com- 
plete a  migration  (minimum  beach  width  divided  by 
maximum  excursion),  the  fraction  of  the  population 
completing  a  seaward  migration  passively  would  be  0.008 
to  0.125.  Therefore,  passive  erosion  could  not  be  used  to 
migrate  seaward  on  these  beaches.  As  a  result  of  active 
seaward  migration,  almost  the  entire  population  has 
moved  to  the  lower  end  of  the  intertidal  at  low  tide  (Figs. 
2  and  3). 

Erosion  undoubtedly  sometimes  forces  clams  out  of 
the  sand.  Erosive  forces  on  a  clam  under  the  sand  are 
occasionally  larger  than  the  force  that  a  clam  can  exert 
with  its  foot  (Ellers.  1988).  Although  erosion  necessitates 


the  ability  to  cope  with  (ride  in)  flow  and  to  dig  in  again, 
it  does  not  necessitate  migration.  D.  variabilis  use  a  sys- 
tematic series  of  rides  in  the  swash  and  the  backwash  to 
migrate. 

This  systematic  series  of  seaward  swash-rides  in  D.  var- 
iabilis ends  about  0.5  to  1  h  before  low  tide.  At  that  time 
the  clams  cease  migrating  and  are  left  at  or  just  shoreward 
of  the  edge  of  the  sea  at  low  tide  (Fig.  3).  Migration  re- 
sumes about  0.5  to  1  h  after  low  tide,  with  clams  then 
moving  in  the  shoreward  direction.  Others  have  also  re- 
ported populations  of  Donax  spp.  that  do  not  necessarily 
follow  the  water's  edge  to  the  low-water  limit  (see  Intro- 
duction). In  some  populations,  the  location  of  the  pop- 
ulation on  the  beach  at  low  tide  follows  a  regular  pattern. 
For  instance.  D.  serra  migrates  following  the  position  of 
the  low-tide  water  table  (Donn  et  al..  1986).  The  water 
table  is  further  seaward  on  the  beach  during  spring  tides 
than  during  neap  tides.  In  laboratory  experiments  with 
D  variabilis,  an  endogenous  rhythm  has  been  observed 
in  which  a  quiescent  period  occurs  around  the  time  of 
low  tide  (Ellers,  1995a).  During  that  period,  clams  do  not 
show  jumping  responses.  The  rhythm  is  thus  a  proximal 
cause  of  the  systematic  cessation  of  migration  close  to  low 
tide  in  D.  variabilis. 

When  migration  resumes  in  the  shoreward  direction, 
many  clams  in  the  population  can  be  seen  simultaneously 
jumping  out  of  the  sand  just  shoreward  of  incoming  waves. 
This  behavior  has  been  reported  for  D.  semigranosus 
(Mori,  1938.  1950)  and  for  D.  variabilis  (Turner  and 
Belding.  1957).  Not  only  do  the  clams  jump  in  front  of 
incoming  waves,  they  jump  only  for  the  biggest  waves. 
Several  measures  of  wave  size  might  be  appropriate:  wave 
height,  breaker  height,  bore  height,  distance  traveled,  and 
maximum  shoreward  position  that  a  swash  wets. 

My  quantitative  data  show  that,  during  the  rising  tide, 
these  clams  ride  waves  shoreward,  using  primarily  the  20% 
of  waves  that  move  the  largest  distance  in  the  shoreward 
direction.  Those  are  not  necessarily  the  swash  that  end 
their  travels  furthest  shoreward;  neither  are  they  neces- 
sarily the  swash  that  start  the  furthest  seaward.  The  con- 
sequence of  riding  these  largest  swash  is  thus  to  be  at 
neither  the  shorewardmost  nor  the  seawardmost  extent 
of  the  swash-zone.  Rather,  riding  the  biggest  swash  max- 
imizes shoreward  distance  traveled  and  minimizes  num- 
ber of  swash-rides  per  migration.  In  terms  of  absolute  dis- 
tance, the  effect  is  remarkable — the  clams  can  choose  a 
swash  that  moves  18  m  shoreward  rather  than  the  1-m 
excursion  of  the  smallest  waves.  By  timing  their  active 
emergence  from  the  sand.  D.  variabilis  clams  control  their 
swash-riding. 

The  clams  choose  swash  with  large  excursion.  E.  But 
they  jump  before  a  shoreward  swash  reaches  them.  What 
cues  the  clams  to  jump  for  these  swash?  Swashes  with 
large  E  are  created  on  dissipative  beaches  by  collapsing 


126 


O.  ELLERS 


bores  of  large  height.  The  initial  velocity,  U0,  of  the  swash 
immediately  following  bore  collapse  is  given  by 

U0  =  U'  +  2  Vgli  ( 1 ) 

where  U'  is  the  speed  of  the  bore  front,  h'  is  bore  height, 
and  g  is  gravitational  acceleration.  The  excursion  of  the 
swash  is 


E  = 


Un 


g  tan 


(2) 


where  /(?  is  the  slope  of  the  beach.  Both  the  velocity  and 
swash  excursion  on  real  beaches  were  within  10%  of  these 
theoretical  predictions  (Bradshaw,  1982).  Thus  if  clams 
could  detect  the  height  of  the  bore,  they  could  predict  the 
swash  excursion.  Larger  bores  presumably  make  louder 
sounds  as  they  collapse.  If  the  clams  listened  to  the  sound 
of  the  bore,  they  could  predict  the  excursion  of  the  swash 
formed  from  it. 

Anecdotal  reports  for  D.  semigranosits  (Mori.  1938, 
1 950)  and  D.  variabi/is  (Turner  and  Belding,  1 957)  suggest 
that  during  rising  tides,  sounds  created  by  incoming  waves 
cue  the  clams  to  emerge  in  front  of  swash.  The  present 
study  demonstrates  that  clams  emerge  in  front  of  swash 
with  large  excursion,  and  eqns  ( 1 )  and  (2)  give  a  mecha- 
nism for  linking  sound  production  by  an  incoming  wave 
to  the  production  of  a  large  swash  excursion.  I  have  dem- 
onstrated appropriate  responses  by  D.  variabi/is  to  wave- 
like  sounds  in  the  laboratory  in  the  absence  of  waves  or 
other  possible  cues  (Ellers,  1995a). 

Behavior  thus  controls  swash-riding  and  migration  by 
D.  variuhilis  on  these  beaches  by  controlling  the  timing 
of  emergence  of  the  clams  from  the  sand.  Morphology 
further  controls  a  clam's  motion  while  it  is  pushed  by 
flow  from  waves  (Ellers,  1995b). 

Rather  than  being  at  the  mercy  of  the  tumultuous  mi- 
lieu of  breaking  waves,  eroding  sand,  and  rip  currents  at 
the  edge  of  a  beach,  these  clams  make  use  of  flows.  Other 
animals  such  as  mole  crabs,  some  gastropods,  and  am- 
phipods  presumably  use  similar  strategies  on  exposed 
beaches.  Unlike  the  familiar  paradigm  for  sessile  organ- 
isms on  rocky  shores,  in  which  organisms  avoid  dislodg- 
ment  primarily  by  avoiding  flow  forces  such  as  stream- 
lining, etc.  (Vogel,  1981;  Denny,  1988),  the  parallel  par- 
adigm for  organisms  that  live  on  shifting  substrata  is  that 
they  use  flow  forces  for  locomotion. 

Acknowledgments 

This  research  is  part  of  the  author's  Ph.D.  dissertation 
completed  at  Duke  University.  NSERC  postgraduate 
scholarships,  Duke  University  teaching  assistantships,  and 
a  Cocos  Foundation  Training  Grant  in  Morphology  sup- 
ported the  author.  The  Lerner-Gray  Fund  for  Marine  Re- 
search (American  Museum  of  Natural  History)  and  a 


Grant-in-Aid  of  Research  from  Sigma  Xi  supported  this 
project.  I  acknowledge  the  Duke  University  Marine  Lab- 
oratory for  use  of  its  facilities.  I  am  grateful  to  the  many 
friends  who  selflessly  volunteered  to  assist  with  field  work 
on  the  beach.  1  also  thank  my  thesis  supervisor,  S.  Vogel, 
and  committee  members,  V.  L.  Roth.  E.  J.  Shaughnessy, 
V.  A.  Tucker,  and  S.  A.  Wainwright. 

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Reference:  Biol.  Bull  189:  128-137.  (October/November,  1995) 


Discrimination  Among  Wave-Generated  Sounds  by  a 

Swash-Riding  Clam 

OLAF  ELLERS* 

Department  of  Zoology,  Duke  University.  Durham.  North  Carolina  27706 


Abstract.  Clams,  Donax  variahilis.  responded  to  sound 
stimuli  presented  to  them  in  a  laboratory  aquarium  by 
jumping  out  of  the  sand,  lying  on  the  sand  for  several 
seconds,  and  digging  in  again.  On  a  beach,  clams  jump 
out  of  the  sand  and  ride  waves,  migrating  shoreward  with 
the  rising  tide  and  seaward  with  the  falling  tide.  Parallels 
between  clam  behavior  on  a  beach  and  that  elicited  in  the 
laboratory  suggest  that  clams  cue  on  wave  sounds  to  jump 
out  of  the  sand.  Three  aspects  of  the  response  to  sound 
were  parallel,  (i)  Clams  were  most  responsive  to  low-fre- 
quency sounds  similar  to  those  produced  on  a  beach  by 
waves  rolling  onto  shore,  (ii)  Clams  were  also  more  re- 
sponsive to  louder  sounds;  on  a  beach,  clams  jump  pref- 
erentially for  the  largest  (loudest)  20%  of  waves,  (iii)  Re- 
sponsiveness in  the  laboratory  had  an  endogenous  tidal 
rhythm,  with  highest  activity  occurring  at  high  tide  and 
no  activity  occurring  at  low  tide;  this  rhythm  corresponds 
to  the  activity  of  clams  on  the  beach  from  which  they 
were  collected.  By  using  sounds  to  identify  large  waves, 
clams  can  ride  selected  waves  and  continuously  maintain 
position  at  the  sea's  edge  as  the  tide  floods  and  ebbs. 

Introduction 

Large  populations  of  the  coquina  clam,  Donax  varia- 
bilis,  migrate  on  sandy  beaches,  shoreward  with  the  rising 
tide  and  seaward  with  the  falling  tide  (as  do  many  other 
clams  in  this  genus).  An  individual  D  variabilis.  which 
can  be  up  to  3  cm  long,  normally  resides  with  the  posterior 
edge  of  its  shell  about  2  to  7  mm  under  the  surface  of  the 
sand.  To  migrate,  several  times  each  tidal  cycle,  it  jumps 
out  of  the  sand  (pushing  its  shell  upward  by  thrusting  two 
to  five  times  downward  with  its  foot)  and  rides  flow  from 


Received  9  September  1994;  accepted  27  July  1995. 
*  Current  address:  Section  of  Evolution  and  Ecology,  Division  of  Bio- 
logical Sciences,  University  of  California,  Davis,  CA  95616. 


waves.  This  method  of  locomotion  has  been  named 
"swash-riding"  (Ellers.  1987,  1988). 

Individual  D  variahilis  control  where  waves  move 
them,  in  pan,  by  choosing  to  ride  specific  waves  (Ellers. 
1995a).  On  a  rising  tide,  clams  jump  out  of  the  sand  pref- 
erentially for  the  biggest  waves;  i.e.,  the  ones  that  drag 
them  the  largest  distance  in  the  shoreward  direction.  As- 
tonishingly, the  clams  jump  out  of  the  sand  before  the 
arrival  of  such  waves,  thus  effectively  predicting  which 
waves  will  carry  them  the  furthest.  The  cues  that  enable 
this  behavior  have  not  been  previously  investigated. 

One  explanation  for  this  discriminatory  and  anticipa- 
tory feat  is  that  clams  might  detect  sounds  from  waves 
and  use  differences  in  the  character  of  these  sounds  to 
select  waves.  Sound  is  a  reasonable  candidate  as  a  cue 
that  enables  clams  to  discriminate  among  waves  because 
a  physical  mechanism  linking  louder  sounds  to  larger 
waves  is  plausible.  On  dissipative  beaches  where  the  clams 
live,  a  breaking  wave  becomes  a  bore  (a  traveling  cliff-like 
structure  of  tumbling  water)  and  the  bore  becomes  swash, 
which  the  clams  ride.  The  higher  the  bore,  the  larger  the 
excursion  of  the  swash  (Bradshaw,  1982).  Since  a  higher 
bore  has  more  potential  energy — it  has  farther  to  fall — it 
tends  to  create  more  intense  vibrations  as  water  falls  off 
its  leading  edge,  thus  emitting  a  louder  sound.  Sound  is 
also  a  reasonable  cue  that  enables  clams  to  anticipate 
waves  since  sound  travels  faster  than,  and  in  front  of,  the 
bore,  thus  announcing  its  arrival.  Species  of  Donax  re- 
spond to  sound  or  vibration  by  either  jumping  out  of  the 
sand  or  burrowing  more  deeply  (Mori,  1938,  1950; 
Loesch.  1 957;  Turner  and  Belding,  1957;  Tiffany,  1971; 
Trueman,  1 97 1 ),  but  no  quantitative  data  describing  either 
wave  sounds  or  sounds  that  elicited  responses  from  clams 
are  available. 

Reports  of  animals  using  flow-induced  sounds  as  be- 
havioral cues  are  very  rare.  Flow-induced  low-frequency 
sounds  and  infrasounds  emanate  from  weather  patterns. 


128 


RESPONSES  OF  CLAMS  TO  WAVE  SOUNDS 


129 


topographic  features,  and  ocean  waves:  such  sounds  can 
travel  thousands  of  kilometers.  Birds  are  able  to  detect 
infrasounds  and  may  be  able  to  orient  relative  to  such 
features  because  birds  can  detect  Doppler  shifts  associated 
with  flying  away  from  or  towards  infrasound  sources 
(Kreithen  and  Quine.  1979).  Detection  of  low-frequency 
sound  and  infrasound  has  also  been  shown  in  fish  (Kar- 
Isen,  1992a,  b)  and  cephalopods  (Packard  el  ai.  1990). 
The  function  of  infrasound  detection  in  these  cases  is  un- 
clear, but  may  be  related  to  orientation,  detection  of  sur- 
face waves,  short-term  inertial  guidance,  or  detection  of 
low-frequency  flows  (Bleckmann  et  a/.,  1991)  from  swim- 
ming motions  of  other  fish.  Avoidance  of  continuously 
vibrating  sound  sources  in  darkness  has  been  demon- 
strated in  herring  (Blaxter  and  Batty.  1985). 

Sound  is  mechanical  energy  (created  by  a  vibration) 
that  propagates  through  a  medium  as  a  result  of  kinetic 
and  potential  energy  being  alternately  stored  and  released 
elastically  by  the  medium.  Sound  propagates  either 
through  the  air  or  through  the  wet  beach  sand  at  the  speeds 
of  sound  in  those  media.  As  the  sound  vibrations  travel 
past  a  point,  they  are  detectable  as  temporal  changes  in 
velocity,  displacement,  or  pressure:  the  latter  can  be  mea- 
sured using  a  hydrophone. 

To  determine  whether  listening  for  and  distinguishing 
among  waves  is  possible  under  the  sand,  I  recorded  wave 
sounds  by  using  a  hydrophone  planted  under  the  sand 
amidst  a  population  of  burrowed  D.  variahilis.  Then,  in 
a  laboratory,  I  tested  whether  clams  would  respond  by 
jumping  out  of  the  sand  when  wave-like  sounds  were  pre- 
sented in  the  absence  of  waves.  I  also  used  recorded  wave 
sounds  and  several  artificial  sounds  of  varying  loudness 
and  frequency  to  test  the  frequency  and  loudness  speci- 
ficity of  clams'  responses. 

The  above  experiments  must  be  considered  in  the  light 
of  suggestions  of  a  tidal  rhythm  of  responsiveness  to  sound. 
Behavior  of  D.  variabilis  on  a  beach  changes  with  time 
of  tide  (Ellers,  1995a),  and  jumping  responses  to  vibration 
were  observed  during  rising  tides  only  (Turner  and  Beld- 
ing,  1957)  or  during  all  tidal  phases  (Tiffany,  1971).  An 
endogenous  rhythm  was  suggested  for  D.  sanigranosiis 
(Mori.  1938,  1950).  Tidal  rhythms  are  often  found  in 
coastal  marine  invertebrates,  particularly  in  crustaceans, 
molluscs,  and  polychaetes  (fora  review,  see  Naylor.  1985). 
An  endogenous  tidal  rhythm  of  shell  gaping  has  been  doc- 
umented in  a  venerid  clam  (Williams  et  a/.,  1993).  There- 
fore, I  also  tested  for  an  endogenous  tidal  rhythm  of  re- 
sponsiveness to  sound. 


Materials  and  Methods 


Specimens 


Immediately  preceding  each  laboratory  experiment, 
fresh  Donax  vuruihilis  were  collected  from  a  beach  (sub- 


sequently "the"  beach)  near  Pine  Knoll  Shores  on  the 
seaward  side  of  Bogue  Bank,  a  barrier  island  off  North 
Carolina.  The  clams  were  transported  with  sand  and  sea- 
water  in  a  thermally  insulated  container  to  the  Duke  Uni- 
versity Marine  Laboratory,  a  half-hour  drive  from  the 
beach.  (An  insulated  container  is  crucial;  if  clams  heat  up 
even  slightly  in  transit,  they  subsequently  show  no  be- 
havioral responses.)  Clams  were  placed  in  an  aquarium, 
where  they  dug  into  sand. 

General  acoustic  tests  and  analyses 

Acoustic  tests  in  the  laboratory  were  done  in  a  glass 
aquarium  (50cm  long  by  26cm  wide  by  30cm  high) 
placed  on  a  plywood  table.  The  bottom  8  cm  of  the 
aquarium  was  covered  with  sand  from  the  beach,  and 
seawater  was  added  to  a  depth  of  28  cm. 

Sounds  were  produced  in  several  ways.  Knocking  with 
knuckles  on  the  plywood  table  produced  a  low-frequency 
sound.  In  addition,  sounds  were  produced  using  an  un- 
derwater loudspeaker  (20  cm  diameter)  made  of  polysty- 
rene foam.  The  loudspeaker,  located  10  cm  from  one  end 
of  the  aquarium  and  partially  buried  in  the  sand,  was 
driven  by  a  tape  recorder.  Sounds  played  were  either  re- 
corded from  waves  on  the  beach  or  synthesized  by  a  sine 
wave  generator  circuit. 

To  determine  the  sound  pattern  reaching  the  clams, 
sounds  were  recorded  by  hydrophones  buried  in  the  sand 
at  various  locations  in  the  aquarium.  The  signal  from  the 
hydrophone  was  amplified  and  digitized  (8-bit  resolution) 
at  a  sampling  rate  of  8192  Hz.  This  sampling  rate  allows 
detection  of  sounds  below  4096  Hz.  Higher  sampling  rates 
were  unnecessary  because  very  little  sound  was  detected 
at  higher  frequencies  when  higher  sampling  rates  were 
tried. 

To  analyze  the  data,  several  standard  methods  were 
used.  Sound  pressure  was  plotted  as  a  function  of  time. 
The  pressure  functions  were  transformed  into  frequency 
spectra  in  which  sound  amplitude  is  plotted  as  a  function 
of  frequency.  This  transformation  was  performed  using 
a  Fast  Fourier  Transform  (FFT)  computer  algorithm 
(Burrus  and  Parks,  1985.  p.  107).  The  FFT  algorithm 
produces  a  complex  number  for  each  frequency:  the  am- 
plitude at  each  frequency  interval  is  twice  the  absolute 
value  of  that  complex  number  divided  by  the  square  root 
of  the  number  of  samples.  Frequency  resolution  is  the 
reciprocal  of  the  time  interval  sampled  (a  0.5-s  sample  of 
sound  resolves  into  2-Hz  intervals). 

I  represented  loudness  of  sound  as  either  the  root-mean- 
square  (RMS)  of  the  pressure  trace  over  an  interval  of 
interest  or  as  the  logarithm  of  that  value.  Loudness  is  a 
subjective  concept;  because  humans  judge  the  relative 
loudness  of  two  sounds  as  the  ratio  of  their  intensities, 
loudness  is  often  represented  using  a  logarithmic  (decibel) 


130 


O.  ELLERS 


scale  (Kinsler  el  at.,  1982).  One  conventional  measure  of 
sound  levels  is  the  intensity  level  (//,): 


IL  =  10  log. 


(1 


where  /  is  the  measured  intensity  and  /ret  is  a  reference 
intensity.  Intensity  is  the  rate  at  which  sound  energy  flows 
through  a  unit  area.  But  for  both  plane  and  spherical 
waves, 

P- 

1=  (2) 

pc 

where  Pc  =  RMS  sound  pressure,  p  =  density,  and  c  =  the 
speed  of  sound  in  the  medium  in  which  the  sound  is  trav- 
eling. Although  an  approximate  speed  of  sound  in  wet 
sand  is  obtainable  from  the  literature,  the  exact  speed  of 
sound  in  the  wet  sand  in  the  present  experiment  is  un- 
known. Therefore,  I  present  Pe  rather  than  the  derived 
quantity,  /. 

Furthermore,  it  is  not  clear,  a  priori,  that  clams  should 
respond  logarithmically  to  sound.  Therefore.  I  present  all 
data  numerically  as  either  pressure  or  />,„  but  relative 
loudness  is  assumed  to  be  represented  by  the  ratios  of  the 
Pe  values  (reflected  in  a  logarithmically  transformed  Pt, 
axis  in  graphs  that  include  several  sounds).  I  also  give  the 
following  conversions  to  a  common  measure  called  the 
sound  pressure  level,  SPL: 


(3) 


where  Prs.t  =  a  reference  pressure.  For  instance,  a  Pe  of 
200  Pa  has  an  SPL  of  200  dB  re  1  n  Pa,  whereas  a  Pf  of 
1  Pa  has  an  SPL  of  120  dB  re  1  n  Pa. 

Wave  sounds  recorded  wider  the  sand  on  a  hcach 

Sounds  of  waves  approaching  the  shore  were  recorded 
on  the  beach  during  several  summer  days.  A  hydrophone 
was  buried  amidst  the  clam  population  at  the  same  depth 
(2-7  mm)  to  which  these  clams  burrow. 

The  hydrophone  was  attached  to  a  tape  recorder  that 
had  two  input-channels:  one  channel  recorded  wave 
sounds:  the  other  channel  simultaneously  recorded  an 
observer's  verbal  description  of  the  waves.  Wave  sounds 
were  thus  matched  with  particular  events  in  the  waves. 
Wave  sounds  were  plotted  as  pressure  versus  time  or  as 
frequency  spectra  calculated  from  0.5-s  intervals  starting 
between  0.5  and  1  s  before  the  arrival  of  swash  at  the 
hydrophone. 

Tidal  variation  in  responsiveness  of  clams  to  sounds 

Clams  were  collected  from  the  beach,  transported  to 
the  laboratory,  and  placed  in  the  aquarium.  There  they 


were  exposed  to  natural  light  and  dark  cycles  through  a 
large  window  on  one  side  of  the  room,  but  the  fluorescent 
room  lights  were  kept  on  constantly  day  and  night  to  allow 
observation  of  clam  behavior.  About  once  each  hour  for 
several  days,  sounds  were  produced  by  knuckle  knocking 
that  was  continued  until  no  new  clams  emerged  for  30  s. 
The  number  of  clams  emerging  in  response  to  each 
knocking  stimulus  was  counted.  A  hydrophone  planted 
in  the  sand  in  the  middle  of  the  aquarium  recorded  the 
sounds  for  subsequent  spectral  analysis.  This  protocol  was 
repeated  for  several  collections  of  between  100  and  170 
clams  each. 

Collections  were  made  on  two  types  of  days.  During 
the  first  type,  high  tide  was  between  noon  and  midnight 
and  between  midnight  and  noon:  during  the  second  type, 
high  tide  coincided  with  midnight  and  noon.  Thus  the 
two  types  of  days  have  noon  and  midnight  falling  either 
in  or  out  of  phase  with  high  tide.  Comparison  of  temporal 
patterns  of  responsiveness  on  the  two  types  of  days  in- 
dicates whether  there  is  a  tidal  or  a  daily  rhythm  of  re- 
sponsiveness. 

A  second  experiment  was  performed  according  to  the 
same  protocol  except  that  the  window  was  blocked  off, 
excluding  sunlight  and  moonlight,  and  the  room  lights 
were  turned  off  except  for  a  few  seconds  during  sampling 
periods. 

Clams'  responsiveness  to  sounds  from  waves 

Clams  were  collected  from  the  beach  and  placed  in  the 
aquarium  in  front  of  the  speaker,  where  they  dug  into  the 
sand.  Recorded  sounds  of  waves  approaching  the  beach 
(about  200  successive  swash)  were  presented  to  these  clams 
during  a  45-min  period  starting  around  the  time  of  high 
tide.  Clam  responses  were  observed.  A  hydrophone  in  the 
sand  5  cm  in  front  of  the  speaker  monitored  the  loudness 
of  the  stimuli  reaching  the  clams.  This  protocol  was  re- 
peated on  three  summer  days  in  an  experiment  involving 
a  total  of  210  clams. 

Frequency  and  amplitude  specificity  of  responses 

To  test  specificity  of  responses  to  sounds,  1  subjected 
clams  buried  in  the  sand  in  the  aquarium  to  specific  syn- 
thesized sound  stimuli  of  different  frequencies  and  loud- 
nesses.  Sounds  were  presented  using  the  underwater 
loudspeaker.  Sounds  were  (i)  broadband  low-frequency 
sound,  (ii)  a  pure  low-frequency  tone  (with  quieter,  higher 
frequency  harmonics),  and  (iii)  a  pure  high-frequency  tone 
(with  quieter,  higher  frequency  harmonics).  Each  pre- 
sented sound  was  recorded  by  a  hydrophone  buried  in 
the  sand  5  cm  in  front  of  the  speaker.  The  RMS  sound 
pressures  (Pc)  and  frequency  spectra  were  calculated. 

Eighty  clams  were  collected  on  the  beach  30  min  before 
high  tide,  transported  to  the  laboratory,  and  placed  hap- 


RESPONSES  OF7  CLAMS  TO  WAVE  SOUNDS 


131 


<D 


Time  (s) 

Figure  I.  The  sound  of  a  wave  approaching  a  beach  as  recorded 
from  a  hydrophone  buried  in  the  sand  amidst  the  clams.  Sound  is  rep- 
resented as  pressure  versus  time,  with  larger  excursions  of  the  pressure 
trace  representing  louder  sounds.  Relative  sound  pressure  is  relative  to 
the  highest  pressure  occurring  in  this  trace.  During  the  time  preceding 
a  wave's  arrival  at  the  hydrophone,  a  low-frequency  rumble  of  increasing 
loudness  is  heard,  culminating  in  a  sudden  increase  in  loudness  when 
the  wave  reaches  the  hydrophone  (arrow).  Between  waves  there  is  relative 
silence. 


hazardly  in  the  aquarium,  between  the  front  of  the  speaker 
and  the  far  end  of  the  aquarium.  Sounds  were  played  to 
the  clams  during  a  35-min  period  starting  10  min  after 
high  tide. 

The  sounds  were  presented  to  the  clams  at  regular  in- 
tervals ( 1  min  silence  between  presentations);  the  signal 
was  always  of  22  ±  1  s  duration.  Each  sound  was  played 
at  a  variety  of  intensities  seven  times  in  a  row.  The  high- 
frequency  signal  was  played  first,  then  the  broadband  low- 
frequency  signal,  then  the  pure  tone  low-frequency  signal. 
The  number  of  clams  jumping  out  of  the  sand  for  each 
stimulus  was  recorded.  Significances  of  differences  in  re- 
sponses were  analyzed  by  regression  analysis  with  dummy 
variables  (Weisberg,  1980).  (Statistical  significance, 
throughout  this  paper,  is  considered  to  occur  when  the 
probability  that  the  null  hypothesis  is  true  is  less  than 
0.05.) 

On  two  other  days,  additional  experiments  were  per- 
formed according  to  a  protocol  that  was  identical  except 
for  the  following.  Instead  of  the  three  stimuli  described 
above,  a  variety  of  synthesized  low-frequency  stimuli  in 
the  range  of  20-100  Hz  were  presented.  The  stimuli  were 
presented  at  irregular,  more  widely  spaced  intervals  in 
random  order  and  over  a  longer  period  of  time  (starting 
40  min  after  and  ending  3  h  and  15  min  after  high  tide). 
The  number  of  clams  responding  was  analyzed  as  a  func- 
tion of  loudness,  log  (Pc),  and  time  after  high  tide. 

Attenuation  of  sound  and  frequency  responsiveness 

The  results  of  the  experiments  on  frequency  and  am- 
plitude specificity  must  be  interpreted  relative  to  the  fre- 
quency-specific attenuation  of  sound.  Attenuation  in  the 


aquarium  was  measured  by  placing  a  hydrophone  in  the 
sand  at  4,  10,  20.  30,  and  40  cm  in  front  of  the  speaker. 
Four  pure  tones,  covering  the  range  of  frequencies  used 
as  stimuli,  were  individually  played  by  the  speaker.  At- 
tenuation of  sound  was  calculated  for  each  as  the  ratio  of 
Pe  at  each  distance  divided  by  Pe  at  4  cm  from  the  speaker. 

Results 

ll'uve  sounds  recorded  under  the  sand  on  a  beach 

Recorded  wave  sounds  had  characteristic  patterns  of 
loudness  and  frequency.  A  bore  approaching  the  shore 
made  a  low  rumbling  sound  that  became  louder  as  the 
bore  approached  the  hydrophone  (Fig.  1).  Bigger  bores 
made  louder  sounds  than  smaller  ones  as  they  approached 
the  hydrophone.  The  rumbling  noise  of  an  approaching 
bore  consists  predominantly  of  low  frequencies  ranging 
from  40  to  300  Hz,  with  the  largest  amplitudes  being  in 
the  range  of  60  to  100  Hz  (Fig.  2 A). 

When  a  bore  or  swash  reached  the  hydrophone,  there 
was  an  additional  sudden  increase  in  loudness  (arrow  in 
Fig.  1 ).  After  a  bore  reached  the  hydrophone,  the  fre- 
quency content  shifted  toward  more  high-frequency 
components.  As  a  bore  or  swash  continued  beachward. 
the  sound  loudness  diminished. 

The  backwash  also  produced  a  distinct  sound.  It  was 
softer  than  the  upwash.  sounded  gurgly  and  uneven  in 
loudness.  and  had  more  high-frequency  components  than 
swash. 

Responses  of  clams  to  knocking  sounds 

Knocking  on  the  plywood  table  under  the  aquarium 
produced  a  low-frequency  sound  with  frequencies  similar 


1.0 


a 
< 


0.5  - 


a. 
•o 


0.0 


1.0 


wave  sound 


200 


400 


600 


800  1000 


1200 


0.5  - 


200 


loud  knocking 


400  600  800 

Frequency  (Hz) 


1000  1200 


Figure  2.  Comparison  of  frequency  spectra  of  wave  and  knocking 
sounds  that  stimulate  clams  to  jump.  (A)  A  typical  spectrum  of  a  wave 
sound  during  a  0.5-s  interval  that  starts  between  0.5  and  I  s  before  swash 
reaches  the  hydrophone.  The  rumbling  sound  that  precedes  the  arrival 
of  swash  at  the  hydrophone  consists  mainly  of  low-frequency  components. 
(B)  The  frequency  spectrum  of  knocking  sounds  presented  to  clams. 
Knocking  contains  low-frequency  sounds  similar  to  those  which  precede 
the  arrival  of  swash  at  a  spot  on  the  beach. 


132 


O.  ELLERS 


to  those  of  a  bore  approaching  the  beach  (Fig.  2).  Donax 
variabilis  clams  often  responded  to  knocking  by  jumping 
out  of  the  sand  (Fig.  3).  After  a  clam  emerged  from  the 
sand,  it  fell  on  one  valve  or  the  other,  often  with  siphon 
and  foot  extended.  This  behavior  mimicked  that  of  clams 
jumping  out  in  front  of  swash  on  the  beach.  Clams  lay 
on  the  sand  for  3  to  30  s  before  digging  in  again.  Occa- 
sionally, individual  clams  only  partially  emerged  from 
the  sand.  Such  hesitant  clams  could  be  induced  to  emerge 
completely  by  increasing  either  the  loudness  or  the  du- 
ration of  knocking  (suggesting  that  clams  sum  sound 
stimuli  over  time). 

Tidal  variation  in  responsiveness  of  clams  to  knocking 
sounds 

The  number  of  clams  that  jumped  out  of  the  sand  de- 
pended on  the  time  of  tide,  not  on  the  time  of  day  (Fig. 
4a).  Similar  results  were  obtained  when  room  lights  were 
off  and  natural  light  cues  were  unavailable  (Fig.  4b).  The 
clams  never  jumped  within  1  to  2  h  of  low  tide;  the  num- 
ber jumping  increased  as  high  tide  approached,  reached 
a  maximum  at  high  tide,  and  decreased  after  high  tide. 
This  tidal  rhythm  persisted  in  the  absence  of  direct  tidal 
cues  (e.g.,  the  aquarium's  water  level  did  not  change),  and 
independent  of  the  light  regime  provided.  The  tidal 
rhythm  persisted  for  three  to  five  tidal  cycles;  subsequently 
no  responses  to  sound  could  be  elicited. 

The  maximum  percentage  of  clams  jumping  at  a  given 
time  was  only  20%.  Jumping  clams  were  not  always  the 
same  individuals  (individuals  are  recognizable  by  distinc- 
tive shell  markings).  Therefore,  the  20%  maximal  response 
rate  was  not  due  merely  to  complete  non-responsiveness 
in  the  other  80%  of  clams.  Thus,  individual  clams  become 
responsive  and  unresponsive  several  times  per  tidal  cycle. 
At  high  tide,  a  larger  fraction  of  the  clams  are  in  a  re- 
sponsive phase  than  at  other  times  of  tide. 

Clams'  responsiveness  to  sounds  /rum  waves 

On  all  3  days,  some  of  the  clams  (maximally  20%  at 
any  given  time)  within  a  15-cm  radius  of  the  speaker 
sometimes  jumped  out  of  the  sand  while  wave  sounds 
were  being  presented.  In  contrast,  during  an  entire  sum- 
mer of  observations,  I  never  observed  clams  jumping  in 
the  absence  of  sound  stimuli. 

Wave  sounds  presented  to  clams  consisted  of  naturally 
occurring,  quiet  periods  several  seconds  long,  interspersed 
with  the  rumble  of  incoming  waves.  Whereas  some  wave 
sounds  elicited  responses,  others  not  obviously  different 
elicited  no  responses.  Because  of  the  complex  nature  of 
wave  sounds,  specific  features  to  which  clams  responded 
could  not  be  identified  unambiguously.  Nevertheless, 
jumps  often  coincided  with  the  pre-arrival  rumble.  The 
loudness,  Pe,  of  0.5-s  samples  of  sound  during  pre-arrival 


Figure  3.  Observed  responses  of  clams  to  sound  stimuli.  In  a  clam 
that  did  not  visibly  respond,  only  the  clam's  exhalant  and  inhalant  (frilled) 
siphon  were  visible  at  the  surface  of  the  sand  (at  left).  Clams  responded 
either  by  elongating  their  siphons  (middle),  usually  in  response  to  a  high- 
frequency  (832-Hz)  tone,  or  they  jumped  out  of  the  sand  and  lay  with 
siphons  and  foot  waving  (at  right).  After  10-30  s.  a  clam  that  had  jumped 
would  again  burrow  into  the  sand. 


rumbles  was  typically  30  Pa,  which  is  similar  to  the  loud- 
ness  of  artificial  sound  stimuli  that  elicited  the  responses 
reported  below. 

Frequency  and  amplitude  specificity  of  responses 

Two  separate  behavioral  responses  to  sound  were  ob- 
served— siphon  elongation  and  jumping  out  of  the  sand 
(Fig.  3).  Siphon  elongation  was  qualitatively  noted  to  oc- 
cur primarily  in  response  to  higher  frequencies  (e.g.,  500 
and  832  Hz  tones).  Such  elongation  of  the  siphons  was 
never  seen  in  the  absence  of  sound  stimuli.  This  is  the 
first  report  in  the  literature  of  siphon  elongation  as  a  re- 
sponse to  sound,  and  its  potential  function  is  unknown. 

The  jumping  responses  were  quantitatively  analyzed. 
The  frequency  spectra  of  the  sound  stimuli  are  shown  in 
Figure  5.  At  all  frequencies,  more  clams  jumped  in  re- 
sponse to  louder  sounds  (Fig.  6).  The  number  of  clams 
jumping  was  linearly  related  to  loudness  as  represented 
by  log  (Pc).  For  both  the  pure  tone  and  the  broadband 
low-frequency  sounds,  this  linear  relationship  was  tight 
(correlation  =  0.98)  and  the  slope  was  significantly  non- 
zero (P  <  0.001),  whereas  for  the  pure  high-frequency 
tone,  the  relationship  was  less  tight  (correlation  =  0.6), 
and  the  slope  was  not  significantly  different  from  zero  (P 
=  0. 1 ).  The  frequency  composition  did  not  affect  respon- 
siveness; both  pure  tone  and  broadband  low-frequency 
sounds  elicited  similar  numbers  of  clams  that  jumped  at 
a  given  loudness  (low-frequency  slopes  not  significantly 
different  from  each  other).  At  a  given  loudness,  more 
clams  jumped  in  response  to  low-  than  to  high-frequency 
sounds  (slopes  of  the  low-frequency  sounds  were  signifi- 
cantly higher  than  the  slope  of  the  high-frequency  tone). 
No  clam  was  ever  observed  jumping  in  the  absence  of  a 
sound  stimulus. 


RESPONSES  OF  CLAMS  TO  WAVE  SOUNDS 


133 


Figure  4a.  Number  of  D  variabilis  clams  in  the  laboratory  jumping  in  response  to  knocking  sounds 
relative  to  time  of  day  and  tide.  Noon  is  n;  midnight  is  m.  Clams  were  most  responsive  around  the  time  of 
high  tide  and  showed  no  response  around  the  time  of  low  tide.  Clams  responded  for  only  three  to  five  tidal 
cycles  after  they  had  been  taken  from  the  beach.  The  pattern  shown  is  a  compilation  of  several  collections 
(represented  by  different  symbols)  taken  on  different  days  from  the  same  beach.  Room  lights  were  continuously 
on  in  this  experiment. 


In  the  2  additional  days  of  experiments,  performed 
during  falling  tides  with  several  other  synthesized  low- 
frequency  stimuli,  jumping  responses  were  consistent  with 
those  already  described.  Specifically,  the  number  of  clams 
responding  increased  with  loudness,  log  (P<,),  and  de- 
creased with  increasing  time  after  high  tide  (multiple 
regression,  P  <  0.001;  loudness  variable:  P  <  0.01,  time 
variable:  P  <  0.01.  n  =  35).  A  cumulative  total  of  147 
responses  were  observed  in  35  trials  during  those  2  days. 

On  these  3  days  of  experiments,  clams  responding  to 
sounds  jumped  only  if  they  were  less  than  14  cm  from 
the  speaker,  and  most  responding  clams  were  less  than 
5  cm  from  the  speaker  face.  The  proximity  of  responding 
clams  to  the  sound  source  raises  the  possibility  of  near- 
field  effects  (complicated  wave  interference  patterns), 
which  would  complicate  interpretation  of  the  responses. 
A  near  field  exists  (close  to  a  vibrating  piston  in  an  infinite 
baffle,  an  approximation  to  the  geometry'  of  the  speaker: 
Kinsler  el  at..  1982.  inter  alia}  only  if  the  diameter  (0.2  m) 
of  the  speaker  is  greater  than  the  sound's  wavelength.  For 
an  832-Hz  sound  the  wavelength  is  1.8  m  (wavelength 
=  speed  of  sound  divided  by  frequency,  speed  of  sound 
in  coarse  silt  sea  bottoms  assumed  =  1540  m/s;  Kinsler 
etai,  1982).  The  speaker  is  even  smaller  compared  to  the 
longer  wavelengths  of  lower  frequency  sounds.  Therefore, 
near-field  effects  are  negligible  in  the  present  experiment. 
The  spatial  response  pattern  of  clams  must,  however,  also 
be  considered  with  respect  to  frequency-dependent  atten- 
uation of  sound. 


m 


September 

Figure  4b.  Number  of  D  variahilix  clams  in  the  laboratory  jumping 
in  response  to  knocking  sounds  relative  to  time  of  day  and  tide.  The 
results  are  similar  to  those  shown  in  Figure  4a,  but  in  this  experiment 
room  lights  were  off  except  for  a  few  seconds  during  sampling. 


1 34 


O.  ELLERS 


Relative  Sound  Pressure  Amplitude 

i.u  - 
0.5- 

ll 

Number  of  Clams  Jumping 

^ 

Q*  _L  _fc 

|  0  01  O  01 

-  Ill 

• 

,.• 

X 

• 

•  X 
X 
X 

0                        o 

o 
•                                  o 
o 

o 
o 

.0                                  \                              i 
In 

0.5- 
On  - 

B 

•U  -|                           i                          i 
1  n 

0.5- 
n  n  - 

I 

A  Mill 

it. 

Till           i          ii 

2         5        10     20        50      100  200 

RMS  Sound  Pressure  (Pa) 

Number  of  clams  mmoine  in  response  to  sounds  of  different 

10 


100 


1000 


Frequency  (Hz) 


Figure  5.  Frequency  spectra  of  three  artificially  produced  sounds 
presented  to  clams  to  test  their  responses  to  different  sounds.  The  pure 
tone,  low-frequency  stimulus  consisted  of  a  72-Hz  tone  with  a  harmonic 
at  1 44  Hz  (A);  the  pure  tone,  high-frequency  stimulus  consisted  of  a  832- 
Hz  tone  (B)  and  a  small  harmonic  at  I664  Hz  (not  shown):  and  the 
broadband,  low-frequency  noise  had  major  frequency  components  rang- 
ing from  20  to  1 40  Hz  (C).  These  frequency  spectra  were  recorded  with 
a  hydrophone  planted  in  the  sand  amidst  the  clams  and  thus  represent 
stimuli  to  which  the  clams  were  exposed.  Responses  of  clams  to  these 
sounds  are  shown  in  Figure  6. 


Attenuation  of  sound  and  frequency  responsiveness 

High  frequencies  attenuated  more  rapidly  with  distance 
from  the  source  than  did  low  frequencies  (Fig.  7),  which 
raises  the  possibility  that  clam  responses  that  appeared  to 
be  frequency  dependent  (Fig.  6)  might  actually  have  been 
attenuation  artifacts.  The  degree  to  which  attenuation  of 
frequency-dependent  responses  is  contributing  to  the  ob- 
served differential  responses  can  be  determined  by  con- 
sidering the  measured  extent  of  attenuation  in  the  aquar- 
ium in  this  experiment.  Two  sounds  (832  and  72  Hz)  of 
equal  Pt,  at  4  cm  from  the  speaker  attenuated  differentially, 
consequently,  at  30  cm,  the  832-Hz  sound  was  only  10% 
as  loud  as  the  72-Hz  sound  (Fig.  7).  At  4  cm,  the  loudest 
832-Hz  stimuli  were  10  times  louder  than  the  loudest  72- 
Hz  stimuli  (Fig.  6);  conservatively  assuming  a  10%  relative 
attenuation,  at  distances  greater  than  4  cm,  the  loudest 
832-Hz  sounds  were  at  least  as  loud  as  the  loudest  72-Hz 
sounds.  Yet  only  half  to  a  third  as  many  clams  responded 
to  the  loudest  high-frequency  sounds  as  responded  to  the 


loudness  and  frequency.  Louder  sounds  have  higher  root-mean-square 
(RMS)  pressures.  Three  distinct  frequency  spectra  (Fig.  5)  were  presented. 
More  clams  jump  at  a  given  loudness  for  both  broadband  (20  to  140  Hz, 
•  )  and  pure  tone  (72-Hz,  •)  low-frequency  sound  than  lor  a  high-fre- 
quency (832-Hz,  O)  sound. 


t 

(fl 

I 

S? 


80  - 


60  - 


20  - 


+ 
x 


O 

D 


O 

D 

40 


Distance  From  Speaker  (cm) 

Figure  7.  Frequency-specific  attenuation  of  sound  in  the  sand  in  the 
aquarium  in  which  acoustic  tests  were  performed  (D  832  Hz:  O  500  Hz; 
>  250  Hz:  +  72  Hz).  The  hydrophone  was  planted  in  the  sand  along  the 
midline  of  the  tank  at  several  distances  in  front  of  the  speaker.  Attenuation 
is  expressed  as  %  RMS  sound  pressure  at  the  given  distance  from  the 
loudspeaker  divided  by  RMS  sound  pressure  4  cm  from  the  face  of  the 
speaker.  (The  slight  increase  in  sound  at  the  point  farthest  from  the 
speaker  is  probably  due  to  sound  patterns  caused  by  partial  reflection  of 
sound  waves  off  the  aquarium  wall.) 


RESPONSES  OF  CLAMS  TO  WAVE  SOUNDS 


135 


loudest  low-frequency  sounds.  Therefore,  although  dif- 
ferential sound  attenuation  must  have  contributed  to  the 
observed  difference  in  responsiveness  to  different  fre- 
quencies, it  cannot  explain  the  entire  difference.  D.  rtir- 
uibilis  clams  are  more  responsive  to  low-frequency  sounds 
in  their  jumping  response. 

Discussion 

Ware  sounds  as  cues  tor  swash-ruling 

The  proposition  that  Dona.\  rariahilis  clams  use  sounds 
from  waves  as  cues  for  swash-riding  is  supported  by  direct 
findings  of  responsiveness  of  clams  to  appropriate  sounds 
and  by  parallels  between  behaviors  elicited  in  the  labo- 
ratory and  observed  on  the  beach.  On  the  beach,  these 
clams  jump  in  response  to  waves:  in  a  laboratory,  in  the 
absence  of  waves,  these  clams  jumped  in  response  to  wave 
sounds  and  to  artificially  produced  sounds  that  contained 
frequencies  similar  to  those  of  wave  sounds.  Wave  sounds 
recorded  in  the  beach  amidst  coquina  clams  show  distinct 
patterns  of  sound  that  allow  the  clams  to  predict  the  size 
and  timing  of  incoming  swash.  For  example,  the  loudness 
of  a  wave's  sound  predicts  the  excursion  of  that  wave's 
swash.  On  the  beach,  clams  preferentially  jump  for  swash 
of  large  excursion  (Ellers,  1995a);  in  the  laboratory,  clams 
were  more  responsive  to  louder  sounds.  Further  corre- 
spondence between  beach  and  laboratory  is  found  in  the 
observed  endogenous  tidal  rhythm.  Clams  in  the  labo- 
ratory, which  were  isolated  from  direct  tidal  cues,  were 
most  responsive  to  sounds  at  high  tide,  and  did  not  re- 
spond at  all  to  sounds  within  1  to  2  h  of  low  tide.  On  the 
beach,  clams  did  not  jump  out  of  the  sand  to  ride  waves 
within  1  to  2  h  of  low  tide  (Ellers,  1995a). 

Taken  together,  these  results  indicate  that  sound  is  a 
major  cue  in  determining  whether  these  clams  jump  out 
of  the  sand  to  swash-ride.  Loud  sounds  from  large  waves 
stimulate  the  clams  to  jump,  and  an  endogenous  rhythm 
of  responsiveness  modulates  the  jumping  behavior  and  is 
thus  a  proximate  cause  of  the  characteristic  tidal  migration 
of  these  clams.  This  is  the  first  report  demonstrating  use 
of  flow-induced  sounds  by  an  invertebrate. 

Detection  oj  pressure  or  velocity  signals  hy  clams 

An  acoustic  effect,  associated  with  how  close  a  re- 
sponding clam  is  to  the  speaker,  gives  insight  into  possible 
mechanisms  by  which  these  clams  may  detect  sounds. 
Sound  can  be  detected  as  temporal  changes  in  either  pres- 
sure or  velocity  of  the  vibrating  medium  at  a  point.  The 
hydrophone  used  in  this  experiment  detects  pressure.  In 
contrast,  organisms  may  use  mechanisms  (tiny  hairs  or 
cilia)  that  detect  motion  (velocity)  rather  than  pressure. 
For  a  sound  wave  far  from  its  source,  pressure  amplitude 
is  directly  proportional  to  velocity  amplitude  (Kinsler  el 


a/..  1 982).  For  sound  close  to  its  source,  however,  velocity 
and  pressure  are  not  directly  proportional,  and  velocity 
attenuates  more  rapidly  than  pressure.  By  convention, 
this  effect  is  considered  important  (Bobber,  1970)  if  the 
distance  from  the  source  to  the  receiver  (hydrophone  or 
clam)  is  less  than  half  a  wavelength  (0.9  and  1 1  m  for 
832-  and  72-Hz  sounds,  respectively).  Therefore,  in  the 
area  where  the  clams  responded  to  sound,  the  velocity 
signal  attenuated  more  rapidly  than  the  pressure  signal. 
If  clams  had  been  detecting  pressure,  then  louder  sounds 
should  have  caused  responses  throughout  the  aquarium. 
For  instance,  72-Hz  sound  attenuated  Pc  by  a  maximum 
of  50%  in  the  aquarium  (Fig.  7):  therefore,  a  signal  with 
twice  the  threshold  Pc  at  4  cm  should  have  caused  re- 
sponses throughout  the  aquarium.  But  clams  more  than 
15  cm  from  the  speaker  never  jumped;  thus,  they  did  not 
jump  appropriately  further  away  when  the  pressure  signal 
was  increased.  Since  the  velocity  signal  attenuates  more 
with  distance  than  the  pressure  signal,  the  locations  of 
responding  clams  suggest  that  the  animals  detected  ve- 
locity rather  than  pressure.  Other  more  complex  possi- 
bilities exist,  however,  regarding  the  transmitting  medium 
and  the  sensor  used.  For  instance,  scorpions  have  detectors 
both  for  compressional  (sound)  waves  under  the  sand  and 
for  waves  traveling  on  the  interface  between  air  and  sand 
(Brownell.  1984). 

Population  versus  individual  responses 

Loudness  and  frequency'  were  not  the  only  factors  de- 
termining whether  clams  jumped  in  response  to  a  sound. 
In  fact,  although  clams  jumped  in  connection  with  specific 
recorded  wave  sounds,  they  ignored  many  waves  of  equal 
loudness.  Similar  indifference  to  otherwise  suitable  stimuli 
was  also  observed  in  the  rhythm  experiments;  a  maximum 
of  20%  of  clams  responded,  even  at  the  maximally  re- 
sponsive times.  Perhaps  the  responsiveness  of  individual 
clams  shifts  several  times  per  tidal  cycle.  The  relative 
length  or  frequency  of  the  responsive  periods  may  change 
with  time  of  tide,  so  that  a  larger  fraction  of  clams  is 
responsive  at  high  tide  than  at  low  tide. 

Endogenous  tidal  rhythm 

An  endogenous  rhythm  should  persist  for  some  time 
in  the  absence  of  cues.  In  the  current  experiment,  the 
tidal  rhythm  in  responsiveness  persisted  without  tidal  cues 
and  irrespective  of  potential  light  cues,  thus  supporting 
the  endogenous  nature  of  the  tidal  rhythm.  But  the  rhythm 
persisted  only  for  three  to  five  tidal  cycles  under  laboratory 
conditions,  suggesting  either  that  the  rhythm  is  a  damped 
oscillator  or  that  a  clock  is  still  running  but  its  expression 
is  blocked.  For  instance,  lack  of  food  may  force  cessation 
of  jumping.  Such  small  numbers  of  persisting  cycles  are 
sometimes  observed  for  tidal  rhythms:  e.g..  a  tidal  rhythm 


136 


O.  ELLERS 


for  phototaxis  in  larval  grass  shrimp  persisted  for  only 
two  to  three  cycles  without  cues  (Douglass  el  a/.,  1992). 

It  was  suggested  that  D  variahilis  has  no  endogenous 
tidal  rhythm  (Tiffany,  1971)  when  clams  transplanted  to 
a  beach  with  a  different  tidal  cycle  were  observed  following 
the  new  tidal  cycle  within  24  h.  Given  the  endogenous 
rhythm  I  observed  (Fig.  4).  an  alternative  interpretation 
is  that  the  clock  or  damped  oscillator  was  rapidly  reset  by 
the  new  conditions. 

Additional  stimuli  on  the  beach  may  further  modify 
responses  in  nature  relative  to  those  observed  in  the  lab- 
oratory. For  instance,  the  rhythm  I  observed  was  sym- 
metrical during  rising  and  falling  tides.  Clam  responses 
were  identical  with  respect  to  sound  occurring  any  given 
time  before  and  after  high  tide,  at  least  within  the  limited 
ability  of  the  present  experiment  to  detect  asymmetries. 
In  contrast,  observations  on  the  beach  show  that  some 
asymmetries  in  behavior  exist.  On  the  beach,  clams 
jumped  in  advance  of  beachward-moving  swash  during 
the  rising  tide,  but  jumped  into  the  backwash  during  the 
falling  tide. 

Asymmetries  may  be  difficult  to  detect:  behavior  in  the 
laboratory  may  not  exactly  mimic  behavior  on  the  beach 
because  only  partial  cues  are  available.  For  instance,  the 
relative  amount  of  time  that  clams  are  covered  by  water 
may  serve  as  a  cue  in  nature.  On  a  beach,  the  water  level 
above  the  clams  is  constantly  changing;  in  the  aquarium, 
the  water  level  remained  unchanged. 

Another  natural  cue  that  may  be  missing  in  the  labo- 
ratory is  the  stimulus  of  being  moved  by  a  wave.  In  the 
aquarium,  after  a  clam  jumped  out  of  the  sand  it  lay  on 
the  sand  and  dug  in  again  after  10-30  s.  If  immediately 
re-exposed  to  the  same  stimulus,  a  responsive  clam  often 
jumped  out  again  (pers.  obs.).  Perhaps,  on  a  beach,  after 
a  clam  has  been  moved  by  a  wave,  its  responsiveness  to 
sound  stimuli  is  reduced  for  some  time. 

Usefulness  of  sound  cues  to  niiihnc  aninuih 

Endogenous  tidal  rhythms  are  common  in  intertidal 
animals.  Another  swash-rider,  the  amphipod  Syncheli- 
c/iitm  sp.  (Enright.  196 la,  1963;  Forward,  1980,  1986), 
has  endogenous  tidal  rhythms  in  response  to  light  (For- 
ward, 1980)  and  pressure  (Enright,  1961b).  Endogenous 
tidal  rhythms  probably  occur  in  other  swash-riders:  e.g., 
mole  crabs,  Emeriia  analoga  (Cubit,  1969)  and  Remipes 
tnincatijronx  (Mori,  1938);  and  gastropods,  Terebra  sal- 
leana  (Kornicker.  1961),  Hastula  inconstans  (Miller, 
1979),  Bullia  sp.  ( Ansell  and  Trevallion,  1969;  McLachlan 
and  Young,  1982),  and  Olivelln  hip/icuta  (Johnson,  1966). 
Other  Donax  spp.  that  swash-ride  (e.g..  D.  incarnalusand 
D.  denticulatiis:  Ansell  and  Trueman,  1973;  D.  serra: 
Donn,  1 987;  and  many  others),  may  also  respond  to  wave 
sounds  on  endogenous  rhythms. 


Whereas  many  populations  of  D.  variabilis  have  been 
reported  as  migratory,  at  least  during  summer  months, 
some  populations  either  do  not  migrate  or  migrate  only 
occasionally.  There  are  many  possible  explanations  for  a 
cessation  of  migration,  although  none  have  been  experi- 
mentally investigated.  The  role  that  sound  plays  in  mi- 
gration should  be  considered  in  that  context.  For  example, 
one  population  of  migratory  D.  variabilis  that  lived  on 
steep-sloped  beaches  has  been  contrasted  with  another 
non-migratory  population  that  lived  on  a  more  gently 
sloping  beach  (Mikkelsen,  1981).  Perhaps  waves  on  the 
gently  sloping  beach  do  not  generate  sounds  that  are  suf- 
ficiently loud  or  sufficiently  distinctive  to  produce  selective 
jumping  responses. 

Finally,  since  sound  and  vibrational  cues  from  waves 
are  so  obvious,  and  so  obviously  useful,  even  non-swash- 
riding  marine  animals  might  use  them.  For  instance,  an 
urchin  might  tighten  its  grip  on  rocks  in  preparation  for 
a  particularly  large  wave,  or  an  anemone  might  adjust  its 
stiffness  in  preparation  for  a  wave  impact.  I  have  observed 
both  an  anemone,  Anthopleura  elegantissima,  and  a  sea 
urchin,  Strongylocentrotus  purpuratus,  to  respond  to  vi- 
brations in  the  frequency  range  generated  by  waves. 

In  any  case,  the  clams  I  studied  are  literally  in  tune 
with  their  environment. 

Acknowledgments 

This  research  is  part  of  the  author's  Ph.D.  dissertation 
completed  at  Duke  University.  I  thank  my  thesis  super- 
visor, S.  Vogel,  and  committee  members,  V.  L.  Roth, 
E.  J.  Shaughnessy,  V.  A.  Tucker,  and  S.  A.  Wainwright. 
NSERC  postgraduate  scholarships.  Duke  University 
teaching  assistantships  and  a  Cocos  Foundation  Training 
Grant  in  Morphology  supported  the  author.  Duke  Uni- 
versity Marine  Laboratory  provided  facilities.  I  also  thank 
S.  A.  Wainwright.  who  generously  funded  acoustic  equip- 
ment; B.  Hunnings.  electronics  technician,  who  built  the 
sine-wave  generator;  and  A.  Johnson  who  critically  read 
the  manuscript. 

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Form  and  Motion  of  Donax  variabilis  in  Flow 

OLAF  ELLERS* 

Department  of  Zoology,  Duke  University,  Durham,  North  Carolina  27706 


Abstract.  The  coquina  clam,  Donax  variabilis,  rides  flow 
from  waves,  migrating  shoreward  during  rising  tides  and 
seaward  during  falling  tides.  This  method  of  locomotion, 
swash-riding,  is  controlled  not  only  behaviorally  but  also 
morphologically.  The  shape  of  this  clam  causes  it  to  orient 
passively;  a  clam  rotates  in  flow,  usually  in  backwash, 
until  its  anterior  end  is  upstream.  Rotation  is  about  a 
vertical  axis  through  a  pivotal  point  where  the  shell 
touches  the  sand.  The  density,  weight  distribution,  and 
wedge-like  shape  are  all  important  in  effecting  orientation. 
Such  orientation  is  significant  because  it  contributes  to 
stability  of  motion.  On  an  unoriented  clam,  upward  lift 
can  be  higher  than  its  underwater  weight — a  circumstance 
that  results  in  uncontrollable  tumbling.  In  contrast,  once 
oriented  with  its  anterior  end  upstream,  a  clam  experi- 
ences downward  lift  that  contributes  to  its  stability  while 
sliding  in  backwash.  Furthermore,  when  the  anterior  end 
is  upstream,  drag  is  reduced  relative  to  when  the  ventral, 
dorsal,  or  posterior  ends  are  upstream.  Since  orientation 
occurs  only  above  a  minimum  velocity,  it  has  the  effect 
of  slowing  a  clam's  motion  over  the  substratum  in  rapid 
flows.  Stability,  drag,  and  speed  reduction  enhance  a 
clam's  ability  to  gain  a  foothold  and  dig  in  after  a  swash- 
ride,  before  wave  flows  can  wash  it  off  the  beach  and  out 
to  sea. 

Introduction 

The  coquina  clam,  Donax  variabilis,  migrates  seaward 
with  the  falling  tide  and  shoreward  with  the  rising  tide  by 
using  a  method  oflocomotion  called  swash-riding  (Ellers, 
1987,  1988).  Swash-riding  involves  jumping  out  of  the 
sand,  being  pushed  by  a  wave  to  a  new  location,  and  dig- 
ging in  again.  For  an  individual  clam,  the  net  movement 
per  swash-ride  depends  in  part  on  behavior.  For  instance. 

Received  9  September  1994;  accepted  27  July  1995. 
*  Current  address:  Section  of  Evolution  and  Ecology.  Division  of  Bio- 
logical Sciences,  University  of  California,  Davis,  CA  95616. 


by  using  sound  to  sense  the  size  and  timing  of  incoming 
waves  (Ellers,  1995b),  these  clams  emerge  to  ride  only  the 
largest  waves  (Ellers.  1995a).  The  net  movement  per 
swash-ride  may  also  depend  on  a  clam's  shape,  just  as 
shape  has  consequences  for  performance  in  other  forms 
oflocomotion  such  as  running  or  flying. 

Consider  events  during  a  shoreward  migration  con- 
sisting of  several  swash-rides.  During  each  swash-ride,  a 
clam  is  pushed  shoreward,  but  does  not  stop  moving  at 
the  most  shoreward  point  of  its  travel  because  backwash 
pulls  it  seaward.  To  make  net  shoreward  progress,  it  must 
gain  a  foothold  while  moving  and  dig  in  before  backwash 
carries  it  seaward  of  its  original  position.  How  far  flow 
moves  a  clam  and  whether  it  gains  a  foothold  depend  on 
forces  the  clam  experiences  in  flow,  which  in  turn  depend 
on  the  clam's  shape.  The  present  study  seeks  to  identify 
shape,  or  form,  that  influences  the  motion  of  a  swash- 
riding  clam. 

The  motion  of  an  object  in  flow  can  be  of  two  dra- 
matically different  types.  An  object  can  orient  to  a  stable 
position  like  a  weather  vane  or  it  can  tumble  chaotically 
like  a  hat.  A  D.  variabilis  clam  moves  like  a  weather  vane. 
If  the  water  flowing  past  an  individual  D  variabilis 
changes  direction,  the  clam  rotates  to  maintain  a  certain 
orientation  with  respect  to  flow  (Fig.  1 ).  The  clam  rotates 
about  a  vertical  axis  through  a  pivotal  point  where  the 
shell  touches  the  sand.  In  the  oriented  position,  the  clam's 
anterior  end  is  upstream  and  the  posterior  end  is  down- 
stream. Once  oriented,  a  clam  slides  stably  before  gaining 
a  foothold  and  digging  into  the  sand  (Fig.  2). 

This  orientation  was  suggested  to  be  caused  by  activity 
of  the  siphons  in  D.  fossor  (Jacobson,  1955)  and  in  D. 
semigranosus  (Mori,  1938).  However,  dead  D.  denticu- 
/<;/;/v(Wade,  1967)  and  dead  D.  variabilis  (Tiffany,  1971; 
Ellers,  1987.  1988)  orient  the  same  way  as  live  ones,  thus 
demonstrating  the  passive  nature  of  orientation. 

Not  every  shape  orients  and  slides  stably  in  backwash. 
For  example,  a  bivalve,  the  cross-hatched  lucine  Divari- 


138 


MOTION  OF  CLAMS  IN  FLOW 


139 


flow 


Figure  I.  A  coquina  clam,  D  variahilix.  orienting  passively  in  flow. 
A  clam  starts  out  in  an  arbitrary  position,  e.g.,  ventral  edge  upstream 
(a).  In  flow  it  rotates,  passing  through  positions  (h)  and  (c)  until  it  reaches 
the  stable  orientation  with  the  anterior  end  upstream  (d).  Orientation  is 
rapid  and  can  occur  during  <0. 1  s.  The  clam  may  slide  downstream 
during  orientation,  as  shown,  or  remain  stationary  while  rotating. 


cella  quadrisculata,  stands  on  edge  and  rolls  like  a  wheel 
(pers.  obs.).  Another  bivalve,  the  sunray  Venus  Macro- 
callisla  nimbosa,  slides  in  a  variety  of  orientations  and 
tumbles  occasionally.  A  very  light  bivalve,  the  tellin  Tel- 
lina  iris  tumbles  chaotically.  A  sand  dollar.  Mellita  quin- 
quiesperforata,  flips  end  over  end  in  surf.  Similar  char- 
acteristic motions  of  these  objects  occur  in  a  flow  tank. 
Therefore,  orientation  by  D.  variabilis  is  a  special  con- 
sequence of  the  shape  of  D.  variabilis,  not  a  general  char- 
acteristic of  bivalves  or  other  invertebrates  that  live  ex- 
posed to  flow. 

Furthermore,  orientation  is  common  among  swash- 
riding  species.  Among  the  swash-riding  gastropods  and 
mole  crabs  for  which  information  is  available,  all  orient 
(Ellers,  1987).  Other  swash-riders  include  a  variety  of  am- 
phipods.  but  observations  of  orientation  are  unavailable 
for  those  species. 

Orientation  in  D.  variabilis  usually  occurs  in  the  back- 
wash rather  than  the  swash,  presumably  because  flow  in 
the  swash  is  too  turbulent.  Swash  forms  from  a  collapsing 
bore  that  itself  originates  from  a  breaking  wave.  The  speeds 


of  resulting  flows  are  relevant  to  the  fluid  dynamics  of  a 
swash-riding  clam.  The  speed  of  the  leading  edge  of  swash 
initially  increases,  then  decreases  as  it  moves  shoreward. 
Maximum  speeds  (Bradshaw.  1982)  ranged  from  4.5  to 
6ms"1  from  waves  with  breaker  heights  of  0.9  to  1.4  m 
on  a  steeply  sloped  beach  (slope  expressed  as  rise-to-run 
ratio  was  0.16);  and  the  maximum  speed  recorded  on  a 
shallow  beach  (slope  of  0.03),  where  bore  collapse  takes 
longer,  was  3  m  s"'.  Backwash  speed  is  typically  less  than 
swash  speed.  Maximum  backwash  speed,  just  shoreward 
of  the  next  incoming  bore,  was  1.2  m  s"1  on  a  beach  with 
slope  of  0.03  (Bradshaw,  1982).  The  average  speed  was 
0.70  m  s"'  among  21  backwashes.  Backwash  generally  in- 
creases in  speed  as  it  flows  seaward.  I  observed  D.  variabilis 
riding  flows  on  beaches  typically  having  a  slope  of  0.1, 
but  varying  from  0.05  at  low  tide  to  0.13  at  high  tide. 


Figure  2.  Coquina  clams.  I)  \'tiruihili\.  in  flow  on  a  beach.  Flow  is 
from  upper  right  to  lower  left  and  is  indicated  by  the  streaks  from  the 
moving  bubbles  on  the  surface  of  the  water.  The  upper  photo  shows 
clams  swash-nding  and  oriented  with  the  anterior  end  upstream.  The 
lower  photo  shows  a  clam  on  a  beach,  having  just  achieved  a  foothold, 
shortly  before  it  burrows  into  the  sand.  The  foot  is  visible  at  the  pointy, 
anterior  end.  and  the  siphons  are  visible  at  the  blunt,  posterior  end. 


140 


O.  ELLERS 


This  study  seeks  to  determine  morphological  features 
of  D  van.ihilis  that  are  important  in  causing  orientation; 
and  to  uciermine  the  functional  consequences  of  orien- 
tation for  movement  of  D.  variabilis  during  swash-riding. 
Relevant  are  forces  and  moments  offerees  due  to  friction 
between  clam  and  sand;  gravity;  buoyancy;  drag;  vertical 
and  horizontal  lifts  (lift  is  denned  as  a  force  normal  to 
flow,  and  there  are  always  two  mutually  perpendicular 
lift  vectors  normal  to  the  flow  direction).  By  combining 
measurements  and  assumptions  about  forces  and  their 
distribution,  I  identify  morphological  features  that  cause 
orientation  and  contribute  to  stability  once  oriented.  By 
comparing  the  forces  on  an  oriented  and  unoriented  clam, 
I  infer  the  function  of  orientation.  In  addition  to  mea- 
suring velocities,  forces,  and  pressures,  I  experimentally 
manipulated  the  weight  distribution  of  a  D.  variabilis  clam 
and  observed  the  resulting  changes  of  the  clam's  motion 
in  flow. 

Materials  and  Methods 

Backwash  speed 

On  a  North  Carolina  beach  with  a  slope  of  0.1,  where 
Donax  variabilis  occurs,  buoyant  plastic  beads  ( 1  cm  di- 
ameter) were  dropped  into  the  backwash  and  photo- 
graphed at  an  exposure  of  l/4s.  On  the  photographs, 
beads  appear  as  streaks,  and  streak  length  was  calibrated 
using  a  series  of  stakes  planted  in  the  beach  at  1-m  intervals 
along  a  transect  perpendicular  to  the  beach  at  the  same 
location.  It  was  assumed  that  the  plastic  beads  moved  at 
the  same  speed  as  the  backwash.  Error  in  the  velocity 
calculated  was  determined  by  a  propagation  or  errors 
analysis  using  estimated  errors  in  the  shutter  speed  and 
distance  measures  (Ellers,  1988). 

Force  distribution,  due  to  flow,  on  an  oriented  and 
unoriented  clam 

To  investigate  the  influence  of  shape  and  orientation 
on  flow  forces  experienced  by  D.  variabilis  clams,  the  dis- 
tribution offerees  acting  normal  to  the  surface  of  a  clam, 
and  their  associated  moments,  were  determined.  Local 
force  normal  to  a  surface  equals  local  pressure  multiplied 
by  local  surface  area  perpendicular  to  the  force.  Moment 
of  a  local  force  about  the  pivotal  point  equals  the  vector 
cross-product  of  location  and  force  (location  is  a  distance 
vector  from  the  pivotal  point  to  the  force).  Thus  pressures, 
areas,  and  locations  must  be  measured. 

The  pressure  distribution  was  estimated  by  measuring 
pressure  at  many  points  on  the  surface  of  a  scaled  model 
clam  that  was  5  times  larger  than  a  real  D.  variabilis  clam. 
A  model  was  used  because  direct  measurement  of  the 
distribution  of  pressures  on  such  small  clams  (maximum 
length  of  real  clams  =  3  cm)  is  not  feasible.  Pressure  mea- 


surements on  the  scale  model  were  made  in  a  wind  tunnel. 
Rows  relevant  to  clams  on  the  beach  were  mimicked  in 
the  wind  tunnel  by  maintaining  dynamic  similarity  (con- 
stant Reynolds  number.  Re).  Due  to  equivalence  of  Euler 
number  in  dynamically  similar  flows  (Shames,  1982), 
forces  on  an  object  determined  in  air  were  translated  to 
forces  on  that  object  in  seawater  using 

2 

PC  =   PW — ~^ — ~ 


where  Fc  and  F,,,  are  the  forces  on  the  template  clam  and 
model,  /j,,.  and  ^,,  are  the  dynamic  viscosities  of  seawater 
and  air,  and  p,,  and  pa  are  the  densities  of  seawater  and 
air.  Assuming  20°C  for  both  air  and  seawater,  the  con- 
version was 


The  model  was  carved  according  to  measurements  of 
the  shape  of  the  outside  of  the  left  valve  of  a  2.2-cm-long 
D.  variabilis  clam.  The  valve  was  attached  to  a  horizontal 
plane  above  which  the  valve  protruded.  A  spring-loaded 
displacement  gage  measured  vertical  distance,  and  cali- 
brated drives  were  used  to  determine  horizontal  coordi- 
nates. This  apparatus  gave  a  grid  of  3-dimensional  coor- 
dinates outlining  the  valve's  shape.  The  grid  had  a  vertical 
measurement  every  millimeter  in  both  horizontal  direc- 
tions with  more  closely  spaced  measurements  taken  in 
regions  where  vertical  measurements  changed  too  rapidly 
with  horizontal  distance. 

A  model  5  times  larger  than  the  measured  valve  and  a 
mirror-image  right  valve  were  fabricated  to  within 
±0.5  mm  (maximum  error)  of  the  scaled  measurements 
of  shape.  When  the  two  valves  were  attached  to  each  other, 
a  hollow  space  existed  inside.  Eighty  holes  were  drilled  in 
the  model  and  a  hollow  steel  pipe,  0.26  mm  in  external 
diameter,  was  attached  to  the  middle  of  the  posterior  end 
such  that  the  interior  of  the  pipe  led  to  the  hollow  inside 
the  model.  The  holes  were  covered  with  tape;  during  pres- 
sure measurements,  one  hole  at  a  time  was  uncovered  till 
pressure  at  all  holes  had  been  measured. 

The  model  was  placed  on  the  floor  of  a  large  wind  tun- 
nel (Tucker  and  Parrott,  1970).  The  pipe  that  protruded 
from  the  posterior  end  of  the  model  was  attached  to  a 
pressure  sensor  via  rubber  tubing.  On  the  other  side  of 
the  pressure  sensor  was  another  rubber  tube  that  con- 
nected to  a  reference  hole  in  a  horizontal  flat  plate  located 
in  and  parallel  to  mainstream  flow  (40  cm  above  the  tun- 
nel floor).  The  reference  hole  was  1  mm  in  diameter  and 
0.1  m  downstream  of  the  leading  edge  of  the  plate.  Mea- 
surement is  made  of  the  difference  between  the  reference 
pressure  at  the  hole  in  the  plate  and  the  pressure  at  the 
open  hole  on  the  model  clam.  The  same  apparatus  was 
previously  used  to  measure  pressures  on  model  squids 
(Vogel,  1985).  The  pressure  signal  was  digitized  (12-bit) 


MOTION  OF  CLAMS  IN  FLOW 


141 


into  a  computer  for  data  analysis.  For  each  hole,  30  rep- 
licate pressure  measurements  were  taken. 

In  the  wind  tunnel,  the  model  was  positioned  on  its 
left  side  at  a  tilt  that  was  within  ±3°  of  the  angle  at  which 
a  live  clam  lies  in  seawater.  In  seawater,  a  live  clam  lies 
with  the  plane  of  symmetry  (between  the  right  and  left 
valves)  at  an  angle  of  1 1  ±  2°  to  the  horizontal  in  both 
the  anterior-posterior  direction  and  the  dorsal-ventral  di- 
rection. These  angles  were  measured  using  a  protractor, 
from  a  photograph  of  a  live  clam  lying  on  the  bottom  of 
a  transparent  aquarium  rilled  with  seawater.  The  photo- 
graph was  taken  from  10  m  away  to  minimize  distortion 
due  to  perspective. 

Six  sets  of  pressure  measurements  were  made.  Two  were 
made  at  a  mainstream  air  speed  of  2.6  m  s~',  the  equiv- 
alent of  0.91  m  s~'  in  20°C  seawater.  The  other  four  sets 
were  made  at  a  wind  speed  of  4.6  m  s~',  the  equivalent 
of  1.6  m  s  '  in  20°C  seawater.  Orientations  of  the  model 
in  the  faster  flows  were  (1)  anterior  end  upstream.  (2) 
posterior  end  upstream,  (3)  ventral  edge  upstream,  and 
(4)  dorsal  edge  upstream.  In  the  slower  flows,  only  ori- 
entations ( 1 )  and  (3)  were  tested. 

When  the  anterior  end  was  upstream,  the  dorsal  edge 
was  at  3.6°  (<  ±3°  error)  counter-clockwise  (when  looking 
from  above)  relative  to  mainstream  flow.  The  other  ori- 
entations were  rotated  90°,  180°,  and  270°  with  respect 
to  that  position. 

Three  perpendicular  components  offeree  are  obtained 
by  multiplying  pressures  by  projected  areas  perpendicular 
to  each  component  (with  appropriate  sign  conventions). 
Projected  areas  were  obtained  from  six  photographs,  par- 
allel to  all  sides  of  a  cube,  of  the  model  taken  from  a 
distance  of  15-20  m  with  a  200-mm  lens  (the  large  dis- 
tance minimizes  systematic  distortion  of  area  resulting 
from  perspective).  Area  was  measured  by  weighing  areas 
cut  out  of  the  photographs  and  also,  for  comparison  and 
estimation  of  errors,  by  digitizing  the  areas  with  a  digitizing 
tablet  and  a  computer.  Error  in  the  area  measurement, 
including  bias  from  area  distortion  (the  difference  between 
the  area  of  calibration  square  centimeters  in  front  of  and 
behind  the  clam  model)  and  imprecision  (estimated  as 
the  standard  deviation  of  repeated  measurements),  was 
always  less  than  ±10%.  For  the  moment  calculations,  the 
3-dimensional  location  coordinates  of  all  holes  were  also 
measured  from  these  photographs.  Location  was  mea- 
sured with  an  error  less  than  ±7%.  The  (0,0,0)  coordinate 
was  placed  at  the  pivotal  point  (the  point  at  which  the 
shell  touches  the  ground). 

A  propagation  of  errors  analysis  was  performed  ac- 
cording to  standard  formulas  (see  p.  28  in  Schulz.  1945; 
Ku,  1969).  Errors  propagated  through  the  moment,  and 
force  calculations  were  the  measured  standard  deviation 
of  pressure  at  each  hole,  and  an  assumed  ±10%  of  the 
area  and  ±7%  of  the  distance  at  each  hole. 


Another  source  of  error,  not  expressed  in  the  propa- 
gation analysis,  is  the  contribution  of  tangential  forces 
acting  on  the  surface  of  the  clam.  Total  force  is  the  sum 
of  forces  normal  and  tangential  to  the  surface,  but  only 
the  normal  forces  are  measured  here.  Tangential  forces 
can  reasonably  be  ignored  because,  for  non-streamlined 
objects  at  the  relevant  Re  values,  tangential  forces  are 
relatively  small.  For  instance,  friction  drag  (due  to  com- 
ponents of  tangential  forces)  is  much  smaller  than  pressure 
drag  (due  to  components  of  normal  forces).  For  a  cylinder 
perpendicular  to  flow,  pressure  drag  is  87%  and  97%  of 
total  drag  at  Re  =  103and  104,  respectively  (Vogel,  1981). 
Also,  for  ellipsoidal  shapes,  with  length-to-diameter  ratios 
ranging  from  2:1  (long  axis  parallel  to  flow)  through  1:1 
(sphere)  to  1:2  (long  axis  perpendicular  to  flow),  pressure 
drag  ranges  from  >80%>  up  to  >95%  of  total  drag,  re- 
spectively, at  Re  =  7  X  104  (Hoerner,  1965).  These  results 
apply  at  subcritical  Re  values;  i.e.,  in  flows  in  which  there 
is  separation  of  flow  on  the  object.  Flow  around  clams  is 
comparable  since  (i)  flow  separates  on  these  clams  (ob- 
served using  dyes  and  inferred  from  pressure  measure- 
ments: see  results),  and  (ii)  clams  have  length-to-diameter 
ratio  of  2:1  or  1:2  depending  on  orientation,  and  (iii) 
measurements  were  made  at  Re  =  1.9  and  3.4  X  104. 
Thus,  components  of  normal  forces  measured  here  will 
tend  to  underestimate  drag  forces,  perhaps  by  as  much  as 
5%-20%. 

The  relative  size  of  the  clam  and  the  boundary  layer 
also  affects  pressure  measurements;  thus  the  velocity  dis- 
tribution existing  in  the  wind  tunnel  under  experimental 
conditions  is  given  for  comparison.  It  was  measured  at  a 
mainstream  wind  of  2.6  m  s"'  using  a  Pilot  tube  and  the 
same  pressure  sensor  as  was  used  for  the  clam.  Velocity 
was  calculated  from 


where  A/7  is  the  pressure  difference  between  the  static  and 
dynamic  openings  of  the  Pilot  tube,  p  is  the  density  of 
the  medium,  and  U  is  the  velocity  of  Ihe  flow  al  that  point 
(Vogel,  1981). 

Density,  weight  and  si~e  of  D.  variabilis  and  density  of 
other  Bivalvia 

Live  D.  variabilis  specimens  of  a  range  of  sizes  were 
weighed  while  they  were  immersed  in  water  and  in  air.  A 
formula  based  on  Archimedes  Principle  was  used  to  cal- 
culate Ihe  density,  pD,  of  D.  variahi/is. 


H" 


PD 


where  p.,  is  the  densily  of  seawaler,  WA  is  Ihe  weighl  of 
Ihe  clam  in  air,  and  H',,  is  Ihe  weight  of  the  clam  in  sea- 


142 


O.  ELLERS 


water.  The  general  shape  of  these  clams  is  also  relevant. 
Using  calipers,  basic  dimension  measurements  were  made 
of  the  anterior-posterior,  ventral-dorsal,  and  left-right 
distances  on  the  same  clams. 

The  densities  of  other  bivalve  species,  chosen  haphaz- 
ardly on  North  Carolina  beaches,  were  determined  in  the 
same  way  (three  specimens  each  of  seven  species  were 
measured).  Specimens  of  the  same  size  as  a  large  individ- 
ual of  D.  variahilis  (2-3  cm  long)  were  used  to  minimize 
potential  allometric  effects  on  the  comparison. 

Density,  weight  distribution,  and  size  effects  on 
orientation 

Motions  of  D.  variabilis  shells  of  various  sizes  with  ex- 
perimentally altered  density  and  weight  distribution  were 
qualitatively  observed  in  steady  flows  up  to  60  cm  s~'  in 
a  recirculating  flow  tank.  Tendency  to  orient  and  the  flow 
speed  at  which  orientation  occurred  were  noted. 

Weight  distribution  was  altered  by  placing  Plasticene 
in  the  posterior  end  and  an  air  bubble  in  the  anterior  end 
(and  vice  versa),  of  empty  D  variabilis  shells.  (Altering 
weight  distribution  also  unavoidably  altered  density.) 
Density  was  altered  by  completely  filling  empty  D.  var- 
iabilis shells  with  candle  wax,  Silicone  rubber  or  Plasti- 
cene; the  resulting  densities  of  filled  shells  were  1.5.  1.7 
and  2.0  (X 103  kg  irT3),  respectively.  (Altering  density  un- 
avoidably alters  weight  distribution  slightly.)  Shells  were 
placed  in  flow  with  four  initial  orientations:  ventral,  dorsal, 
posterior,  or  anterior  edges  upstream. 


20  -, 


o 


C3 

HI 


10  - 


2.2      2.4       2.6      2.8 


WIND  VELOCITY  (ms-1) 


Figure  .V  The  wind-speed  distribution  in  the  tunnel  in  which  pressure 
measurements  on  the  clam  model  were  made.  The  mainstream  velocity 
was  equivalent  to  0.91ms  'in  seawater.  The  clam  was  well  inside  the 
boundary  layer,  and  is  shown  for  scale  (it  was  not  in  the  tunnel  while 
the  speed  distribution  was  measured).  Error  bars  show  I  SD. 


10-, 


5- 


n 
Q. 


CO 
LLJ 

cc 

Q. 


4  - 

2 

0 

-2  - 
-4  - 


flow 


468 
POSITION  (cm) 


10 


12 


Figure  4.  Pressure  measurements  taken  along  transects  with  the  clam 
oriented  with  the  ventral  edge  upstream  and  the  anterior  end  upstream. 
Positive  pressures  indicate  forces  acting  inward  normal  to  the  surface; 
negative  ones  indicate  outward  forces.  Dashed  line  joins  upper  (right) 
valve  measurements;  solid  line  joins  lower  (left)  valve  measurements. 
Error  bars  are  I  SD.  (For  comparison,  mainstream  wind  velocity  was 
4.6  m  s~',  which  corresponds  to  a  dynamic  pressure  of  13  Pa.) 


Results 


Backwash  speed 


The  average  speed  of  33  beads  was  0.76  ±  0.20  m  s  ' 
standard  deviation.  Speed  ranged  from  1 . 1  to  0.38  m  s~'. 
Inaccuracy  bounds  (>95%  confidence  intervals)  from  a 
propagation  of  errors  estimate  are  ±11%  for  the  higher 
speeds,  and  ±26%  for  the  lower  speeds. 

Force  distribution  due  to  /low  on  an  oriented  and 
unoriented  clam 

In  the  wind  tunnel,  a  clam  model  was  located  within 
the  gradient  of  speeds  of  the  boundary  layer  (Fig.  3).  The 
model's  shape  altered  the  velocity  distribution  around  it, 
which  caused  pressures  on  the  model.  Observed  pressure 
distributions  (Fig.  4)  are  similar  to  those  typical  of  a  bluff 
body  such  as  a  cylinder  perpendicular  to  flow  in  that,  first. 


MOTION  OF  CLAMS  IN  FLOW 


143 


on  the  upstream  side,  pressures  were  high  (causing  forces 
pushing  downstream);  second,  partway  downstream, 
pressures  became  negative  (causing  outward  forces)  as  flow 
sped  up  around  the  clam;  and  third,  at  the  downstream 
end,  pressures  did  not  recover  (did  not  become  positive 
as  they  would  have  done  in  an  ideal  fluid  or  on  a  stream- 
lined shape),  thus  giving  rise  to  pressure  drag  and  signaling 
separation  of  flow  on  the  model. 

Unlike  the  pressure  distribution  on  a  cylinder,  however, 
time-averaged  pressures  on  the  upper  and  lower  surfaces 
were  unequal  and  resulted  in  lift.  Different  characteristic 
pressure  distributions  were  observed  when  the  anterior 
end  was  upstream,  as  in  other  orientations.  When  the 
ventral  edge  was  upstream,  the  upper  valve  always  ex- 
perienced lesser  pressures  than  the  lower  valve,  thus  giving 
rise  to  upward  lift.  In  contrast,  when  the  anterior  end  was 
upstream,  the  pressure  was  alternately  lesser  on  the  lower 
and  then  on  the  upper  surface  as  flow  proceeded  down- 
stream; forces  from  these  pressures  summed  to  downward 
lift. 

The  overall  effects  of  the  pressure  distributions  in  ori- 
ented and  unoriented  clam  models  are  summarized  by 
the  resulting  forces  and  moments  (Table  I).  Lift  was 
downward  when  the  anterior  end  was  upstream  and  up- 
ward when  the  model  was  in  any  other  orientation.  Drag 
when  oriented  with  the  anterior  end  upstream  was  less 
than  drag  in  any  other  orientation. 

When  oriented  with  the  anterior  end  upstream,  the 
model  clam  experienced  a  moment  tending  to  force  ro- 
tation of  the  anterior  end  into  the  sand  (Table  1;  Fig.  5). 
In  other  orientations  there  was  also  an  analogous  moment 
tending  to  push  the  anterior  end  into  the  sand.  Orientation 
towards  the  stable  position  with  the  anterior  upstream 
may  be  enhanced  by  such  a  moment  because  it  tends  to 
cause  tilting;  tilting  moves  the  pivotal  point  anteriorly. 


thus  increasing  rotational  moments  about  the  vertical, 
pivotal  r-axis. 

Even  without  such  tilting,  when  oriented  with  the  ven- 
tral, dorsal,  or  posterior  ends  upstream,  there  were  large 
moments  tending  to  rotate  the  clam  model  about  the  ver- 
tical axis  and  towards  an  orientation  with  the  anterior 
end  upstream  (Fig.  6).  In  contrast,  when  the  anterior  end 
was  upstream,  the  clam  model  experienced  much  smaller 
moments  about  the  vertical,  r-axis.  The  anterior  upstream 
orientation  is  rotationally  stable. 

Density,  weight,  and  size  of  D.  variabilis  and  density  of 
other  Bivalvia 

D.  variabilis  is  one  of  the  densest  bivalves  measured 
(Table  II).  Size  and  weight  are  described  for  20  specimens. 
The  (ventral-dorsal  distance)  =  0.51  (anterior-posterior 
distance)  +  0.081.  r  =  0.995;  and  the  (left-right  distance) 
=  0.36  (anterior-posterior  distance)  +  0.028.  r2  =  0.98, 
with  all  distances  in  centimeters.  The  anterior-posterior 
distances  ranged  from  0.5 1  to  2.3  cm,  and  corresponding 
weights  ranged  between  7.6  10~5  and  6.8  10"3  N.  The 
(weight  in  seawater)  =  6.2  10~4  (anterior-posterior  dis- 
tance)29. /•-  =  0.99,  with  weight  in  N  and  distance  in  cen- 
timeters. 

Density,  weight  distribution,  and  size  effects  on 
orientation 

The  tendency  to  orient  with  the  anterior  end  upstream 
in  flow  is  affected  by  a  combination  of  density,  weight 
distribution,  and  size  (Table  III).  When  shells  oriented, 
they  rotated  around  a  vertical  axis  through  the  pivotal 
point  and  did  so  while  either  sliding  downstream  or  re- 
maining at  their  original  location.  Weight  distribution  af- 
fected the  location  of  the  pivotal  axis:  the  more  relatively 


Table  I 

Forces  mid  moment'*  due  In  How-induced  normal  forces  al  lm>  speeds  in  seawater  (converted  from  wind  tunnel  measurements  using 
dynamic  similarity) 


End  Upstream 

Speed  m  s~  ' 

Drag  X  10"4  N 

LiftH  *  lO'4  N 

Lifty  X  1Q-"  N 

M,  x  ID'6  Nm 

My  •»•  10""  Nm 

Mz  x  10"6  Nm 

Anterior 

0.91 

72  ±    2.8 

-39+    2.9 

-22  ±    4.6 

16  ±    2.6 

-3.0  ±    3.3 

14+    2.6 

Anterior 

1.6 

310+  10 

-130  +  11 

-94  +  17 

17  +  11 

-31     ±14 

49  ±  10 

Posterior 

1.6 

600+  15 

-330  +  17 

470  +  26 

160  ±22 

570     ±  21 

340  ±  17 

Dorsal 

1.6 

960  ±  24 

340  ±  19 

810  ±  41 

-220  ±  26 

530     ±  24 

140  ±  13 

Ventral 

0.91 

200  +    6.6 

66  +    6.2 

54  +  12 

11  ±    7.8 

65     ±    7.3 

-88  ±    4.6 

Ventral 

1.6 

560  ±  17 

120  ±  14 

500  +  29 

67  ±  17 

270     +  15 

-240  ±  10 

These  measurements  apply  to  a  D  variabilis  clam  with  an  anterior-posterior  distance  of  2.2  cm.  For  comparison,  this  clam  weights  61  x  10  "  N 
in  seawater.  The  coordinate  system  used  for  the  moments  is  nght-handed*  and  the  positive  .v-axis  is  downstream,  the  positive  --axis  is  up.  Errors, 
determined  b\  propagation  of  errors  analysis,  that  approximate  1  SD  are  shown.  LiftH  is  horizontal  lift;  Liftv  is  vertical  lift;  Mx  is  the  moment  about 
the  .v-axis:  M>  is  the  moment  about  the  y-axis;  M;  is  the  moment  about  the  r-axis. 

*  Sign  convention  for  moments:  if  you  point  your  right-hand  thumb  in  the  positive  direction  along  the  axis  about  which  a  moment  is  tending  to 
cause  rotation,  then  a  positive  moment  tends  to  cause  rotation  in  the  direction  that  your  fingers  are  pointing.  A  negative  moment  tends  to  cause 
rotation  in  the  opposite  direction. 


144 


O.  ELLERS 


anterior     upstream 


flow 


posterior     upstream 


flow 


ventral     upstream 


dorsal      upstream 


Figure  5.  Vertical  lift  forces  and  moments  tending  to  tilt  a  clam's 
anterior  end  downward  in  flow  in  four  different  orientations.  (Magnitudes 
not  to  scale.)  The  dot  indicates  flow  out  of  the  page;  the  .Y-axis  is  positive 
downstream.  For  other  symbol  and  axis  definitions  see  Table  I. 


heavy  the  anterior  end,  the  closer  the  pivotal  axis  was  to 
the  anterior  end  and  the  more  likely  the  clam  was  to  orient 
with  the  anterior  end  upstream.  If  the  posterior  end  was 
sufficiently  heavy,  then  the  pivotal  axis  lay  closer  to  the 
middle  and  no  orientation  was  stable.  Denser  or  larger 
clams  oriented  with  the  anterior  end  upstream  at  higher 
flow  speeds.  The  densest  filled  shells  tested  did  not  orient 
with  the  anterior  upstream  at  any  of  the  tested  speeds. 

Discussion 

Morphological  causes  of  passive  orientation  to  a 
rotationally  stable  position 

A  clam  orients  with  the  anterior  end  upstream  because 
moments  about  a  vertical  axis  through  the  pivotal  point 
tend  to  rotate  the  anterior  end  upstream  (Table  I;  Fig.  6). 
Once  the  anterior  end  is  upstream,  the  moment  about 
that  vertical  axis  is  smaller.  Further,  rotational  moments 
reverse  direction  if  a  clam  rotates  past  that  position;  thus, 
once  the  anterior  end  is  upstream,  it  tends  to  stay  there. 

Larger  moments  that  occur  when  posterior,  ventral,  or 
dorsal  edges  are  upstream  are  a  combined  result  of  the 
wedge-like  shape  of  D.  variabilis  and  the  location  of  the 
pivotal  point.  The  wedge-like  shape  creates  larger  mo- 
ments by  having  unequal  projected  areas,  subject  to  pres- 
sure drag,  on  either  side  of  the  vertical  axis  through  the 
pivotal  point.  Likewise,  if  the  pivotal  point  is  located  close 
to  one  end  of  the  clam,  then  unequal  projected  areas  exist 
on  either  side  of  the  vertical  axis. 

The  location  of  the  pivotal  point  is  determined  both 
by  a  clam's  weight  distribution  and  by  forces  from  flow. 


Forces  from  flow  tilt  a  clam  such  that  the  anterior  end  is 
pushed  into  the  sand  (Table  I;  Fig.  5),  which  moves  the 
pivotal  point  anteriorly.  Tilting  thus  further  increases  the 
projected  area  on  one  side  of  the  pivotal  axis  and  increases 
the  moment,  tending  to  cause  orientation  with  the  anterior 
end  upstream.  Experimentally  changing  the  tilt  by  chang- 
ing the  weight  distribution  (using  an  air  bubble  and  Plas- 
ticene  in  opposite  ends)  either  enhanced  or  prevented  ori- 
entation. When  the  posterior  end  was  heavier  and  tilted 
down,  the  pivotal  point  moved  posteriorly  and  orientation 
was  prevented  because  the  projected  area  that  gives  rise 
to  orienting  moments  was  nearly  equal  on  either  side  of 
the  vertical  axis.  Conversely,  when  the  anterior  end  was 
heavier  and  tilted  downward,  orientation  occurred  in  the 
flow  tank  even  in  relatively  slow  flow. 

In  general,  orientation  depends  on  flow  speed.  A  min- 
imum flow  speed  is  required  because  static  friction  be- 
tween clam  and  sand  prevents  rotation  below  critical 
forces  and  speeds.  Equations  modeling  force  balances  just 
prior  to  rotation  show  that  larger  or  denser  clams  require 
higher  flow  speeds  to  start  rotating  (Ellens,  1987.  1988). 
Similarly,  experiments  showed  that  higher  minimum  flow 
speeds  are  required  for  orientation  of  larger  and  denser 
shells  (Table  III). 

Density  is  also  crucial  in  keeping  the  pivotal  point  on 
the  ground.  If  a  shell  is  too  light,  upward  lift  can  tem- 
porarily raise  the  shell,  which  results  in  tumbling  rather 
than  orientation.  For  example,  less  dense  bivalves,  such 


flow 


Figure  6.  Moments  tending  to  rotate  a  clam  about  the  vertical,  r- 
axis  through  the  pivotal  point.  Four  initial  orientations  are  shown.  Mo- 
ments tend  to  rotate  a  clam  towards  a  position  with  the  anterior  end 
upstream  (upper  left).  That  orientation  is  stable  because  rotational  mo- 
ments are  nearly  zero  and  rotational  moments  change  sign  (and  direction) 
if  a  clam  rotates  past  that  orientation.  (Magnitudes  not  to  scale.) 


MOTION  OF  CLAMS  IN  FLOW 


145 


Table  II 

Densities  <>l  \everal  \/vr/o  nfclam\  li'inui  mi  \nilh  (.'arnlina  beaches; 
most  />m;/rip  \/>ivio  .;/v  /ON  c/cvnr  than  D  \ariabilis.  which  has  a 
density  ,'t  I  65  ±  0.5  SD.  n  =  JO 


Species 


Density  x  10'  kg  m 


C'hii'iie  cancellata 

.66 

Men  cihina  nii'nciitiria 

.54 

Spi\uUi  raveneli 

.47 

Miicrocallixlti  nimhosa 

.53 

Divancella  quadrisculata 

.40 

Tellina  iris 

.26 

Tagettus  plebeius 

.17 

as  Tell i mi  iris  or  Tagelhts  plebeius.  tumbled  when  placed 
in  backwash.  Thus,  the  high  density  of  D.  variabilix  among 
bivalves  (Table  II).  may  be  crucial  for  orientation  during 
swash-riding.  A  D.  variabilis  clam  is  in  danger  of  being 
lifted  off  the  sand  in  flows  from  waves:  the  underwater 
weight  of  a  clam.  2.2  cm  long,  is  less  than  its  upward  lift 
when  it  is  oriented  with  the  posterior,  ventral,  or  dorsal 
edge  upstream  (Table  I)  in  a  flow  of  1.6  m  s~'.  In  contrast, 
at  0.91  m  s~',  this  clam  is  slightly  heavier  than  the  lift  it 
experiences',  orientation  to  a  stable  position  could  occur. 

Forces  exerted  on  a  D.  variabilis  clam  at  flow  speeds 
encountered  in  backwash  can  be  expected  to  cause  ori- 
entation: observed  speeds  in  the  backwash  averaged 
0.76  m  s~ '.  and  60^  of  the  observed  speeds  were  between 
0.56  and  0.96  m  s  '.  Indeed,  these  clams  often  orient  in 
backwash  (Fig.  2).  Flow  speeds  of  swash  are  usually  higher, 
reaching  a  maximum  of  3  m  s~'  even  on  shallow-sloped 
beaches  (Bradshaw.  1982):  thus  forces  exerted  on  D  var- 
iabi/is  at  flow  speeds  encountered  in  swash  can  cause 
tumbling.  Indeed,  on  a  beach,  clams  often  tumble  in 
swash,  usually  orienting  with  the  anterior  end  upstream 
when  the  swash  slows  as  it  reaches  its  maximum  beach- 
ward  position,  or  in  slower  flow  in  the  backwash. 

These  measurements  and  observations  suggest  that  the 
shape  and  density  of  D.  variabilis  clams  are  a  complex  of 
morphological  characters  that  act  in  concert  and  that  are 
crucial  in  creating  orientation  in  flow.  Further,  there  is  a 
range  of  flow  speeds  in  which  D.  variabilis  clams  can  rotate 
to  a  stable  orientation,  and  speeds  in  that  range  are  com- 
mon on  beaches  on  which  these  clams  live. 

Consequences  oj  orientation:  a  stable,  slower  ride 

When  a  D.  variabilis  clam  is  oriented  with  the  anterior 
end  upstream,  it  experiences  less  drag  than  in  any  other 
orieni  ition  (Table  I).  Furthermore,  it  experiences  down- 
ward if,.  Lower  drag  is  primarily  attributable  to  the  lower 
projei  ted  surface  area  exposed  to  pressure  drag  when  a 
clam's  iong  axis  is  parallel  to  flow.  Downward  lift  is  pri- 


marily due  to  the  downward  tilt  of  the  anterior  end  of  the 
clam  relative  to  the  posterior  end.  which  effectively  creates 
a  negative  angle  of  attack  relative  to  flow.  Lower  drag  and 
downward  lift  result  in  a  stable,  slower  ride. 

Lower  drag  resulting  in  slower  speed  relative  to  the 
substratum  may  seem  initially  counterintuitive  because 
drag  is  most  commonly  encountered  as  a  force  that 
impedes  motion.  During  swash-riding,  however,  drag  is 
a  force  that  propels  a  clam.  A  higher  drag  results  in  a 
sliding  speed  more  nearly  water  speed,  and  therefore  a 
greater  ground  speed.  Conversely,  lower  drag  and  down- 
ward lift  (higher  friction  with  the  ground)  cause  slower 
speeds  of  swash-riding  relative  to  the  substratum. 

During  slower,  stable  sliding  with  the  anterior  end  up- 
stream, the  foot,  which  protrudes  from  the  anterior  end. 
has  a  better  chance  of  achieving  a  foothold  than  if  the 
clam  were  tumbling,  rotating,  or  oriented  any  other  way. 
After  gaining  a  foothold,  the  sand  upstream  of  a  clam 
tends  to  get  scoured  out  (Fig.  2).  which  assists  a  clam  in 
digging  into  the  sand.  On  a  beach,  dead  shells  oriented 
with  the  anterior  end  upstream  were  sometimes  observed 
to  sink  passively  into  the  scoured  sand  just  upstream  of 
their  shells.  Thus,  orientation  with  the  anterior  end  up- 
stream enhances  a  clam's  ability  to  stop  moving  and  to 
burrow  into  the  sand  after  a  swash-ride. 

A  clam's  ability  to  maintain  position  on  the  beach  or 
to  make  net  shoreward  progress  during  shoreward  migra- 
tion depends  on  its  being  able  to  establish  a  foothold  before 
being  washed  out  to  sea.  Gaining  a  foothold  while  the 
backwash  is  flowing  is  therefore  crucial.  Orientation  with 
the  anterior  end  upstream,  with  its  concomitant  reduced 
drag  and  downward  lift,  may  be  the  decisive  factor  in 
making  migration  by  swash-riding  possible. 

Table  III 

Qualitative  obsen-ations  of  the  effects  of  weight  distribution,  density, 
and  size  on  the  tendency  of  D.  variabilis  shells  to  orient  passively  with 
the  anterior  end  upstream 


Variable 


Level 


Final  Orientation  in  Flow 


Weight  Light  anterior  No  orientation 

distribution        Light  posterior  Anterior  upstream  always 

Density  1.5  x  103  kg  irT3       Anterior  upstream  usually 

1.7  X  103  kg  m~3        Anterior  upstream  occasionally 
2.0  x  103  kg  m'3        No  orientation 
Size*  2.2  cm  Anterior  upstream  above 

40  cm  s"1 
1.5cm  Anterior  upstream  above 

30cm  s-' 

1.2cm  Anterior  upstream  above 

25cm  r1 

Shells  were  experimentally  altered  as  indicated  and  were  placed  in  a 
flow  tank  with  one  of  the  initial  orientations  (anterior,  posterior,  ventral, 
or  dorsal  edge  upstream).  Max.  flows  tested  were  60  cm  s"'. 

*  Numbers  shown  are  for  filled  shells  of  density  1.5  x  103  kg  m'3. 


146 


O.  ELLERS 


Morphological  and  behavioral  control  of  swash-riding 

That  shape  can  affect  movement  of  clam-like  objects 
in  waves  has  been  demonstrated  previously  (Lever,  1958; 
Lever  et  al.  1961,  1964,  1968).  Wave-induced,  passive, 
net  movement  of  many  thousands  of  manufactured  model 
Donax  villains  valves  were  observed  over  one  tidal  cycle. 
Right  valves  were  swept  seaward,  whereas  left  valves  re- 
mained on  the  beach;  less  dense  valves  moved  further 
than  more  dense  ones;  larger  shells  remained  on  the  beach 
longer  than  smaller  ones.  Thus,  net  passive  movement  of 
valves  by  waves  depends  on  density,  size,  and  shape. 
Likewise,  the  movements  of  whole,  live  clams  also  depend 
on  these  variables.  D.  variabilis  clams  control  where  waves 
move  them  by  using  a  combination  of  behavior  and  a 
complex  of  morphological  characters:  the  wedge-like 
shape,  high  density,  and  anteriorly  located  pivotal  point. 

A  D.  Yiiricibilix  clam  can  modulate  its  speed  on  the 
beach  relative  to  the  speeds  of  the  swash  and  backwash 
occurring  under  a  range  of  wave  conditions.  On  days  with 
only  small  waves  and  slow  swash  and  backwash  speeds, 
clams  protract  foot  and  siphons  (pers.  obs.),  thus  increas- 
ing drag  and  clam  speed  by  exposing  more  area  to  flow. 
On  days  with  higher  waves,  clams  keep  foot  and  siphons 
retracted,  decreasing  their  speed  relative  to  flow.  If  flow 
speeds  are  sufficiently  high  to  cause  orientation,  a  clam 
orients  with  the  anterior  end  upstream,  which  reduces 
drag  and  thus  again  reduces  clam  speed  relative  to  flow 
speed.  If  flow  speeds  are  so  high  that  the  clam  tends  to 
tumble  before  it  can  orient,  then  the  clam  tumbles  in  flow 
and  moves  nearly  at  flow  speeds  until  the  flow  slows  down, 
as  it  usually  does  in  the  backwash.  Once  the  flow  is  suf- 
ficiently slow  for  orientation,  a  clam  can  regain  a  foothold. 

Ecological  and  evolutionary  con.scc/ncncex 

Net  movement  in  waves,  the  number  of  swash-rides 
required  for  migration,  and  hence  the  energy  cost  of  mi- 
gration (Ansell  and  Trueman.  1973),  depend  on  both  the 
prevailing  wave  conditions  and  the  shape  and  behavior 
of  a  swash-riding  clam.  Flow  speeds  from  waves  typically 
depend  on  beach  structure  and  wave  conditions.  Whether 
D.  variabilis  clams  can  live  on  a  given  beach  will  depend 
on  the  prevalence  of  suitable  flow  speeds  on  the  beach. 
Flow  speeds  must  typically  be  fast  enough  to  overcome 
friction  between  clam  and  sand,  but  slow  enough  that  lift- 
off does  not  occur  in  the  backwash. 

The  functional  morphology  of  swash-riding  thus  sug- 
gests that  the  swash-riding  performance  of  a  Donct.x  species 
should  be  related  to  typical  flows  on  the  beaches  on  which 
it  lives.  An  intriguing  comparison  species  is  the  largest 
(7  cm  long)  Donax  species,  Donax  serra.  which  swash- 
rides  but  migrates  on  a  semilunar  cycle  (Donn  et  al.,  1986) 
rather  than  a  tidal  cycle.  Zonation  patterns  of  different 
populations  of  D.  serra  were  found  to  be  statistically  ex- 


plained by  the  morphological  characters  weight,  surface 
area,  and  elongation;  populations  found  higher  in  the  in- 
tertidal  had  thicker  valves  and  higher  density  than  those 
found  lower  in  the  intertidal  (Donn,  1990). 

Similarly,  the  ecology  of  sandy  beaches  may  depend 
on  the  flow  regime  of  swash  and  backwash.  The  physical 
environment,  described  by  wave-regime  parameters,  was 
found  to  control  species  richness  of  sandy  beach  fauna 
(McLachlan  et  al..  1993).  Body  size  and  means  of  loco- 
motion (such  as  swash-riding)  were  suggested  as  important 
parameters  in  determining  the  extent  to  which  organisms 
were  able  to  live  on  beaches  as  the  swash  "climate"  became 
harsher. 

The  functional  morphology  of  swash-riding  clams  also 
suggests  characters  that  may  have  been  important  in  the 
evolution  of  swash-riding  in  donacid  bivalves.  Since 
swash-riding  is  enabled  by  high  density,  a  wedge-like 
shape,  and  an  anteriorly  located  pivotal  point,  evolution 
of  these  characters  is  predicted  to  be  correlated  with  swash- 
riding.  A  large  range  of  donacid  species  inhabiting  a  range 
of  beach  types  exists  in  the  world.  Independent  contrasts 
methods  (Harvey  and  Pagel.  1991)  and  morphological 
comparisons  using  functional  morphospaces  (Filers  and 
Telford.  1991;  Moore  and  Filers,  1993)  could  be  applied 
to  test  whether  these  characters  are  evolutionarily  corre- 
lated with  swash-riding. 

Acknowledgments 

This  research  is  part  of  the  author's  Ph.D.  dissertation 
completed  at  Duke  University.  I  thank  my  thesis  super- 
visor, S.  Vogel,  and  committee  members,  V.  L.  Roth. 
E.  J.  Shaughnessy.  V.  A.  Tucker,  and  S.  A.  Wainwright. 
NSERC  postgraduate  scholarships.  Duke  University 
teaching  assistantships,  and  a  Cocos  Foundation  Training 
Grant  in  Morphology  supported  the  author.  I  thank 
A.  S.  Johnson  for  reading  the  manuscript  and  S.  Vogel 
for  writing  the  digitizer  communication  code. 

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Morphology  and  Physiology  of  the  Thoracic  and 

Abdominal  Stretch  Receptors  of  the  Isopod 

Crustacean  Ligia  exotica 

AKIYOSHI  NIIDA,  YOSHIKO  TAKATSUKI*.  AND  TSUNEO  YAMAGUCHI 

Department  oj  Biology.  Family  of  Science,  Okayama  University.  Tsushima,  Okayama  700.  Japan 


Abstract.  In  the  terrestrial  isopod  Ligia  exotica,  paired 
stretch  receptors,  each  comprising  a  separate  rapidly  and 
slowly  adapting  receptor  cell,  were  found  in  the  third  to 
eighth  thoracic  segments  and  first  five  abdominal  seg- 
ments. The  dendritic  endings  of  the  two  sensory  cells  in 
each  receptor  terminate  on  a  common  receptor  muscle; 
the  cross-striation  of  this  fiber  is  homogeneous  throughout 
the  segments.  But  the  dendritic  endings  of  the  receptor 
cells  differ:  the  rapidly  adapting  cell  has  a  club-shaped 
ending  restricted  to  the  middle  of  the  receptor  muscle, 
whereas  the  slowly  adapting  receptor  cell  has  a  bifurcating 
ending  that  extends  along  the  entire  length  of  the  muscle. 
Stretch  applied  to  the  receptor  muscle  evokes  character- 
istically different  responses  in  the  two  sensory  cells.  The 
slowly  adapting  receptor  cell  has  a  lower  firing  threshold 
and  fires  continuously  for  the  duration  of  the  stretch,  while 
the  rapidly  adapting  receptor  cell  has  a  higher  threshold 
and  fires  a  brief  burst  at  the  beginning  of  the  stimulus. 
However,  application  of  an  intense  stimulus  will  evoke 
continuous  firing  of  the  rapidly  adapting  receptor,  which 
then  changes  to  intermittent  bursts.  The  adaptive  signif- 
icance of  such  a  response  is  not  known,  nor  is  it  likely  to 
occur  in  nature.  However,  this  unusual  response  is  in- 
trinsic to  the  rapidly  adapting  cell,  as  it  can  be  evoked  by 
current  injection.  In  the  second  thoracic  segment,  instead 
of  rapidly  and  slowly  adapting  cells,  we  found  a  single 
slowly  adapting  cell  with  a  long  robust  dendrite  attached 
to  the  extensor  muscle. 

Introduction 

Phylogenetically.  Ligia  and  the  pill  bug  Armadillidum 
vnlgare  belong  to  the  same  suborder  (Oniscoidea)  of 

Received  26  July  1994;  accepted  2h  July  1995. 
*  Present  address:  Department  of  Oral  Science.  Kyushu  Dental  College. 
Manazuru  2-61-1,  Kokura,  Kita-Kyushu  803,  Japan. 


Isopoda.  Both  show  a  similar  segmental  pattern:  a  mobile 
thorax  occupying  a  large  part  of  the  body  and  a  reduced 
abdomen.  The  two  animals  also  show  distinct  segmental 
movements  of  the  body.  The  pill  bug  sluggishly  rolls  up 
in  a  spherical  shape  in  response  to  noxious  stimuli  to  its 
body  or  to  the  removal  of  its  substratum.  Ligia.  in  con- 
trast, cannot  roll  up  in  this  manner  in  response  to  such 
stimuli;  rather,  as  in  its  swimming  behavior,  it  shows  the 
rapid  upward  and  downward  movements  of  the  thoracico- 
abdominal  segments.  Niida  et  al.  (1990)  studied  the  stretch 
receptors  that  might  correlate  with  the  pill  bug's  sluggish 
conglobating  behavior,  and  demonstrated  that  all  the 
stretch  receptors  throughout  thoracic  and  abdominal  seg- 
ments were  of  the  slowly  adapting  type.  Alexander  ( 1 97 1 ) 
recorded  rapidly  adapting  discharges  from  the  thoracic 
stretch  receptors  of  Ligia  oceanica:  but  slowly  adapting 
stretch  receptors,  such  as  those  in  the  abdomen  of  the 
crayfish  Procamhanis  clarkii  (Wiersma  et  al.,  1953),  have 
not  been  reported  in  Ligia. 

The  existence  of  slowly  adapting  stretch  receptors  in 
Ligia  is  strongly  suggested  by  the  behavior  described 
above,  which  surely  requires  postural  controls.  In  addition, 
two  types  of  stretch  receptors — slowly  and  rapidly  adapt- 
ing— occur  commonly  in  the  abdomens  of  the  decapod 
(Wiersma  et  til..  1953)  and  stomatopod  (Pilgrim,  1964). 
Even  the  N-cells.  which  are  located  in  the  most  anterior 
segment  of  the  thorax  and  have  been  considered  as  rem- 
nants of  retrograde  stretch  receptors  in  the  abdomen 
(Wiersma  and  Pilgrim.  1961).  show  slowly  adapting  im- 
pulse discharges  in  response  to  imposed  stimuli.  We  thus 
assume  that  the  slowly  adapting  stretch  receptor  should 
also  predominate  in  Ligia. 

The  goal  of  this  research  is  to  understand  the  functional 
roles  of  the  thoracic  stretch  receptor,  especially  anteriorly 
located  ones,  which  would  be  closely  related  to  segmental 


148 


STRETCH  RECEPTORS  OF  LIU  I A 


149 


movements.  The  present  study  thus  characterized  the 
stretch  receptors  of  Ligia  exotica  both  morphologically 
and  physiologically.  Some  of  the  results  presented  here 
were  reported  in  an  earlier  abstract  (Takatsuki  el  al..  1992). 


Materials  and  Methods 


Animals 


Specimens  of  Ligia  exotica,  30-35  mm  in  total  length, 
were  collected  at  the  coast  of  the  Seto  Inland  Sea  near 
Ushimado  Marine  Laboratory.  Faculty  of  Science,  Okay- 
ama  University,  Japan.  They  were  kept  under  a  photo- 
periodic  regime  of  12  h  light:  12  h  dark  at  20°C.  Both 
males  and  females  were  used  in  the  experiments. 

Identification  of  stretch  receptors 

Conventional  vital  staining  with  methylene  blue  was 
used,  as  well  as  axonal  filling  with  nickel  chloride.  In  the 
latter  staining  technique,  the  cut  distal  stump  of  the  dorsal 
nerve  of  the  third  nerve  root  in  the  thoracic  ganglion  was 
introduced  into  a  glass  capillary  filled  with  0.2  M  NiCli. 
The  preparation  was  stored  at  4°C  for  12-24  h  to  allow 
diffusion  of  the  NiCl:,  which  was  precipitated  by  the  ad- 
dition of  rubeanic  acid.  Stretch  receptors  identified  by 
both  staining  methods  were  isolated  and  mounted  in  gel- 
atin on  glass  slides. 

Preparation  for  recording 

The  responses  of  the  stretch  receptors  to  imposed  stim- 
uli were  recorded  in  situ  and  //;  vitro.  The  following  three 
types  of  preparations  were  used. 

1.  A  semi-intact  preparation  was  used  when  flexion 
was  imposed  in  situ.  After  animals  were  anesthetized  in 
cold  seawater  and  decapitated,  the  legs  and  the  6th  ab- 
dominal segment  were  cut  off.  The  viscera  were  then  dis- 
sected away  from  the  cut  end  of  the  6th  abdominal  seg- 
ment, and  the  nerve  cord  was  left  intact.  Such  preparations 
were  immediately  flushed  with  seawater  to  prevent  the 
deterioration  of  stretch  receptors  and  nervous  tissue  by 
endogenous  digestive  enzymes. 

2.  A  consecutive  tergite  preparation  was  used  for  im- 
posed stretch  experiments.  The  semi-intact  preparation 
described  above  was  cut  with  scissors  along  the  midline 
of  the  sternite  so  that  the  trunk  was  bisected  into  two 
stripes  of  hemisegments  from  which  the  nerve  cord  was 
removed.  These  hemisegment  preparations  were  then 
further  cut  into  pieces  of  two  consecutive  tergites  each. 

3.  A  preparation  of  isolated  stretch  receptor  was  used 
for  in  vitro  experiments.  The  dorsal  nerve  containing  the 
axons  of  the  stretch  receptors  was  cut  at  its  proximal  end. 
The  stretch  receptor  was  then  isolated  by  cutting  the  re- 
ceptor muscle  near  its  insertion. 


Each  of  these  three  preparations,  when  complete,  was 
then  transferred  to  an  experimental  chamber  filled  with 
seawater.  Most  experiments  were  carried  out  in  seawater 
cooled  to  1 5- 1 8°C.  But  at  times  a  physiological  saline  for 
Ligia,  prepared  by  Yamagishi  (1985)  based  on  the  com- 
position of  Ligia  serum  (Parrey,  1953),  was  also  used.  We 
found  no  remarkable  difference  in  impulse  discharges  for 
at  least  5  h  between  seawater  and  physiological  saline. 

Stimulation  and  recording 

Flexion  experiments.  For  extracellular  recording  from 
the  stretch  receptors  in  the  7th  thoracic  segment,  all  the 
anterior  tergites  up  to  the  6th  thoracic  segment  were  fixed 
ventral  side  up  on  a  silver  plate  with  instantaneous  ad- 
hesive, while  the  free  movable  tergite  of  the  6th  abdominal 
segment  was  pierced  with  a  hook-shaped  needle  connected 
to  the  vertically  moving  central  pin  of  a  vibrator  device 
(Fig.  1  A).  The  vibrator  device,  with  a  frequency  response 
from  DC  to  200  Hz,  was  driven  by  applying  a  ramp-and- 
hold  pulse  by  which  flexion  size  of  the  abdomen  to  the 
horizontal  was  varied  from  0°  to  60°.  The  flexion-induced 
responses  were  recorded  from  the  dorsal  nerve  of  the  3rd 


Figure  I.  Experimental  setup  for  recording  responses  from  stretch 
receptors.  (A)  In  vivo  preparation.  A  flexion  stimulus  was  delivered  by 
a  vibration  device  with  an  L-shaped  arm  that  moves  upward.  In  the 
experiments  reported  here,  the  flexion  was  produced  by  flexing  the  ab- 
domen to  60°  to  the  horizontal.  Resultant  responses  were  recorded  with 
a  tungsten  hook  electrode  attached  to  the  3rd  nerve  root.  (B)  In  situ 
preparation.  One  end  of  a  pair  of  the  bisected  tergites  was  fixed  with 
insect  pins,  and  the  other  was  connected  to  a  vibration  device  that  de- 
livered the  stretch  stimulus.  Stretch-induced  activities  of  the  receptor 
cells  were  recorded  through  a  suction  electrode  attached  to  the  distal  cut 
end  of  the  dorsal  nerve  of  NR3.  NR3,  3rd  nerve  root:  VD.  vibration 
device. 


150 


A.  NIIDA  ET  AL 


nerve  root  of  the  6th  thoracic  ganglion  with  a  tungsten 
hook  electrode  insulated  by  addition  of  mineral  oil. 

Imposed  stretch  experiments.  The  tergite  just  anterior 
to  the  segment  containing  the  stretch  receptors  to  be  stud- 
ied was  fixed  with  an  insect  pin  ventral  side  up.  The  free 
end  posterior  to  the  segment  to  be  studied  was  connected 
to  the  horizontally  moving  pin  of  the  vibrator  device 
through  the  hook-shaped  needle  in  the  same  manner  as 
described  above  (Fig.  IB).  A  controlled  stretch  stimulus 
was  thus  delivered  to  the  receptor  muscle  in  the  relevant 
segment,  and  the  resulting  responses  were  obtained  from 
the  dorsal  nerve  of  the  3rd  nerve  root:  the  dorsal  nerve 
was  cut  distally  and  introduced  into  a  suction  electrode 
made  of  a  glass  capillary. 

In  vitro  experiments.  Intracellular  recordings  were  made 
for  two  purposes.  The  first  was  to  determine  which  of  the 
two  receptor  cells  was  responsible  for  a  given  response: 
i.e.,  the  slowly  or  rapidly  adapting  response  to  stretch 
stimuli.  Each  of  the  receptor  cells  was  impaled  with  a 
glass  microelectrode  filled  with  3  M  KC1.  Subsequently, 
imposed  stimuli  were  delivered:  each  end  of  the  receptor 
muscle  of  an  isolated  stretch  receptor  was  gripped  with  a 
clamp  mounted  on  a  micromanipulator  with  which  the 
receptor  muscle  was  manually  stretched.  The  second  pur- 
pose was  to  analyze  the  characteristics  of  the  intermittent 
discharges  (described  later)  specific  to  the  rapidly  adapting 
stretch  receptors  of  L.  exotica.  A  bridge  circuit  was  used 
in  the  analysis  of  the  intermittent  bursts,  so  current  was 
injected  into  the  receptor  cell  and  the  concomitant  re- 
sponses were  recorded  through  a  single  microelectrode 
that  was  filled  with  3  M  KC1  and  had  an  impedance  of 
20-30  MO. 

Results 

As  described  below,  segmental  stretch  receptors  occur 
bilaterally  in  the  thoracic  and  the  abdominal  segments  of 
L.  exotica.  For  simplicity,  the  results  will  be  described 
from  one  side  only,  and  we  refer  to  the  thoracic  and  the 
abdominal  stretch  receptors  as  TSR  and  ASR,  respec- 
tively. This  study  demonstrated  the  existence  of  slowly 
and  rapidly  adapting  stretch  receptors  differing  both 
physiologically  and  morphologically.  A  few  animals  did 
not  respond  to  the  stretch  stimuli,  where  current  injection 
into  the  stretch  receptor  cells  produced  a  response  com- 
parable to  that  obtained  by  stretching.  In  Ligia,  therefore, 
mechanical  transduction  might  be  greatly  influenced  by 
the  mechanical  deformation  that  occurs  during  the  dis- 
section of  stretch  receptors. 

Spatial  organization  of  stretch  receptors 

The  segmental  trunk  of  L.  exotica  is  composed  of  eight 
thoracic  and  six  abdominal  segments:  because  the  first 
thoracic  segment  is  fused  with  the  head,  the  main  part  of 


the  thorax  forms  seven  segments,  i.e.,  the  2nd  to  8th  seg- 
ments. Figure  2  A  shows  the  spatial  organization  of  stretch 
receptors  in  the  thoracic  and  abdominal  segments,  where 
each  stretch  receptor,  except  for  TSR-1  (Fig.  2A),  com- 
prises a  set  of  paired  receptor  cells  and  a  single  specialized 
receptor  muscle.  The  TSR-1  possesses  no  specialized  re- 
ceptor muscle;  instead,  the  musculature  associated  with 
the  TSR-1  is  an  ordinary  dorsal  extensor  muscle.  The 
dendrite  of  the  TSR-1,  in  its  course,  is  partially  attached 
to  the  articular  membrane  of  the  anterior  ridge  of  the  3rd 
thoracic  segment  and  runs  toward  its  insertion  in  the  an- 
terior edge  of  the  extensor  muscle  of  the  2nd  thoracic 
segment. 

The  characteristic  organization  of  stretch  receptors  ap- 
pears in  TSR-2,  which  is  located  between  the  3rd  and  4th 
thoracic  segments.  A  long  receptor  muscle  (ca.  5  mm  in 
3.5  cm  body  length)  has  its  posterior  insertion  on  the  an- 
terior ridge  of  the  5th  segment  and  runs  through  the  4th 
segment  to  the  articular  membrane  of  the  anterior  ridge 
of  the  3rd  segment.  A  pair  of  functionally  differentiated 
receptor  cells  terminates  on  this  receptor  muscle  within 
the  4th  thoracic  segment. 

In  the  5th  thoracic  segment,  the  anterior  and  posterior 
insertions  of  the  receptor  muscle  lie  on  the  individual 
anterior  ridge  of  the  5th  and  6th  thoracic  segments  (Fig. 
2 A).  This  arrangement  of  the  anterior  and  posterior  in- 
sertions also  occurs  in  the  receptor  muscle  of  the  6th  tho- 
racic segment.  The  arrangement  in  the  most  posterior  of 
the  thoracic  segments  is  different  again:  anterior  insertions 
of  the  receptor  muscles  of  the  7th  and  8th  thoracic  seg- 
ments are  in  the  connective  tissue  of  each  leg  muscle  of 
the  7th  and  8th  thoracic  segments,  whereas  their  posterior 
insertions  occurred  on  the  anterior  ridges  of  the  8th  tho- 
racic and  1st  abdominal  segments,  respectively  (Fig.  2A). 

TSR-1,  as  can  be  seen  in  Figure  2A,  lies  medially  in 
association  with  the  extensor  muscle,  but  TSR-2  and  sub- 
sequent stretch  receptors  lie  somewhat  dorsolaterally,  and 
much  more  laterally  than  abdominal  stretch  receptors  in 
the  crayfish.  The  thoracic  receptor  muscles  are  shorter  in 
successively  more  posterior  segments,  whereas  abdominal 
receptor  muscles  become  longer  posteriorly  (Fig.  2A). 

Axonal  pathway  of  stretch  receptors 

The  3rd  nerve  root  of  each  thoracic  ganglion  branches 
complexly:  the  dorsal  nerve  in  this  3rd  root  provides  a 
common  pathway  both  for  the  central  projection  of  the 
axons  of  stretch  receptor  cells  and  for  efferents  to  the  ex- 
tensor muscle  (Fig.  3A).  A  pair  of  the  axons  of  the  thoracic 
stretch  receptor  cells  (rapidly  and  slowly  adapting  cells) 
bifurcate  at  the  3rd  nerve  root  and  course  in  two  directions 
in  pairs;  one  runs  towards  the  subesophageal  ganglion 
and  the  other  towards  the  6th  abdominal  ganglion  (unpub. 
obs.).  In  the  abdomen,  the  axons  of  the  stretch  receptor 


STRETCH  RECEPTORS  OF  Utit.l 

A                        TS 

AS 

s                          ^ 
23          45678 
^—  ?  ^  —  ~~j-  , 

151 


TS-8 


AS 


AG 


Figure  2.  Organization  of  thoracic  and  abdominal  stretch  receptors  (A)  and  the  central  connection  of 
abdominal  stretch  receptors  (B).  (A)  and  (B)  are  viewed  from  the  ventral  side,  and  the  viscera  were  removed 
as  well  as  all  muscles,  except  for  those  with  which  the  stretch  receptors  are  associated.  In  (A),  head  and  legs 
are  removed,  and  the  first  thoracic  segment  is  not  depicted  because  it  is  fused  with  the  head.  AG.  abdominal 
ganglion;  AN.  abdominal  nerve:  AS.  abdominal  segment;  ASR,  abdominal  stretch  receptor;  BP.  basal  pro- 
podite;  TG-8.  8th  thoracic  ganglion;  TS-8.  8th  thoracic  segment:  TSR.  thoracic  stretch  receptor.  Numerals 
after  TSR  and  ASR  indicate  position  in  the  sequence  of  the  segmental  stretch  receptors. 


cells  run  through  the  abdominal  nerve  (Fig.  2B)  and  enter 
the  several  fused  abdominal  ganglia. 

Morphological  characteristics 

Thoracic  stretch  receptors.  TSR- 1  has  an  extremely 
long  dendritic  process  extending  from  a  bipolar  receptor 
cell  located  in  the  ventral  surface  of  the  medial  extensor 
muscle  of  the  3rd  thoracic  segment  (Fig.  2A).  The  den- 
dritic process  is  quite  stout  in  the  anterior  ridge  of  the 


3rd  thoracic  segment,  but  as  it  extends  forward,  it  grad- 
ually thins,  running  in  close  contact  with  the  extensor 
muscle.  The  dendrite  is  attached  to  the  muscle  at  its 
anterior  extremity  in  the  2nd  thoracic  segment.  Thus, 
although  the  length  of  the  dendritic  process  depends  on 
the  total  body  length,  in  animals  3  to  4  cm  long,  it  mea- 
sures 2  to  3  mm  from  the  receptor  cell  soma.  Running 
posteriorly,  a  thin  strand  originates  from  the  initial  part 
of  the  stout  dendrite,  but  its  insertion  could  not  be  traced 
precisely. 


152 


A.  NIIDA  ET  AL 


A 


NR3 


AM 


B 


m  • 


Figure  3.  (A)  Spatial  arrangement  of  stretch  receptors  in  the  6th  and 
7th  thoracic  segments  and  the  central  connections  of  stretch  receptors. 
The  figure,  viewed  from  the  ventral  side,  shows  hemisegments  with  part 
of  the  lateral  side  of  the  tergites  not  depicted.  NR 1  and  2  run  towards 
the  pereopod,  whereas  the  dorsal  nerve  of  NR3  provides  a  common 
pathway  for  the  central  projection  of  the  axon  of  the  stretch  receptors 
and  for  the  efferent  fiber  to  the  extensor  muscle  (EM).  Note  that  the 
anterior  insertion  of  receptor  muscle  of  the  7th  thoracic  segment  occurs 
in  the  leg  muscle  of  the  same  thoracic  segment.  (B)  Photomicrograph  of 
a  thoracic  stretch  receptor  (TSR-3)  stained  by  axonal  filling  with  nickel 
chloride.  Thick  arrowhead  indicates  an  efferent  to  the  extensor  muscle. 
Thin  white  and  black  arrowheads  indicate  C-type  (rapidly  adapting)  and 
B-type  (slowly  adapting)  stretch  receptors,  respectively.  Scale  bar,  200  ^m. 
ANT,  anterior;  AM.  articular  membrane;  BP.  basal  protopodite;  EM, 
extensor  muscle;  FM.  flexor  muscle;  NR1.  1st  nerve  root;  NR2.  2nd 
nerve  root;  RC,  receptor  cells;  RM.  receptor  muscle;  POST,  posterior. 


With  the  exception  of  TSR-1,  the  receptor  cells  of  the 
stretch  receptors  in  both  thoracic  and  abdominal  segments 
were  classified  morphologically  into  two  different  types 
on  the  basis  of  their  dendrites:  club-shaped  cells  (C-type) 
and  bifurcating  cells  (B-type)  (Fig.  3B).  The  characteristics 
of  these  cell  types  emerge  from  schematic  illustrations  of 
the  stretch  receptors  in  the  2nd  to  8th  thoracic  segments 
(Fig.  4).  Each  C-type  cell  shows  a  stout  dendrite  attached 
to  the  central  part  of  the  receptor  muscle.  In  contrast,  the 
branching  dendrites  of  B-type  cells  run  in  both  directions 
along  the  total  length  of  the  receptor  muscles.  The  den- 
dritic processes  of  both  C-  and  B-type  cells  are  much  longer 
in  TSR-2  than  those  of  the  stretch  receptors  in  other  tho- 
racic segments.  Another  characteristic  of  the  stretch  re- 
ceptors is  the  homogeneous  striation  of  the  receptor  mus- 
cles throughout  thoracic  and  abdominal  segments.  Al- 
though'systematic  measurements  were  not  made,  the 
sarcomere  length  of  the  receptor  muscle  in  the  7th  thoracic 
segment  was  3.6  ±  0.18  (mean  ±  SD)  /urn.  Of  course,  this 
muscle — in  every  segment — is  shared  by  slowly  and  rap- 
idly adapting  stretch  receptor  cells.  In  the  crayfish,  how- 
ever, the  sarcomeres  are  short  (3.3  jum)  in  the  receptor 
muscle  of  the  rapidly  adapting  stretch  receptor,  and  long 
(6.5  ;nm)  in  that  of  the  slowly  adapting  stretch  receptor 
(Komuro,  1981). 

Abdominal  stretch  receptors.  The  morphology  of  the 
abdominal  stretch  receptors  (Fig.  5)  is  similar  in  general 
to  that  of  the  thoracic  receptors:  i.e..  there  are  C-type  and 
B-type  receptor  cells,  and  they  are  attached  to  the  single 
receptor  muscles  with  homogeneous  striations.  But  the 
B-type  receptor  cells  of  the  2nd  and  3rd  abdominal  seg- 
ments show  morphological  variations  in  the  manner  of 
the  bifurcation  of  their  dendrites.  Generally,  the  dendrites 
of  the  B-type  receptor  cells  in  the  2nd  and  3rd  abdominal 
segments  bifurcated  in  close  contact  with  the  receptor 
muscle,  as  in  ASR-1  (Fig.  5),  but  some  receptor  cells  show 
a  dendritic  branching  pattern;  e.g..  in  ASR-4  the  dendrite 
branches  distally  to  the  receptor  muscle.  Another  differ- 
ence from  the  TSR  is  that  the  dorsal  extensor  muscle 
closely  parallels  the  abdominal  receptor  muscle.  This  an- 
atomical arrangement  closely  resembles  that  of  the  cray- 
fish. Procamharus  clcirkii  (Wiersma  el  a/..  1953). 

In  situ  response  of  stretch  receptors  to  imposed  flexion 

Figure  6  shows  a  representative  in  situ  recording  from 
TSR-5  of  the  7th  thoracic  segment  in  response  to  ab- 
dominal flexion  in  the  ventral  direction  (upward  im- 
posed flexion).  The  flexion  was  imposed  with  a  vibrator 
device  driven  by  a  ramp-and-hold  pulse  of  0.05  Hz;  the 
animal  was  ventral  side  up,  and  the  abdominal  flexion 
was  60°  from  the  horizontal  axis  (Fig.  1  A).  The  evoked 
responses  showed  slowly  and  rapidly  adapting  impulse 
discharges  or  phasic  and  tonic  responses,  as  shown  in 


STRETCH  RECEPTORS  OF  Z./G7.I 


153 


TSR-2 


TSR-3 


TSR-4 


Figure  4.  Schematic  drawings  of  thoracic  stretch  receptors.  The  drawings  in  this  and  the  next  figures 
were  based  on  specimens  stained  with  methylene  blue.  B,  B-type  cell:  C,  C-type  cell.  Note  that  the  cross- 
striation  within  every  receptor  muscle,  from  TSR-2  to  TSR-6,  is  homogeneous.  Scale  bar,  100  ^m. 


the  inset  of  Figure  6.  The  adaptive  time  courses  of  im- 
pulse discharges  from  rapidly  and  slowly  adapting 
stretch  receptors  are  also  shown  in  Figure  6a  and  b.  The 
phasic  response  has  longer  latency  due  to  the  slow  rise 
of  the  stimulus  delivered  at  0.05  Hz:  the  phasic  quality 
indicates  dependence  on  the  velocity;  i.e.,  the  rate  of 
displacement  (angle/s)  of  the  thoracic  segment  by  the 


flexion  stimulus  (Fig.  7).  Similar  responses  accompanied 
by  tonic  impulse  discharges  were  recorded  from  the 
TSR-5  upon  abdominal  extension  (downward  imposed 
flexion)  (data  not  shown).  Extension  produced  much 
lower  impulse  frequencies  than  flexion,  even  when  the 
degree  of  the  applied  stimulus  was  the  same.  In  the 
presence  of  motor  activities  of  the  extensor  neuron. 


ASR-1 


ASR-2 


RM 


Figure  5.     Schematic  drawings  of  abdominal  stretch  receptors.  Homogeneous  cross-striation  is  similarly 
noted  in  the  abdominal  receptor  muscles.  B.  B-type  cell;  C.  C-type  cell.  Scale  bar,  100  urn. 


154 


A.  NIIDA  ET  AL. 


0       2       4       6       8      10      12 
Time  (sec) 

Figure  6.  Response  of  TSR-5  (in  xilu  preparation)  to  imposed  flexion, 
(a)  and  (b):  the  time  courses  of  impulse  discharges  in  the  rapidly  and 
slowly  adapting  stretch  receptors,  respectively.  Inset:  responses  from  the 
two  types  of  stretch  receptors.  Lower  trace,  flexion  amplitude  (60°  from 
horizontal).  Time  scale,  I  s. 


however,  impulse  frequencies  of  the  TSR-5  were  some- 
what increased  (data  not  shown). 

Response  of  thoracic  stretch  receptors  to  stretch  stimuli 

Both  slowly  and  rapidly  adapting  impulse  discharges 
were  evoked  from  each  stretch  receptor  in  the  3rd  to  8th 
thoracic  segments  by  an  imposed  stretch  stimulus.  In 
contrast,  TSR-l  in  the  most  anterior  segment  is  a  simple 
stretch  receptor  and  its  response  is  only  slowly  adapting 
(not  shown).  To  represent  activities  of  the  stretch  receptors 
with  both  slowly  and  rapidly  adapting  cells,  the  records 
from  TSR-2  are  shown  in  Figure  8,  and  two  kinds  of 
impulse  discharges  differing  in  their  frequency  and  am- 
plitude can  be  seen  (Fig.  8 A).  One  was  derived  from  a 
slowly  adapting  receptor  cell  and  showed  a  tonic  impulse 
discharge  that  gradually  adapted  as  long  as  the  receptor 
muscle  was  stretched.  In  this  particular  receptor,  the  on- 
going tonic  impulse  discharges  appeared  before  the  stretch 
stimulus  because  we  extended  the  receptor  muscle  slightly 
while  securing  the  thoracic  segment  with  insect  pins  to 
the  cork  platform  in  the  experimental  chamber.  In  this 
experiment,  therefore,  we  took  the  initial  length  of  the 
receptor  muscle  with  the  slight  extension  as  its  apparent 
zero  length. 


When  the  receptor  muscle  was  stretched  in  increments 
of  0.03  mm  (Fig.  8B),  a  notable  phasic  response  occurred 
at  an  increment  of  0.25  mm  from  the  relative  zero  length 
of  the  receptor  muscle  (Fig.  8B).  This  indicates  that  the 
cells  showing  a  phasic  response  possess  a  higher  threshold 
for  a  given  length  of  stretch  than  cells  showing  a  tonic 
response,  and  they  might  be  more  sensitive  to  transient 
segmental  movement.  On  the  other  hand,  tonic  cells  might 
serve  as  positional  detectors:  thus,  when  the  receptor 
muscle  was  stretched  in  steps  of  0.05  mm,  up  to  0.9  mm, 
a  linear  relationship  was  observed  between  impulse  fre- 
quency and  the  length  of  stretch  in  the  range  of  0.45  to 
0.9  mm  (Fig.  8C).  This  relationship  holds  good  only  in 
the  dynamic  range  of  the  stretch  receptor;  i.e.,  the  impulse 
discharge  saturates  when  the  stretch  stimulus  is  much 
larger  (see  Fig.  lOb). 

Identification  of  particular  response  characteristics  to 
either  B-type  or  to  C-type  cells  was  demonstrated  by  in- 
tracellular  recording.  A  microelectrode  was  used  to  pen- 
etrate either  B-type  or  C-type  cells  that  had  been  identified 
under  a  binocular  microscope.  When  stretch  stimuli  or 
current  injections  were  applied,  a  slowly  adapting  response 
was  recorded  from  the  B-type  (not  shown),  and  the  rapidly 
adapting  response  was  recorded  from  the  C-type  receptor 
cells  (Fig.  9B). 


200 


o 
0) 


OT 

CD 

v>  100 

3 
Q. 

E 


o 


0      100    200     300   400     500 

Velocity  (7  sec) 


600 


Figure  7.  Effect  of  the  velocity  of  imposed  flexion  on  the  response 
of  stretch  receptors.  These  data  were  obtained  by  varying  the  ramp  slopes 
in  the  experiment  shown  in  Figure  6.  Points  with  vertical  bars  represent 
mean  ±  SD. 


STRETCH  RECEPTORS  OF  /./(,/.! 


155 


B 


lil" 


5- 


1sec 


01 

0) 


a. 

I   10 


0 


biA*:uTvi    ' 
iWr^'Vfi^ 

c 


J 


0.4         0.6        0.8         1.0    (mm) 

Length  of  Stretch 

Figure  8.  (A)  Typical  stretch-induced  response  of  the  in  \iiii  thoracic 
stretch  receptor.  TSR-2.  showing  both  rapidly  and  slowly  adapting  re- 
sponses. Stretch  amplitude.  0.3  mm.  (B)  Responses  of  TSR-2  in  the  same 
specimen  as  in  (A)  to  various  lengths  of  stretching.  In  (A)  and  (B).  the 
lower  record  in  each  pair  represents  the  relative  amplitude  of  the  applied 
flexion.  B,,  Omm;  B2,  0.03mm:  B3.  0.06mm;  B4.  0.09mm;  B5. 
0.12  mm;  B6.  0.25.  Arrows  indicates  beginning  and  cessation  of  stretch 
stimulus.  (C)  Relationship  between  length  of  stretch  and  frequency  of 
impulses.  These  data  were  taken  from  an  in  situ  preparation  of  TSR-5 
obtained  from  a  specimen  different  from  that  in  (A).  Inset  shows  some 
of  the  responses  and  stimulus  amplitude  (lower  traces)  that  were  plotted, 
(a)  to  (d)  correspond  to  stretch  of  0.5.  0.6.  0.7.  and  0.8  mm,  respectively. 
Time  scale  (inset).  2  s.  In  (A).  (B).  and  (C).  recordings  were  made  extra- 
cellularly  through  a  suction  electrode. 

A  closer  examination  of  the  phasic  response  led  to  un- 
expected results.  When  the  receptor  muscle  was  stretched 
beyond  a  certain  length,  the  usual  pattern  of  the  phasic 


response  changed  to  maintained  discharge  of  intermittent 
bursts  (Fig.  9A).  In  this  case,  the  receptor  (TSR-5)  was 
stimulated  with  a  0.6-mm  stretch.  Within  8.4  s  after  the 
onset  of  stimulus,  a  stretch-induced  response  with  the 
usual  impulse  discharge  pattern  of  a  rapidly  adapting 
stretch  receptor  occurred.  But  by  8.6  s  after  the  onset  of 
the  stimulus,  intermittent  bursting  began  and  lasted  for 
the  duration  of  the  stretch  stimulation.  In  Figure  9A,  two 
groups  of  impulse  bursts  appear  at  the  rising  phase  of 
stimulation  (arrowheads).  This  was  caused  by  the  un- 
evenness  of  manually  imposed  stretch  stimulus.  Inter- 
mittent bursts  equivalent  to  those  evoked  by  stretch  stim- 
ulus were  also  produced  by  intracellular  injection  of  elec- 
trical current  (Fig.  9B,  5n.A  in  this  case).  The  occurrence 
of  this  phenomenon  is  illustrated  graphically  in  Figure 
9C  and  D.  No  intermittent  bursts  occurred  after  a  current 
injection  of  3nA  (Fig.  9C)  and  the  evoked  responses  ceased 
in  about  4  s.  But  at  8nA  (bottom  line  in  Fig.  9D),  and 
about  18  s  after  current  injection,  intermittent  bursts  ap- 
peared and  lasted  for  40  s.  The  outcome  of  this  experiment 
is  shown  in  the  inset  of  Figure  9D.  with  intermittent  bursts 
still  occurring  in  the  penultimate  10  s  of  a  5-min  stimulus. 
However,  the  question  remained:  Could  this  phenom- 
enon be  produced  by  a  much  stronger  imposed  stimulus 
beyond  the  functional  range  of  the  receptor?  This  possi- 
bility might  be  excluded  by  the  observation  that  the  fre- 
quency of  intermittent  bursts  increased  linearly  until 
0.8  mm  (Fig.  lOa).  Because  we  adopted  the  stretch  stim- 
ulus of  0.6  mm.  we  could  exclude  the  above  possibility. 
This  stretch  amplitude  was  also  within  physiological  range 
of  the  tonic  response  cell.  The  tonic  response  cell,  which 
was  simultaneously  activated  (since  both  type  of  receptor 
cells  have  a  common  single  receptor  muscle),  responded 
with  an  increase  of  impulse  discharges,  even  up  to  1 .0  mm 
(Fig.  lOb),  corresponding  to  a  30%  increase  in  the  total 
length  of  the  receptor  muscle. 

Response  of  the  abdominal  stretch  receptor 

As  can  be  seen  in  Figure  1 1 ,  slowly  adapting  and  rapidly 
adapting  responses  also  appeared  in  the  abdominal  stretch 
receptors  (inset  of  Fig.  1 1 ).  These  responses  are  similar  to 
those  in  the  thorax,  but  the  rapidly  adapting  receptor  cells 
of  the  abdomen  never  showed  the  intermittent  bursts  ob- 
served in  those  of  the  thorax.  As  in  the  thorax,  instead  of 
the  stretch,  a  certain  amount  of  current  injection  could 
cause  an  equivalent  response  in  the  stretch  receptors.  In- 
jection of4nA  (Fig.  1  la),  or  even  the  much  larger  current 
of  8nA  (Fig.  1  Ib),  induced  no  intermittent  bursts  in  the 
abdomen.  Although  a  current  of  8nA  was  sufficient  to 
evoke  intermittent  bursts  in  thoracic  receptors,  injection 
even  beyond  8nA  generated  no  intermittent  bursts  in  the 
abdominal  stretch  receptors. 


156 


A.  NIIDA  ET  AL 


246 

Time  (sec) 


10  20  30 

Time  (sec) 


40 


Figure  9.  (A)  Intermittent  bursts  in  response  to  an  imposed  stretch,  recorded  extracellularly  from  the 
rapidly  adapting  receptor  cell  in  an  in  situ  preparation  of  TSR-5  to  an  imposed  stretch.  Stretch  amplitude 
(lower  trace),  0.6  mm.  Thin  arrows  indicate  impulse  bursts  caused  by  an  uneven  stretch  stimulus  imposed 
manually.  The  small  impulse  discharges  indicate  the  activities  of  the  slowly  adapting  stretch  receptor.  (Bl 
Similar  intermittent  impulse  discharges  produced  by  current  injection  applied  intracellularly;  recordings 
were  made  intracellularly  from  a  C-type  cell.  Thick  arrows  in  (A)  and  (B)  indicate  the  onset  of  stretch 
stimulus  and  current  injection,  respectively.  (C)  A  current  of  3  nA  caused  no  intermittent  bursts.  (D)  At 
8  nA,  intermittent  bursts  occurred  about  20  s  after  beginning  current  injection:  they  were  maintained 
throughout  a  5-min  stimulus  (inset). 


Discussion 

Intrinsic  response  property  of  the  rapidly  adapting 
stretch  receptor 

The  intermittent  bursts  of  the  rapidly  adapting  receptor 
cell  that  were  observed  during  imposed  stretch  experi- 
ments were  dependent  on  the  extent  of  a  stretch  stimulus. 
As  can  be  seen  in  Figure  lOa,  stretching  within  the  range 
of  0.6  to  0.8  mm  would  not  be  unusual,  because  concom- 
itant impulse  discharges  increased  with  the  increment  of 
stretch.  This  type  of  response  is  clearly  specific  to  the  tho- 
racic rapidly  adapting  stretch  receptor  cells,  because  ap- 
plication of  electrical  current  to  the  rapidly  adapting 
stretch  receptors  of  the  abdomen  did  not  alter  their  re- 
sponse pattern  and  evoked  no  intermittent  bursts  (a,  b  in 
Fig.  1 1 ).  Such  a  stable  response  pattern  as  that  in  the 
abdomen  of  L.  exotica  occurs  also  in  the  rapidly  adapting 
abdominal  stretch  receptors  of  crayfish  (Nakajima  and 
Onodera,  1969),  which  showed  a  phasic  response  with 
any  intensity  of  applied  electrical  current. 


On  the  other  hand,  segmental  //;  situ  flexion  induced 
no  intermittent  bursts  (Fig.  6).  One  reason  for  this  incon- 
sistency might  be  the  absence  or  presence  of  inhibitory 
inputs  from  central  neurons  to  the  stretch  receptor  cells; 
specimens  for  stretch-imposed  experiments  are  isolated 
from  the  central  connection.  An  unequal  stimulus  am- 
plitude between  imposed  flexion  and  stretch  experiments 
might  also  account  for  the  difference  in  response. 

Segincniiil  mobility  and  response  type  of  stretch  receptor 

Unlike  the  segments  in  large  crustaceans  such  as  cray- 
fish, all  isopod  segments  are  mobile,  suggesting  that  all  of 
the  stretch  receptors  should  be  equipped  with  a  specialized 
receptor  muscle  on  which  the  dendrite  of  the  receptor  cell 
terminates.  This  assumption  is  derived  from  a  concept  by 
Bush  and  Laverack  (1982):  in  the  Crustacea,  evolution 
progresses  with  increasing  sclerotization.  and  thoracic 
segments  are  consequently  immobilized;  anterior  rapidly 
adapting  stretch  receptors  are  lost  first,  followed  by  slowly 


STRETCH  RECEPTORS  OF  L1GIA 


157 


0.0      0.2      0.4      0.6      0.8     1.0      1.2 

Length  of  stretch  (mm) 

Figure  10.  Frequency  plots  based  on  recording  from  two  receptor 
cells  (TSR-5)  to  various  lengths  of  stretch,  (a)  and  (h):  rapidly  and 
slowly  adapting  receptor  cells,  respectively.  Paired  slowly  and  rapidly 
adapting  receptor  cells  were  simultaneously  activated,  because  they  have 
a  single  common  receptor  muscle.  Points  with  vertical  bars  represent 
mean  ±  SD. 


adapting  stretch  receptors;  these  are  finally  replaced  by 
N-cells,  which  have  no  specialized  receptor  muscles.  The 
stretch  receptor  of  the  2nd  thoracic  segment  of  Ligia  has 
no  specialized  receptor  muscles,  a  lack  also  reported  in 
pill  hugs  (Niida  ft  ai.  1990).  This  type  of  stretch  receptor 
may  be  equivalent  to  the  N-cells  of  large  decapods  (Al- 
exandrowicz,  1952;  Wiersma  and  Pilgrim,  1961)  and  of 
Squilla  mantis  (Crustacea,  Stomatopoda)  (Pilgrim,  1964). 
S.  mantis  has  "free"  thoracic  segments  that  are  mobile; 
thus,  in  contrast  to  the  Decapoda,  this  species  contains  a 
complete  set  of  stretch  receptors,  each  with  a  specialized 
receptor  muscle  and  a  receptor  cell,  from  the  abdominal 
segment  up  to  the  5th  thoracic  segment. 

In  Squilla,  the  N-cell,  termed  SR-«  (Wiersma  and  Pil- 
grim. 1961),  lies  only  in  the  2nd  thoracic  segment.  This 
segmental  organization  is  the  same  as  that  in  Ligia.  but 
the  responses  of  the  stretch  receptors  of  the  3rd  thoracic 
segment  differ  in  these  two  animals.  The  response  in 
Squilla  is  only  of  the  slowly  adapting  type,  whereas  the 
receptors  in  Ligia  show  both  slowly  and  rapidly  adapting 
responses.  Therefore,  the  3rd  thoracic  segment  may  be 
more  mobile  in  Ligia  than  in  Squilla.  The  appearance, 
within  the  Isopoda,  of  a  segmentally  arranged  series  of 


stretch  receptors  comprising  sensory  cells  of  two  types 
thus  further  supports  the  hypothesis  by  Alexandrowicz 
(1967):  the  organization  of  the  thoracic  stretch  receptors 
is  closely  related  to  the  mobility  of  the  thorax. 

Comparison  of  the  structure  of  receptor  muscle  in  other 
Crustacea 

As  already  stated,  the  receptor  muscle  of  Ligia  is  a  single 
structure  throughout  each  segment.  In  large  Crustacea, 
such  a  single  receptor  muscle  appears  in  the  anterior  tho- 
racic segments;  e.g.,  Astactts  has  it  in  the  7th  thoracic 
segment,  Homalus  in  the  7th  thoracic  segment,  and 
Squilla  in  the  3rd  and  4th  thoracic  segments  (for  review, 
see  Bush  and  Laverack,  1982).  The  more  posterior  tho- 
racic segments  and  successive  abdominal  segments  of  each 
animal  have  two  separate  receptor  muscles.  In  the  pill  bug 
(Niida  el  ai,  1990;  Niida  et  ai,  1991),  unlike  Ligia.  the 
receptor  muscle  that  spans  the  3rd  and  4th  thoracic  seg- 
ments separates  completely,  and  from  the  5th  to  8th  tho- 
racic segments  each  pair  of  receptor  muscle  runs  closely 
together — but  Moser's  observation  (1976)  is  somewhat 
different  from  ours.  In  the  abdomen,  parallel  receptor 
muscles  connect  tightly  with  each  other  in  the  anterior 
ridge  of  a  targum  and  run  toward  the  adjacent  segment, 
separating  into  two  muscle  components.  Thus  the  vari- 
ation in  the  organization  of  the  receptor  muscle  might  be 
difficult  to  account  for  on  the  basis  of  evolutionary  se- 
quence alone  in  a  limited  number  of  animals;  adaptive 
behaviors  specific  to  the  relevant  animal  should  be  also 
considered. 


u 

0> 

CM 
CD 


10 


0> 
_W 

Q. 


(b) 


(a 


10 

Time  (sec) 


20 


Figure  11.  Inset:  Extracellularly  recorded  responses  from  rapidly  and 
slowly  adapting  abdominal  stretch  receptors  ( ASR-2).  Lower  trace,  stretch 
amplitude  (0.22  mm).  Time  scale,  2  s.  Graph:  Time  courses  of  impulse 
discharges  by  the  rapidly  adapting  receptor  cell  of  ASR-2.  Impulse  dis- 
charges recorded  intracellularly  were  evoked  by  intracellular  current  in- 
jection, (a),  4  nA;  (b),  8  nA. 


A.  NIIDA  ET  AL. 


The  sarcomere  length,  as  one  of  the  characteristics  of 
the  differentiated  receptor  muscle,  may  be  noted;  because 
in  crayfish,  a  slowly  adapting  receptor  cell  and  a  rapidly 
adapting  receptor  cell  have  receptor  muscles  with  a  long 
sarcomere  and  a  short  one,  respectively  (Komuro,  1981 ). 
The  same  is  the  case  with  Squilla  (Alexandrowicz,  1967). 
In  contrast,  in  Ligia,  microscopic  observation  of  the  cross- 
stnation  of  the  receptor  muscles  (not  measured  except  in 
the  6th  thoracic  segment)  did  not  reveal  any  difference  in 
sarcomere  length  in  each  segment.  Accordingly,  the  dif- 
ferentiated function  of  stretch  receptors  in  Ligia  should 
be  attributable  to  the  specific  morphology  of  receptor  cells 
(B-type,  C-type  cells)  coupled  with  their  response  prop- 
erties, rather  than  to  sarcomere  length. 

When  this  receptor  muscle  is  passively  stretched,  both 
of  the  receptor  cells  associated  with  it  (B-type  and  C-type) 
should  be  synchronously  stimulated,  but  the  behavioral 
significance  is  difficult  to  evaluate. 

Adaptive  behavior  and  thoracic  stretch  receptor 

The  slowly  adapting  stretch  receptor  of  the  crayfish  has 
been  regarded  as  a  positional  detector  of  abdominal  flex- 
ion, whereas  the  rapidly  adapting  stretch  receptor  is  be- 
lieved to  function  when  the  fast  muscular  system  is  ac- 
tivated, such  as  during  swimming  and  escape  (Wiersma 
and  Pilgrim,  1961).  In  L.  exotica,  the  abdominal  and  pos- 
terior thoracic  segments,  as  well  as  the  uropod  styles,  flex 
in  the  dorsal  and  ventral  directions.  This  segmental 
movement  is  related  to  swimming,  but  is  also  sequentially 
elicited  by  another  key  stimulus:  When  pereopods  of  L. 
exotica  are  dipped  in  a  large  quantity  of  water,  the  animal 
attempts  to  stand,  elevating  its  body  and  beginning  to 
raise  and  lower  its  styles  to  the  substrate.  This  behavior 
might  be  coupled  with  the  water-conducting  system  that 
has  been  extensively  studied  (for  review,  see  Warburg, 
1993;  Hoese,  1984).  In  this  system,  water  is  taken  up  from 
water  droplets  by  the  capillary  action  of  the  pereopods 
and  enters  the  marsupium;  the  extra  water  is  released  by 
the  touch  of  the  styles  to  the  ground.  In  performing  this 
behavior,  the  animals  must  obtain  continuous,  momen- 
tary information  about  the  position  of  their  styles  with 
respect  to  the  ground  and  about  the  velocity  of  flexion. 
In  the  anterior  thoracic  segments,  although  the  functional 
roles  of  the  rapidly  and  slowly  adapting  stretch  receptors 
are  unclear,  both  types  of  stretch  receptors  would  be  re- 
quired for  sophisticated  segmental  movements.  For  in- 
stance, when  opening  the  breeding  pouch,  which  occurs 
in  the  2nd  to  posterior  thoracic  segments  of  females,  the 
animal  presses  its  anterior  thoracic  segments  against  the 


substrate  and  simultaneously  lifts  its  posterior  segment 
by  supporting  the  abdominal  segments  with  the  styles. 

Acknowledgments 

This  work  was  supported  by  Ryoubi  Teien  Foundation 
and  in  part  by  a  Grant  in  Aid  from  the  Ministry  of  Ed- 
ucation, Science  and  Culture  of  Japan  to  TY  for  scientific 
research. 

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Reference:  Biol  Hull  189:  159-167.  (October/November,  1995) 


Transport  and  Metabolism  of  Alanine  and  Palmitic 

Acid  by  Field-Collected  Larvae  of  Tedania  ignis 

(Porifera,  Demospongiae):  Estimated  Consequences 

of  Limited  Label  Translocation 

WILLIAM  B.  JAECKLE 

Smithsonian  Marine  Station  at  Link  Port,  5612  Old  Dixie  Highway.  Fort  Pierce,  Florida  34946 


Abstract.  The  epidermis  of  larvae  of  Tedania  ignis 
(Porifera,  Demospongiae)  is  uniformly  ciliated  except  for 
the  posterior  pole.  The  epidermal  cells  are  long,  columnar, 
and  monociliate;  each  cilium  arises  from  an  epidermal 
crypt;  symbiotic  bacteria  were  not  observed  in  larval  cells. 
These  lecithotrophic  ("nonfeeding")  larvae  can  feed  by 
assimilating  dissolved  organic  materials  (DOM)  from 
seawater.  Larvae  transported  both  the  amino  acid  alanine 
(mean  =  2.73  pmol  larva"1  h~';  [S]  =  1  ^M )  and  the  fatty 
acid  palmitic  acid  (mean  =  16.27  pmol  larva"1  h~';  [S] 
=  1  p.\f)  from  seawater.  Following  assimilation,  the  label 
from  alanine  was  recovered  primarily  in  small  molecular 
weight  compounds;  the  label  from  palmitic  acid  was  lo- 
calized chiefly  in  the  lipid  fraction.  Estimates  of  the  con- 
tribution of  transport  to  metabolism  (mean  respiration 
rate  =  940.7  pmol  O:  larva"1  h"')  reveal  that  alanine 
transport  is  energetically  insignificant.  Palmitic  acid 
transport,  in  contrast,  could  account  for  2 1%-55%  of  lar- 
val metabolism.  Autoradiographic  analysis  of  the  distri- 
bution of  the  label  in  larvae  suggests  that  epidermal  cells 
are  the  chief  recipients  of  the  assimilated  materials.  Thus, 
the  contribution  of  transport  to  whole-larva  metabolism 
may  underestimate  the  tissue-specific  value.  At  palmitic 
acid  concentrations  of  1  and  0.25  nM,  the  contribution 
of  transport  to  the  estimated  metabolism  of  the  epidermis 
would  be  131%  and  33%  of  energy  requirements.  Thus, 
the  potential  benefits  of  DOM  to  larvae  are  dependent 
not  only  on  the  nature  of  the  epidermal  transporters  and 


Received  I  July  1993;  accepted  II  July  1995. 

Current  address:  Friday  Harbor  Laboratories.  620  University  Road. 
Friday  Harbor.  WA  98250. 
Contribution  #384  to  the  Smithsonian  Marine  Station  at  Link  Port. 


the  solute  concentration,  but  also  the  degree  to  which 
materials  are  distributed  among  tissues. 

Introduction 

The  energy  requirements  for  development  of  "non- 
feeding"  (lecithotrophic)  larvae  of  marine  invertebrates 
have  historically  been  thought  to  be  solely  derived  from 
the  catabolism  of  maternally  provided  stores  (Chia,  1974; 
Crisp,  1974;  Day  and  McEdward,  1984).  In  recent  years, 
however,  it  has  been  shown  that  nonfeeding  embryos  and 
larvae  can  obtain  energy  from  the  environment  through 
the  transport  of  dissolved  organic  materials  (DOM)  from 
seawater  (Reish  and  Stephens,  1969;  Jaeckle  and  Mana- 
han,  1989a;  Manahan  et  a/..  1989;  Welborn  and  Mana- 
han,  1990;  Jaeckle,  1994).  Prefeeding  embryos  of  plank- 
totrophic  (feeding)  larvae  can  also  assimilate  organic  ma- 
terials from  seawater  (e.g..  Monroy  and  Tolis,  1 96 1 ;  Tyler 
et  a/..  1966;  Epel,  1972;  Karp  and  Weems,  1975;  Mana- 
han, 1983a;  Schneider  and  Whitten,  1987). 

Analyses  of  the  energetics  of  larval  development  indi- 
cate that  DOM  transport  by  nonfeeding  larvae  and  em- 
bryos may  be  important.  The  contribution  of  organic  sol- 
ute transport  to  metabolic  processes  can  be  estimated  by 
comparing  joules  supplied  (through  transport)  with  joules 
expended  (metabolic  rate)  (Stephens.  1963;  Wright,  1981: 
Manahan  et  ai.  1983;  Jaeckle  and  Manahan,  1989a). 
These  comparisons  reveal  that  the  potential  energetic 
benefits  of  DOM  transport  vary  among  both  transported 
compounds  and  larval  forms.  In  general,  for  compounds 
at  a  concentration  of  1  nM,  the  estimated  contribution  of 
transport  to  the  metabolism  of  nonfeeding  larvae  and 
prefeeding  embryos  ranges  from  <1%  to  ca.  35%  for  free 
amino  acids  and  sugars  (Jaeckle  and  Manahan,  1989a, 


159 


160 


W.  B.  JAECKLE 


1992;  Jaeckle,  1994)  and  from  ca.  20%  to  70%  for  the 
fatty  acid  palmitic  acid  (Jaeckle,  1994).  Thus  the  potential 
nutritional  and  energetic  value  of  DOM  in  seawater  to 
nonfeeding  life  history  stages  of  invertebrates  is  a  function 
of  both  the  quantity  and  the  quality  of  the  organic  com- 
pounds present  and  the  physiological  capacities  of  the 
larva. 

Most  published  studies  on  DOM  transport  report  the 
capabilities  of  embryos  and  larvae  of  temperate-water 
species  to  exploit  this  potential  source  of  nutriment  and 
energy  (see  Manahan,  1990,  for  a  recent  review).  Of  these 
studies,  few  (Karp  and  Weems,  1975;  DeBurgh  and  Burke, 
1983;  Manahan  and  Crisp,  1983)  have  examined  the  spa- 
tial distribution  of  a  label  (initially  associated  with  the 
assimilated  molecules)  within  the  larval  body  and  how 
the  distribution  pattern  of  the  label  changes  over  time. 
Further,  where  translocation  of  materials  has  been  sug- 
gested, the  larvae  used  were  planktotrophic  and  the  ap- 
pearance of  label  in  interior  cells,  i.e.,  the  digestive  system, 
cannot  be  attributed  solely  to  the  assimilatory  activity  of 
the  ectoderm  and  subsequent  translocation  to  interior  cells 
via  a  blood-vascular  system  (e.g.,  Ruppert  and  Carle, 
1983). 

The  objectives  of  this  study  were  to  measure  transport 
and  metabolism  of  an  amino  acid  and  a  fatty  acid  from 
seawater,  to  determine  the  rates  of  oxygen  consumption, 
and  to  follow  the  distribution  of  a  3H  label  within  the 
larval  body  (using  light-microscopic  autoradiography)  in 
field-collected  parenchymula  larvae  of  the  demosponge 
Tedania  ignis.  The  results  of  these  experiments  reveal  that 
for  larvae  of  T.  ignis  the  calculated  contribution  of  DOM 
to  whole-larva  metabolism  is  highly  dependent  on  the 
available  solute  in  solution.  Transport  and  metabolism 
of  palmitic  acid  ([S]  =  1  pM)  could  account  for  an  average 
of  37%  of  the  metabolic  demand,  while  <1%  of  the  met- 
abolic rate  could  be  supplied  through  alanine  transport. 
Following  transport,  the  distribution  of  the  label  in  larval 
tissue  is  not  uniform;  most  of  the  label  was  detected  in 
the  epidermis  after  a  2-h  continuous  exposure  to  the  label. 
Comparison  of  the  rates  of  DOM  transport  to  estimates 
of  the  metabolic  rate  of  the  epidermis  reveals  that  the 
energetic  significance  of  DOM  transport  to  the  epithelium 
apparently  responsible  for  material  assimilation  can  be 
very  high  (>90%  compensation  of  the  estimated  metabolic 
rate  of  the  epidermis). 

Materials  and  Methods 

Collection  and  handling  of  larvae 

Larvae  of  Tedania  ignis  were  collected  from  general 
plankton  samples  taken  from  the  Fort  Pierce  Inlet  (ca. 
21°  28'  N;  80°  18'  W)  during  April-June  of  1991.  All 
samples  were  collected  during  Hooding  tides  by  deploying 
a  0.5-m  plankton  net  with  202-jum  (mesh  size)  netting  in 


the  tidal  flow  for  10-15  minutes.  Samples  were  sorted  at 
the  Smithsonian  Marine  Station  at  Link  Port  as  soon  as 
possible  (<1  h)  after  collection.  Larvae  of  T.  ignis  were 
placed  in  0.2-^m  (pore  size)  filtered  seawater  (hereafter 
termed  seawater)  and  held  at  a  temperature  of  22.5°C. 

Measurement  of  morphological  and  physical 
characteristics 

Before  the  linear  dimensions  of  Tedania  larvae  were 
measured,  individuals  were  fixed  by  immersion  in  1% 
OsO4  in  seawater  for  1  h,  washed  in  seawater,  and  then 
measured  (±0.5  urn)  using  a  compound  microscope 
equipped  with  an  ocular  micrometer. 

For  morphological  inspection,  larvae  of  Tedania  ignis 
were  processed  in  a  number  of  ways.  For  examination  of 
surface  structures,  larvae  were  fixed  in  1%  OsO4  in  sea- 
water  for  1  h.  washed  with  seawater.  dehydrated  with  an 
ascending  ethanol  series,  and  critical-point  dried  using 
CO:  as  the  transition  fluid.  The  specimens  were  mounted 
on  stubs,  coated  with  a  gold-palladium  mixture,  and  ex- 
amined using  a  Novascan  30  scanning  electron  micro- 
scope. For  light  microscopic  histology  and  autoradiog- 
raphy and  transmission  electron  microscopy,  specimens 
were  initially  fixed  in  2.5%  glutaraldehyde  in  seawater 
and  then  post-fixed  in  2%  OsO4  in  a  1.25%  solution  of 
NaHCO3.  This  material  was  dehydrated  using  ethanol, 
transferred  into  propylene  oxide,  and  embedded  in  an 
epoxy  resin  (Epon  812).  Thick  sections  (ca.  1  ^m)  were 
cut  with  a  glass  knife,  stained  with  "Richardson's  stain" 
(Richardson  ct  at..  1960)  and  examined  with  a  compound 
microscope.  Thin  sections  (ca.  60  nm)  were  cut  with  a 
diamond  knife,  stained  with  saturated  aqueous  solutions 
of  lead  citrate  and  of  uranyl  acetate,  and  examined  with 
a  Zeiss  EM-9S  transmission  electron  microscope. 

For  determinations  of  larval  organic  weight  (biomass), 
larvae  were  processed  using  the  procedures  described  in 
Jaeckle  and  Manahan  (1989b). 

Measurement  oj  oxygen  consumption 

The  respiration  rate  of  Tedania  ignis  larvae  was  mea- 
sured following  the  procedures  outlined  in  Jaeckle  (1994) 
at  a  temperature  of  22.5  ±  0.05°C.  All  measured  respi- 
ration values  were  corrected  for  the  self-consumption  rate 
of  the  electrode  (<9%  of  the  larval  respiration  rate).  The 
rate  of  oxygen  consumption  (mol  O;  larva"1  h"1)  was  cal- 
culated as  the  slope  of  a  regression  line  of  the  collected 
data,  divided  by  the  number  of  larvae,  and  multiplied  by 
60  min/h.  The  measured  rates  of  oxygen  consumption 
were  converted  to  the  energy  units  by  using  an  oxyen- 
thalpic  equivalent  of  480  kJ  mol  O2~'  (the  average  oxy- 
enthalpic  equivalent  for  protein  [527  kJ  mol  O;  '],  lipid 
[441  kJ  mol  O:  '].  and  carbohydrate  [473  kJ  mol  O:  '], 
all  from  Gnaiger  [1983]). 


DOM  TRANSPORT  AND  METABOLISM  IN  SPONGE  LARVAE 


161 


Alanine  and  palmitic  acid  transport 

Larvae  were  transferred  to  10  ml  of  seawater  in  an  au- 
toclave-sterilized 20-ml  scintillation  vial  (for  experiments 
with  palmitic  acid,  the  vial  was  previously  silanized  with 
Sihue  [SDS  Coatings,  Inc.]).  All  transport  was  measured 
at  larval  concentration  of  <4  larvae/ml  and  an  added  sol- 
ute concentration  of  1  n\l.  After  the  addition  of  the  label 
(3H-alanine  or  3H-palmitic  acid,  New  England  Nuclear, 
specific  activities  70  or  84  Ci/mmol  and  60  Ci/mmol,  re- 
spectively) and  cold  carrier,  the  vial  was  mixed  by  inver- 
sion and  the  first  sample  removed.  Each  sample  of  larvae 
(<5  larvae  per  sample)  was  treated  following  the  methods 
described  in  Jaeckle  and  Manahan  ( 1989a).  For  one  ex- 
periment, the  rate  of  alanine  transport  was  measured  as 
the  accumulation  of  radioactivity  in  larvae  after  1  h  of 
continuous  exposure  to  the  label.  For  all  other  experi- 
ments, the  measured  amount  of  radioactivity  per  larva 
(corrected  for  signal  quenching)  was  converted  to  moles 
of  material  per  individual,  and  the  rate  of  transport  was 
calculated  as  the  slope  of  a  regression  line  describing  the 
relationship  between  moles  of  material  per  larva  and  time. 

Alanine  and  palmitic  acid  metabolism 

Larvae  remaining  after  the  1-h  incubation  in  the  so- 
lution of  either  3H-alanine  or  palmitic  acid  (see  above) 
were  removed  and  pipetted  onto  a  glass-fiber  filter.  The 
sample  of  larvae  was  gently  washed  twice  with  20  ml  each 
of  cold  (5°C)  seawater.  and  the  number  of  individuals  on 
the  filter  was  counted.  After  washing,  the  filters  were  then 
placed  into  a  -70°C  freezer  to  stop  all  metabolic  activity 
of  the  larvae.  The  sample  of  larvae  was  then  lyophilized 
for  8  h  (<10  nm  Hg).  5  ml  of  distilled  water  was  added, 
and  the  larval  tissue  was  homogenized  using  an  ultrasonic 
tissue  disrupter  (Fisher  model  #300).  Samples  of  the  tissue 
homogenate  were  separated  into  general  biochemical 
fractions  (protein,  lipid.  and  small  molecular  weight  com- 
pounds) using  the  methods  described  in  Jaeckle  and 
Manahan  ( 1989b).  Each  resulting  fraction  and  a  sample 
of  the  intact  homogenate  were  dissolved  in  tissue  solu- 
bilizer,  and  the  radioactivity  in  each  sample  was  measured 
48  h  after  the  addition  of  scintillation  cocktail.  The  mea- 
sured amount  of  radioactivity  per  fraction  (corrected  for 
signal  quenching)  was  converted  to  a  percentage  of  the 
total  by  dividing  the  radioactivity  in  each  fraction  by  the 
amount  of  radioactivity  in  the  sample  of  tissue  homoge- 
nate. 

Localization  of  the  label  following  assimilation  within 
larvae 

Light-microscopic  autoradiography  was  used  to  deter- 
mine the  location  of  the  3H-label  in  larvae.  Larvae  were 
continuously  exposed  to  radiolabeled  alanine  and  palmitic 


acid  (each  at  1  ^M  added  concentration)  for  10,  60,  or 
120  min.  At  the  end  of  each  exposure,  the  larvae  were 
washed  twice  with  seawater  ( 10  ml  each  time)  and  fixed 
and  processed  as  described  above.  Serial  thick  sections 
were  cut,  then  secured  onto  acid-cleaned  microscope 
slides.  The  slides  were  immersed  into  a  liquid  photo- 
graphic emulsion  (II ford  #Kd.5).  air  dried  for  24  h,  and 
stored  in  a  light-tight  box  at  5°C.  The  slides  were  devel- 
oped according  to  manufacturer  specifications,  and  the 
autoradiograms  were  examined  and  photographed  with 
a  compound  microscope. 

Results 

Physical  characteristics 

Field-collected  parenchymula  larvae  of  Tedania  ig- 
nis are  orange-red  in  coloration  and  averaged  818.5 
±  17.5  urn  in  length  and  576.3  ±  17.7  ^m  in  width  (both 
mean  ±  1  standard  error  (SE);  «  =  16  larvae).  The  average 
length:width  ratio  for  these  larvae  was  1.4  ±  0.1  (mean 
±  1  SE,  n  =  16  larvae).  With  the  sole  exception  of  the 
posterior  pole  (assigned  as  the  trailing  pole  during  swim- 
ming), the  larvae  were  uniformly  ciliated  (Fig.  1 ).  The 
epidermis  is  composed  primarily  of  long,  thin,  monociliate 
cells  (Figs.  2,  3);  each  cilium  emerges  from  the  cell  body 
through  an  epidermal  crypt  or  pit  (Fig.  3).  This  morpho- 
logical examination  of  the  epidermis  did  not  reveal  either 
intra-  or  extracellular  bacteria  (not  shown);  hence  the 
measured  rates  of  solute  transport  (below)  represent  the 
physiological  activity  of  larval  cells  alone. 

The  average  weight  of  a  Tedania  parenchymula  larva 
was  16.99  ±  0.72  ^g/larva  (mean  ±  1  SE.  n  =  14  groups 
of  larvae  @  <7  larvae/group). 

Alanine  and  palmitic  acid  transport 

Both  alanine  and  palmitic  acid  were  transported  from 
seawater  by  larvae  of  Tedania  ignis,  but  the  rates  of  trans- 
port differed  between  the  two  compounds.  Alanine  was 
transported  at  rates  that  averaged  2.73  ±  0.6  pmol  alanine 
larva"1  h"1  (mean  ±  1  SE,  »  =  3  experiments).  The  rates 
of  palmitic  acid  transport  were  nearly  6X  higher  and  av- 
eraged 16.27  ±  2.3  pmol  palmitic  acid  larva"1  h~'  (mean 
±  1  SE,  n  =  4  experiments).  The  fate  of  the  radioactive 
label  in  larval  tissue  also  differed  between  the  two  com- 
pounds. For  larvae  exposed  to  3H-alanine,  most  of  the 
label  (64%)  was  recovered  in  the  small  molecular  weight 
compound  fraction,  e.g..  soluble  in  cold  5%  trichloroacetic 
acid  (TCA).  The  remaining  label  was  found  in  the  TCA- 
insoluble  (macromolecular)  fraction  (23%)  and  in  lipoic 
materials  (14%,)  localized  in  the  CHCVsoluble  fraction. 
For  larvae  exposed  to  3H-palmitic  acid,  most  of  the  ma- 
terial was  recovered  in  the  CHCl,-soluble  fraction  (79%). 
and  the  remaining  radioactivity  was  divided  between  the 


162 


W.  B.  JAECK.LE 


V 


Figure  1.  Scanning  electron  micrograph  of  a  lateral  view  of  a  field-collected  larva  of  Tedania  ignis.  The 
antenor  (A)  and  posterior  (P)  regions  of  the  larvae  were  designated  as  the  leading  and  trailing  poles  during 
swimming.  Scale  bar  =  50  pm. 

Figures  2  and  3.     Light  and  transmission  electron  micrographs  of  the  epidermis  of  parenchymulae  of 

Tcdailia  I'.ifH/V. 

Figure  2.  Light  micrograph  of  a  larva  of  Tedania  ignis.  The  epidermis  (EP)  is  composed  primarily  of 
thin,  columnar,  ciliated  cells.  Subapically  a  continuous  line  (large  arrowhead)  is  present  and  is  suggestive  of 
intercellular  junctional  complexes.  The  presence  of  epidermal  crypts  from  which  the  cilia  (C)  arise  at  the 
apices  of  the  ciliated  cells  is  denoted  by  the  small  arrowheads.  Scale  bar  =  10  nm. 

Figure  3.  Transmission  electron  micrograph  of  the  apical  region  of  a  ciliated  epidermal  cell  ot  a  larva 
of  Tedania  ignis.  A  single  cilium  (C)  can  be  seen  emerging  from  the  epidermal  crypt  of  the  epidermal  cell. 
Scale  bar  =  0.25  pm. 


TCA-soluble  fraction  (14%)  and  the  TCA-insoluble  frac- 
tion (8%). 

Larval  respiration 

The  respiration  rate  (O:  consumption)  of  larvae  of  Te- 
dania ignis  was  variable  among  the  groups  of  larvae  ex- 
amined. Values  ranged  from  846.8  to  1113.9pmol  O2 


larva  '  h  ',  with  an  average  of  940.7  ±  70.0  pmol  O: 
larva~'  h~'  (mean  ±  1  SE.  n  =  5  independent  collections 
of  larvae). 

Autoradiographic  analysis  of  the  distribution  of  the 
3H-lahel 

The  biochemical  nature  (macromolecular  or  small 
molecular  weight)  of  the  molecules  containing  the  label 


DOM  TRANSPORT  AND  METABOLISM  IN  SPONGE  LARVAE 


163 


cannot  be  ascertained  by  light-microscopic  autoradiog- 
raphy  of  glutaraldehyde-fixed  material.  Glutaraldehyde  is 
a  good  preservative  of  cellular  details,  in  part  because  it 
acts  by  cross-linking  primary  amines.  This  activity  may 
result  in  a  false  intracellular  localization  of  the  label,  if 
the  label-bearing  molecule  resides  in  the  extracellular 
space  (e.g..  Peters  and  Ashley.  1967).  However,  the  au- 
toradiographic  analysis  presented  here  was  designed  to 
ascertain  whether  the  label,  after  transport,  was  distributed 
throughout  the  entire  larval  body,  not  to  determine  the 
pathway  of  material  movement  (paracellular  or  transcel- 
lular). 

After  10  min  of  exposure  to  either  3H-alanine  or  pal- 
mitic acid,  the  label  is  found  in  or  around  the  cells  of  the 
epidermis  (Fig.  4A),  as  evidenced  in  the  autoradiograms 
by  the  appearance  of  silver  granules  overlying  these  cells. 
Even  though  larvae  were  continuously  exposed  to  the  label 
for  up  to  2  h.  examination  of  the  autoradiograms  (Fig. 
4A-C)  indicates  that  most  of  the  label  remained  associated 
with  the  cells  of  the  epidermis. 

Discussion 

Most  research  on  the  larvae  of  demosponges  has  focused 
on  their  morphological  or  behavioral  characters  (e.g.. 


Berquist  et  «/..  1970;  Woollacott,  1990,  1993;  Kaye  and 
Reiswig,  1 99 1 ).  The  morphology  of  the  epidermis  of  larvae 
of  Teiiania  ignis  (subclass  Poecilosclerida)  closely  ap- 
proximates that  described  for  larvae  of  the  haplosclerid 
demosponge  Haliclonn  tnhijera  (Woollacott,  1993).  For 
both  species,  the  epidermis  is  composed  chiefly  of  elongate 
columnar  cells  (each  with  a  single  cilium  arising  from  an 
epidermal  crypt).  The  posterior  pole  is  aciliate  in  both, 
but  the  enlarged  ciliary  band  that  exists  at  the  intersection 
of  the  lateral  and  posterior  surfaces  in  H.  ttibifera  larvae 
is  wanting  in  larvae  of  T.  ignis.  The  physiological  signif- 
icance of  the  epidermal  crypts  remains  unknown,  but 
these  depressions  in  the  larval  epidermis  do  increase  the 
apical  surface  area  of  the  cells  and  represent  a  potential 
morphological  correlate  to  solute  transport  (Oschman, 
1978). 

Although  parenchymula  larvae  of  Tedania  ignis  lack 
a  functional  digestive  system,  these  larvae  have  the  phys- 
iological capacity  to  acquire  nutrients  and  energy  from 
their  environment  through  the  transport  of  DOM  from 
seawater.  A  comparison  of  the  energy  acquired  through 
transport  with  the  metabolic  rate  indicates  that  the  po- 
tential energetic  importance  of  alanine  and  palmitic  acid 
transport  differs  (Table  I).  The  energy  supplied  through 


/ 

••-  A 


5 


•• 


-. 


- 

- 


. 


• 

-- 


B 


^-w^ 
i- 


Figure  4.  Light  microscopic  audioradiograms  depicting  the  distribution  of  the  radioactive  label  in  trans- 
verse sections  of  larvae  of  Tcdaniu  ignis  that  were  exposed  to  3H-palmitic  acid  for  different  periods  of  time. 
(A)  After  10  min  of  exposure  to  3H-palmhic  acid,  the  presence  of  latent  images  (reduced  silver  grains)  overlies 
only  the  cells  of  the  epidermis  (arrow).  Scale  bar  =  303  urn.  (B)  After  a  1-h  exposure,  the  number  of  latent 
images  over  the  epidermis  has  increased,  but  no  label  is  detectable  over  other  regions  of  the  section.  Scale 
bar  =  303  Mm.  (C)  After  2  h  of  continuous  exposure  to  'H-palmitic  acid,  the  latent  images  are  more  intense 
and  the  label  primarily  overlies  the  epidermis,  but  there  is  evidence  for  the  presence  of  radioactive  materials 
in  interior  regions  of  the  larva.  Scale  bar  =  303  ^m. 


164  W.  B.  JAECKLE 

Table  I 

(.'tinipiiri.\i'H  nl  ilic  t'liiculitil  contribution  of  palmitic  acid  and  alanine  transport  ([S]  =  I  fiM)  to  the  energy  metabolism  of  Tedania  ignis  larvae 


Solute 


Transport' 


CK  demand2 


Metabolism' 


Compensation4 


[S]  for  100%  compensation5 


Palmitic  acid 

13.19 

317.17 

940.66 

33.7 

3.0 

22.54 

518.42 

940.66 

55.1 

1.8 

11.86 

272.78 

940.66 

20.9 

3.4 

16.27 

374.30 

940.66 

39.8 

2.5 

avg. 

16.27 

370.67 

37.4 

2.7 

s.d. 

4.66 

106.91 

14.2 

0.7 

Alanine 

1.77 

5.31 

940.66 

0.6 

166.7 

3.87 

11.61 

940.66 

1.2 

83.3 

2.53 

7.59 

940.66 

0.8 

125.0 

avg. 

2.72 

8.17 

0.6 

166.7 

s.d. 

1.06 

3.19 

0.3 

41.7 

1  Transport  rate  =  pmol  x  larva  '  h  '. 

;  Oi  demand  =  the  transport  rate  X  the  mol  CK  required  for  complete  catabolism  of  palmitic  acid  (23  mol  O2/mol  Pal)  and  alanine  (3  mol 
mol  Ala). 

3  Metabolism  =  the  average  respiration  rate  per  larva  (pmol  O:  x  larva'1  h"1). 

4  %  Compensation  =  the  oxygen  demand  -H  the  metabolic  rate  X  100. 

5  [S]  for  complete  compensation  =  100  -i-  %  compensation  of  the  metabolic  rate. 


alanine  transport  ([S]  =  1  pM)  could  supply  <1%  of  met- 
abolic demands,  and  complete  compensation  of  the  met- 
abolic rate  from  alanine  transport  would  require  ecolog- 
ically unrealistic  alanine  concentrations  for  seawater 
samples  (range:  83-167  ^Af).  In  contrast,  the  energy  con- 
tribution supplied  through  palmitic  acid  transport  ([S] 
=  1  nM)  may  be  quantitatively  important.  The  average 
transport  rate  of  palmitic  acid  is  nearly  6X  that  found  for 
alanine  transport  and,  owing  to  the  difference  in  energy 
content  of  the  two  compounds,  the  energy  acquired 
through  palmitic  acid  transport  could  supply  between  21% 
and  55%  (mean  ±  1  SE  =  37.4%  ±  7.1;  Table  I)  of  the 
metabolic  demand.  A  palmitic  acid  concentration  ranging 
from  2  to  3  pM  would  be  necessary  if  all  of  the  energy 
requirements  of  Tedania  larvae  were  supplied  solely 
through  the  transport  and  catabolism  of  this  compound. 
Following  transport  of  both  alanine  and  palmitic  acid, 
the  3H-label  was  recovered  in  each  of  the  measured  bio- 
chemical fractions  (protein,  lipids,  and  small  molecular 
weight  compounds).  Because  a  3H-label  was  used  in  the 
experiments,  the  pathways  that  place  the  label  in  each  of 
the  three  fractions  remain  unresolved.  Yet  a  comparison 
of  results  of  experiments  using  either  3H-alanine  or  pal- 
mitic acid  revealed  a  differential  distribution  of  the  label 
among  the  biochemical  fractions.  After  alanine  transport, 
most  of  the  label  was  recovered  in  the  small  molecular 
weight  fraction  followed,  in  sequence,  by  the  macromo- 
lecular  and  lipid  fractions.  This  pattern  of  label  distri- 
bution following  alanine  transport  is  consistent  with  pre- 
viously published  accounts  in  which  I4C  served  as  the 
radioactive  label  (Manahan,  1983b;  Jaeckle  and  Manahan, 
1989a,c).  The  distribution  of  label  recovered  in  larvae  ex- 


posed to  3H-palmitic  acid  was  different;  most  of  the  label 
was  found  in  the  lipid  fraction,  with  the  remainder  being 
localized  in  the  small  molecular  weight  compounds  and 
macromolecule  fractions.  This  pattern  of  label  distribution 
compares  well  with  that  described  for  adult  Stauronereis 
ntdo/p/ii  (Annelida:  Polychaeta)  after  exposure  to  I4C-1- 
palmitic  acid  (Testerman,  1972). 

The  concentrations  of  both  free  amino  acids  and  free 
fatty  acids  in  seawater  vary  from  below  detection  (<  O/) 
to  low  pM  levels  (Testerman.  1972;  Bunde  and  Fried, 
1978;  Mopper  and  Lindroth,  1982;  Carlucci  el  a/.,  1984; 
Fuhrman  and  Bell,  1985;  Laanbroek  et  al,  1985).  Thus 
the  estimates  of  energetic  contribution  presented  above 
are  dependent  upon  the  physiological  state  of  the  larvae 
and  the  concentration  of  the  organic  materials  in  seawater. 
For  larvae  of  Tedania  ignis,  even  when  exposed  to  high 
concentrations  of  amino  acids  in  surface  waters  (^A/),  the 
net  energetic  benefit  is  likely  to  be  small.  The  concentra- 
tion of  free  fatty  acids  in  subtropical  Floridian  waters  was 
reported  to  be  50-80  ^g  1~',  a  range  that  is  equivalent  to 
about  0.25  nM  palmitic  acid  (Bunde  and  Fried,  1978).  At 
this  concentration,  assuming  that  the  K+  of  the  palmitic 
acid  transporter  is  greater  than  1 .0  ^M.  the  energetic  con- 
tribution of  transport  to  the  larva  would  be,  on  average, 
9.4%  of  the  metabolic  demand. 

Attempts  to  quantify  the  energetic  importance  of  the 
transport  of  organic  solutes  from  seawater  usually  involve 
a  comparison  of  the  energy  gained  (through  transport)  to 
the  metabolic  rate.  Material  assimilation,  however,  is  a 
regional  process  restricted  to  the  outer  epithelium  of  le- 
cithotrophic  larvae  and,  in  contrast,  metabolic  rate  sums 
over  the  metabolic  activities  of  all  cells.  Comparison  of 


DOM  TRANSPORT  AND  METABOLISM  IN  SPONGE  LARVAE 


165 


energy  supply  (transport)  and  demand  (O2  consumption) 
for  lecithotrophic  larvae  is  based  on  the  assumption  that 
the  transported  organic  solutes  are  distributed  throughout 
the  entire  larval  body.  For  larvae  of  Tedania  ignis,  the 
assumption  of  translocation  to  parenchyma!  tissues  after 
assimilation  by  the  outer  epithelium  does  not  seem  to  be 
true.  Examinations  of  autoradiograms  of  larvae  exposed 
to  radiolabeled  alanine  and  palmitic  acid  for  up  to  2  h 
reveals  that  nearly  all  the  label  remains  with  the  cells  pu- 
tatively  responsible  for  transport. 

The  suggestion  that  DOM  transport  may  be  a  regionally 
important  source  of  nutrition  and  energy  is  not  new.  Ear- 
lier researchers  (e.g..  Pequignat,  1966;  Ferguson.  1967. 
1970)  working  on  DOM  transport  in  adult  invertebrates 
suggested  that  the  epidermis  may  be  the  sole  recipient  of 
the  assimilated  materials  and  that  there  is  little  translo- 
cation of  materials  from  the  adult  endoderm  to  the  ec- 
toderm. Later  studies  (e.g.,  Pearse  and  Pearse,  1973;  Pe- 
quignat. 1973;  Ferguson,  1980;  Chien  and  Rice,  1985; 
Rice  and  Stephens.  1987)  indicated  that  there  could  be 
translocation  of  material  to  interior  cells  and  that  the  vi- 
ability of  the  epidermis  was  not  dependent  upon  exoge- 
nously  supplied  nutrients.  In  his  review  of  integumentary 
transport  by  invertebrates.  Wright  (1988)  reported  that 
"the  nutritional  impact  of  DOM  uptake  may  vary  with 
the  specific  integumental  site  of  transport:  over  much  of 
the  surface  of  the  integument,  accumulated  substrates  will 
support  the  nutritional  needs  of  those  cells;  uptake  into 
other  integumental  regions  may  result  in  a  rapid  trans- 
cellular  movement  of  accumulated  materials  to  the  he- 
molymph  for  transport  to  deeper  tissues.  To  the  extent 
that  this  type  of  specialized  "partitioning"  of  accumulated 


Table  II 

The  contribution  of  palmitic  acid  and  alanine  transport  to  the 
metabolism  ol  the  larval  epidermis  at  substrate  «>miv)/ra//"/n  <>l  I  / 
and  0.25  ^M 


Solute 

O2  demand 
(1  pM,  0.25  fiM)' 

%  Compensation 
(1  nM,  0.25  itM)~ 

Palmitic  acid 
Alanine 

370.7,  92.7 
8.2,  2.0 

131%.  33% 
3%,  <1% 

The  estimated  metabolic  rate  of  the  epidermis  (282.2  pmol  O2  X  epi- 
dermis"1 h"1)  is  calculated  as  the  estimated  weight  fraction  of  the  epi- 
dermis (0.3)  times  the  average  metabolic  rate  of  the  intact  larvae  (940.7 
pmol  O2  x  larva"1  h"'). 

1  O2  demand  ([S]  =  1  n\l)  =  the  transport  rate  •  the  mol  O2  required 
for  complete  catabolism  of  palmitic  acid  (23  mol  Oj/mol  Pal)  and  alanine 
(3  mol  O2/mol  Ala).  O2  demand  ([S]  =  0.25  /iA/)  is  calculated  by  dividing 
the  O2  demand  (1  jjA/)  by  4  (Pal  and  Ala  transport  is  assumed  to  be 
first-order  at  substrate  concentrations  of  1  pM  or  lower). 

2  %  Compensation  of  the  metabolic  rate  of  the  epidermis  is  calculated 
by  dividing  the  O2  demand^,^,™,  by  the  estimated  metabolic  rate,^,.,™, 
and  multiplying  by  100. 


& 

UJ 


500  , 


450. 


400. 


350  . 


300. 


I 
o 
U 


200. 


150. 


50 


B. 


10 


Metabolic  Activity  Ratio  (MAR) 

Figure  5.  The  change  in  the  estimated  contribution  of  palmitic  acid 
(A)  and  alanine  (B)  transport  ([S]  =  1  nM  each)  from  seawater  to  the 
metabolism  of  the  epidermis  of  larvae  of  Tedania  ii>nix.  The  range  in 
metabolic  activity  ratios  (MARs)  represents  the  relative  difference  in  the 
metabolic  activities  of  epidermal  cells  and  internal  cells. 


DOM  occurs  in  the  integument,  the  calculation  of  the 
nutritional  potential  that  have  been  described  here  will, 
of  course,  under-  or  overestimate  the  nutritional  potential 
of  uptake  for  a  given  organism  or  tissue." 

The  estimated  contributions  of  DOM  transport  to  the 
metabolism  of  Tedania  ignis  larvae  are  detailed  in  Table 
I.  If,  however,  most  of  the  assimilated  DOM  from  seawater 
is  metabolized  solely  by  the  cells  responsible  for  transport, 
then  evaluations  of  the  energetic  importance  of  this  form 
of  nutrient  acquisition  underestimate  the  potential  epi- 
dermis-specific value.  In  larvae  of  T.  ignis,  assimilated 
materials  apparently  remain  within  the  epidermis;  to  es- 
timate the  importance  of  transport  to  the  metabolism  of 
the  epidermis  of  Tedania  larvae,  the  following  analysis 
was  done.  Cross-sections  ( 1  nm  thick)  of  Tedania  larvae 
were  photographed  and  the  negatives  printed.  From  the 


166 


W.  B.  JAECKLE 


photographs,  the  cross-sections  of  three  larvae  were  cut 
out  and  weighed  on  an  analytical  balance.  The  area  of 
the  section  representing  the  epidermis  was  then  cut  out 
and  the  remaining  photographic  images  of  the  internal 
cells  were  reweighed.  If  it  is  assumed  that  larvae  of  T. 
ignis  are  cylindrical  and  that  all  cells  have  the  same  weight 
density,  then  the  areal  proportion  of  the  epidermis  in  the 
photograph  is  equal  to  the  proportion  of  a  larva's  organic 
weight  that  is  represented  by  the  cells.  On  the  basis  of  this 
analysis,  the  epidermis  of  T.  ignis  larvae  represents,  on 
average,  30%  of  the  total  larval  biomass  (ca.  5  yug).  If  all 
cells  of  a  larva  have  the  same  respiration  rate,  then  the 
metabolic  rate  of  the  epidermis  is  282.2  pmol  O: 
larva'1  IT1  (0.3  •  940.7  O:  larva  '  h~').  The  energetic 
contribution  of  palmitic  acid  transport  ([S]  =  1  nM  and 
0.25  nAI)  to  the  metabolism  of  the  larval  epidermis  is 
potentially  important  (Table  II).  It  seems  unlikely,  how- 
ever, that  the  metabolic  rate  is  constant  among  all  cells, 
given  the  morphological  differences  among  cell  types  in 
sponge  larvae  (Woollacott.  1990.  1993).  The  theoretical 
contribution  from  transport  to  metabolism  can  be  ad- 
justed to  account  for  the  differences  in  cellular  metabo- 
lism. Changes  in  the  potential  contribution  of  alanine  and 
palmitic  acid  transport  to  metabolism  as  a  function  of 
the  relative  activities  of  the  external  and  internal  cells  are 
shown  in  Figure  5.  In  this  figure,  a  metabolic  activity  ratio 
(MAR)  of  1  represents  the  condition  in  which  the  meta- 
bolic rate  of  the  epidermal  cells  is  directly  proportional 
to  their  weight-fraction  of  larval  tissue;  i.e..  all  cells  respire 
at  the  same  weight-specific  rate.  A  metabolic  activity  ratio 
of  2  equals  the  condition  in  which  the  metabolic  rate  of 
the  epidermal  cells  is  twice  that  of  their  interior  counter- 
parts. Complete  compensation  of  the  estimated  metabolic 
demand  of  the  epidermis  would  be  provided  through  pal- 
mitic acid  transport  ([S]  =  1  n.M)  at  a  MAR  <  1.3.  For 
alanine  transport  alone,  100%  compensation  of  the  met- 
abolic rate  could  not  be  accomplished. 

Sponge  parenchymula  larvae  can  live  in  plankton  for 
periods  of  time  lasting  from  hours  to  days  (e.g..  Berquist 
el  at.  1970;  Woollacott.  1990,  1993;  Kaye  and  Reiswig, 
1991).  During  their  planktonic  existence  they  are  func- 
tionally incapable  of  ingesting  paniculate  forms  of  food. 
Despite  this  inadequacy,  these  lecithotrophic  larvae  are 
physiologically  capable  of  assimilating  DOM  from  sea- 
water  across  their  epidermis;  hence  they  are  not  nutri- 
tionally independent  of  their  environment.  The  potential 
benefits  obtained  through  DOM  transport  and  metabo- 
lism are  not,  however,  dependent  only  on  the  substrate 
concentration  and  the  class  and  species  of  the  organic 
materials  present  in  seawater,  but  also  on  whether  the 
assimilated  materials  are  distributed  among  all  larval  cells. 

Acknowledgments 

I  thank  Dr.  Shirley  Pomponi  (Harbor  Branch  Ocean- 
ographic  Institution)  for  identifying  juveniles  of  the  larvae 


used  in  this  study,  Ms.  Julie  Piriano  for  assisting  with  the 
scanning  electron  microscopy,  and  Dr.  Mary  Rice 
(Smithsonian  Marine  Station  at  Link  Port)  for  providing 
the  equipment  necessary  to  complete  this  project.  This 
manuscript  was  significantly  improved  by  the  comments 
of  Dr.  E.  J.  Balser  and  two  anonymous  reviewers.  This 
research  was  supported  by  a  fellowship  from  the  Smith- 
sonian Institution  to  W.  B.  Jaeckle. 

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Role  of  Digestive  Gland  in  the  Energetic  Metabolism 

of  Penaeus  setifems 


CARLOS  ROSAS',  ANDREA  BOLONGARO-CREVENNA2,  ADOLFO  SANCHEZ1, 
GABRIELA  GAXIOLA1.  LUIS  SOTO2,  AND  ELVA  ESCOBAR2 

lLaboratorio  de  Ecofisiologia.  Deplo.  de  Biologia,  Fac.  de  Ciendas  UN  AM,  Mexico  04510, 

D.F.  Mexico,  and  2Laboratorio  de  Eco/ogia  del  Bentos,  Institnto  de  Cicndas  del  Mar 

y  Limnologia,  UNAAI,  Mexico  04510.  D.F.  Mexico 


Abstract.  We  determined  the  role  of  the  digestive  gland 
in  the  respiratory  metabolism  of  Penaeus  setifems  adult 
males  as  a  step  toward  proposing  a  feeding  schedule  based 
on  the  cycle  of  activity  in  the  digestive  gland.  We  mea- 
sured pre-  and  postprandial  values  for  oxygen  consump- 
tion rate  and  hemolymph  glucose  concentrations  in  live 
animals,  and  oxygen  consumption  rate  and  glycogen 
concentration  in  excised  digestive  gland.  After  the  ani- 
mals were  fed,  which  enhanced  general  metabolic  activ- 
ity, these  indices  changed.  There  was  a  high  correlation 
between  the  oxygen  consumption  rate  of  the  animal  and 
the  glucose  concentration  in  the  hemolymph,  and  be- 
tween the  oxygen  consumption  rate  by  the  digestive  gland 
and  the  glycogen  concentration  in  the  digestive  gland, 
all  in  relation  to  time  after  feeding.  Correlations  support 
the  hypothesis  that  the  energy  demand  depends  upon 
the  metabolic  substrate  concentration.  In  this  theory, 
glucose  sustains  muscle  activity  (during  ingestion  of  food) 
and  glycogen  is  the  product  of  the  digestive  gland  during 
food  assimilation.  Our  observations  of  metabolic  dy- 
namics during  the  feeding  period  allowed  us  to  examine 
the  feeding  process.  The  metabolic  activity  of  the  diges- 
tive gland  was  highest  6  h  after  feeding.  This  could  mean 
that  assimilation,  having  started  2  h  after  food  intake, 
peaked  6  h  after  feeding.  Eight  hours  after  feeding,  the 
oxygen  consumption  rate  of  the  digestive  gland  decreased 
and  fell  to  values  similar  to  those  recorded  for  animals 
subjected  to  72  h  of  fasting. 


Received  23  September  1994;  accepted  29  June  1995. 
Abbreviations:  AMI,  apparent  heat  increment:  afdw,  ash-free  dry 
weight:  dw,  dry  weight. 


Introduction 

The  digestive  gland  (also  known  as  the  midgut  gland 
or  hepatopancreas)  of  decapod  crustaceans  serves  the  dual 
role  of  secreting  enzymes  and  absorbing  digested  food. 
This  gland  is  composed  of  embryonic  (E)  cells,  which  give 
rise  to  two  basic  cell  types:  R  cells  (Restzellen),  which 
store  nutrients,  and  F  cells  (Fibrillenzellen),  which  secrete 
enzymes  (Hirsch  and  Jacobs,  1930).  The  F  cells  develop 
into  B  cells  (Blasenzellen),  a  more  mature  secretory  stage 
with  a  large  vacuole  containing  digestive  enzymes  (Gibson 
and  Barker,  1979).  The  overall  functions  of  the  digestive 
gland,  including  the  temporal  relationship  of  secretion 
and  absorption  to  food  intake,  have  been  assessed  in  sev- 
eral species.  Because  many  of  those  studies  used  histo- 
chemical  methods,  the  results  are  difficult  to  interpret. 

Gibson  and  Barker  ( 1 979)  reported  that  in  the  digestive 
gland  ofHoimirus  aincncainis.  B  cells  were  replaced  12  h 
after  food  ingestion,  and  in  Penaeus  semisiilcatus  the 
highest  activity  of  proteolytic  enzymes  was  evident  within 
7  to  10  h.  Al-Mohanna  and  Nott  (1987)  detected  in  the 
latter  species  a  cycle  of  maximum  enzymatic  activity  6  h 
after  food  intake,  with  production  of  feces  containing  B 
cells,  membranous  remains,  and  paniculate  matter  after 
24  h.  Hopkin  and  Nott  (1980)  found  that  in  Cardmis 
nuwnas,  digestion  and  absorption  took  about  12  h  after 
feeding  and  were  followed  by  an  excretory  phase  lasting 
from  about  12  to  48  h  after  feeding. 

Despite  the  amount  of  information  published  on  the 
activity  and  characteristics  of  the  crustacean  digestive 
gland,  little  is  known  about  its  role  in  respiratory  activity 
during  feeding.  Several  authors  (e.g..  Beamish  and  Trippel, 
1990)  recognized  that  the  apparent  heat  increment  (AHI; 


I6S 


DIGESTIVE  GLAND  ACTIVITY  IN  PKKAECS  SKTIl-'KRL'S 


169 


previously  referred  to  as  specific  dynamic  action,  SDA) 
is  an  indicator  of  the  mechanical  and  biochemical  pro- 
cesses associated  with  the  ingestion  and  assimilation  of 
food.  Although  muscular  tissue  is  responsible  for  the  me- 
chanical activity,  the  digestive  gland  is  the  site  of  metabolic 
functions  that  break  the  stomach  contents  down  bio- 
chemically. Hence,  the  AHI  may  result  from  addition  of 
the  energy  used  in  the  above  two  processes:  this  constitutes 
a  considerable  percentage  of  the  daily  energy  budget  in 
aquatic  organisms  (Du-Preez  et  ai,  1992;  Chakraborty  el 
a!.,  1993). 

In  aquaculture,  AHI  has  been  used  in  the  selection  of 
diets  for  raising  shrimp;  thus  it  is  imperative  to  determine 
the  magnitude  of  energy  costs  associated  with  feeding  ac- 
tivity. No  previous  studies  have  correlated  this  energy  cost 
to  digestive  gland  metabolism  during  food  ingestion. 
Therefore,  no  approximations  have  been  made  that  allow 
the  differentiation  of  components  of  the  AHI  and  the  role 
of  the  digestive  gland  in  these  processes.  Our  study  was 
aimed  at  determining  the  role  of  the  digestive  gland  in 
the  respiratory  metabolism  ofPenaeus  setiferus.  At  various 
stages  while  the  shrimp  were  ingesting  and  assimilating 
food,  we  measured  the  rate  of  oxygen  consumption  in  live 
animals  and  in  the  digestive  gland;  the  content  of  glucose 
in  hemolymph:  and  the  content  of  glycogen  in  the  diges- 
tive gland. 


Materials  and  Methods 


Animals 


Thirty-nine  sexually  mature  male  shrimp  (P.  setiferus; 
37.57  ±  0.54  g  wet  weight)  were  caught  on  the  continental 
shelf  off  Laguna  de  Terminos.  Campeche,  Mexico.  In  the 
laboratory,  the  shrimp  were  placed  in  1000-1  flow-through 
tanks,  with  aerated  seawater,  under  a  light/dark  cycle  of 
14/10  h.  After  24  h  of  conditioning,  shrimp  were  left 
without  food  for  72  h  to  provide  fasting  conditions.  During 
the  experiment,  salinity  was  kept  at  32%o  and  temperature 
at  28  ±  1°C. 

Oxygen  consumption  rale  in  whole  live  shrimp 

After  the  fasting  period,  6  shrimp  were  placed  in  a 
1-1  chamber  connected  to  a  flow-through  respirometer 
(0.1  1/min)  (Martinez-Otero  and  Diaz-Iglesia.  1975),  in 
which  they  were  acclimated  for  8  h  before  the  experiments 
were  conducted.  Oxygen  consumption  rate  was  estimated 
by  the  difference  in  oxygen  concentration  in  the  input 
and  output  of  the  chamber.  The  difference  was  multiplied 
by  the  flow  rate  and  corrected  for  a  control  chamber  with- 
out organisms.  Metabolic  rate  was  recorded  at  time  0  (an- 
imals fasting  72  h)  and  at  1,2,  4,  6,  and  24  h  after  a  meal 
of  1  g  squid  meat  (Loligo  brevis)  was  given  and  totally 


ingested.  These  times  were  selected  on  the  basis  of  the 
finding  that  the  major  activity  of  the  digestive  gland  in  P. 
semisiilcatiis  occurs  between  1  and  6  h  after  feeding  (Al- 
Mohanna  and  Nott,  1987). 

At  the  end  of  the  experimental  phase,  all  animals  were 
sacrified  and  fresh  weight,  dry  weight  (dw),  and  ash-free 
dry  weight  (afdw)  determined.  Results  of  oxygen  con- 
sumption measurements  were  expressed  in  milligrams  of 
oxygen  per  gram  per  hour  afdw  (Sanchez  el  ai,  1991). 
AHI  was  estimated  as  the  difference  between  feeding  and 
fasting  rates  of  oxygen  consumption  (Du  Preez  et  ai, 
1992).  This  difference  was  transformed  using  the  exoca- 
loric  coefficient  of  3.53  cal/mg  O2  consumed  (Elliot  and 
Davison,  1975),  and  expressed  in  relation  to  a  mean  afdw 
of  11.4g/(animal-24  h). 

Digestive  gland  oxygen  consumption  rate 

A  total  of  15  shrimp  were  used  for  this  experiment. 
Fasted  (72  h)  animals  were  placed  in  a  600-1  tank  with 
filtered  seawater.  The  digestive  glands  of  animals  chosen 
at  random  were  dissected  and  placed  in  physiological 
solution  for  crustaceans  (Prosser,  1973).  This  solution 
was  made  with  NaCl  (26.42  g/1).  KC1  (1.12g/l).  CaCl2 
(2.78  g/1),  MgCl:  (0.32  g/1),  MgSO4  (0.49  g/1),  H3BO3 
(0.53  g/1),  and  NaOH  (0.192  g/1)  with  a  pH  of  7.6.  Each 
digestive  gland  was  cut  in  two,  and  each  half  was  consid- 
ered a  duplicate  of  the  other.  Rate  of  oxygen  consumption 
was  measured  in  fasting  shrimp  (72  h)  and  at  1,2,  6,  and 
8  h  after  feeding.  Each  piece  of  digestive  gland  was  placed 
in  a  microrespirometer  chamber  with  2  ml  of  previously 
aerated  physiological  solution.  The  oxygen  concentration 
in  the  chambers  was  measured,  under  gentle  agitation, 
with  a  Strathkelvin  Model  781  oxygen  meter  equipped 
with  a  high-sensitivity  membrane  (12.5^m)  electrode. 
This  system  was  connected  to  a  thermostat  that  kept  tem- 
perature at  28  ±  0.0 1°C  during  the  experiment.  Mea- 
surements lasted  for  3  to  5  min,  recording  oxygen  varia- 
tions every  10  s.  Due  to  the  uniformity  of  readings,  only 
the  results  obtained  30  s  after  sectioning  the  digestive  gland 
were  used. 

Glvcogen  concentrations  in  digestive  gland  and  glucose 
in  hemolymph 

Glycogen  was  measured  in  digestive  gland  sections  from 
18  shrimp  at  time  0  (after  72  h  fasting),  and  at  1,2,  4.  6, 
and  24  h  after  feeding.  Glycogen  was  extracted  with  an- 
throne  reagent.  This  reagent  consisted  of  a  solution  of 
0.05%  anthrone,  1%  thiourea,  and  72%  H2SO4  (Carroll  et 
ai,  1956).  The  digestive  gland  was  first  homogenized  in 
trichloroacetic  acid  (TCA;  5%)  for  3  min.  After  centrifu- 
gation  (3000  rpm)  the  supernatant  was  filtered  (acid-free 
paper)  and  quantified.  This  procedure  was  performed 


170 


C.  ROSAS  ET  AL. 
Table  I 


Oxygen  cuiMiination  rate  (VOJ.  blood  glucose  concentration,  and  digestive  gland  glycogen  concentration  ofPenaeus  setiferus  in  relation  to  time 
after  feeding 


Intact  animals 


Digestive  gland 


Time  H 

V02 
mgO2/(g  afdw-h) 

Glucose 

mmol/1 

Wet  weight 

g 

V02 

mgO2/(g  afdw-h) 

Glycogen 
mg/  1  00  g  dw 

0 

1.01 

1.66 

0.62 

1300 

1.70 

(0.22) 

(0.01) 

(0.01) 

(130) 

(0.14) 

1 

1.56 

5.46 

1.28 

1310 

2.08 

(0.09) 

(0.40) 

(0.17) 

(107) 

(0.43) 

2 

1.25 

5.71 

1.02 

1507 

4.10 

(0.75) 

(0.01) 

(0.07) 

(204) 

(0.43) 

4 

1.45 

5.67 

0.80 

— 

12.06 

(0.19) 

(0.19) 

(0.01) 

(0.84) 

6 

1.12 

5.45 

0.92 

2027 

17.41 

(0.10) 

(0.02) 

(0.05) 

(112) 

(0.14) 

8 

— 

— 

— 

1250 

— 

112 

24 

0.93 

1.28 

0.59 

— 

0.74 

(0.07) 

(0.01) 

(0.06) 

(0.06) 

N  by  measurement 

6 

3 

3 

3 

3 

Total 

6 

IS 

15 

Values  as  mean.  SEM  in  parentheses. 


three  times.  One  ml  of  TCA  filtrate  was  pipetted  into  a 
Pyrex  centrifuge  tube  and  mixed  with  5  volumes  of  95% 
ethanol.  The  tubes  were  placed  in  a  water  bath  at  37°C 
for  3  h.  After  precipitation  occurred,  the  tubes  were  cen- 
trifuged  at  3000  rpm  for  15  min.  The  packed  glycogen 
was  dissolved  by  addition  of  2  ml  of  distilled  water.  Ten 
ml  of  anthrone  reagent  was  delivered  into  each  tube  with 
vigorous  blowing,  and  the  tubes  were  placed  in  a  cold 
(4°C)  tap  water  bath.  Later  all  tubes  were  placed  in  a 
boiling  water  bath  for  15  min.  The  contents  of  the  tubes 
were  transferred  to  a  colorimeter  tube  and  read  at  620  nm 
after  the  instrument  was  adjusted  with  the  reagent  blank 
(distilled  water  plus  anthrone  reagent).  A  standard  was 
prepared  by  adding  2  ml  of  standard  glucose  solution 
containing  0.1  mg  of  glucose  to  anthrone  reagent. 

Glucose  concentration  in  the  hemolymph 

Glucose  was  measured  in  hemolymph  from  the  same 
shrimp  used  for  the  glycogen  determination.  Before  the 
digestive  gland  was  excised.  200  ft]  of  hemolymph  was 
extracted  from  the  pericardium  of  each  shrimp.  A  12.5% 
solution  of  sodium  citrate  was  used  to  prevent  clotting 
(Martin  ci  ai,  1991).  The  glucose  concentration  in  the 
hemolymph  was  measured  with  a  commercial  kit  for 
medical  diagnosis  ( Merckotest  3306,  Rosas  et  ai.  1992a). 

Statistical  analysis 

Analysis  of  variance  (ANOVA)  was  used  to  test  the 
significance  of  the  results  obtained.  Duncan's  multiple 


range  test  (Zar,  1974)  was  used  to  determine  differences 
in  the  means  of  oxygen  consumption  of  whole  animals, 
oxygen  consumption  of  digestive  gland,  glycogen  concen- 
tration in  digestive  gland,  and  glucose  concentration  in 
hemolymph.  For  all  groups,  an  analysis  of  covariance  was 
performed  between  the  rate  of  oxygen  consumption  by 
the  animal  and  the  concentration  of  glucose  in  hemo- 
lymph and  between  the  rate  of  oxygen  consumption  by 
the  digestive  gland  and  the  concentration  of  glycogen  in 
the  hemolymph. 

Results 

Respiratory  metabolism  and  levels  of  glucose  and  gly- 
cogen changed  with  time  after  feeding  (Table  I).  The  oxy- 
gen consumption  rate  of  live  organisms  was  higher  be- 
tween 1  and  4  h  after  feeding  (p  <  0.05)  than  at  time  zero. 
A  respiratory  rate  increase  of  54%  and  an  AHI  of  1.95  cal/ 
(g  afdw-h),  equivalent  to  533.3  cal/(l  1.4  g  afdw-day), 
were  obtained  (Table  II).  Daily  AHI  was  8.5%-  of  the  energy 
of  the  ingested  food  (Table  II).  Subsequently  there  was  a 
reduction  of  about  28%>  in  oxygen  consumption  rate  (as 
observed  at  6  h  after  feeding),  and  the  oxygen  consump- 
tion rate  returned  to  the  initial  level  by  24  h  after  feeding 
(Table  I). 

Digestive  gland  weight  increased  after  1  h.  from  0.62 
to  1 .28  g  dw/animal,  then  diminishing  gradually  in  the  2 
and  6  h  observations.  The  lowest  value  was  obtained  24  h 
after  feeding  (Table  I).  Digestive  gland  oxygen  consump- 


DIGESTIVE  GLAND  ACTIVITY  IN  PE.\AEL'S  SETIFERUS 


171 


Table  II 

Apparent  heat  increment  f.-tHI)  calculated  for  Penaeus  setiferus 

mg  O:/  cal/  cal/ 

(gafdw-h)       (gafdw-h)      (1 1.4  gafdw-h) 

AHI  0.55  ±  0.03       1.95  +  0.09        533.5  ±  26.7 

AHI  °~v  of  the  energy 

of  the  ingested  food  8.5 

Values  as  mean  ±  SEM.  Shrimp  wet  weight:  37.57  ±  0.51  g:  shrimp 
ash-free  dry  weight:  1 1 .4  ±  0. 16  g:  energy  content  of  Loligo  hreris:  6300 
cal/g  afdw. 


tion  rate  remained  constant  between  time  zero  and  1  h. 
with  an  average  of  1305  mg  O:/(g  dw-h)  (Table  I).  A 
gradual  increase  was  detected  until  it  reached  its  highest 
level.  6  h  after  feeding,  which  was  56%  higher  than  for 
fasting  animals  (Table  I)  (p  <  0.05).  The  oxygen  con- 
sumption rate  of  the  digestive  gland  was  returned  to  fasting 
levels  8  h  after  feeding. 

Hemolymph  glucose  concentration  showed  a  significant 
increase  by  1  h  after  feeding  (Table  I).  Recorded  values 
were  1.66  mmol/1  in  starved  animals  and  5.46  mmol/1  in 
fed  shrimp.  The  hemolymph  glucose  level  of  fed  shrimp 
remained  stable  between  1  and  6  h,  the  average  value  being 
5.5  mmol/1.  Twenty-four  hours  after  feeding,  glucose 
concentration  had  fallen  to  1.28  mmol/1.  observed  in 
starved  animals  (p  <  0.05). 

Glycogen  in  digestive  gland  showed  a  gradual  increase 
after  2  h  of  feeding,  reaching  a  maximum  10.2  times  larger 
than  fasting  animals  at  6  h  (Table  I).  Twenty-four  hours 
after  feeding,  glycogen  levels  were  significantly  lower  than 
those  observed  before  feeding. 

The  oxygen  consumption  rate  of  the  animal  was  cor- 
related with  hemolymph  glucose  (r  =  0.78).  and  the  ox- 
ygen consumption  rate  of  the  digestive  gland  was  corre- 
lated with  glycogen  concentration  (r  =  0.99:  Table  III). 
In  both  cases,  values  of  r  and  p  confirm  a  positive  rela- 
tionship between  responses,  which  are  positive  and  linear 
(p<Q.05). 

Discussion 

The  use  of  mature  male  shrimp  in  this  study  excludes 
the  effect  of  biochemical  processes  related  to  gonadal 
maturation,  thus  assuring  that  the  results  were  due  solely 
to  the  activity  of  the  digestive  gland.  In  previous  studies. 
Rosas  el  al.  (1992a,  b)  showed  that  in  a  24-h  cycle,  the 
oxygen  consumption  rate  and  the  hemolymph  glucose 
concentration  of  P.  setiferus  were  highest  between  9  and 
16  h  after  feeding,  which  assures  an  8-h  interval  of  general 
metabolic  stability.  In  the  present  study  we  used  previous 
results  to  select  a  time  period  for  observation  of  metabolic 


changes  due  to  feeding,  thus  eliminating  possible  effects 
of  circadian  rhythm  upon  metabolic  activity. 

Apparent  heat  increment  (AHI)  is  related  to  an  increase 
in  oxygen  consumption  rate  induced  by  locomotory  ac- 
tivity, capture,  ingestion  and  digestion  of  food,  and  bio- 
chemical activity  related  to  absorption  of  material 
(Beamish  and  Trippel,  1990).  These  expenditures  of  en- 
ergy can  constitute  a  high  percentage  of  the  energy  used 
by  shrimp.  If  we  consider  organisms  with  an  average 
weight  of  40  g  dw  ( 1  1 .4  g  afdw).  a  squid  diet  with  a  caloric 
value  of  1890  cal/g  afdw  (Del  Barco.  1975).  and  an  AHI 
of  533.5  cal/(  1 1 .4  g  afdw  •  day),  it  is  possible  to  infer  that 
the  AHI  corresponds  to  8.5%  of  the  daily  metabolized 
energy  (Table  II).  Although  the  AHI  levels  might  change 
depending  on  the  quality  and  quantity  of  food,  our  results 
can  be  applied  to  squid  (Loligo  brevis)  diets  normally  given 
to  reproductive  shrimp.  Du  Preez  el  al.  (1992)  reported 
an  AHI  of  2.4%  to  19.5%  of  ingested  energy  for  juveniles 
of  Penaeus  monodon  fed  shrimp  muscle,  and  2%  to  17% 
for  shrimp  fed  with  commercial  balanced  feed.  In  another 
study.  Nelson  et  al.  ( 1977)  reported  that  in  juvenile  Ma- 
cmbrachiwn  rosenbergii,  the  AHI  fluctuates  from  7.4% 
to  27.5%  of  available  energy,  depending  on  the  type  of 
feed,  with  the  highest  level  found  in  those  fed  on  tubifid 
worms. 

From  the  results  of  this  study  it  is  possible  to  isolate 
some  components  of  the  energy  costs  associated  with  AHI, 
and  shed  some  light  on  utilization  and  assimilation  (Table 
IV).  Because  of  the  difficulty  in  estimating  each  AHI 
component  directly,  we  attempted  to  differentiate  them 
on  the  basis  of  their  respective  times.  Once  food  was  pro- 
vided, the  animals  displayed  intensive  muscular  activity 
(pleopod  motion),  which  contrasted  with  the  no-motion 
behavior  observed  within  the  respirometer  chamber  dur- 
ing the  8-h  acclimatization  period.  As  the  first  three  pairs 
of  pereiopods  secure  the  food,  it  is  fragmented  and  passed 
onto  the  mouth  parts  for  ingestion.  Contact  digestion  then 
begins  (Gibson  and  Barker,  1979;  Al-Mohanna  and  Nott, 
1987)  (Table  IV).  This  behavior  occurred  during  the  first 
hour  after  feeding  and  coincided  with  the  elevation  of 
hemolv  mph  glucose  concentration  and  oxygen  consump- 

Table  III 

Oxygen  consumption  rale  Hug  O,/(g  afdw  •  h))  and  concentrations  of 
hemolymph  glucose  (mmol/1)  correlation  (A)  and  digestive  gland 
oxygen  consumption  rale  (mg  O2/(g  d\v  •  /ill  and  digestive  gland 
glycugen  (mg/gj  correlation  IB)  <>/"Penaeus  setiferus 


A 
B 


0.83 
1185.30 


0.09 
0.70 


0.78 
0.99 


0.05 

o.oo: 


=  a  +  h.\'  Values  from  all  groups. 


172 


Feeding  schedule  of  Penaeus  setiferus 


C.  ROSAS  ET  AL. 
Table  IV 


Stage 


Activity 


Source 


Associated 
time 


Metabolic  substrate 


IV 

V 


Excitation.  Ingestion,  and  Contact  digestion  (Stomach)        Maximum  VO,  (AHI) 


Absorption  of  small  particles  and  Chyme  digestion 
(Lumen) 

Assimilation  and  Synthesis 


Feces  production  and  Digestive  gland  metabolic  rate 

reduction 
General  metabolic  reduction 


Weight  increment  of  DG 
VO:DG 
VO,  AHI 

Maximum  VCK  DG  Glycogen 
(17.41  mg/lOOgdw) 


1  Glucose  (5.5  mmoi/1) 

1-2  Glucose  (5.5  mmol/1) 

Proteins  (?) 
Lipids  (?) 

6  Glucose  (5.5  mmol/1) 

Proteins!?) 
Lipids  (?) 


24  Less  glucose  than  T0 

57%  less  glycogen  than  in  T0 


VO2  (AHI)  is  the  oxygen  consumption  rate  of  whole  animals;  VO:  DG  is  the  digestive  gland  oxygen  consumption  rate;  DG  is  the  digestive  gland. 
This  schedule  integrated  all  results  obtained. 


tion  noted  1  h  after  feeding  (Table  I).  Taking  into  account 
that  the  oxygen  consumption  of  the  digestive  gland  re- 
mained constant,  we  attribute  the  increase  in  oxygen  con- 
sumption to  the  mechanical  aspects  of  feeding  (muscle 
excitement,  ingestion,  and  contact  digestion).  During  this 
time  glycogen  reserves  in  muscular  tissue  and  digestive 
gland  provide  glucose  in  hemolymph  as  fuel  for  these  ac- 
tivities. The  correlation  between  oxygen  consumption  rate 
and  glucose  level  in  hemolymph  reported  for  crustaceans 
in  this  and  other  works  can  be  used  as  an  indicator  of  this 
process  (Table  III)  (Ramos  and  Fernandez,  1981:  Brito 
and  Diaz-Iglesia,  1987;  Diaz-Iglesia  ct  al.,  1987;  Rosas  et 
at..  1992a). 

Digestive  gland  weight  increased  as  a  function  of  time 
after  feeding.  A  maximum  weight  of  1.28  g  was  reached 
1  h  after  feeding;  this  value  was  twice  as  high  as  that  re- 
corded for  fasting  animals.  If  we  attribute  this  difference 
in  weight  to  the  amount  of  food  in  the  digestive  gland 
(Al-Mohanna  and  Nott,  1987),  we  can  evaluate  the  effi- 
ciency of  incorporation  of  ingested  squid.  Considering 
that  1  g  of  food  was  available  per  shrimp  and  using  initial 
weight  of  the  digestive  gland,  we  estimate  an  efficiency  of 
66%  of  ingested  food.  In  view  of  this  result  and  those 
reported  by  Al-Mohanna  and  Nott  (1987),  for  aquaculture 
purposes  it  is  the  activity  of  the  digestive  gland  rather 
than  the  ingestion  of  the  food  that  should  be  considered 
in  establishing  a  feeding  schedule  for  P.  setiferus. 

Once  the  food  is  digested  in  the  gut.  the  chyme  and 
fine  particles  are  digested  in  the  lumen  and  absorbed  by 
diffusion  to  the  inner  portions  of  the  digestive  gland  tu- 
bules, thus  initiating  the  accumulation  of  glycogen  (Al- 
Mohanna  and  Nott,  1987;  Hopkin  and  Nott,  1980).  The 
140%  increase  in  the  glycogen  concentration  in  the  diges- 
tive glands  that  took  place  2  h  after  feeding  could  indicate 


the  onset  of  glucogen  synthesis  (Tables  I  and  IV).  Because 
these  processes  require  energy,  we  would  expect  the  oxy- 
gen consumption  of  the  digestive  gland  to  increase.  In 
fact,  a  56%  increase  in  oxygen  consumption  was  recorded 
in  the  digestive  gland  of  P.  setiferus  after  6  h  (Table  I). 
This  increase  can  be  correlated  to  the  calorigenic  effect 
induced  by  the  food  in  the  digestive  gland.  In  this  study, 
the  oxygen  consumption  rate  of  the  digestive  gland  was 
1287%>  higher  than  that  of  intact  animals.  Although  we 
have  no  explanation  for  such  a  high  consumption  rate, 
these  results  are  similar  to  those  obtained  by  other  authors. 
Conceicao  (1993)  and  Diaz-Iglesia  et  al.  (1995)  recently 
found  that  in  feeding  Panulirus  argus.  the  oxygen  con- 
sumption rate  of  the  digestive  gland  was  312%  higher  than 
that  observed  in  living  lobsters.  The  lack  of  endogenous 
controls  during  //;  vitro  experiments  could  account  for  the 
high  metabolic  rate  found  for  Penaeus  setiferus  and 
Piimtlinis  argus.  Schmidt-Nielsen  ( 1984)  stated  that  "the 
metabolic  rate  in  homologous  tissues  (liver,  for  example) 
is  relatively  constant,  irrespective  of  body  size,  but  this 
rate  is  restricted  or  depressed  in  the  large  animals  by  some 
'central'  control  or  other  'organismic'  factor  resident  in 
the  intact  organism."  Although  this  observation  was  based 
on  data  for  mammals,  it  might  apply  equally  well  to 
shrimp  and  explain  metabolism-depressing  factors  in  the 
digestive  gland.  Hormones  from  the  eyestalks  could  also 
be  responsible  for  the  metabolic  control  of  the  digestive 
gland  in  living  animals  (Silverthorn,  1975a,  b:  Kleinholz, 
1976;  Madyastha  and  Rangneker.  1976;  Mauviot  and 
Castell,  1976;  Radakrishnan  and  Vijakumaran.  1984; 
Rosas  ct  a/.,  1991).  The  presence  of  elevated  glycogen 
levels  concomitant  with  an  increase  in  the  oxygen  con- 
sumption rate  by  the  digestive  gland  may  point  to  the 
synthesis  of  reserves  during  this  period  mirroring  the  as- 


DIGESTIVE  GLAND  ACTIVITY  IN  PENAEUS  SETIh'ERl'S 


173 


similation  of  ingested  food  (Table  IV).  This  hypothesis  is 
supported  by  the  correlation  between  metabolic  activity 
and  glycogen  concentrations  (Table  III). 

Major  activity  of  the  digestive  gland  has  been  reported 
6  h  after  feeding  activity  in  P.  semisulcatus  (Al-Mohanna 
and  Nott,  1987).  This  elapsed  time  could  mirror  the  high- 
est respiratory  activity  in  the  digestive  gland  of  P.  setifems 
(Table  I)  and  indicate  that  assimilation,  having  started 
2  h  after  food  intake,  would  peak  6  h  after  feeding.  Eight 
hours  later,  the  oxygen  consumption  of  the  digestive  gland 
could  decrease  and  fall  to  values  similar  to  those  recorded 
for  digestive  gland  tissue  from  animals  subjected  to  72  h 
of  fasting  (Table  I).  Although  the  amount  of  energy  lost 
as  heat  cannot  be  precisely  accounted  for  in  all  the  pro- 
cesses in  this  study,  the  largest  amount  of  energy  con- 
sumed was  associated  with  the  mechanical  processes  of 
feeding,  as  evidenced  by  the  oxygen  consumption  of  living 
animals  1-2  h  after  feeding  (Table  I). 

The  accumulation  of  glycogen  as  storage  material  can 
also  be  used  as  an  indicator  of  the  energetic  potential  of 
the  diet,  because  glycogen  is  the  source  of  glucose  for 
metabolic  use  and  for  the  synthesis  of  chitin  (Owing  and 
Stevenson,  1979;  Chan  el  at.  1988).  Considering  that 
molting  is  an  important  factor  in  shrimp  growth,  the  dy- 
namics of  glucose  could  be  useful  in  determining  the  diet 
for  shrimp  species. 

Acknowledgments 

The  experimental  work  was  done  at  the  Centro  de  In- 
vestigaciones  Pesqueras  (CRIP)  of  Campeche.  of  the  In- 
stituto  Nacional  de  la  Pesca.  under  a  collaborative  pro- 
gram with  the  Faculty  of  Science,  UNAM.  The  project 
was  partially  financed  by  DGAPA  project  IN-201292 
given  to  Dr.  Luis  A.  Soto  and  Dr.  Carlos  Rosas.  Our  rec- 
ognition for  their  support  in  laboratory  work  goes  to  M. 
Eugenia  Chimal  and  Mauricia  Borja. 

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Reference:  Bio/  Bull  189:  175-184.  (October/November, 


Bioassay  and  Preliminary  Characterization  of 

Ovigerous-Hair  Stripping  Substance  (OHSS)  in 

Hatch  Water  of  Crab  Larvae 


MASAYUKI  SAIGUSA 

Okayama  University.  Faculty  of  Science,  Department  of  Biology, 
Tsushima  2-1-1.  Okayama  700.  Japan 


Abstract.  Hatch  water  (the  filtrated  medium  into  which 
zoea  larvae  have  been  released)  of  the  estuarine  terrestrial 
crab  Sesamia  haematocheir  (akate-gani)  contains  a  sub- 
stance that  causes  premature  detachment  of  embryos  from 
ovigerous  females.  Detachment  occurs  when  the  ovigerous 
hairs  along  the  female's  ovigerous  setae  slip  out  of  the 
investment  coat  that  binds  them  to  the  embryos  through 
stalks,  or  funiculi.  The  active  factor,  which  I  call  ovigerous- 
hair  stripping  substance  (OHSS),  is  released  outside  of  the 
egg  capsule  at  the  time  of  hatching,  and  is  not  secreted 
by  the  female.  This  study  describes  the  results  of  a  quan- 
titative assay  for  measuring  the  activity  of  OHSS.  Activity 
is  measured  as  the  percentage  of  hairs  on  a  seta  that  can 
be  induced  to  slip  out  of  the  coat  without  damage.  Ex- 
periments with  an  extract  of  crushed  embryos  indicated 
that  OHSS  is  present  up  to  2  days  before  hatching.  Its 
activity  was  destroyed  by  heat  and  trypsin,  suggesting  that 
it  is  a  protein.  Its  molecular  size  was  estimated  by  gel 
filtration  to  be  1 5-20  kDa  in  51.  haematocheir  and  30  kDa 
in  S.  pictum.  Reciprocal  tests  among  different  species  in- 
dicated that  OHSS  occurs  widely  in  intertidal  and  estu- 
arine crabs. 

Introduction 

After  oviposition,  the  embryos  of  decapod  crustaceans 
are  wrapped  in  a  thick  membrane  and  clustered  on  the 
ovigerous  setae  beneath  the  abdomen  of  the  female.  Many 
fine  hairs  (i.e..  ovigerous  hairs)  are  arranged  along  the 
seta,  and  the  embryos  are  attached  to  these  hairs  by  a 
stalk:  the  funiculus.  The  mechanism  by  which  the  fertil- 
ized egg  is  attached  to  the  ovigerous  hairs  and  the  source 

Received  21  December  1992;  accepted  25  July  1495. 


of  the  material  that  makes  up  the  funiculus  and  the  egg 
capsule  have  been  subjects  of  controversy  for  many  years 
(e.g..  Andrews,  1906;  Yonge,  1937,  1946;  Mawson  and 
Yonge,  1938;  Linder,  1960;  Suko,  1961;  Cheung,  1966; 
Fisher  and  Clark,  1983;  Goudeau  and  Lachaise,  1980, 
1983;  Goudeau  el  a/..  1987;  Talbot  and  Demers,  1993). 

In  addition  to  the  funiculus,  the  embryo  attachment 
system  involves  a  clear  coat  that  wraps  around  (invests) 
the  ovigerous  hairs  (Saigusa.  1994).  The  funiculus  is 
therefore  not  connected  to  the  ovigerous  hairs  directly, 
but  indirectly  through  the  coat — a  fact  not  previously  re- 
ported. The  investing  coat  may  be  composed  of  the  same 
materials  that  make  up  the  funiculus  and  the  outer  layer 
of  the  egg  capsule,  but  this  notion  has  not  been  tested 
adequately. 

While  attached  to  the  ovigerous  hairs,  the  embryos  are 
ventilated  by  the  movement  of  pleopod  setae.  When  de- 
velopment is  completed,  the  egg  capsule  breaks,  and  the 
zoeas  hatch  and  are  released  into  the  water  by  a  special 
fanning  movement  of  the  female's  abdomen  (larval  release 
is  described  in  Saigusa,  1982).  After  the  larvae  have  been 
released,  the  empty  egg  cases,  funiculi,  and  investing  coats 
remain  attached  to  the  ovigerous  hairs.  Soon,  however,  a 
substance  released  at  the  time  of  hatching  causes  the  ovi- 
gerous hairs  to  slip  out  of  the  investment  coat,  detaching 
the  funiculi  and  empty  egg  cases;  the  active  factor  is  called 
'ovigerous-hair  stripping  substance'  (OHSS)  (Saigusa, 
1 994).  Within  a  few  hours,  the  ovigerous  hairs  are  cleaned 
without  damage,  and  in  a  few  days  a  new  clutch  of  fer- 
tilized eggs  is  attached  to  the  hairs.  Because  OHSS  is  re- 
leased at  the  time  of  hatching,  one  might  suppose  it  to  be 
directly  involved  in  hatching.  But  there  is  no  evidence 
that  this  factor  is  a  hatching  enzyme  of  the  sort  known 
in  many  groups  of  animals  (Saigusa,  1994). 


175 


176 


M.  SAIGUSA 


If  the  medium  in  which  hatching  and  larval  release  has 
occurred  is  tillered,  the  resulting  solution — called  hatch 
water — can  affect  other  ovigerous  females,  causing  all  of 
their  embryos  to  slip  off  the  ovigerous  hairs  and  out  of 
the  brooding  space  without  hatching.  This  effect  on  whole 
crabs  was  used  in  the  preceding  experiments  (Saigusa. 
1994)  to  assay  the  activity  of  the  OHSS  contained  in  hatch 
water.  To  investigate  the  properties  of  this  substance  fur- 
ther, however,  an  assay  that  would  require  only  small 
amounts  of  active  material  was  essential. 

In  this  study,  therefore,  I  have  used  only  segments  of 
ovigerous  setae  with  their  attached  embryos  in  a  quanti- 
tative assay  of  the  effect  of  OHSS.  This  substance  is  a 
protein  released  into  the  medium  at  the  time  that  the  egg 
capsule  breaks.  The  molecular  weight  of  OHSS  was  esti- 
mated by  gel  nitration.  Reciprocal  tests  with  several  species 
suggest  that  OHSS  occurs  widely  in  intertidal  and  estua- 
rine  crabs. 

Materials  and  Methods 

Handling  of  ovigerous  females  far  assays 

Individuals  of  the  terrestrial  red-handed  crab,  Sesanna 
haematocheir,  were  collected  from  the  thicket  along  a 
small  estuary  at  Kasaoka,  Okayama  Prefecture.  Japan,  in 
1992  and  1993.  After  collection,  the  crabs  were  quickly 
brought  into  the  experimental  rooms,  where  they  were 
kept  in  several  plastic  containers  (70  cm  long,  40  cm  wide, 
and  25  cm  high)  containing  shallow  water  (about  1  cm 
deep)  and  hiding  spaces.  The  light  and  temperature  were 
controlled,  respectively,  at  LD  15:9  and  24  ±  1°C.  The 
assay  is  sensitive  and  seemed  to  be  influenced  by  even 
very  small  quantities  of  OHSS  mixed  into  the  water.  So 
when  one  or  more  females  released  their  larvae  into  a 
container,  all  the  females  kept  in  that  container  were  re- 
turned to  the  field.  To  avoid  this  problem,  females  were 
usually  used  for  assay  within  a  few  days  after  collection. 

Preparation  of  hatch  water 

As  reported  elsewhere  (Saigusa,  1994),  OHSS  is  con- 
tained in  hatch  water,  i.e.,  the  filtered  medium  in  which 
zoea  larvae  have  hatched.  The  color  of  the  embryos  carried 
by  females  changes  from  dark  brown  to  brownish  green 
according  to  the  stage  of  development,  which  can.  there- 
fore, be  estimated  by  visual  inspection.  To  obtain  hatch 
water,  females  with  mature  embryos  (brownish  green)  that 
were  due  to  hatch  within  a  few  days  were  collected  in  the 
field  (i.e.,  in  Kasaoka)  (Saigusa.  1982). 

As  shown  earlier  (e.g.,  Saigusa.  1988),  hatching  and 
larval  release  by  estuarine  crabs  are  under  the  control  of 
a  circatidal  rhythm  the  phase  of  which  can  be  shifted  in 
the  laboratory  by  the  24-h  light  cycle.  When  these  females 
were  kept  under  a  day-night  cycle  in  phase  with  that  in 


the  field  (i.e.,  light-on  at  0500  and  light-off  at  2000),  the 
larvae  hatched  at  night  at  about  the  time  of  high  tide.  The 
ovigerous  females  were  placed  individually  in  glass  or 
plastic  beakers  (8.5  cm  in  diameter,  12  cm  in  height)  con- 
taining 30  ml  of  diluted  seawater  (10  ppt)  or  the  same 
quantity  of  distilled  water.  The  solution  was  aerated  for 
1  day  before  use.  The  medium  was  changed  every  day  if 
larval  were  not  released. 

Hatching  of  estuarine  crabs  is  highly  synchronized;  all 
of  the  embryos  may  hatch  within  about  5-30  min  in  the 
laboratory  (see  Saigusa,  1992,  1993).  As  soon  as  hatching 
was  complete  and  the  female  had  released  all  of  her  zoeas 
into  the  medium,  she  was  removed,  and  the  medium  was 
filtered  through  nylon  mesh  to  remove  the  zoeas.  This 
filtered  medium  was  immediately  transferred  to  a  small 
bottle,  and  was  stored  at  -15°C  until  used:  at  this  tem- 
perature, the  activity  of  OHSS  is  maintained  for  at  least 
half  a  year.  But  almost  all  of  the  experiments  were  done 
with  hatch  water  that  had  been  collected  within  the  month. 

Assay  of  OHSS  activity 

I  have  not  yet  found  an  artificial  substrate  that  is  acted 
upon  by  OHSS,  so  an  efficient  biological  assay  was  de- 
veloped, as  follows.  Female  crabs  have  four  pairs  of  ab- 
dominal appendages,  each  of  which  consists  of  plumose 
and  ovigerous  seta.  Embryos  are  attached  to  the  ovigerous 
hairs  arranged  along  the  ovigerous  seta,  by  the  funiculi 
(for  details,  see  Saigusa,  1994).  Several  ovigerous  setae 
with  their  clusters  of  embryos  still  attached  were  cut  off 
from  many  females  and  subdivided,  usually  into  six  seg- 
ments, under  the  stereomicroscope  (Fig.  la.  b).  Each  seg- 
ment with  its  cluster  of  embryos — hereinafter  called  an 
egg  cluster — (Fig.  la)  was  immersed  in  0.5  ml  of  10  ppt 
seawater  (SW).  or  the  same  quantity  of  hatch  water.  The 
medium  and  egg  clusters  were  incubated  for  various  times 
in  a  plastic  culture  dish  with  24  wells,  each  1.6cm  in 
diameter  and  1 .7  cm  in  height.  This  dish  was  shaken  back 
and  forth  (3-4  cm)  at  100-120  times  per  min  in  the  ex- 
perimental rooms. 

After  the  incubation,  each  egg  cluster  was  again  placed 
in  a  glass  dish  with  10  ppt  SW  or  distilled  water.  The  dish 
was  put  under  a  stereomicroscope,  and  fine  forceps  were 
used  to  pull  the  embryos  gently  away  from  their  seta. 
When  an  egg  cluster  incubated  in  10  ppt  SW  was  pulled, 
about  90%  of  the  ovigerous  hairs  were  broken  away  from 
the  seta  (Fig.  Ic).  But  of  the  clusters  incubated  with  hatch 
water,  about  80%  slipped  off  easily  and  without  damage 
( Fig.  1  d ).  The  activity  of  OHSS  was  therefore  taken  as  the 
percentage  of  the  hairs  stripped  clean  and  undamaged  to 
the  total  number  of  hairs  along  the  segment  of  seta. 

Time  course  of  the  effect  of  OHSS  with  diluted  hatch 
water 

Hatch  water  collected  from  a  single  female  was  diluted 
3,  9,  and  27  times,  and  the  time  course  of  OHSS  activity 


CRAB  HATCHING  SUBSTANCE 


177 


1mm 


1mm 


Figure  1.  A  method  for  assessing  the  activity  of  OHSS.  (a)  One  of  the  setal  segments  (ss)  subdivided 
before  hatching,  with  its  developing  embryos  (cm)  still  attached,  (b)  An  ovigerous  seta  is  cut  into  six  pieces 
(1-6).  The  arrow  shows  where  the  tip  of  the  seta  is  removed  (see  text  for  details).  This  figure  shows  a  seta 
just  after  the  zoeas  have  hatched,  leaving  the  empty  egg  cases  (<r)  still  attached  to  ovigerous  hairs  which, 
however,  are  visible,  (c)  Ovigerous  hairs  (oh)  incubated  in  10  ppt  SW.  and  the  attached  embryos  then  pulled 
gently  away  with  forceps,  (d)  The  same,  but  the  ovigerous  hairs  (oh)  incubated  in  hatch  water.  Note  that 
many  of  the  hairs  incubated  in  seawater  are  broken  off  (compare  c  and  d). 


was  compared  with  that  of  undiluted  samples.  This  ex- 
periment was  carried  out  with  hatch  water  of  S.  haema- 
tochcir  and  S.  picluin. 

Appearance  of  OHSS  activity  in  living  females 

To  determine  when  OHSS  is  released,  either  by  the 
larvae  or  by  the  female,  one  or  two  ovigerous  setae  were 
detached  from  the  female  before  and  after  hatching.  Before 
hatching,  unhatched  embryos  were  attached  to  the  setae, 
but  after  the  release  of  larvae,  only  the  remnants  (i.e.. 
broken  egg  capsules,  funiculi.  and  the  investment  coat) 
remained  on  the  setae.  These  setae  were  subdivided,  and 
the  embryos  or  the  remnants  were  pulled  with  forceps. 
Time  of  hatching  and  larval  release  was  determined  and 
recorded  by  the  photoelectric  switch  method  described 
earlier  (Saigusa,  1992,  1993). 

Presence  of  OHSS  activity  before  hatching 

Embryonic  development  of  5.  haematocheir  can  be  di- 
vided into  four  stages  by  visual  inspection:  the  early  stage 


(10  days),  from  the  start  of  cleavage  to  just  before  the 
formation  of  eye  pigments;  the  intermediate  stage 
(10  days),  from  eye  pigment  formation  to  completion  of 
the  compound  eyes;  the  late  stage  ( 1  week),  in  which  the 
egg  color  changes  from  brown  to  brownish  green;  and  the 
final  stage  (i.e..  mature  embryos),  in  which  hatching 
should  occur  within  a  few  days. 

To  examine  whether  active  OHSS  is  present  before 
hatching,  egg  clusters  of  various  developmental  stages  were 
examined:  i.e..  two  or  three  ovigerous  setae  with  their 
attached  embryos  were  detached  from  the  females  and 
crushed  by  hand  for  a  few  minutes,  with  3  ml  of  10  ppt 
SW  per  one  seta.  Aliquots  (0.5  ml)  of  this  embryo  extract, 
uncentrifuged.  were  immediately  pipetted  into  the  wells 
of  a  culture  dish.  Freshly  detached  egg  clusters  were  placed 
into  this  solution,  and  OHSS  activity  was  monitored  4  h 
later.  Time  of  hatching  and  larval  release  by  the  females 
that  had  yielded  the  experimental  egg  clusters  was  also 
monitored  by  the  photoelectric  switch  method. 


178 


M.  SAIGUSA 


Susceptibility  of  OHSS  to  trypsin 

Two  milligrams  of  trypsin  (porcine  pancreatic  "trypsin 
1:250,"  Difco  Laboratories)  was  dissolved  in  20ml  of 
hatch  water.  The  solution  was  divided  into  aliquots  and 
incubated  at  35°C  for  either  75  min  or  3  h.  These  solu- 
tions were  then  transferred  to  room  temperature  (about 
25°C).  Egg  clusters  were  placed  into  this  solution,  and 
OHSS  activity  was  examined  for  the  next  4  h.  Further- 
more, to  test  whether  trypsin  itself  causes  the  wrapping 
coat  to  slip  off  the  ovigerous  hair,  egg  clusters  were  in- 
cubated for  4  h  at  25°C.  with  0.5  ml  of  10  ppt  SW  con- 
taining only  trypsin,  and  at  the  same  concentration. 

Gel  filtration 

Hatch  water  collected  from  several  females  was  cen- 
trifuged  at  15,000  rpm  for  30  min  at  5°C  to  remove  the 
solid  materials.  The  supernatant  was  freeze-dried  and  was 
then  reconstituted  in  1  ml  of  10  m^/Tris-HCl  buffer  (pH 
7.5).  This  test  sample,  containing  Blue  Dextran  (Phar- 
macia) and  1  M  NaCl  for  calibration,  was  applied  to  a 
Sephacryl  S-200  (Pharmacia)  column  (45  cm  X  1.3  cm 
i.d.),  and  fractions  (0.8  ml)  were  collected  at  10-min  in- 
tervals. The  column  was  eluted  with  the  Tris-HCl  buffer, 
and  the  protein  in  each  fraction  was  monitored  with  a 
Beckman  DU-65  spectrophotometer  at  O.D.  280  nm. 

The  activity  of  OHSS  in  the  fractions  from  gel  nitration 
was  determined  by  the  method  of  Shirai  (1986),  as  follows. 
A  series  of  threefold  dilutions  of  each  active  fraction  was 
prepared,  and  an  egg  cluster  was  immersed  in  each  dilu- 
tion and  tested  by  gentle  pulling  with  forceps.  The  re- 
sponse— the  percentage  of  stripped,  undamaged  hairs  in 
each  solution — was  then  plotted  against  the  log  of  the 
dilution  (Fig.  2).  The  potency  of  a  fraction  was  expressed 
as  the  dilution  producing  a  half-maximal  effect  (ED50). 
But  because  the  maximal  response  in  this  assay  is  about 
80%  and  the  minimum  is  about  10%  (dashed  lines  in  Fig. 
2),  the  ED5(j  was  taken  as  the  dilution  producing  45% 
stripped  ovigerous  hairs  (shown  as  -  •  —  •  -  in  Fig.  2). 

OHSS  in  other  species.  Females  of  S.  erythrodactylum. 
S.  pictuin,  and  S.  dehaani  bearing  embryos  that  appeared 
likely  to  hatch  within  a  few  days  were  collected  from  the 
field  at  Kasaoka  and  brought  into  the  laboratory.  Hatch 
water  from  these  crabs  was  obtained  in  the  same  way  as 
from  5.  haemal ocheir.  although  the  quantity  and  salinity 
of  the  medium  varied  with  the  body  size  of  each  species 
and  the  ambient  water  into  which  larvae  would  normally 
be  released  (i.e..  20  ml  of  20  ppt  SW  for  S.  pictum;  15  ml 
of  20  ppt  SW  for  S.  erythrodactylum:  and  40  ml  of  10  ppt 
SW  for  S.  dehaani).  The  filtered  medium  was  frozen  at 
-15°C  until  it  was  used. 

Hatch  waters  of  these  additional  three  species  and  S. 
haematocheir  were  applied  to  the  unhatched  embryos  of 
six  species  (listed  in  Table  II).  Females  carrying  unhatched 


100 -, 


Q. 
CL 


(D 

.C 


50- 


3° 


32  33  34 

12  46 

Degree  of  dilution 


3s 


Figure  2.  Dilution-response  curves  of  crude  and  concentrated  hatch 
water;  the  degree  of  dilution  is  scaled  logarithmically  (base  3).  Broken 
lines  show  the  maximum  and  minimum  percentages  of  ovigerous  hairs 
that  were  stripped  off  the  investing  coat  without  damage.  The  dilution- 
response  curves  for  crude  hatch  water  (open  triangles)  and  concentrated 
hatch  water  (black  circles)  were  drawn  by  hand  without  statistical  treat- 
ment. Activity  units  are  taken  as  the  values  of  the  dilution  at  which  each 
dose-response  curve  intersects  the  45%  level  (downward  arrows):  i.e..  12 
(crude)  and  46  (concentrated)  units  in  these  preparations. 


embryos  were  collected  from  each  habitat,  and  the  ovi- 
gerous setae  with  their  attached  egg  clusters  were  cut  into 
2-6  pieces  according  to  the  size  of  the  crab.  Experimental 
procedures  were  the  same  as  for  S.  haematocheir.  Time 
of  incubation  was  4-6  h.  depending  upon  the  species  that 
provided  the  egg  clusters. 

To  examine  further  whether  the  effect  of  OHSS  is  dif- 
ferent among  species,  the  hatch  water  collected  from  sev- 
eral females  of  S.  pictum  was  pooled,  and  assayed  with 
egg  clusters  of  S.  haematocheir.  S.  erythrodactylum.  and 
Hemigrapsus  sanguineus.  The  time  course  of  the  effect 
of  OHSS  was  monitored  every  15  min  or  1  h. 

Results 

Development  of  the  assay 

The  following  preliminary  experiments  were  aimed  at 
understanding  the  variables  in  the  assay  and  thus  im- 
proving its  reliability.  The  ovigerous  setae  with  their  egg 
clusters  were  cut  off  and  subdivided,  usually  into  six  seg- 
ments (Fig.  la).  As  shown  earlier  (Saigusa,  1994),  most 
ovigerous  hairs  are  arranged  in  whorls  along  the  ovigerous 
seta.  The  length  of  these  hairs  depends  upon  their  position 
along  the  seta  (Fig.  Ib):  i.e..  the  hairs  along  positions  2- 
5  are  longest;  those  at  the  tip  of  the  seta  (position  6)  are 
shortest;  and  those  at  the  base  (position  1 )  are  of  inter- 
mediate length  (see  also  Fig.  6B  in  Saigusa,  1994). 

The  first  question  was  whether  the  length  of  the  hairs 
would  affect  the  ease  with  which  they  slip  out  of  the  in- 
vesting coat.  When  incubated  with  10  ppt  SW,  the  shortest 


CRAB  HATCHING  SUBSTANCE 


179 


N=I20 


20 


50 


100 


%  hairs   stripped 
per   one   setal    segment 

Figure  3.  Distribution  of  the  number  of  segments  in  which  the  per- 
centage of  the  ovigerous  hairs  that  slipped  out  of  the  coat  without  damage 
was  estimated,  (a)  Data  from  experiments  in  which  the  tip  of  the  seta 
remained  (incubation  in  10  ppt  SW).  (b)  Data  from  experiments  in  which 
the  seta  was  cut  at  its  tip  as  shown  in  Figure  la  (incubation  with  10  ppt 
SW).  (c)  Embryo  clusters  treated  with  hatch  water.  N  =  Total  number 
of  subdivided  setal  segments  with  their  attached  egg  clusters.  The  per- 
centage for  each  setal  segment  is  shown  at  intervals  of  KK. 

hairs  certainly  tended  to  slip  out  most  easily.  This  ten- 
dency was  especially  marked  at  the  tip  of  the  seta  (Fig. 
3a).  So  in  most  of  the  experiments,  the  tip  of  the  ovigerous 


seta  was  cut  away  before  use  (arrow  in  Fig.  Ib).  On  av- 
erage, only  10%  of  the  hairs  were  stripped  clean  without 
damage  (Fig.  3b).  In  contrast,  about  80%  of  the  egg  clusters 
slipped  off  when  the  preparation  was  placed  into  hatch 
water  (Fig.  3c). 

A  further  difficulty  is  that  the  response  to  a  given  con- 
centration of  OHSS  is  considerably  different  even  among 
the  egg  clusters  produced  by  the  same  female.  Figure  4 
shows  the  response  of  two  groups  of  the  egg  clusters  that 
were  separated  from  one  female  and  both  immersed  in 
hatch  water  with  the  same  concentration  of  OHSS.  Al- 
though the  percentage  of  stripped  hairs  fluctuated  consid- 
erably, it  clearly  increased  with  time,  reaching  a  maximum 
in  1-3  h  (e.g..  Fig.  4).  In  contrast,  control  egg  clusters 
immersed  in  10%  seawater  showed  no  such  increase  in 
the  percentage  of  stripping.  The  fluctuation  in  the  re- 
sponses suggests  that  the  adhesion  between  the  investing 
coat  and  the  ovigerous  hairs  is  also  variable,  even  in  the 
same  female. 

Concentration-dependence  of  OHSS  activity 

Unhatched  embryos  detached  from  two  females  of  S. 
haematocheir  were  incubated  with  a  series  of  threefold 
dilutions  of  hatch  water  collected  from  one  female,  and 
the  time  course  of  the  effect  of  OHSS  was  monitored  (Fig. 


180 


Time  of    incubation  (min) 


Figure  4.  A  typical  time  course  of  the  effect  of  S.  haematocheir  hatch 
water  on  the  stripping  of  unhatched  embryos  of  the  same  specimen.  Two 
egg  clusters  were  incubated  in  hatch  water  from  one  female  (•  —  •  and 
A  —  A),  and  one  incubated  in  10  ppt  SW  (••  •  ••).  These  egg  clusters 
were  all  separated  from  a  single  female.  The  broken  line  shows  the  re- 
sponse curve  of  the  mean  value  between  the  two  preparations  immersed 
in  hatch  water. 


180 


M.  SAIGUSA 


100-. 


•D 

CD 
Q. 
Q. 


l/) 

«  50 


I  2  3 

Time  of   incubation   (h) 


lOOn 


Q. 
Q. 


U1 

co  50- 

<T> 

_C 


/ 
;    a 


o        /  ' 

•^M 


/' 


1  2 

Time  of   incubation  (h) 


Figure  5.  Time  course  showing  the  effect  of  diluting  of  hatch  water  with  10  ppt  SW.  (a)  5  haematocheir 
embryos  incubated  with  5.  haematocheir  hatch  water,  (b)  51.  pictum  embryos  incubated  with  5  picluni 
hatch  water.  No  dilution  (O-  •  •  O):  3-fold  dilution  (A A):  9-fold  dilution  (D — D);  27-fold  dilution 


5a).  In  the  groups  of  embryos  placed  into  undiluted  and 
threefold-dilute  hatch  water,  half-maximal  activity  of 
OHSS  was  reached  in  20-30  min  and  was  largely  com- 
pleted (about  85%)  after  60-80  min  of  treatment.  A  nine- 
fold dilution  of  hatch  water  became  equiactive  to  the  un- 
diluted solution  in  an  hour,  and  the  maximum  response 
was  decreased  to  60%-70%.  Finally,  the  maximum  activity 
of  a  27-fold  dilute  solution  was  reduced  about  30%. 

The  effect  of  dilution  of  hatch  water  was  also  tested  in 
5.  pictum  (Fig.  5b).  In  this  experiment,  embryos  detached 


hatching  and 
larval  release 


lOO-i 


IT) 


X 
O 


50- 


> 

o 

< 


.  A 
•     A 


A 

A 


I 
48  h  24  0 

Hours  before  hatching  and   larval  release 

Figure  6.  OHSS  activity  within  the  embryos  before,  and  just  after, 
hatching  and  larval  release.  Solid  circles  (•):  percentage  of  stopped  hairs 
on  setae  detached  from  the  females.  These  setae  were  taken  from  females 
with  unhatched  embryos  (before  hatching  and  larval  release),  as  well  as 
those  with  remnants  (after  the  release  of  larvae).  They  are  not  treated 
with  hatch  water.  Open  triangles  (A):  hairs  on  setae  that  were  incubated 
in  a  solution  of  crushed  embryos  for  4  h.  and  then  pulled  with  forceps. 


from  two  females  of  S.  pictum  were  placed  into  a  series 
of  threefold  dilutions  of  5.  pictum  hatch  water.  Although 
the  percentage  of  slipped  hairs  also  fluctuated  in  this  spe- 
cies, the  trends  in  Figure  5b  were  very  similar  to  those  of 
Figure  5a. 

Appearance  oj  the  activity  in  the  embryo  and  the  timing 
oj  release  outside  the  egg  membrane 

To  determine  the  time  that  OHSS  has  an  effect  on  living 
females,  the  activity  of  the  substance  before  the  time  of 
hatching  and  larval  release  was  compared  with  the  activity 
afterward.  Until  just  before  hatching,  most  ovigerous  hairs 
were  broken  when  the  egg  cluster  was  detached  and  pulled 
with  a  forceps;  but  just  after  larval  release,  the  hairs  easily 
slipped  out  of  the  coat  (solid  circles  in  Fig.  6).  This  in- 
dicates that  OHSS  is  released  at  the  time  of  hatching, 
either  by  the  zoeas  or  by  the  female. 

Furthermore,  to  determine  whether  the  OHSS  activity 
appears  in  the  embryos  before  hatching,  freshly  detached 
embryos  were  incubated  with  the  material  containing 
crushed  embryos.  No  activity  was  detected  in  the  embryos, 
even  at  late  stages  of  development  (data  not  shown).  But 
activity  was  clearly  detected  in  embryos  that  should  have 
hatched  within  48  h  (triangles  in  Fig.  6).  Thus,  OHSS 
seems  to  accumulate  in  the  embryos  before  hatching.  Be- 
cause the  activity  is  not  detected  outside  of  the  egg  case 
just  before  hatching,  we  can  suppose  that  its  release  is 
associated  with  breakage  of  the  egg  capsule. 

Gel  filtration 

Lyophilized  hatch  water  collected  from  four  specimens 
of  5.  haematocheir  was  redissolved  and  subjected  to  gel 


CRAB  HATCHING  SUBSTANCE 


181 


I50n 


Table  I 

The  e/Jci'l  nl  hoiliiiK  anil  iryp.iin  treatment  on  /lie  OHSS  activity 
oj  hatch  water  from  Sesarma  haematocheir 


0J 


10  20 

Fraction  number 


Figure  7.  Gel  filtration  of  hatch  water,  (a)  5.  haemdtocheir.  Lyoph- 
ilized  hatch  water  collected  from  four  specimens  was  reconstituted  and 
passed  through  a  Sephacryl  S-200  column  equilibrated  with  10  rrbl/  Tris- 
HC1  buffer  (pH  7.5).  Egg  clusters  of  S.  haematocheir  were  incubated 
with  each  fraction  for  1.5  h,  and  theOHSS  activity  was  assayed  (protocol 
in  text).  Downward  arrows  show  elution  peaks  of  blue  dextran  (BD). 
bovine  serum  albumin  (BA),  carbonic  anhydrase  (CA).  cytochrome  c 
(CC).  and  NaCl.  These  markers  were  passed  through  the  column  without 
hatch  water,  (b)  S  pictum.  Test  sample  was  obtained  from  three  females 
of  5.  pictum.  and  passed  through  the  same  column  after  reconstitution 
with  the  same  buffer.  Embryos  of  S.  haematocheir  were  incubated  for 
5  h,  and  the  OHSS  activity  was  then  assayed.  Note  species  differences. 


filtration  on  a  column  of  Sephacryl  S-200.  The  activity 
of  each  fraction  was  assayed  with  unhatched  embryos  of 
the  same  species.  As  shown  elsewhere  (e.g..  Fig.  5),  activity 
is  variable,  so  the  experiments  were  repeated  12  times, 
always  with  similar  results.  Figure  7a  shows  one  of  these 
experiments.  The  activity  of  OHSS  extends  over  a  wide 
range  of  fractions.  The  molecular  size  of  OHSS  was  es- 
timated to  be  15-20  kDa  by  a  comparison  of  its  elution 
volume  with  those  of  standard  proteins:  i.e.,  bovine  serum 
albumin  (66  kDa),  carbonic  anhydrase  (29  kDa),  and  cy- 
tochrome c  (12.4  kDa). 

Hatch  water  collected  from  three  females  of  S.  pictum 
was  subjected  to  the  same  gel  filtration  protocol  that  was 
used  with  5.  haematocheir,  and  the  activitv  was  also  as- 


Experiments 


Percentage  of  stripped  hairs 
(X  ±  SD)* 


Dilute  SW  (10  ppt)  0    (±0) 

Trypsin  solution  2.6  (±4.7) 

Hatch  water  82.2  (±9.8) 
Boiling  of  hatch  water  3.6  (±2.5) 

Trypsin  treatment  of  hatch  water  (75  min)  69.1  (±11.8) 
Trypsin  treatment  of  hatch  water  (4  h)  8.3  (±6.8) 

*  In  all  cases,  the  number  of  setal  segments  tested  was  12. 


saved  with  embryos  of  .S.  haematocheir.  This  separation 
was  repeated  four  times  with  similar  results.  As  shown  in 
Figure  7b,  OHSS  of  S.  pictum  eluted  as  a  single  peak,  and 
the  molecular  size  was  estimated  by  comparison  with 
standards  to  be  about  30  kDa. 

Effect  oftrypsm 

A  few  tests  were  conducted  to  examine  the  nature  of 
OHSS.  As  indicated  in  Table  I,  its  activity  was  eliminated 
when  hatch  water  was  heated  for  5  min.  The  activity  was 
also  somewhat  decreased  when  the  hatch  water  was  treated 
with  trypsin  for  75  min,  and  was  completely  lost  with  3  h 
of  treatment.  On  the  other  hand,  trypsin  itself  did  not 
affect  the  coat,  although  this  enzyme  often  decomposed 
the  basement  of  the  ovigerous  hairs.  These  experiments 
suggest  that  OHSS  is  a  protein. 

Species  distribution  of  OHSS  activity 

Table  II  summarizes  the  reciprocal  tests  in  which  hatch 
waters  from  four  species  of  Sesarma  were  applied  to  the 


Table  II 

Reciprocal  tests  on  the  e'lecl  ft' OHSS 


Percentage  of  stripped  hairs 
(X  ±  SD)« 

Source  of  hatch 

Source  of  cluster  of 

water 

embryos 

Hatch  water 

KlpptSW 

Sesarma  dehaani 

5.  haematocheir 

81.9  ±    9.2(12) 

3.8 

4.1  (12) 

S  erythrodactylum 

S  erylhrodaaylum 

84.5  ±13.1  (6) 

5.2 

5.7(6) 

S  I'Hiutn 

89.3  ±    6.3  (4) 

2  2 

3.8(4) 

S-  haematocheir 

61.7  ±  18.6(6) 

3.7 

5.1  (6) 

S  haematocheir 

S  bidm\ 

99.2  ±    1.3(4) 

8.4 

5.5  (4) 

Gucncc  depressits 

49.7  ±    8.9  (6) 

1.9 

2.1  (6) 

S  piclit/u 

Hemigrapsm  sangiimeus 

41.7  ±  22.5(6) 

1.6 

1.0(6) 

S  erythrodactylum 

94.1  ±    3.7(6) 

3.7 

3.5(6) 

S  haematocheir 

60.8  ±  12.1  (4) 

2.6  ±  2.6(4) 

S  pu'litm 

64.1  ±  11.5(6) 

5.0  ±6.3  (6) 

*  Number  of  setal  segments  tested  is  in  parentheses. 


182 


M.  SAIGUSA 


embryos  of  six  species  of  crab.  All  species  were  affected 
by  all  types  of  hatch  water,  suggesting  that  OHSS  occurs 
wideh  in  intertidal  and  estuarine  crabs. 

But  there  was  a  clear  difference  in  the  time  course  of 
activity  among  species.  Figure  8a.  b  summarizes  the  results 
of  experiments  in  which  S  pictum  hatch  water  with  the 
same  OHSS  concentration  was  applied  to  the  embryos  of 
S.  erythrodactylum,  S.  haematocheir,  and  Hemigrapsus 
sangiiineus.  In  S.  erythrodactylum,  the  maximal  activity 
was  reached  after  1  h  of  incubation  (Fig.  8a),  but  more 
than  5  h  was  needed  for  H.  xangiiim'iis  (Fig.  8b). 

Discussion 

Hatch  water  of  the  estuarine  terrestrial  crab  Sesanna 
haematocheir  contains  a  substance  that  causes  premature 
detachment  of  embryos  from  ovigerous  females.  The  ac- 
tive factor — ovigerous-hair  stripping  substance  (OHSS) — 
is  released  outside  of  the  egg  case  at  the  time  of  hatching, 
and  is  not  released  by  the  female.  Its  molecular  size  was 
estimated  by  gel  filtration  to  be  15-20kDa  in  S  hae- 
matocheir and  30  kDa  in  S.  pictuni  These  results  raise 
the  following  five  issues  in  relation  to  the  OHSS  activity 
and  its  function:  species  specificity,  timing  of  synthesis 
and  secretion,  mode  of  action,  characterization,  and 
composition  of  the  investment. 

Species  specificity  of  OHSS  activity 

As  shown  in  Figure  7b,  OHSS  activity  of  S.  haemato- 
cheir can  be  assayed  with  egg  clusters  of  5.  pictum.  Re- 
ciprocal tests  among  different  species  indicate  that  OHSS 


occurs  widely  in  intertidal  and  estuarine  crabs  (Table  II). 
Thus,  the  hatch  water  from  one  species  of  crab  has  an 
effect  on  the  embryos  of  other  species.  On  the  other  hand, 
a  given  concentration  of  hatch  water  from  one  species 
can,  in  its  action  on  crabs  of  other  species,  have  very  dif- 
ferent time  courses  (Fig.  8).  This  disparity  suggests  that 
the  response  to  OHSS  differs  among  species.  For  example, 
the  length  of  the  ovigerous  hairs  differs  among  species,  so 
the  strength  of  the  bond  between  the  coat  and  the  ovi- 
gerous hairs  might  also  differ,  causing  the  variation  in 
time  course.  Moreover,  the  molecular  size  of  OHSS  is 
different  in  S.  haematocheir  saA  S.  pictum  (Fig.  7).  This 
suggests  that  the  molecular  structure  of  OHSS  might  also 
vary,  and  it  might  have  a  different  effect  when  applied  to 
the  embryos  of  other  species. 

Timing  of  OHSS  synthesis  and  secretion 

As  shown  elsewhere  (Saigusa,  1992).  clusters  of  5.  hae- 
inatocheii'  embryos  that  are  detached  from  the  female 
within  48-49.5  h  of  larval  release  all  hatch  successfully; 
but  embryos  that  have  been  detached  from  the  female  for 
longer  periods  do  not  hatch  at  all,  though  they  are  ob- 
viously alive  and  have  the  potential  of  hatching  (see  also 
Saigusa.  1993).  These  results  suggests  that  the  hatching 
process  is  a  distinct  program  initiated  near  the  end  of 
embryonic  development  (Saigusa,  1992). 

No  activity  appeared  outside  of  the  egg  case  until 
hatching  occurred.  But  a  homogenate  of  embryos  clearly 
showed  that  the  activity  was  present  in  embryos  that 
should  have  been  in  the  process  of  hatching  (Fig.  6).  Be- 
cause no  activity  was  detected  in  earlier  developmental 


100n 


"D 


Q. 
CL 


<T 


1         1.5         2         2.5         3 
Time  of  incubation  (h) 


10CH 


CL 

CL 


"> 


50- 


0* 


3.5 


I I I I I I I I '» 

2          A          6          8 
Time  of    incubation  (h) 


Figure  8.  Time  course  of  the  effect  of  OHSS  when  hatch  water  from  S  pictum  was  applied  to  the 
embryos  of  other  species,  (a)  Embryos  of  51.  erythrodactylum  (solid  circles)  and  S.  haematocheir  (solid 
triangles);  (b)  Embryos  of  Hemigrapsus  sanguineus  (solid  circles). 


CRAB  HATCHING  SUBSTANCE 


183 


stages,  we  can  speculate  that  OHSS  begins  to  be  produced 
only  immediately  before  the  start  of  hatching.  Further 
studies  are  required  to  determine  the  exact  timing  of 
OHSS  synthesis  and  secretion  in  the  unhatched  embryos. 

Mode  of  action  of  OHSS 

The  embryos  that  slip  off  the  ovigerous  hairs  are  always 
associated  with  the  funiculus  and  the  wrapping  coat,  and 
scanning  electron  microscopy  revealed  no  morphological 
changes,  either  on  the  coat  or  the  hair  (Saigusa,  1994). 
Goudeau  and  Lachaise  (1983)  showed  a  cross  section  of 
the  wrapping  coat  in  the  shore  crab  Carcinux  maenas. 
Their  figure  suggests  that  the  material  of  the  funiculus  is 
coiled  around  the  ovigerous  hair  without  any  additional 
substance.  If  this  is  the  case  in  S.  haematoctieir.  it  is  not 
plausible  that  OHSS  invades  the  space  between  the  coat 
and  the  ovigerous  hair.  So  I  suppose  that  OHSS  works 
directly  on  the  coat,  perhaps  softening  it.  so  that  the  hairs 
can  separate  from  the  investment  coat.  But  what  is  the 
mechanism  by  which  this  substance  softens  the  coat? 

Identity  of  OHSS 

One  possibility  is  that  OHSS  is  a  protease  that  partially 
digests  the  layers  of  the  investment  coat.  Embryos  of  many 
groups  of  animals  (e.g.,  fishes,  sea  urchins,  and  amphib- 
ians) release  a  protease  that  digests  egg  membrane  or  egg 
capsule  to  cause  hatching  (for  a  review,  see  Saigusa,  1994). 
DeVries  and  Forward  (1991)  also  reported  a  proteolytic 
enzyme  released  near  the  time  of  hatching  in  three  species 
of  estuarine  crabs.  It  is  not  known  whether  OHSS  is  a 
proteolytic  enzyme,  nor  whether  it  acts  as  a  hatching  en- 
zyme. But  if  so,  then  OHSS  may  partially  degrade,  not 
only  the  egg  capsule,  but  also  the  investment  coat,  causing 
them  to  slip  off  the  ovigerous  hairs. 

Another  possibility  is  that  OHSS  is  a  type  of  chitinase 
such  as  is  known  in  many  kinds  of  animals  and  plants. 
Chitin  is  a  stable  sugar  complex  constituting  the  exocu- 
ticle,  endocuticle,  and  membraneous  layer  in  crustaceans. 
It  is  hydrolyzed  by  two  enzymes  in  sequence:  chitinase, 
which  converts  the  long  chitin  polymers  into  small  oli- 
gosaccharides;  and  chitobiase  (/i-Ar-acetylglucosamini- 
dase).  which  hydrolyzes  these  chito-oligomers  into  jY-ace- 
tyl-D-glucosamine  (Lunt  and  Kent.  I960;  Jeuniaux,  1966; 
O'Brien  el  til..  1993).  These  enzymes  might  be  degrading 
the  coat  wrapping  the  ovigerous  hairs  until  it  is  able  to 
slip  off  the  hairs.  The  problem  with  this  notion  is  that 
such  enzymes  might  also  degrade  the  skin  of  the  zoeas. 

Composition  of  the  investment  coat,  funiculus,  and  egg 
envelope 

In  crabs,  the  funiculus  extends  to  the  coat  investing  the 
ovigerous  hair,  which  suggests  that  the  same  material 


forms  both  the  funiculus  and  the  investment  coat  (Saigusa, 
1994).  As  shown  in  Goudeau  and  Lachaise  (1980).  the 
structure  and  formation  of  the  egg  envelope  of  the  shore 
crab  Carcimts  nuieiKis  are  complex.  Cheung  (1966) 
showed  that  the  egg  envelope  and  funiculus  of  this  species 
are  not  affected  by  pepsin  or  trypsin,  though  the  yolk  of 
the  embryos  is  easily  digested.  Cheung  concluded  that  at 
least  the  outer  layer  (i.e..  trichromatic  membranes  in  her 
paper)  consists  largely  of  non-proteinous  substances.  Fur- 
thermore, our  pictures  by  the  transmission  electron  mi- 
croscopy indicate  that  this  material  makes  up  the  surface 
layer  of  the  egg  envelope  of  crabs  (unpub.  data).  If  OHSS 
softens  the  tissue  of  the  investment  coat,  it  would  also 
work  on  the  outer  layer  of  the  egg  envelope,  softening  the 
envelope.  This  might  contribute  to  the  case  of  hatching 
and  might  enhance  hatching  synchrony.  In  any  event, 
morphological  and  experimental  studies  on  this  coat 
might  help  in  identifying  OHSS. 

Acknowledgments 

Gel  nitration  chromatography  was  done  at  Ushimado 
Marine  Laboratory.  Okayama  University.  I  thank  Dr. 
Tadashi  Akiyama  for  technical  assistance.  I  am  also  in- 
debted to  Dr.  Hiroko  Shirai  for  her  frequent  advice  on 
experimental  procedures. 

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Reference:  JJ/o/  Hull  189:  185-262.  (October/November, 


Reports  of  Papers  Presented  at  the  General  Scientific  Meetings 
of  the  Marine  Biological  Laboratory 

August  14  to  16,  1995 

Program  Chairs: 

Robert  Paul  Malchow,  University  of  Illinois  at  Chicago 
Anne  Giblin,  Ecosystems  Center,  MBL 
Kathleen  Shvicki,  Swarthmore  College 

Special  Editorial  Board 


Donald  Abt,  Laboratory  for  Marine  Animal  Health. 

MBL 

Peter  Armstrong.  University  of  California,  Davis 
Conxita  Avila.  CEAB,  Spain 
Robert  Barlow,  Syracuse  University 
Michael  V.  L.  Bennett.  University  of  California. 

Berkeley 

David  Bodznick.  Wesleyan  University 
Barbara  Boyer.  Union  College 
Robert  Bullis.  Laboratory  for  Marine  Animal  Health. 

MBL 

Richard  Chappell.  Hunter  College 
Thomas  Cleland,  University  of  California.  San  Diego 
Larry  Cohen,  Yale  University 
Neal  Cornell.  MBL 

Leah  Devlin.  Pennsylvania  State  University 
Barbara  Ehrlich,  University  of  Connecticut  Health 

Center 

Harvey  Fishman.  University  of  Texas  Medical  Branch 
Kenneth  Foreman,  Ecosystems  Center,  MBL 
Robert  Garritt,  Ecosystems  Center,  MBL 
Leah  Haimo,  University  of  California.  Riverside 
Ferenc  Harosi,  MBL 

Susan  Douglas  Hill.  Michigan  State  University 
John  Hobbie.  Ecosystems  Center.  MBL 
Charles  Hopkinson.  Ecosystems  Center.  MBL 
Tom  Humphreys,  University  of  Hawaii 
William  J.  Kuhns,  Hospital  for  Sick  Children,  Canada 
Joseph  G.  Kunkel.  University  of  Massachusetts. 

Amherst 


Alan  Kuzirian.  MBL 

Stephen  C.  Land,  National  Vibrating  Probe  Facility, 

MBL 

Mark  Q.  Martindale.  University  of  Chicago 
James  McClelland.  Boston  University  Marine  Program. 

MBL 
Judith  McDowell,  Woods  Hole  Oceanographic 

Institution 

Lisa  Moore.  Albert  Einstein  College  of  Medicine 
Stephen  Moorman.  University  of  North  Texas  Health 

Science  Center 
John  Murray.  University  of  Pennsylvania  School  of 

Medicine 

Estela  O'Brien.  The  Rockefeller  University 
George  Pappas.  University  of  Illinois  at  Chicago 
Chris  Passaglia,  Syracuse  University 
James  Prechtl,  University  of  California,  San  Diego 
James  Quigley.  State  University  of  New  York 
Robert  F.  Rakowski.  The  Chicago  Medical  School 
Thomas  Reese.  National  Institutes  of  Health 
Harris  Ripps.  University  of  Illinois  at  Chicago 
Joan  Ruderman.  Harvard  Medical  School 
Rafael  Sarda.  CEAB.  Spain 
Christian  Sardet.  Station  Zoologique.  Villefranche  sur 

Mer,  France 
Robert  Silver.  MBL 
Roger  Sloboda,  Dartmouth  College 
Peter  J.  S.  Smith,  National  Vibrating  Probe  Facility, 

MBL 


185 


Ig6  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Roxanna  Smolowitz,  Laboratory  for  Marine  Animal  Ivan  Valiela,  Boston  University  Marine  Program, 

Health,  MBL  MBL 

Antoinnette  Steinacker,  Washington  University  Joseph  Vallino,  Ecosystems  Center,  MBL 

Joel  Tabb,  Dartmouth  College  Matt  Wachowiak,  The  Whitney  Laboratory 

Sidney  Tamm,  Boston  University  Marine  Program,  James  Walker,  University  of  Cambridge 

MBL  Jerome  Wolken,  Carnegie  Mellon  University 

Mark  Terasaki,  University  of  Connecticut  Health  Seymour  Zigman,  University  of  Rochester  Medical 

Center  School 

Jane  Tucker,  Ecosystems  Center,  MBL  Steve  Zottoli,  Williams  College 


LIST  OF  MBL  REPORTS 

Contents 


187 


Featured  Articles: 
A  Resurgence  of  Experimental  Embryology 

Martindale,  Mark  Q.,  and  Jonathan  (,).  Henry 
Diagonal  development:  Establishment  of  the  anal 
axis  in  the  ctenophore  Mncniiopsis  k-iilyi    . .  .        190 

Henry,  Jonathan  Q.,  and  Mark  Q.  Martindale 
The  experimental  alteration  of  cell  lineages  in 
the  nemertean  Cerebratulus  lactcux:  Implications 
for  the  precocious  establishment  of  embryonic 
axial  properties    192 

Henry,  Jonathan  Q.,  Mark  Q.  Martindale,  and 

Barbara  C.  Boyer 

Axial  specification  in  a  basal  member  of  the  spir- 
alian  clade:  Lineal  relationships  of  the  first  four 
cells  to  larval  body  plan  in  the  polyclad  turbel- 
larian  Hoploplana  inqitilina  194 

Development 

Mizell,  Merle,  Eric  Romig,  William  Hartley,  and 
Arunthavarani  Thiyagarajah 

Sex  on  the  brain  but  the  heart  is  not  really  in  it: 
Developmental  heart  defects  associated  with 
aquatic  pollution  and  microinjection  of  hexa- 
chlorobenzene  into  the  Japanese  medaka  em- 
bryo .  196 

Kunkel,  Joseph  G.,  and  Ellen  Faszeweski 

Pattern  of  potassium  ion  and  proton  currents  in 
the  ovariole  of  the  cockroach,  Periplaneta  anier- 
icana,  indicates  future  embryonic  polarity  197 

Fukui,  Voshio,  and  Shinya  Inoue 

Chemotaxis.  aggregation  behavior,  and  foot  for- 
mation in  Dictyostelium  discoideum  amoeba 
controlled  by  microbeam  uncaging  of  cyclic- 
AMP  .  198 

Keefe,    David,   John    Pepperell,    Paulo    Rinaudo, 

Joseph  Kunkel,  and  Peter  Smith 

Identification  of  calcium  flux  in  single  preim- 
plantation  mouse  embryos  with  the  calcium- 
sensitive  vibrating  probe  200 

Troll,  Walter,  Naoko  Sueoka,  Eisaboro  Sueoka, 

Jeffrey  D.  Easkin,  and  Diana  E.  Heck 

Inhibitors  of  protein  phosphatases  (okadic  acid 
and  tautomycin)  block  sea  urchin  development  201 

Cell  Cycle 

Suzuki,  Keisuke,  Fabrice  Roegiers,  Phong  Tran,  and 
Shinya  Inoue 

Reversible  regression  of  cytokinesis  induced  by 
Ca:+  ionophore  201 


Silver,  Robert  B. 

Leukotriene  B4  induces  release  of  calcium  from 
endomembrane  stores  in  vivo  in  eggs  and  second 
cell  blastomeres  of  the  sand  dollar  Echinaracnim 
pcmna  203 

Krendel,  Mira,  and  Shinya  Inoue 

Anaphase  spindle  dynamics  under  D:O-en- 
hanced  microtubule  polymerization  204 

Tran,  Phong,  E.  D.  Salmon,  and  Rudolf  Oldenbourg 
Quantifying  single  and  bundled  microtubules 
with  the  polarized  light  microscope  206 


Calcium 

Devlin,  C.  Leah,  and  Peter  J.  S.  Smith 

Acetylcholine-induced  Ca2+  flux  across  the  sar- 
colemma  of  an  echinoderm  smooth  muscle  207 

Fishman,  Harvey  M.,  Todd  L.  Krause,  Andrew  L. 

Miller,  and  George  D.  Bittner 

Retardation  of  the  spread  of  extracellular  Ca2+ 

into  transected,  unsealed  squid  giant  axons     .        208 

Smith,  Peter  J.  S.,  Katherine  Hammar,  and  Michael 

Tytell 

Effect  of  exogenous  heat  shock  protein  (hsp70) 

on  neuronal  calcium  flux  209 


Neurobiology 

Edds-Walton,  Peggy  L.,  and  Richard  Fay 

Regional  differences  in  directional  response 
properties  of  afferents  along  the  saccule  of  the 
toadfish  (Opsanus  tan)  211 

O'Brien,  Estela  V.,  and  Robert  B.  Barlow 

Optical  imaging  of  intrinsic  signals  from  the 
Liinulus  optic  nerve  212 

Passaglia,  C.  L.,  F.  A.  Dodge,  and  R.  B.  Barlow 

Limuhm  tuned  into  its  visual  environment    ...     213 

Prechtl,  James  C. 

Flutter-like  response  in  visual  cortex  of  the  semi- 
isolated  turtle  brain  215 

Metuzals,  J.,  H.  M.  Fishman,  and  I.  A.  Robb 
The  neurofilamentous  network-smooth  endo- 
plasmic  reticulum  complex  in  transected  squid 
giant  axon  216 

Eddleman,  C.  S.,  C.  M.  Godell,  H.  M.  Fishman, 

M.  Tytell,  and  G.  D.  Bittner 

Fluorescent  labeling  of  the  glial  sheath  of  giant 
nerve  fibers  2 1 8 


188 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


Biophysics 

Demurest,  Jeffery  R.,  and  James  L.  M.  Morgan 

Effect  of  pH  buffers  on  proton  secretion  from 
gastric  oxyntic  cells  measured  with  vibrating  ion- 
selective  microelectrodes  .  219 

Novales  Flamarique,  Inigo,  Rudolf  Oldenbourg,  and 

Ferenc  I.  Harosi 

Transmission  of  polarized  light  through  sunfish 
double  cones  reveals  minute  optical  anisotropies  220 

Comparative  Physiology  and  Biochemistry 

Zigman,  Seymour,  Nancy  S.  Rafferty,  and  Mark 
Schultz 

Dogfish  (Mitstelits  amis)  lens  catalase  reduces 
H:O2-induced  opacification  222 

kuhns,  William  J.,  Max  M.  Burger,  and  Gradimir 

Misevic 

Sulfotransferase  activities  in  the  marine  sponge 
Microciona  prolifera:  Correlation  with  sul fated 
glycan  adhesive  structures 223 

Aimes,  Ronald  I.,  James  P.  Quigley,  Snehasikta 

Swarnakar,  Dudley  K.  Strickland,  and  Peter  B. 

Armstrong 

Preliminary  investigations  on  the  scavenger  re- 
ceptors of  the  amebocyte  of  the  American  horse- 
shoe crab,  Limulits  polyphemus  225 

Swarnakar,  Snehasikta,  Ralph  Melchior,  James  P. 

Quigley,  and  Peter  B.  Armstrong 

Regulation  of  the  plasma  cytolytic  pathway  of 
Limulits  polyphemus  by  «:-macroglobulin  .  .  226 

Cornell,  Neal  W .,  Mark  E.  Hahn,  and  Holly  A. 

Martin 

Characterization  and  use  of  isolated  toadfish  he- 
patocytes  for  studies  of  heme  synthesis  and  uti- 
lization    227 

Land,  S.  C,  and  P.  J.  S.  Smith 

Suppression  of  Ca:+  flux  during  the  transition  to 
anoxia  in  turtle  hepatocytes  revealed  by  a  non- 
invasive  Ca:+-selective  vibrating  probe  228 

Smolowitz,  Roxanna  M. 

Immunohistochemical  localization  of  saxitoxin 
in  the  siphon  epithelium  of  the  butter  clam,  Sax- 
idomus  giganteus  229 

Behavior 

Consi,  T.  R.,  F.  Grasso,  D.  Mountain,  and  J.  Atema 

Explorations  of  turbulent  odor  plumes  with  an 
autonomous  underwater  robot  ...  ....      231 

Dittmer,  Kevin,  Frank  Grasso,  and  Jelle  Atema 

Effects  of  varying  plume  turbulence  on  temporal 
concentration  signals  available  to  orienting  lob- 
sters 232 


Physiological  Ecology  and  Behavior 

Hill,  Richard  \\ '.,  John  \V.  H.  Dacey,  David  K.  D. 
Hill,  Judith  E.  McDowell,  and  Dale  F.  Leavitt 

Accumulation  and  retention  of  dimethylsulfon- 
iopropionate  by  bivalve  molluscs:  High  and 
nonnormal  variation  233 

White,  Bradley  A.,  Richard  W.  Hill,  and  John 

W.  H.  Dacey 

Accumulation  of  dimethylsulfoniopropionate  in 
Geukensia  demissa  depends  on  trophic  interac- 
tions    235 

Bumann,  Dirk 

Localization  of  digestion  activities  in  the  sea 
anemone  Haliplanella  luciae  236 

A  vila,  Conxita,  and  Alan  M.  Kuzirian 

Natural  diets  for  Hermissenda  crassicornis  man- 
culture  237 

Brazik,  David  C.,  and  Robert  A.  Bullis 

The  effect  of  temperature  on  the  relationship  be- 
tween a  ciliated  protozoon,  Trichodina  cottida- 
nim,  and  the  longhorn  sculpin,  Myoxocephalus 
octodecemspinosus  239 

W  intermyer,  M.  L.,  D.  Leavitt,  and  J.  McDowell 
A  settlement  bioassay  assessing  the  response  of 
soft  shell  clam  larvae  to  sediments  from  various 
sites  in  Massachusetts  Bay    240 


Ecology:  Fish  and  Invertebrates 

Ahern,  Jenny,  Julie  Lyons,  James  McClelland,  and 
Ivan  Valiela 

Invertebrate  response  to  nutrient-induced 
changes  in  macrophyte  assemblages  in  Waquoit 
Bay  241 

Preisser,  Matthew  C.,  and  Linda  A.  Deegan 
Effect  of  changing  plant  morphology  on  inver- 
tebrate susceptibility  to  predation  in  eelgrass  beds     242 

Drake,  Chaka,  Peter  J.  Behr,  and  Ivan  Valiela 
Effect  of  algal  cover  on  size-selective  predation 
ofGammarus  mucronatus  by  the  striped  killifish, 
Fnmlitlus  majalis    243 

Martinez,   Nicole,  Jennifer  Hauxwell,  and   Ivan 

Valiela 

Effect  of  macroalgal  species  and  nitrogen-loading 
rates  on  colonization  of  macroalgae  by  herbiv- 
orous amphipods 244 

Sarda,  Rafael,  Kenneth  Foreman,  and  Ivan  Valiela 
Differences  in  benthic  invertebrate  assemblages 
in  two  estuaries  in  Waquoit  Bay  receiving  dis- 
parate nutrient  loads 245 

O'Neil,  Jonathan  S.,  and  Ilene  M.  Kaplan 

Impact  on  marine  species  of  New  England  rec- 
reational fishing  policies  246 


LIST  OF  MBL  REPORTS 


189 


Ecology:  Biogeochemistry  and  Nutrient  Cycling 

Chaplin,  Sue  Ann,  Catherine  Hunter  MacGregor, 
Ivan  Yaliela,  Kenneth  Foreman,  and  Lori  Soucy 

The  effect  of  residential  and  forested  watershed 
land  cover  on  nutrient  loading  to  Hamblin  and 
Jehu  Ponds,  Waquoit  Bay.  Massachusetts  .  247 

MacGregor,  Catherine  Hunter,  Sue  Ann  Chaplin, 

and  Ivan  Yaliela 

Land  cover  effects  on  inorganic  nutrients  in 
groundwater  entering  estuaries  of  Waquoit  Bay, 
Massachusetts  248 

Alderman,  Derrick,  Brian  R.  Balsis,  Ishi  D.  Buffam, 

Robert  H.  Garritt,  Charles  S.  Hopkinson  Jr.,  and 

Joseph  J.  Vallino 

Pelagic  metabolism  in  the  Parker  River/ Plum 
Island  Sound  estuarine  system  250 

Balsis,  Brian,  Derrick  VV.  M.  Alderman,  Ishi  D. 

Buffam,  Robert  H.  Garritt,  Charles  S.  Hopkinson 

Jr.,  and  Joseph  J.  Yallino 

Total  system  metabolism  of  the  Plum  Island 
Sound  estuarine  system  252 

Callaway,  David  W.,  Ivan  Yaliela,  Kenneth  Fore- 
man, and  Lori  Soucy 

Effects  of  nitrogen  loading  and  salt  marsh  habitat 
on  gross  primary  production  and  chlorophyll  a 
in  estuaries  of  Waquoit  Bay  254 

Lyons,  Julie,  Jenny  Ahern,  James  McClelland,  and 

Ivan  Yaliela 

Macrophyte  abundances  in  Waquoit  Bay  estu- 
aries subject  to  different  nutrient  loads  and  the 
potential  role  of  fringing  salt  marsh  in  ground- 
water  nitrogen  interception 255 

L'hlenhopp,  Amy  G.,  John  E.  Hobbie,  and  Joseph 

J.  Yallino 

Effects  of  land  use  on  the  degradability  of  dis- 
solved organic  matter  in  three  watersheds  of  the 
Plum  Island  Sound  Estuary  256 

Tomasky,  Gabrielle,  and  Ivan  Yaliela 

Nutrient  limitation  of  phytoplankton  growth  in 
Waquoit  Bay,  Massachusetts  257 

Sheridan,    Cecelia    C.,    Ivan    Y'aliela,    Kenneth 

Foreman,  and  Lori  A.  Soucy 

Effect  of  nutrient  enrichment  on  phytoplankton 
growth  in  Waquoit  Bay.  Massachusetts  .  ...  258 


Bohrer,  Travis,  Amos  Wright,  Jennifer  Hauxwell, 
and  Ivan  Yaliela 

Effect  of  epiphyte  biomass  on  growth  rate  of 
Zostera  marina  in  estuaries  subject  to  different 
nutrient  loading 260 

\Yright,  Amos,  Travis  Bohrer,  Jennifer  Hauxwell, 

and  Ivan  Yaliela 

Growth  of  epiphytes  on  Zostera  marina  in  es- 
tuaries subject  to  different  nutrient  loading  ...  261 

Wolfe,  Cheryl  Ann,  Carol  Rietsma,  and  Ivan  Valiela 
Foliar  release  of  ammonium  and  dissolved  or- 
ganic nitrogen  by  Spartina  alternijlora 262 


ABSTRACTS 

In  addition  to  the  work  described  here  in  Short  Reports, 
the  following  papers  were  also  presented.  The  abstracts  of 
these  papers  are  available  from  the  Marine  Biological 
Laboratory  Archives,  Woods  Hole,  MA  02543. 

Basil,  Jennifer,  Frank  Grasso,  and  Jelle  Atema 

High  resolution  odor  measurements  from  freely  moving 
lobsters  in  turbulent  odor  plumes 

Grasso,  Frank  W.,  Jennifer  A.  Basil,  and  Jelle  Atema 
Dual  sensor  information  in  turbulent  odor  plumes  on 
the  spatial  and  temporal  scale  of  the  lobster  lateral  an- 
tennules 

Heck,  Diane  E.,  Walter  Troll,  Seymour  Zigman,  and 

Jeffrey  D.  Laskin 

Role  of  oxidants  in  the  activation  of  sperm  from  Ar- 
bacia  pitnctii/ata 

Nixon,  Jennifer,  N.  Tay  Evans,  and  Jelle  Atema 

Effects  of  female  lobster  (Ilomarus  americanus)  urine 
on  male  aggression  at  the  shelter  entrance 

Ong,  Yea-Ling,  Jeffrey  S.  Seewald,  and  Lorraine   B. 

Eglinton 

An  experimental  investigation  of  vitrinite  reflectance 

Porcello,  Darrell  M.,  and  Robert  B.  Barlow 

Is  histamine  the  transmitter  for  lateral  inhibition  in  the 
Limiilus  eye? 

Walker,  James,  and  Nancy  Standart 

Regulation  of  protein  synthesis  in  the  early  develop- 
ment of  the  surf  clam  (Spisu/a  solidissima) 


Reference:  Biol  Bull  189:  190-195.  (October/November,  199?) 


Introduction  to  Featured  Articles: 
A  Resurgence  of  Experimental  Embryology 


Around  the  last  turn  of  the  century,  outstanding  biologists,  such  as  E.  B.  Wilson,  E.  G.  Conklin,  F.  R. 
Lillie,  T.  H.  Morgan.  andJ.  Loeb,  came  to  the  Marine  Biological  Laboratory  to  study  the  details  of  embryonic 
development  of  a  rich  diversity  of  marine  organisms.  These  workers  were  searching  for  homologies  in  the 
pattern  of  cleavage  and  development  and  in  the  origin  of  the  organ-forming  regions  of  these  embryos.  The 
information  that  was  compiled  then  has  had  a  powerful  and  persistent  influence  on  our  thinking  about  the 
evolution  of  development,  and  about  the  role  of  development  in  the  evolution  ofmetazoan  body  plans. 

Although  the  successes  of  the  early  experimental  embryologists  were  far-reaching,  decades  were  to  pass 
before  the  mechanisms  underlying  the  events  they  had  observed  were  discovered.  Indeed,  our  understanding 
of  cellular  and  molecular  processes  has  increased  rapidly  in  recent  years,  and  many  of  the  central  questions 
asked  a  century  ago — and  never  answered — have  now  become  tractable.  In  particular,  the  evolution  of  de- 
velopment— long  the  subject  of  speculation — is  now  becoming  one  of  the  most  exciting  areas  ^/"experimental 
biology. 

This  vear's  featured  reports  illustrate  how  new  disciplines  and  methods  can  be  applied  with  great  effectiveness 
to  lough,  old,  but  still  very  attractive  biological  problems.  The  authors  of  these  reports — Jonathan  Q.  Henry, 
Mark  Q  Martindale,  and  Barbara  C  Boyer — use  modern  techniques  to  analyze  cell  lineage  in  diverse  marine 
animals.  Their  experiments  are  aimed  at  the  mechanisms  that  generate  form  and  pattern  during  embryogenesis 
and  the  modification  of  those  mechanisms  in  organisms  that  have  evolved  from  a  common  ancestor.  These 
reports  provide,  in  brief,  new  information  about  "the  ways  in  which  embryos  are  put  together  and  how 
they  work. " 

— Michael  J.  Greenberg 
Editor-in-Chief 


Reference:  Biol.  Bull.  189:  190-192.  (October/November.  1995) 

Diagonal  Development:  Establishment  of  the  Anal  Axis  in  the  Ctenophore  Mnemiopsis  leidyi 

Mark  Q.  Martindale  (University  of  Chicago,  Department  of  Organismal  Biology  ana"  Anatomy, 
Chicago,  Illinois  60637)  and  Jonathan  Q.  Henry 


The  Ctenophora  is  a  phylum  of  biradially  symmetrical  marine 
carnivores,  the  comb  jellies.  These  animals  possess  an  outer  epi- 
dermis that  is  separated  from  an  inner  gastrodermis  by  a  largely 
acellular  mesoglea.  Although  ctenophores  have  been  thought  to 
be  diploblastic,  definitive  muscle  cells  reside  in  the  mesoglea 
and  in  association  with  both  epithelial  layers.  The  major  body 
axis — the  oral-aboral  axis  (Fig.  la) — is  the  intersection  of  two 
orthogonally  situated  planes  of  mirror  symmetry:  the  sagittal 
plane  passes  through  the  plane  of  the  flattened  stomadeum;  and 
the  tentacular  plane  passes  through  the  two  tentacle  pouches. 
These  planes  of  symmetry  define  four  quadrants,  each  containing 
two  of  the  eight  ctene  rows,  half  of  a  tentacular  apparatus,  and 
one  quarter  of  the  apical  sensory  organ.  The  biradial  symmetry 
of  these  animals  is  reflected  in  their  embryonic  development  in 
which  the  first  cleavage  plane  corresponds  to  the  sagittal  plane 


and  the  second  to  the  tentacular  plane  (1,  2).  Thus,  each  of  the 
first  four  blastomeres  generates  one  of  the  four  body  quadrants. 
At  the  aboral  end  of  all  ctenophores,  two  anal  canals  open  at 
the  anal  pores  in  two  diametrically  opposed  quadrants  (Fig.  Ib). 
The  location  of  these  pores  appears  to  be  conserved  in  all  cten- 
ophore  species  and  provides  another  axis  of  rotational  (but  not 
mirror)  symmetry,  which  we  refer  to  as  the  anal  axis.  The  oblique 
orientation  of  the  anal  axis  suggests  that  it  might  arise  asym- 
metrically during  the  development  of  the  four  cell  quadrants. 
We  have  investigated  the  ontogeny  of  the  anal  axis  by  performing 
cell  lineage  studies  in  the  Ctenophore  Mnemiopsis  leidyi.  One 
or  more  identified  blastomeres  up  through  the  32-cell  stage  were 
impaled  with  glass  microelectrodes  and  injected  with  the  flu- 
orescent lipophilic  dye.  Dil  (Molecular  Probes  Inc..  Eugene,  OR) 
dissolved  in  vegetable  oil  (3).  A  100  mg/ml  Dil  stock  was  made 


190 


A  RESURGENCE  OF  EXPERIMENTAL  EMBRYOLOGY 


191 


in  ethanol  and  diluted  20-fold  in  soybean  oil.  Injected  cells  con- 
tinued to  divide  normally  and  the  injections  had  no  observable 
effect  on  development.  Embryos  were  reared  at  19°C;  and  after 
24  to  48  h,  the  cydippid  larvae  were  examined  by  fluorescence 
microscopy  for  the  presence  of  labeled  anal  canals. 


Four-cell  stage  embryos  divide  to  give  rise  to  four  middle  (M) 
cells  and  four  end  (E)  cells.  Our  injections  of  blastomeres  at  the 
32-cell  stage  show  that  the  anal  canals  are  derived  from  the  en- 
dodermal  derivatives  of  the  second  order  macromeres  of  the  M 
lineage,  the  2M  blastomeres  (Fig.  Ic,  d).  Injections  of  2M  bias- 


a 


Pharynx 


CircumpharyngeaJ 
muscles 


Clene  row 


Longitudinal 

muscles 


Anal  Canal 


T— 


Circumpharynceal 

and 
Longitudinal  muscles 


-T 


Circumphatyngeal    / 

and  Ana!  Canal 

Longitudinal  muscles 

Figure  I.  Development  of  the  anal  axis  in  the  ctenophore  Mnemiopsis  leidyi.  (a)  Side  view  at  a  cydippid  larva-  The  two  tentacles  define  the 
tentacular  plane  of  mirror  symmetry  (i.e..  in  the  plane  of  the  paper):  an  apical  sensory  organ  is  \ilualed  at  the  aboral  pole,  (b)  Aboral  view  of  the 
apical  organ  showing  the  location  of  the  two  anal  pores-  (c)  Aboral  view,  showing  the  lour  2M  and  2E  macromeres.  ol  a  32-cell  stage  Mnemiopsis 
embryo.  The  24  aboral  micromeres  are  not  shown  lor  the  sake  of  clarity.  Two  diagonally  opposed  puns  ol  2M  macromeres.  one  situated  top  left  and 
bottom  right,  and  the  other  lop  right  and  bottom  left,  can  be  identified.  Each  pair  gives  rise  to  distinct  structures  in  the  cydippid  larva.  The  diagonal 
fates  ol  these  macromeres  are  indicated,  (d)  and  (e)  Color  fluorescence  micrographs  of  cydippid  larvae,  oriented  the  same  way  as  in  Figure  la  (aboral 
pole  up,',  in  which  two  different  diagonal  pairs  ol  2M  macromeres  were  miccled  with  lineage  tracer.  Note  that  there  is  staining  associated  with  the 
tentacle  bulbs  (tb)  and  endoderm  under  the  dene  rows  in  both  photos  Id)  The  unlabeled  apical  organ  sils  in  the  crotch  of  the  two  diagonally  labeled 
anal  canals  (act.  but  there  is  no  staining  associated  with  the  pharynx  at  the  oral  end  of  the  larva,  (e)  In  contrast,  there  is  no  staining  of  the  anal  canals 
in  the  vicmilr  ol  the  apical  organ  (ao)  in  this  larva,  but  the  circiimpharyngeal  muscles  (cpm)  are  labeled.  See  Fig.  Ic  lor  the  positions  of  the  two  pairs 
o]2M  cells  injected.  T  -  the  tentacular  plane:  S  =  the  sagittal  plane  Scale  bar  =  50  nm 


192 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


tomeres  show  that  only  two  of  the  four  2M  macromeres  generate 
anal  canals.  These  cells  are  located  diagonal  to  one  another  (Fig. 
Ic)  and  do  not  generate  any  other  cell  types  present  in  the  me- 
soglea.  The  remaining  two  2M  macromeres  produce  longitudinal 
and  circumpharyngeal  muscle  cells  (Fig.  Ic,  e).  Thus,  the  four 
quadrants  of  the  ctenophore  embryo  are  not  identical.  Two  cells 
at  the  four-cell  stage  are  capable  of  forming  circumpharyngeal 
muscle  cells  but  not  anal  canals,  whereas  the  other  two  cells  are 
capable  of  forming  anal  canals  but  not  circumpharyngeal  mus- 
cles. Cell  deletion  experiments  confirm  this  precocious  specifi- 
cation (Henry  and  Martindale,  unpub.  data),  indicating  that  de- 
velopmental potential  is  being  segregated  asymmetrically  as  early 
as  the  second  division  in  these  embryos.  No  bilaterian  embryos 
we  know  display  features  of  diagonal  development.  The  presence 
of  the  two  developmental  axes  specifying  anal  canals  and  cir- 
cumpharyngeal muscles,  in  addition  to  the  anatomically  defined 
sagittal  and  tentacular  axes,  warrants  a  reassessment  of  the  "bi- 
radial"  condition  in  these  animals,  and  the  alignment  of  the 
Ctenophora  with  their  probable  sister  taxon.  the  Cnidaria,  as  a 
group  of  radially  symmetrical  animals,  the  Coelenterata  (4.  5). 


This  paper  is  dedicated  to  the  memory  of  Dr.  Sebastian  Beroe 
on  the  20th  anniversary  of  his  tragic  death  from  cholera.  The 
authors  appreciate  the  generosity  of  the  Marine  Biological  Lab- 
oratory community  in  facilitating  these  studies,  and  S.  Q.  Irvine 
for  help  with  the  collection  of  animals.  J.Q.H.  (J.J.H.)  was  sup- 
ported as  an  MBL  Associates  Fellow  and  a  Lemann  Fellow. 
M.Q.M.  was  supported  by  the  NSF  and  the  Illinois  chapter  of 
the  American  Cancer  Society. 


Literature  Cited 


1.  Reverberi,  G.,  and  G.  Orlolani.  1963.     Ada  Embryo/.  Morphol. 
Exper.  6:  175-190. 

2.  Freeman,  G.  1976.     Develop.  Biol  49:  143-177. 

3.  Terasaki,  M.,  and  L.  Jaffe.  1991.     J.  Cell  Biol.  114:  929-940. 

4.  Christen,  R..  A.  Ratio,  A.  Baroin,  R.  Perasso.  K.  G.  Grell,  and  A. 
Adoutte.  1991.     EMBO  J.  10:  499-503. 

5.  Morris.  S.  C.  1993.     Nature  361:  219-225. 


Reference:  Biol.  Bull  189:  192-193.  (October/November,  1995) 


The  Experimental  Alteration  of  Cell  Lineages  in  the  Nemertean  Cerebratulus  lacteus:  Implications  for 
the  Precocious  Establishment  of  Embryonic  Axial  Properties 

Jonathan  Q.  Henry  (University  of  Illinois.  Department  of  Cell  and  Structural  Biology. 
Urbana.  Illinois  61801)  and  Mark  Q.  Martindale 


Spiralians,  including  the  molluscs,  annelids,  and  nemerteans. 
are  characterized  by  a  highly  stereotypic  pattern  of  embryonic 
cell  divisions,  which  is  based  on  the  establishment  of  four  discrete 
cell  quadrants.  In  molluscan  and  annelid  embryos,  these  are  the 
dorsal,  ventral,  left-  and  right-lateral  quadrants,  and  they  generate 
respective  portions  of  the  larval  and  adult  body  plan.  In  some 
species  the  first  and  second  cleavage  divisions  are  equal,  and  the 
blastomeres  of  the  four-celled  embryo  are  identical  in  size.  Ex- 
periments with  equally  cleaving  molluscan  embryos  reveal  that 
any  one  of  the  first  four  blastomeres  can  assume  the  fate  of  the 
dorsal  quadrant,  which  in  turn  directs  the  development  of  the 
other  quadrants.  Quadrant  identities,  and  the  dorsoventral  axis, 
are  established  relatively  late  through  cell-cell  interactions  fol- 
lowing the  fifth  cleavage  division.  In  other  spiralian  embryos, 
the  first  two  cleavages  are  unequal,  so  the  dorsoventral  axis  is 
established  precociously,  at  the  four-cell  stage,  through  the  dif- 
ferential segregation  of  developmental  determinants  within  the 
dorsal  cell  quadrant  ( 1 ). 

Nemertean  embryos  typically  display  equal  cleavage;  but  the 
identity  of  the  four  cell  quadrants  differs  from  those  described 
above.  The  first  and  second  cleavage  divisions  normally  corre- 
spond to  the  frontal  plane  and  the  plane  of  bilateral  symmetry, 
generating  left-  and  right-ventral  and  left-  and  right-dorsal  quad- 
rants. In  some  embryos  the  first  cleavage  plane  corresponds  to 
the  frontal  plane,  and  the  second  to  the  plane  of  bilateral  sym- 


metry. In  other  embryos  the  order  is  reversed.  In  either  case, 
the  end  result  is  the  same  (2). 

Are  quadrant  identities  and  the  dorsoventral  axis  in  nemer- 
teans established  late  by  virtue  of  cell-cell  interaction,  as  in  other 
equal-cleavers,  or  are  they  set  up  precociously  as  a  consequence 
of  the  early  cleavage  divisions,  perhaps  relative  to  some  under- 
lying axial  properties?  We  have  examined  this  question  by  shift- 
ing the  orientation  of  the  spindle,  thus  altering  the  plane  of  the 
first  cleavage.  Fertilized  eggs  of  Cerebratulus  lacteus  were  com- 
pressed to  about  1 70%  of  their  normal  diameter,  from  just  after 
first  polar  body  formation  but  before  the  formation  of  the  first 
cleavage  spindle,  until  first  cleavage  was  completed  (Fig.  la). 
The  cleavage  spindle  is  forced  to  align  parallel  to  the  plane  of 
compression.  As  cytokinesis  always  occurs  perpendicular  to  the 
cleavage  spindle  (3).  the  first  cleavage  plane  was  altered  in  some 
embryos  relative  to  the  animal-vegetal  axis.  Compression  was 
released  after  first  cleavage  and  one  cell  was  microinjected  with 
Fluoro-Rubv  ( 10.000  MW  rhodamine-conjugated  dextran.  Mo- 
lecular Probes,  Eugene,  OR).  Because  the  boundary  between 
labeled  and  unlabeled  ectodermal  domains  corresponded  to  the 
first  cleavage  plan,  the  fluorescent  label  made  it  possible  to  an- 
alyze the  relationship  between  the  first  cleavage  plane  and  the 
dorsoventral  axis  in  the  resulting  pilidium  larva.  Cell  divisions 
occurred  equally,  and  successive  cleavages  appeared  to  proceed 
normally.  In  the  majority  of  treated  embryos  (64  cases)  cleavage 


A  RESURGENCE  OF  EXPERIMENTAL  EMBRYOLOGY 


193 


a 


Injection 


Altered  Patterns 


Figure  1.  Experimental  alteration  of  cell  lineages  in  ilie  nemertean  Cerebratulus  lacteus.  (a)  Procedure  employed  to  shift  the  plane  of  the  first 
cleavage  division.  Fertilized  eggs  were  compressed  to  orient  the  spindle  parallel  to  the  plane  of  compression.  After  first  cleavage,  the  coverslip  was 
removed  and  one  blastomere  was  injected  with  a  fluorescent  lineage  tracer  at  the  two-cell  stage,  (b)  Diagrams  showing  left  lateral  and  apical  views  of 
the  pilidium  larva.  The  dorsal  side  is  to  the  right,  and  the  ventral  side  to  the  left,  (c)  Normal  and  altered  labeling  patterns  observed  in  this  study  (apical 
views  shown).  The  line  dividing  the  larvae  in  half  corresponds  to  the  boundary  between  labeled  and  unlabeled  eclodermal  domains,  and  thus  to  the 
hr\i  cleavage  plane,  \umbers  indicate  the  number  of  cases  displaying  each  of  the  indicated  labeling  patients.  L.  left:  R.  right:  D.  dorsal:  I',  ventral: 
L  \  'O,  left  ventral  oblique:  RDO.  right  dorsal  oblique:  R I  'O.  right  ventral  oblique:  LDO,  left  dorsal  oblique,  labeling  patterns.  A  wide  range  of  oblique 
labeling  patterns  was  actually  observed:  but  they  are  lumped  into  four  general  categories  here  for  the  sake  of  simplicity,  (d)  Combined  DIC  and 
fluorescence  micrographs  showing  one  ol  the  four  normal  labeling  patterns  (R.  right),  in  which  the  first  cleavage  plane  corresponds  to  the  plane  of 
bilateral  symmetry.  I  'iew  is  from  the  oral  pole,  opposite  the  apical  organ.  Line  demarcates  the  labeled  and  unlabeled  eclodermal  domains.  Arrows 
mark  the  plane  of  bilateral  symmetry.  Dorsal  side  is  to  the  right,  gt.  gut.  le)  A  n  altered  labeling  pattern  IRI'O.  right  ventral  oblique),  in  which  l/ic  tir\t 
cleavage  plane  passed  obliquely  relative  to  the  plane  of  bilateral  symmetry  mid  the  frontal  plane.  Scale  bar  equals  50  nm. 


and  subsequent  development  to  the  pilidium  larva  (Fig.  Ib)  were 
normal.  Forty-one  of  these  cases  displayed  one  of  the  four  normal 
patterns  (Fig.  le,  d).  indicating  that  there  was  no  dissociation 
between  the  early  cleavage  planes  and  the  dorsoventral  axis  in 
these  cases.  On  the  other  hand.  23  larvae  displayed  altered  re- 
lationships in  which  the  first  cleavage  plane  was  oriented 
obliquely  to  the  larval  dorsoventral  axis  (Fig.  le.  e). 

The  altered  relationships  observed  in  these  experiments  in- 
dicate that  the  plane  of  first  cleavage  does  not  play  a  causal  role 
in  establishing  the  dorsoventral  axis  in  the  nemertean  C.  lucieti^ 
Thus,  quadrant  identity  is  established  precociously  relative  to 
an  underlying  system  of  axial  properties  present  in  the  fertilized 
egg  before  first  cleavage.  Normally,  to  generate  the  four  typical 
nemertean  cell  quadrants,  some  mechanism  must  link  the  early 
cleavage  divisions  to  this  scaffold  of  axial  information.  It  is  re- 
markable that  alternate  cell  lineages  were  produced  without  dis- 
turbing larval  development,  and  in  some  cases  quadrant  rela- 
tionships similar  to  those  found  in  annelids  and  molluscs  were 
generated.  These  results  support  the  argument  proposed  by 


Martindale  and  Henry  (4)  that  cleavage  geometry  may  have  been 
uncoupled  from  an  underlying  scaffold  of  axial  information, 
leading  to  the  generation  of  different  cell  lineages  during  the 
course  of  spiralian  evolution. 

The  authors  thank  the  generous  community  of  the  Marine 
Biological  Laboratory.  J.Q.H.  was  supported  as  an  MBL  Asso- 
ciates Fellow  and  a  Lemann  Fellow.  M.Q.M.  was  supported  by 
NSF  and  the  Illinois  chapter  of  the  A.C.S. 

Literature  Cited 

1.  van  den  Biggelaar,  J.  A.  M.,  and  P.  Guerrier.  1983.     Pp  179-213 
in  The  Mollusca.  N.  H.  Verdonk.  J.  A.  M.  van  den  Biggelaar,  and 
A.  S.  Tompa.  eds.  Academic  Press.  New  York. 

2.  Henry,  J.  Q.  and  M.  Q.  Martindale.  1994.     De\:  Genetics  15:  64- 
78. 

3    Rappaport,  R.  1986.     //;/.  Rev.  Cytol.  105:  245-281. 
4.  Martindale.  M.  Q..  and  J.  Q.  Henry.  1995.     Development  121:31 75- 
3185. 


194  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Biol  Bull.  189:  194-195.  (October/November.  1995) 

Axial  Specification  in  a  Basal  Member  of  the  Spiralian  Clade:  Lineage  Relationships  of  the  First  Four 
Cells  to  the  Larval  Body  Plan  in  the  Polyclad  Turbellarian  Hoploplana  inquilina 

Jonathan  Q.  Henry,  Mark  Q.  Martindale,  and  Barbara  C.  Bayer  (Department  of  Biology, 
Union  College,  Schenectady,  New  York  12308) 


The  Spiralia  comprises  several  invertebrate  phyla  including 
the  molluscs,  annelids,  nemerteans.  sipunculids,  echiurans,  and 
some  turbellarian  platyhelminths.  These  animals  share  many 
common  features  of  embryonic  development  including  a  ste- 
reotypic  pattern  of  cell  divisions  referred  to  as  spiral  cleavage. 


In  most  molluscs  and  annelids,  first  cleavage  occurs  oblique  to 
the  future  plane  of  bilateral  symmetry  and  generates  two  blas- 
tomeres,  designated  AB  and  CD.  At  second  cleavage,  these  cells 
divide  to  produce  the  four  primary  embryonic  cells,  or  quadrants, 
called  A.  B.  C,  and  D,  which  give  rise  respectively  to  the  left. 


Dorsal  View 


Anal  Tuft 


D 


Left  Side  View 


Stomodeum 


Right  Side  View 


Posterior  View 


Ventral  View 

Key: 
|    A  Quadrant 

B  Quadrant 
|  C  Quadrant 
|  D  Quadrant 


Figure  1.  Ketodermal  territories  ol  the  A.  B.  C.  and  D  cell  quadrants  in  the  Mailer's  larva  ol  Hoploplana  inquilina.  The  central  illustration  portrays 
the  5-diiy  Mitllc:  \  larva  in  an  oblique,  right-frontal  3-D  perspective.  Line*  indicate  the  anterior-posterior  (A-P).  dorsal-ventral  (D-  \ ')  and  right-left  (R- 
L)  axes.  The  apical  lufl.  anal  tuft,  and  sloinodeinn  are  also  labeled.  Flanking  colored  illustrations  portray  the  v/v  different  surfaces  of  the  lart'a  as 
indicated.  Each  ol  the  lour  eaodenmil  territories  derived  from  the  four  cell  quadrants  is  colored  as  shown  in  the  key.  The  apical  organ  corresponds 
In  the  embryonic  animal  pule,  and  the  stomodeum  corresponds  to  the  embryonic  vegetal  pole.  The  A  and  C  quadrants  contribute  to  the  formation  of 
the  apical  lull,  whereas  all  lour  quadrants  contribute  to  the  formation  of  the  stomodeum.  During  development,  the  various  ectodermal  territories 
become  somewhat  distorted  through  the  formation  of  the  oral  hood  and  the  various  larfal  lobes.  The  oblique  orientation  of  the  first  cleavage  plane 
relative  to  the  larval  dorm  neutral  a\i\  is  shown  for  the  apical  view.  Thus,  the  locations  of  the  four  cell  quadrants  (A.  B.  C.  and  D)  are  similar  to  those 
found  in  poh'chaelc  annelids  and  molluscs  Anterior  and  posterior  views  are  oriented  with  the  dorsal  surface  upward  and  the  ventral  surface  downward 
Dorsal,  ventral,  right,  and  left  views  are  oriented  with  the  anterior  siir/uce  upward  and  the  posterior  surface  downward 


A  RESURGENCE  OF  EXPERIMENTAL  EMBRYOLOGY 


195 


ventral,  right,  and  dorsal  regions  of  the  larval  and  adult  body 
plan.  Cell  lineage  studies  have  -evealed  such  homology  in  the 
fates  of  the  four  embryonic  quadrants  in  representatives  of  several 
of  the  spiralian  phyla  (1-3):  but  recent  evidence  in  both  mol- 
luscan  and  nemertean  embryos  indicates  significant  differences 
in  the  fates  of  the  embryonic  quadrants  in  certain  members  of 
the  Spiralia  (4.  5). 

Polyclad  turbellarians  are  thought  to  represent  basal  members 
of  the  spiralian  clade.  An  early  study  indicated  that  their  devel- 
opment is  based  on  a  quadrant  system  similar  to  that  of  annelids 
and  molluscs  (6).  but  more  recent  experimental  analyses  have 
suggested  that  assignment  of  cell  fate  may  not  be  strictly  cor- 
related with  specific  quadrants  (7,  8,  9).  To  resolve  this  contro- 
versy, we  have  examined  the  fates  of  the  first  four  blastomeres — 
and  hence  the  relationship  of  the  first  two  cleavage  planes — to 
the  larval  body  of  the  polyclad  Uoploplana  inqnilina.  The  first 
cell  division  generates  two  equally  sized  blastomeres,  but  the 
second  division  in  both  cells  is  unequal,  producing  a  four-cell 
stage  in  which  the  two  larger  cells  meet  at  the  vegetal  pole  (vegetal 
cross-furrow).  We  labeled  the  outside  surface  of  individual  blas- 
tomeres at  the  two-  and  four-cell  stages  with  a  tiny  drop  of  soy- 
bean oil  containing  5.0  mg/ml  Dil  (Molecular  Probes,  Inc.,  Eu- 
gene, OR)  delivered  with  a  pressurized  glass  micropipet  (Chris 
Q.  Doe,  pers.  comm.).  The  embryos  were  raised  at  22°C,  and 
the  labeling  patterns  were  examined  5-6  days  later. 

Figure  1  is  a  summary  of  ectodermal  labeling  patterns  from 
38  cases  labeled  at  the  two-cell  stage  and  60  cases  at  the  four- 
cell  stage.  That  the  first  two  cleavage  planes  are  oblique  to  the 
plane  of  bilateral  symmetry  is  clearly  seen.  The  orientation  of 
the  first  cleavage  plane  is  indicated  on  the  anterior  view  shown 
in  Figure  I .  Although  a  relatively  complex  pattern  of  morpho- 
genesis takes  place  during  the  development  of  the  Miiller's  larva, 
driven  in  part  by  the  dorsolateral  expansion  of  the  dorsolateral 
ectodermal  domains  and  the  formation  of  the  oral  hood  and 
various  larval  lobes,  we  have  clearly  identified  A,  B,  C,  and  D 
quadrants  similar  in  relationship  to  those  of  annelids  and  mol- 
luscs. 

One  of  the  vegetal  cross-furrow  blastomeres  was  labeled  in 
22  four-cell  stage  embryos:  of  these  only  one  did  not  generate  a 


ventral  (B)  or  dorsal  (D)  quadrant.  Similarly,  of  the  38  embryos 
in  which  a  non-cross-furrow  cell  was  marked,  all  but  one  pro- 
duced a  labeled  left  (A)  or  right  (C)  quadrant.  These  results  in- 
dicate that  the  vegetal  cross-furrow  cells  are  reliable  indicators 
of  the  dorsoventral  axis,  and  the  non-cross-furrow  blastomeres 
of  the  left-right  axis.  This  is  similar  to  the  situation  found  in 
annelids  and  molluscs  (3). 

These  results  confirm  and  extend  the  classical  work  of  Surface 
on  Hoploplana  (6),  though  he  did  not  map  the  fates  of  the  four 
quadrants  as  far  as  the  Miiller's  larva  stage.  The  identification 
of  the  A,  B,  C.  and  D  cell  quadrants  with  a  consistent  relationship 
to  the  axes  of  bilateral  symmetry  in  this  basal  member  of  the 
spiralian  clade  suggests  that  this  association  may  represent  the 
ancestral  condition  for  this  large  group  of  protostome  inverte- 
brates. 

J.Q.H.  (J.J.H.)  was  supported  as  an  MBL  Associates'  Fellow 
and  a  Lemann  Fellow.  M.Q.M.  was  supported  by  NSF  and  the 
Illinois  Chapter  of  the  American  Cancer  Society.  B.C.B.  was 
supported  by  the  Union  College  Faculty  Research  Fund. 


Literature  Cited 

1 .  Wilson,  E.  B.  1898.     Pp.  2 1  -42  in  Biological  Lectures  of  the  Marine 
Biological  Laboratory,  Woods  Hole.  MA.  Ginn  and  Co..  Boston. 

2.  Wilmer,  P.  1990.    Pp.  199-222  in  Invertebrate  Relationships,  Pat- 
terns in  Animal  Evolution.  Cambridge  University  Press,  Cambridge. 

3.  Verdonk,  N.  H.,  and  J.  A.  M.  van  den  Biggelaar.  1983.     Pp.  91- 
122  in  The  Mollusca  N.  H.  Verdonk,  J.  A.  M.  van  den  Biggelaar, 
and  A.  S.  Tompa,  eds.  Academic  Press,  New  York. 

4.  Damen,  P.  1994.     Cell  lineage,  and  specification  of  developmental 
fate  and  dorsoventral  organisation  in  the  mollusc  Patella  vulgata. 
Thesis  Llniversiteit   Utrecht.   CIP-DATA    KONINKLIJKE    BIB- 
LIOTHEEK,  DEN  HAAG. 

5.  Henry,  J.  Q.,  and  M.  Q.  Martindale.  1994.  Dev.  Genetics  15:  64- 
78. 

6.  Surface,  F.  M.  1907.     Pnic.  Acad.  Nal.  Sci.  Phil.  59:  514-559. 

7.  Boyer,  B.  C.  1986.     Int.  J  Invert  Repro.  Dev.  9:  243-251. 

8.  Boyer,  B.C.  1987.     Roux's  Arch  Dev  Biol  196:  158-164. 

9.  Boyer,  B.  C.  1989.     Biol.  Bull.  Ill:  338-343. 


196  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Bioi  Hull.  189:  1%-I97.  (October/November.  1995) 

Sex  on  the  Brain  but  the  Heart  Is  Not  Really  In  It:  Developmental  Heart  Defects  Associated  with 
Aquatic  Pollution  and  Microinjection  of  Hexachlorobenzene  into  the  Japanese  Medaka  Embryo 

Merle  Mizell  (Center  for  Bioenvironmental  Research,  'Department  of  Cell  and  Molecular  Biology.  Tulane 

University,  New  Orleans,  Louisiana.  70118),  Eric  Roinig.  William  Hartley. 

and  Anmthavarani  Thiyagarajah 


Hexachlorobenzene  (HCB)  was  found  in  high  concentration 
[280  mg/Kg  (ppm)]  in  the  sediment  of  the  Mississippi  River 
Basin  (MRB)  near  Baton  Rouge,  Louisiana,  in  an  area  known 
as  Devil's  Swamp.  Such  pollution  has  created  the  need  for  an 
effective  biomarker  or  sentinel  species  for  ecological  and  health 
risk  assessment  ( 1 ).  Embryos  of  the  Japanese  medaka  (Ory:ias 
latipes)  are  transparent,  and  this  feature  together  with  a  new 
method  of  microinjection  provides  an  efficient  and  sensitive 
means  of  delivering  chemicals  and  observing  the  systemic  and 
genotoxic  effects  during  embryonic  differentiation.  Recently 
fertilized  medaka  embryos  were  collected  from  our  breeding 
colony  at  the  Tulane  University  Center  for  Bioenvironmental 
Research.  The  embryos  were  cleaned,  staged,  and  then  placed 
in  a  plastic  injection  slide  specially  machined  at  the  Marine  Bio- 
logical Laboratory  for  this  project.  HCB  was  dissolved  in  vege- 
table oil  and  made  up  in  several  concentrations.  The  range  of 
injected  concentrations  was  100  mg/1  (ppm)  to  0.1  /ig/1  (ppm) 
of  HCB.  Vegetable  oil  lacking  HCB  was  injected  as  a  negative 
control.  The  volume  of  the  injected  droplet  was  always  40  nl. 
Embryonic  growth  and  differentiation  was  monitored  daily  in 
these  transparent  embryos,  and  deviations  from  normal  devel- 
opment were  videorecorded.  The  fish  were  grown  out  for  either 
6  months  or  one  year,  and  then  sacrificed  for  histopathological 
examination. 

Although  our  histopathology  study  is  still  in  progress,  we  have 
encountered  a  curious  finding.  One  of  the  embryos  injected  with 
0. 1  jug/1  HCB  possessed  ectopic  gonads  in  its  cranial  cavity.  This 
female's  ectopic  gonad  contained  well-developed  testicular  tissue: 
although  not  arranged  in  tubules,  it  consisted  of  primordial  germ 
cells,  including  well-differentiated  spermatocytes  and  spermatids. 
None  of  the  other  animals  examined  in  this  experimental  series, 
exhibited  such  structures. 

However  careful  routine  monitoring  of  our  breeding  colony 
revealed  that  2  out  of  86  fish  sacrificed  due  to  disease  or  injury 
contained  similar  ectopic  hermaphroditic  gonads  in  the  cranial 
cavity  (2).  Therefore,  the  ectopic  cranial  gonads  observed  in  the 
current  study  cannot  be  attributed  to  the  HCB  microinjection. 
Our  breeding  colony  was  maintained  in  dechlorinated  New  Or- 
leans municipal  water  that  was  further  treated  by  five  separate 
filtering  units  and  met  the  standards  of  the  American  Public 
Health  Association  for  culture  and  toxicity  testing  of  aquatic 
organisms.  All  of  our  animals  are  now  housed,  bred,  and  main- 
tained in  recirculating  spring  water. 

Ectopia  is  an  anomaly  due  to  errors  of  morphogenesis  that 
position  an  organ  or  tissue  outside  its  normal  anatomical  locus. 
Ectopic  tissues  have  been  reported  in  all  classes  of  vertebrates. 
Indeed,  ectopic  thyroid  tissue  often  undergoes  proliferation  in 
response  to  organochlorines  (3).  Nevertheless,  prior  to  these  three 
cases,  discussed  above,  ectopic  gonads  in  the  cranial  cavity  had 
never  been  reported  in  vertebrates.  Moreover,  all  three  cases  of 


ectopic  consisted  of  hermaphrodidic  gonads.  The  events  (or  lack 
of  events)  leading  to  these  anomalous  animals  undoubtedly  are 
of  fundamental  importance  and  environmental  pollution  may 
be  involved  in  its  occurrence.  We  are  currently  attempting  to 
uncover  the  cause  of  the  ectopic  gonads  in  our  microinjection 
studies.  We  are  also  monitoring  the  Devil's  Swamp  resident  fish 
for  similar  gonadal  anomalies. 

HCB  was  microinjected  into  the  perivitelline  space  of  embryos 
either  24  h  or  1 28  h  post  fertilization.  Embryos  exposed  to  HCB 
at  24  h  after  fertilization  exhibited  profound  circulatory  defects. 
Fifty  embryos  were  exposed  to  each  concentration  of  HCB.  At 
0. 1  ppm  HCB  there  was  a  2%  incidence  of  circulatory'  defects. 
And  at  1.0  ppm  there  was  a  6%  incidence;  at  10  ppm  the  inci- 
dence increased  to  8%.  At  100  ppm  the  incidence  of  circulatory 
defects  plateaued  at  12.5%.  Control  embryos  that  were  injected 
with  oil  or  were  uninjected  lacked  circulatory  defects.  The  af- 
fected embryos  invariably  had  reduced,  or  even  were  devoid  of, 
extra-  or  intraembryonic  circulation.  The  heart,  in  a  typical  case, 
was  little  more  than  a  thin  tube  stretching  between  the  yolk  sac 
and  the  embryo,  so  this  defect  was  easily  visualized  upon  ex- 
amination (4).  Embryos  with  severe  circulatory  defects  also  ex- 
hibited pericardia!  edema  which  displaced  the  yolk  sac  to  one 
side  (Fig.  1).  Heart  rate  was  markedly  decreased.  Histopatho- 
logical examination  revealed  that  the  cardiac  musculature  was 
often  reduced  to  sparce.  abnormal  trabeculae  and  other  gross 
heart  malformations  which  were  frequently  accompanied  by  di- 
lated kidney  tubules  and  cystic  kidneys. 

The  HCB  concentration  in  the  Devil's  Swamp  sediment  was 
three  times  greater  than  the  highest  dose  of  HCB  injected  into 


Figure  I.  Typnal  lie.\aelilor<>hen:eue  /HCB)  cardiovascular  malfor- 
mation in  a  meilaka  einhryo  which  mis  microinjected  24  h  after  tcrlil- 
1:11111111  The  single  ilose  »l  HCB  ailnunistereil  mis  0  I  n.1/1  Ippm)  The 
emhrvo  mis  nnahle  In  hatch  anil  thus  was  manually  removed  from  Us 
chonnii  and  photographed  21  ft  h  alter  mieclion  The  heart  eonsisis  nl  a 
I  Inn  inhe  (arrow)  surrounded  by  an  enlarged,  edematous  pericardia!  cavity. 


DEVELOPMENT 


197 


the  embryos.  HCB  has  been  shown  to  cause  circulatory  defects 
common  to  several  other  aromatic  compounds:  toluene,  car- 
baryl.  parathion.  tolbutamide.  dinitrophenol,  and  2,4,5-trichlo- 
rophenoxN acetic  acid  (4,  5,  6.  7).  The  abnormalities  observed 
in  the  developing  medaka  heart  are  similar  in  several  respects 
to  those  seen  in  zebrafish  embryos  treated  with  retinoic  acid  (8). 
The  anterior-posterior  axis  of  the  heart  is  truncated,  especially 
affecting  the  anterior  region,  as  in  zebrafish  (and  \cnopus)  heart 
development  (8).  These  effects  seem  both  dose-dependent  as  well 
as  stage-dependent.  Because  the  circulatory  defects  uncovered 
in  our  studies  are  lethal,  resident  aquatic  species  of  Devil's 
Swamp  that  are  exposed  to  HCB  during  embryonic  development 
may  experience  appreciable  early  mortality.  These  aquatic  pop- 
ulations are  currently  being  followed. 


Supported  by  grants  from  the  Department  of  Energy  and  the 
Department  of  Defense. 


Literature  Cited 


1.  Abel,  P.  D.  1989.     fli-wVirv  on  Environmental  Health  8:  1 19-155. 

2.  Hartk>.  \\ ..  A.  Thiyagarajah,  and  M.  Mizell.  1995.     J.  Aquatic 
Animal  Health  1:  172-177. 

3.  Hoover,  K.  L.  198-4.     \atl  Caitecr  lust   Monograph  65:  275-289. 

4.  \Veis,  P.,  and  J.  \Veis.  1974.     Tmili>hKy  10:  263-268. 

5.  Schreiweis,  D.  O.,  and  G.  J.  Murray.  1976.     Teratology  14:  287- 
268. 

6.  Smithbcrg,  M.  1962.     Am.  J.  Anat.  Ill:  205-213. 

7.  Wilde,  C.  E.  Jr.,  and  R.  B.  Crawford.  1966.     Exp.  Cell  Res.  44: 
471-488. 

8.  Stanier,  D.  V.,  and  M.  C.  Fishman.  1992.     Dev.  Btoi  153:  91-101. 


Reference:  Bin/.  Bull.  189:  197-198.  (October/November.  1995) 

Pattern  of  Potassium  Ion  and  Proton  Currents  in  the  Ovariole  of  the  Cockroach,  Periplaneta  amencana, 

Indicates  Future  Embryonic  Polarity 

Joseph  G.  Kunkel  and  Ellen  Fasiewski  (National  Mbraling  Probe  Facility.  Marine  Biological  Laboratory) 


Ionic  currents  are  associated  with  developing  patterns  in  var- 
ious organisms  ( 1 )  and  are  ascribed  to  the  movement  of  various 
ions.  The  function  of  these  currents  in  each  system  is  still  unclear. 
We  previously  reported  a  pattern  of  ionic  current  about  the  vi- 
tellogenic  follicles  of  cockroaches  and  termites  (?).  This  group 
of  insects  is  particularly  interesting  because  of  the  simplicity  of 
their  ovarian  follicles:  a  large  oocyte  surrounded  by  a  single  cell 
layered  follicle  epithelium.  The  observed  pattern  of  currents, 
which  we  investigated  with  the  older  wire  probe  technology, 
indicates  the  location  of  the  future  embryonic  germ  band.  We 
now  report  the  identity  of  the  ions  involved,  which  we  investi- 
gated using  the  recently  developed  non-invasive  ion  selective 
electrode  technology  at  the  National  Vibrating  Probe  Facility, 
MBL.  Woods  Hole  (3).  Microelectrodes  with  tips  of  2  ^m  were 
filled  with  15  fjm  columns  of  liquid  ion  exchanger  (L1X)  cock- 
tails. Potassium-sensitive  LIX  (Fluka  Potassium  lonophore  I- 
cocktail  A)  and  proton-sensitive  LIX  (Fluka  Hydrogen  lono- 
phore I-cocktail  A)  were  used.  The  microelectrodes,  oscillated 
10  Mm  in  the  X-.  Y-,  and  Z-directions  to  measure  ^V  gradients 
in  those  dimensions,  were  propelled  by  stepper  motors  controlled 
by  computer  software.  3DVIS,  designed  to  measure  3-D  patterns. 
Total  flux  was  calculated  by  vector  addition  of  the  measured 
X-.  Y-  and  Z-^V  difference  components.  The  efficiency  of  the 
K+  electrode  to  measure  K+  flux  was  80%;  that  of  the  proton 
electrode  is  also  assumed  to  be  high,  but  the  effects  of  buffering 
in  physiological  salines  are  unclear.  We  therefore  report  our  pro- 
ton flux  in  terms  of  ^V  drop  over  a  measured  distance  which 
can  be  interpreted  as  pH  difference.  Ovaries  of  the  cockroach 
were  dissected  into  cockroach  Ringer,  and  the  individual  ova- 
rioles  were  separated  from  connective  tissue.  Single  ovarioles 
were  transferred  to  a  measurement  chamber  bathed  in  an  ap- 
propriate saline.  For  measuring  potassium,  the  Peripliint'in 
Ringer  of  Smith  was  used  (157  m3/Na+,  3  mA/K+,  2  m,UCa+  +  , 


2  rruUMg++,  165  mA/Cl  and  8.6  m,A/ Hepes,  pH  7.2).  For  pro- 
tons, the  same  Ringer,  but  with  a  weaker  buffer  (Hepes, 
0.96  mA/)  was  used  to  prevent  the  dampening  of  proton  fluxes. 
We  measured  substantial  outward  K+  and  proton  gradients 
at  the  anterior  end  of  each  vitellogenic  follicle  within  an  ovariole. 
Figure  1A.  The  pattern  of  both  the  proton  and  K+  gradients 
were  largely  identical,  outward  about  an  anterior  polar  cap,  with 
the  exception  that  a  generalized  lower  level  outward  proton  cur- 
rent was  observed  about  the  entire  follicle.  This  low  level  outward 
current  may  reflect  a  generalized  respiratory  secretion  of  CO? 
from  the  tissue  in  general.  No  ion  gradients  were  detected  around 
previtellogenic  follicles  or  around  follicles  close  to,  or  after,  cho- 
rion  formation  (Fig.  IB).  The  major  gradients  of  ions  exit  the 
follicle  through  a  tight  epithelium  of  follicle  cells  that  form  a 
cap  over  the  anterior  pole  of  the  follicle.  The  follicle  anterior 
pole  can  be  thought  of  as  the  vegetal  pole  of  the  Periplaneta 
oocyte;  this  is  because  the  embryonic  germ  band  will  develop 
at  the  posterior  pole.  The  location  of  the  germ  band  can  be 
considered  the  animal  pole.  Aside  from  the  vegetal  polar  cap  of 
'tight'  epithelium,  the  remainder  of  the  follicle  cell  epithelium 
around  the  vitellogenic  follicle  is  'patent'  (4).  allowing  the  bathing 
medium  to  reach  the  oocyte  surface.  The  extent  to  which  the 
observed  currents  are  electrically  coupled  between  the  follicle 
cells  and  oocyte  is  unknown;  but  TEM  sections  show  that  all 
follicle  cells  are  morphologically  coupled  to  the  oocyte  via  gap 
junctions.  We  suggest  that  the  tight  cap  of  follicle  cells  at  the 
anterior  pole  act  as  a  polarized  epithelium,  responsible  for  the 
pumping  of  ions,  which  we  see  (Fig.  1 ).  In  many  insects  the  V- 
type  ATPase  is  responsible  for  pumping  of  protons.  This  pump 
is  sensitive  to  the  inhibitor  Bafilomycin  Al  from  Streptomyces 
tfmi'H.v.  In  several  oocytes,  which  we  treated  with  1  nAf  Bafilo- 
mycin, the  peak  proton  flux  seen  at  the  anterior  cap  of  the  follicle 
was  inhibited  by  up  to  60%  over  a  period  of  1 5  min.  This  finding 


198 

200 

150 

100. 

UV<  50. 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


A.  Pattern 


of  proton  flux  from  penultimate  follicle 


scan-; 


1000 
500 


Y-axis 
(urn) 


-500 
-1000 
-1500 


B.  XY-posltions  of  scans  in  Z-axis 


posterior 


anterior 


-2500     -2000    -1500    -1000     -500 
X-axis  (urn) 


500      1000 


Figure  1.  Pattern  of  proton  movement  about  a  vilellogenic  ovarian 
follicle  of  Periplaneta  americana.  The  animal  (A)  and  vegetal  (\'l  pole 
positions  of  the  penultimate  follicle  are  indicated  in  both  IA  and  IB  A. 
Total-iiV  drop  (iiVJ  over  the  oscillation  distance  (10  nm)  in  three-di- 
mensions. The  scan-axis  represents  the  linear  additive  distance  from  the 
(0.0)  XY-loca/ion  at  the  anterior  end  of  the  penultimate  follicle  to  other 
locations  along  the  scan  route  as  depicted  in  Figure  I B  (58  m  I  'represents 
I  pH  unit  of  proton  concentration  difference  with  a  measured  background 
pH  of  7.1).  B  Outline  of  a  section  ofovariole  including  the  anterior  tip 


suggests  that  a  portion  of  the  observed  gradients  are  generated 
by  the  V-type  proton  pump  and  the  established  proton  gradient 
might  be  further  utilized  by  a  K+/H+  exchange  pump  to  produce 
the  outward  K+  flux  at  the  anterior  pole.  If  a  gradient  of  ions  is 
being  created  through  or  around  the  enclosed  oocyte  by  electrical 
coupling  with  the  follicle  cell  layer,  a  force  to  influence  the  po- 
larity of  the  future  embryo  may  be  at  play. 

The  hospitality  and  advice  of  Peter  J.  S.  Smith  at  the  National 
Vibrating  Probe  Facility  was  a  deciding  factor  in  our  progress 
on  this  project.  We  also  thank  Jeffery  R.  Demerest  for  sharing 
his  experience  with  proton  LIXs.  This  research  was  supported 
by  grants  to  the  National  Vibrating  Probe  Facility  and  from  the 
Biotechnology  Program  of  the  University  of  Massachusetts  at 
Amherst. 

Literature  Cited 

1.  Jaffe,  L.  F.  1991.     Phil.  Trans.  R  Soc.  London  295:  553-556. 

2.  Kunkel,  J.  G.  1991.     In  I'm,  5:  443-456. 

3.  Smith,  P.  J.  S.,  R.  H.  Sanger,  and  L.  F.  Jaffe.  1994.     Mali.  Cell 
Bin/  40:  115-134. 

4.  Zhang,  V.  and  J.  G.  Kunkel.  1992.     Tissue  &  Cell  24:  905-9 1 7. 


ot  the  terminal  follicle  (labeled  1.  chorionated  and  scheduled  to  ovulale 
in  I  day),  the  entire  penultimate  follicle  (labeled  2,  vilellogenic  and  to  be 
ovulated  in  4  days),  and  a  portion  of  ihe  pen-penultimate  follicle  (labeled 
3.  as  yet  non-vilellogenic  and  to  be  ovulated  in  7  days).  The  14  XY- 
posttions  of  7  3-D  scans  in  the  Z-axis  are  indicated.  These  positions  were 
transformed  into  scan  axis  locations  corresponding  to  the  14X7  mesh 
of  points  plotted  versus  measured  \i  I  '-difference  in  Figure  I  A.  The  anterior 
and  posterior  polarity  of  the  ovariole  is  labeled. 


Reference:  Biol.  Bull.  189:  198-199.  (October/November.  1995) 

Chemotaxis,  Aggregation  Behavior,  and  Foot  Formation  in  Dictyostelium  discoideum  Amoeba 
Controlled  by  Microbeam  Uncaging  of  Cyclic-AMP 

Yoshio  Fukui  and  Shinya  Inoue  (Marine  Biological  Laboratory) 


At  a  certain  stage  of  development,  amoebae  of  the  cellular 
slime  mold  Dictyostelium  discoideum  signal  to  each  other  by 
secreting  c-AMP  [cyclic-3',5'  adenosine  monophosphate  (1,2, 
3)] — and  then  aggregate.  We  analyzed  the  responses  of  aggre- 
gation-competent amoebae  to  brief  applied  pulses  of  c-AMP 
under  high-resolution  video  DIG  (differential  interference  or 
Nomarski  contrast)  microscopy. 

Miniature  sources  of  c-AMP  pulses  were  generated  by  illu- 
minating caged  c-AMP  (4)  with  a  366-nm-wavelength  UV  (ul- 
traviolet) microbeam  delivered  as  3-ms  flashes  repeated  every 
0.65  s;  we  had  added  the  caged  c-AMP  to  the  buffer  and  agar 
layer  overlying  the-  amoebae  (5).  A  Zeiss  Ultrafluar  (UV-  and 
visible  light-transmitting.  100X/1.25  NA,  glycerol  immersion 
objective)  lens  equipped  with  a  DIC  prism  replaced  a  conven- 
tional condenser  to  focus  a  highly  reduced  image  of  a  first-surface 
micromirror,  placed  in  front  of  the  field  diaphragm,  superim- 


posed with  the  DIC  image  of  the  specimen.  The  UV-reflecting 
micromirror  was  located  at  the  focus  of  the  UV  source,  an  aux- 
iliary 1 00- Watt  Hg-arc  lamp  with  quartz  collector,  366-nm  band- 
pass filter,  and  electrically  activated  shutter.  The  2.2  X  3.0  /im2/ 
UV  image  can  be  seen  as  a  bright  rectangle  at  the  tip  of  the  dark 
shadow  of  the  mirror  support  in  Figure  1A  and  B,  slightly  off 
center  from  the  visible  (546  nm)  light  image  of  the  specimen  in 
DIC.  Moving  the  micromirror  or  specimen  carrier  placed  the 
source  of  c-AMP  in  different  locations  relative  to  one  or  more 
amoebae. 

Migrating  aggregation-stage  amoebae  responded  to  the  c-AMP 
pulses  by  turning  towards  the  source  (Fig.  IA)  and  migrating  it. 
The  first  amoeba  to  reach  the  source  engulfed  it,  and  the  others 
spiraled  and  aggregated  around  this  first  amoeba,  which  remained 
at  the  source  (Fig.  1  B).  When  the  artificial  source  of  c-AMP  was 
removed  by  shutting  off  the  UV  flashes,  all  the  amoebae  dispersed 


DEVELOPMENT 


199 


Figure  1 .     Response  of  aggregation-competent  slime  mold  amoebae  to  366-nm  microbeam  uncaging  of  caged  c-AMP.  The  microbeam  was  positioned 
near  the  side  ol  this  amoeba,  and  the  I T  flashes  were  sinned  I  ininule  27  seconds  before  I  lie  frame  A.  See  le.\l.  Time  in  minutes:seconds  after  A 


and  headed  towards  their  natural  source,  an  aggregate  formed 
by  many  amoebae  located  outside  the  induced  territory. 

Early  during  aggregation,  most  amoebae  remained  in  loose 
contact  with  each  other  and  slowly  spiralled  around  the  center 
of  the  aggregate.  Whether  in  a  naturally  formed  aggregate  made 
up  of  many  scores  of  cells  or  in  an  artificially  induced  aggregate 
made  up  of  only  a  few  cells,  the  amoeba  located  in  the  center 
became  stationary  relative  to  the  substratum.  Through-focus 
observations  indicated  that  such  an  amoeba  anchored  itself  to 
the  soft  substrate  by  protruding  knobby  "feet"  into  the  agar 
surface.  In  the  artificially  induced  aggregate,  the  central  amoeba 
generated  and  maintained  its  feet  (small  arrows  in  Fig.  1C;  the 
UV  micromirror  is  retracted  and  focus  is  now  on  the  agar  surface) 
so  long  as  the  c-AMP  pulses  (UV  flashes)  were  continued.  Once 
the  UV  flashes  were  turned  off,  the  feet  were  gradually  retracted 
(Fig.  ID).  If  the  UV  flashes  were  restarted  within  a  minute  or 


so,  the  feet  reformed,  and  the  departing  outer  amoebae  rejoined 
the  aggregate.  The  process  was  completely  reversible  and  could 
be  repeated  many  times.  Thus  we  have  established  a  tool  for 
investigating  localized  and  reversible  c-AMP-mediated  modu- 
lation of  chemotaxis  and  cell  response. 

Supported  by  NIH  grants  R01  GM  39548  to  YF  and  R37 
GM  31617  to  SI. 


Literature  Cited 

1-   Bonner.  J.  T.  1971.     Ann   Rev Microlnol  25:  75-92. 

2.  Gerisch,  G.  1987.     Anmi.  Rev  Biochem.  56:  853-879. 

3.  Devreotes,  P.  1989.     Science  IAS:  1054-1058. 

4.  Nerbonne,   J.   M.,   S.   Richard,   J.   Nargcot   and   H.   A.   Lester. 
1984.     Nature  310:  74-76. 

5.  Fukui,  V.  and  S.  Inoue.  1991.     Cell  Afiilil.  Cvtoskel.  18:  41-54. 


200  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  /?•      Rutt.  189:  200.  (October/November.  1995) 

Identification  of  Calcium  Flux  in  Single  Preimplantation  Mouse  Embryos 
with  the  Calcium-Sensitive  Vibrating  Probe 

David  Keefe  (Yale  University  School  of  Medicine.  Department  of  Obstetrics  and  Gynecology), 
John  Pepperell,  Paulo  Rinaudo,  Joseph  Kunkel,  and  Peter  Smith 


Although  aging  affects  nearly  every  aspect  of  female  repro- 
duction, oocytes  are  believed  to  be  major  targets.  Oocytes  are 
long-lived,  post-mitotic  cells  and  donation  of  these  cells  by  young 
females  to  older  ones  clearly  ameliorates  the  effects  of  aging  on 
reproductive  success  ( 1 ).  Elucidation  of  the  mechanisms  under- 
lying reproductive  aging  is  of  basic  importance  because  the  se- 
nescence of  oocytes  provides  a  model  of  aging  of  other  long- 
lived,  post-mitotic  cells.  Moreover,  many  women  now  delay 
marriage  and  childbearing  until  their  late  thirties,  when  the  effects 
of  aging  on  fertility  can  become  clinically  significant  (2).  A  non- 
invasive  technique  that  could  be  used  to  assay  the  developmental 
potential  of  an  embryo  before  implantation  would  both  facilitate 
the  diagnosis  of  reproductive  senescence  and  help  middle-aged 
women  decide  whether  to  depend  on  their  own  oocytes  and 
pursue  costly  reproductive  therapies,  or  to  pursue  alternatives 
such  as  adoption  or  oocyte  donation. 

Disruption  of  intracellular  calcium  [Ca],  regulation  is  an  im- 
portant mechanism  underlying  senescence  in  many  long-lived 
cells  (3).  Normally,  the  plasma  membrane  has  at  least  two  sys- 
tems that  contribute  to  maintaining  low  [Ca],:  a  Na/Ca  exchanger 
powered  by  the  Na-K  ATPase,  and  a  Ca-ATPase  (4).  Both  of 
these  systems  have  been  implicated  in  cell  injury  and  senescence 
(3.  4).  Inhibition  of  the  Na-K  ATPase  by  ouabain  alters  in  vitro 
development  of  mouse  preimplantation  embryos  (7). 

In  this  study  we  employed  the  calcium-selective  vibrating 
probe  to  test  directly  the  hypothesis  that  mouse  preimplantation 
embryos  exhibit  steady-state  calcium  currents.  Moreover,  to  de- 
velop the  vibrating  probe  as  a  non-invasive  assay  of  the  devel- 
opmental potential  of  such  embryos,  we  have  begun  to  map 
steady  state  calcium  flux  in  mouse  embryos  with  differing  de- 
velopmental potential. 

After  hybrid  matings  (B6C3F1  X  B6D2F1),  mouse  embryos 
were  removed  surgically  at  the  two  cell  stage  and  either  studied 
at  this  stage  or  cultured  to  the  four-  or  eight-cell  stages  in  M2 
medium  supplemented  with  0.4%  BSA  at  37°C  in  5%  CO,.  The 
two-cell  stage  embryos  were  washed  at  least  twice  in  a  modified 
M2  medium  containing  only  50  pM  calcium  and  then  trans- 
ferred in  the  same  medium  to  petri  dishes  coated  with  high- 
molecular  weight  polylysine  to  which  the  embryos  adhered.  Em- 
bryos were  then  examined  for  a  calcium  flux  at  room  temperature 
(=;220C).  Measurement  of  the  voltages  associated  with  steady- 
state  transmembrane  calcium  flux  were  done  as  previously  de- 
scribed (8).  The  calcium-sensitive  electrodes,  after  calibration  to 
determine  their  Nernstian  characteristics,  were  positioned  within 
about  1  ^m  of  an  embryo's  zona  pellucida.  The  distribution  of 
the  efflux  was  mapped  by  moving  the  probe  to  at  least  four 
quadrants  of  the  embryo's  circumference.  Images  of  the  embryos 
were  recorded  with  a  video  printer  so  that  morphology  could  be 
correlated  with  steady-state  calcium  flux.  After  experimentation 


embryos  were  routinely  returned  to  normal  M2  medium  and 
kept  at  37°C  in  5%  CO,,  where  cell  division  was  monitored. 

Morphology  combined  with  growth  allowed  us  to  divide  the 
two  cell  stage  embryos  into  two  classes: 

1 .  Morphologically  normal  or  capable  of  further  cleavage. 

2.  Morphologically  fragmented  or  incapable  of  further  cleavage. 

In  the  case  of  the  first  class  of  embryos,  there  was  a  strong  calcium 
efflux  signal  measured  in  all  cases.  In  10  preparations  this  signal 
had  an  amplitude  of  -2 1 .22  juV  ±  5.7  (mean  ±  standard  devia- 
tion). Initial  observations  from  the  four  quadrants  did  not  exhibit 
any  differences  in  the  microvolts  recorded.  In  class  2  embryos 
there  was  no  measurable  calcium  efflux  signal.  Of  5  embryos 
examined,  the  signal  at  the  plasma  membrane  was  -1.75  ^V 
±  5.28.  Background  was  2.07  MV  ±  2.24. 

We  conclude  that  mouse  preimplantation  embryos,  which 
retain  their  developmental  potential  (Class  1),  exhibit  a  steady 
state  transmembrane  calcium  efflux  as  measured  by  the  non- 
invasive,  vibrating  calcium  selective  electrode.  The  efflux  mapped 
in  a  symmetrical  pattern  about  the  embryo,  with  no  polarity 
observed.  Embryos  with  impaired  developmental  potential,  as 
measured  either  by  a  fragmented  morphology  or  subsequent 
failure  to  divide  (Class  2),  failed  to  exhibit  a  steady  state  calcium 
efflux  equivalent  to  that  observed  in  Class  1  embryos.  The  ab- 
sence of  an  equivalent  efflux  in  those  embryos,  which  subse- 
quently failed  to  grow  further,  suggests  that  the  steady  state  cal- 
cium efflux  may  be  a  viable  assay  of  the  health  of  the  embryo. 

Supported  by  NIH  K.08HD01099,  the  American  Society  for 
Reproductive  Medicine  (O.K.)  and  NIH  National  Center  for 
Research  Resources,  P41RROLW  (P.J.S.S.). 

Literature  Cited 

1.  vom  Saal,  F.  S.,  C.  E.  Finch,  and  J.  S.  Nelson.  1994.     Pp  1213- 
1314  in  Tin-  Physioliw  nl  Reproduction.  E.  Knobil,  J.  Neil,  eds. 
Raven  Press  New  York. 

2.  Keefe,  D.,  T.  Niven-Fairchild.  S.  Powell,  and  S.  Buradagunta. 
1995.     Fertil.  Slen/64:  577-583. 

3.  Rasmussen,  H.  1986.     Arir  Kiit-l.  ./.  Med.  314:  1094-1164. 

4.  Eckert,  A.,  H.  Hartmann,  H.  Forsll,  and  W.  E.  Muller.  1994.     Life 
Sci.  25:2019-2029. 

5.  Robinson,  D.  H.,  and  D.  J.  Bvnos.  1991.     Current  Topics  in  Mem- 
branes 319:  121-150. 

6.  Overstrom,  E.  VV.,  D.  J.  Benos,  and  J.  D.  Biggers.  1989.     J  Repwd. 
Ferl  85:  283-295. 

7.  Dumoulin,  J.  C.  M.,  A.  H.  J.  Michiels,  M.  Bras,  M.  H.  Pieter, 
J.  P.  Geraedts,  and  J.  L.  H.  Evers.  1993.     Human  Rtpmd.  8:  1469- 
1476. 

8.  Smith,  P.  J.  S.,  R.  H.  Sanger.  L.  F.  Jaffe.  1994.     Pp.  1 15-134  in 
Methods  in  Cell  Biology  40:  R.  Nuccitelli,  ed.  Academic  Press,  San 
Diego. 


DEVELOPMENT 


201 


Reference:5/o/.  Bull  189:  201.  (October/November, 


Inhibitors  of  Protein  Phosphatases  (Okadaic  Acid  and  Tautomycin)  Block  Sea  Urchin  Development 

II 'alter  Troll  (\e\\-  York  L'nivcrsity  Medical  Center),  Naoko  Sueoka,  Eisaboro  Sueoka, 

Jeffrey  D.  Luskin,  and  Diane  E.  Heck 


Protein  phosphor,  lation.  regulated  by  protein  kinases  and 
phosphatases.  is  critical  for  cell  growth,  regulation,  and  devel- 
opment ( 1-3).  Phosphatase  inhibitors,  which  prolong  the  phos- 
phorylated  slate  of  proteins,  can  often  perturb  cell  development. 
Our  laboratory  has  been  interested  in  utilizing  two  of  these  com- 
pounds, okadaic  acid,  isolated  from  the  marine  sponge  Hali- 
chondria  okadai,  and  tautomycin.  isolated  from  Streptomyces 
spirover  ticillatits.  to  examine  the  role  of  phosphatases  in  sea 
urchin  development.  Previous  work  demonstrated  that  these 
compounds  are  effective  inhibitors  of  serine  and  threonine 
phosphatases.  in  particular,  phosphatases  1  (PP-1)  and  2  A  (PP- 
2A)  (4).  Okadaic  acid  is  more  selective,  preferentially  inhibiting 


100 


10  100  1000 

Concentration  (nM) 


10000 


Figure  I.  Effects  of  phosphalase  inhibitor-,  on  the  viubilny  <>/  uv/ 
urchin  embrvos.  Eggs  obtained  from  Arbacia  punctulata  were  fertilized 
in  vitro  in  the  presence  of  increasing  concentrations  of  okadaic  acid  (broken 
line)  or  tautomycin  (solid  line).  The  percentage  <>/  viable  e»ibryn\  IUM 
calculated  from  the  mimhei  "I  embryos  that  were  freely  rotating  24  h 
after  fertilization 


PP-2A.  whereas  tautomycin  inhibits  PP-1  and  2A  with  equal 
effectiveness  (4).  Recent  studies  indicate  that  PP-1  may  be  im- 
portant in  the  initial  responses  of  sea  urchin  eggs  to  fertilization 
(5).  In  the  present  studies  we  sought  to  determine  whether  oka- 
daic acid  and  tautomycin  could  differentially  modify  fertilization 
and  early  development  in  the  sea  urchin  Arbacia  punctulata. 

In  initial  experiments,  sea  urchin  sperm  and  eggs  were  in- 
cubated with  increasing  concentrations  of  okadaic  acid  and  tau- 
tomycin ( 1-1000  n.\f).  We  found  that  these  compounds  had  no 
effect  on  sperm  activation  or  on  early  events  of  fertilization  (not 
shown).  Thus,  in  all  cases.  >95%  of  the  eggs  raised  fertilization 
membranes  and  formed  embryos.  We  also  found  that  blastula 
rotation,  representing  a  later  stage  of  differentiation,  was  selec- 
tively inhibited  by  tautomycin  but  not  okadaic  acid.  Tautomycin 
was  a  potent  inhibitor  of  the  transition  into  the  rotating  blastula 
stage  and  was  dose  dependent  at  concentrations  of  100-1000  nM 
(Fig.  1).  Embryos  treated  with  okadaic  acid,  however,  did  not 
exhibit  this  effect  (Fig.  1 ).  No  embryos  treated  with  tautomycin 
(100  nM-10  nM)  developed  into  plutei  (data  not  shown).  Only 
very  high  concentrations  of  okadaic  acid  (\-\Q  pAI)  inhibited 
the  hatching  of  plutei.  consistent  with  the  small  effect  of  this 
compound  on  the  initial  development  of  motility. 

In  conclusion,  tautomycin.  but  not  okadaic  acid,  is  an  effective 
inhibitor  of  sea  urchin  development,  acting  on  the  maturation 
into  rotating  blastula.  We  speculate  that  the  preferential  inhi- 
bition of  PP-1  by  tautomycin  may  reflect  differential  roles  of 
PP-1  and  PP-2A  in  this  process. 


Literature  Cited 


1.  Racker,  E.  1992.     Curt:  Top  Ceil  Regnl.  33:  127-143. 

2.  Kreimer,  D.  I.,  and  V.  S.  Khotimchenko.  1995.     Comp.  Biochem. 
Physiol.  A  Comp.  Physinl.  110:  95-105. 

3.  Ribot,  H.  D.,  E.  A.  Eisenmann,  and  VV.  H.  Kinsey.  1984.     J  Biol 
Chem.  259:  5333-5338. 

4.  Suganuma,  M.,  II.  Fujiki,  S.  Okabe,  S.  Nishiwaki,  D.  Brautigan, 
T.  S.  Ingebritsen,  and  M.  R.  Rosner.  1992.     Toxicon  30:  873-878. 

5.  Tosuji,  H.,  I.  Mabuchi,  N.  Eusetani,  and  T.  Nakazawa.  1992.     Proc. 
\till.  Acad.  Sci.  i'SA  89:  10613-10617. 


Reference:  Biol.  Bull.  189:  201-202.  (October/November.  1995) 

Reversible  Regression  of  Cytokinesis  Induced  by  Ca2+  lonophore 

Keisukc  Suzuki,  Fabrice  Roegiers,  Phong  Tran,  and  Slrinya  Inoue  (Marine  Biological  Laboratory) 


Transient  changes  in  intracellular  free  Ca2+  concentration  are 
spatially  and  temporally  coupled  to  specific  cell  cycle  events 
such  as  mitosis  and  cytokinesis  (e.g..  1.  2).  In  the  sea  urchin 
embryo,  an  endogenous  rise  in  Ca:+  precedes  cytokinesis  (3): 


and  in  the  medaka  embryo,  this  transient  Ca2+  increase  is  lo- 
calized to  the  cleavage  furrow  (4).  Injection  of  Ca2+  chelators 
into  the  sand  dollar  embryo  prior  to  cytokinesis  arrests  devel- 
opment and  inhibits  furrow  formation  (5).  We  have  examined 


202 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


Figure  1 .     Lytc\  atus  etnhryo  al  early  stages  of  development 

imaged  with  polarized  '<'\<v>/>r  (A)  Embryo  al  first  cell  division 

cycle,  with  cleavage Jurrow.  at  the  time  of'A23 1 87 perfusion.  (B)  Cleavage 
furrow  regression  indue,;!  ;>i  A23I87.  (C)  Cytokinesis  resumed  at  the 
same  site  when  the  A23IH~  mi,-,  washed  out  with  ASW.  (D)  The  same 
embryo  continued  through  cell  division  and  development  at  the  next  cycle 
after  a  single  A23 187  treatment  ami  AS\\'  washing.  Bar  =  50  urn. 


further  the  roles  that  Ca2+  plays  in  cytokinesis,  specifically 
downstream  of  cleavage  furrow  formation;  our  approach  was  to 
induce  a  global  Ca:+  increase  within  the  sea  urchin  embryo. 

Lylec/iinus  variegutits  eggs  and  sperm  were  collected  by  elec- 
trically stimulating  the  sea  urchins.  The  eggs  were  fertilized  and 
the  embryos  developed  in  artificial  seawater  (ASW)  containing 
about  10  mA/Ca:+.  The  fertilization  envelope  was  removed  from 
the  fertilized  eggs  by  washing  with  a  digestion  mix  containing 
1  mAl  DTT  and  15  /ig/ml  pronase  at  pH  8.9.  The  eggs  were 
then  place  in  a  wedge-profiled  perfusion  chamber  and  observed 
with  polarized  light  microscopy. 

At  18°C,  the  fertilized  sea  urchin  eggs  completed  the  first  cell 
division  in  ~2  h,  with  the  cleavage  furrow  appearing  at  ~  1 :45 
h.  After  the  first  few  minutes  of  furrow  formation,  we  perfused 
the  eggs  with  ASW  containing  5  Mg/ml  A23 1 87,  a  Ca2+  ionophore 
and  an  effective  transporter  that  presumably  causes  a  global  in- 
flux of  extracellular  Ca2+  into  the  dividing  egg. 

Figure  1 A  shows  a  sea  urchin  egg  with  cleavage  furrow  just 
before  A23187  perfusion.  Furrow  progression  in  the  egg  im- 
mediately stopped  and  the  furrow  regressed  outwardly  (Fig.  IB) 
until  the  egg  returned  to  its  original  spherical  shape.  This  furrow 
regression  was  reversible,  and  cytokinesis  resumed  al  the  same 
xitc  when  the  A23187  was  washed  out  with  ASW  within  10-20 
min  of  the  initial  A23187  perfusion  (Fig.  1C).  The  cycle  of 
cleavage  furrow  regression  in  A23 1 87  followed  by  the  resumption 
of  cytokinesis  after  washing  could  be  repeated  two  to  three  times 
with  the  same  cell.  After  two  cycles  of  A23187  application  and 
ASW  washing,  the  embryo  continued  to  divide  and  develop. 
Cells  exposed  to  5  ng/m\  of  A23187  for  more  than  15-20  min 
did  not  survive  the  treatment. 

To  our  knowledge,  this  is  the  first  example  of  a  reversible 
regression  of  cytokinesis  induced  by  Ca2+  ionophore  A23187. 
A  previous  study  by  Arnold  (6)  showed  that  A23187  enhances 
furrowing  in  the  squid  embryo,  but  the  concentration  of  iono- 
phore used  in  those  experiments  was  not  stated.  In  our  study, 
the  reversible  regression  of  cytokinesis  was  dependent  on  tem- 
perature, extracellular  Ca2+  concentration,  ionophore  concen- 
tration, stage  of  the  cell  cycle,  and  ionophore  exposure  time. 
We  have  not  yet  determined  the  exact  mechanism  underlying 
the  reversible  regression  of  cytokinesis  induced  by  A23187. 
However,  we  propose  that  A23187  causes  a  global  increase  in 
free  Ca2+  that  may  activate  actin  severing  proteins  or  deactivate 
actin  cross-linking  proteins;  or  alternatively,  Ca2+  modulates 
binding  of  myosin  to  actin  filaments  directly  or  via  a  Ca2+ 
sensitive  kinase  such  as  myosin  light  chain  kinase  or  protein 
kinase  C. 

We  gratefully  acknowledge  the  support  of  members  of  the 
MBL  Architectural  Dynamics  in  the  Living  Cell  Program;  sup- 
port from  Olympus  Corporation  to  K.S.;  support  from  CNRS/ 
NSF  to  C.  Sardet  for  F.R.;  and  support  from  MBL  Eric  F.  Fries 
Fellowship  to  P.T.  and  NIH  grant  GM-31617  to  S.I. 

Literature  Cited 

1.  Silver,  R.  B.  1990.     Ann.  New  York  Acad.  Sci.  582:  207-221. 

2.  Hepler,  P.  K.  1992.     Inl  Rev.  Cyln  138:  239-268. 

3.  Ciapa,  B.,  D.  Pesando,  M.  Wilding,  and  M.  \\hitaker.  1994.     Nature 
368:  875-878. 

4.  Fluck,  R.  A.,  A.  L.  Miller,  and  I..  F.  Jaffe.  1991.     /  Cell  Biol.  115: 
1259-1265. 

5.  Silver,  R.  B.  1989.     De\:  Biol.  131:  1 1-26. 

6.  Arnold,  J.  M.  1975.     Cytobiologie  11:  1-9. 


CELL  CYCLE 


203 


Reference:  Biol.  Bull.  189:  203-204.  (October/November.  199?) 

Leukotriene  B4  Induces  Release  of  Calcium  From  Endomembrane  Stores  In  I  ivo  in  Eggs  and  Second 
Cell  Cycle  Blastomeres  of  the  Sand  Dollar  Echinaracnius  parma 

Robert  B.  Silver  (Marine  Biological  Laboratory) 


Nuclear  envelope  breakdown  (NEB)  is  preceded  by  a  large 
signal  of  intracellular  tree  calcium  (Ca,2+)  composed  of  several 
thousand  individual  events  of  elevation  of  Ca,2+  concentration. 
Each  event  represents  a  release  of  Ca,2+  from  endomembrane 
stores  to  the  cytoplasm,  and  we  have  described  them  as  quantum 
emission  domains  (Ca,2+-QEDs)  (1,2,  5-7;  Silver  el  al.,  unpub- 
lished). Individual  calcium  release  events  were  visualized  as 
bright  observable  blobs  in  aequorin  labeled  cells  (6,  7),  and  they 
occur  within  restricted  regions  of  space  called  microdomains  (6. 
7).  1  reasoned  that  an  agonist  capable  of  evoking  such  events 
should  be  generated  locally,  should  be  present  for  a  very  brief 
period  of  time,  and  should  thus  trigger  NEB  or  other  processes 
that  are  signaled,  initiated,  coordinated,  or  controlled  by  ele- 
vations of  intracellularly  derived  Ca,2+  concentration.  And,  in 
fact,  this  laboratory  has  shown  that  the  reduced  form  of  leu- 
kotriene  B4  (LTB4)  could  induce  release  of  Ca2+  from  endo- 
membranes  isolated  from  unfertilized  eggs  and  from  second  cell 
cycle  blastomeres  of  the  sand  dollar  (Echinaracnius  parma)  (8). 
The  release  of  Ca2+  from  isolated  endomembranes  by  LTB4  in 
vitro  was  similar  to  that  observed  with  1 ,4,5-inositol  trisphosphate 
(8):  but  oxidized  LTB4  was  ineffective.  Arachidonic  acid  (AA) 
derivatives  play  a  role  in  a  wide  variety  of  cellular  processes  (9- 
13),  including:  vascular  contraction  cycles,  neutrophil  activation, 
activation  of  DNA  synthesis,  aggregation  of  marine  sponge  cells, 
and  activation  of  tumor  necrosis  factor. 

In  the  current  study,  the  potential  role  of  AA  or  major  AA- 
derived  metabolites  as  putative  agonists  for  release  of  Ca2+  from 
endomembrane  stores  was  tested  in  vivo.  AA  metabolites  were 
microinjected  into  aequorin-loaded  (6.  7.  15)  sand  dollar  (Echi- 
naracnius parma)  eggs  or  mitotic  second  cell  cycle  blastomeres. 
and  the  emission  of  Ca2+-dependent  photon  signals  was  followed 
as  previously  described  (6-7;  Silver  et  al.  unpublished). 

Candidate  agonists  were  tested  at  pipette  concentrations 
ranging  between  10~9  A/and  1CT3  Al;  the  final  equilibrium  di- 
lution factor  was  estimated  at  about  104(1.  14).  so  the  estimated 
intracellular  equilibrium  concentrations  were  between  10""  to 
10~7  M.  I  used  the  microinjection  method  (1,2)  originally  de- 
veloped by  Hiramoto  (16):  the  aqueous  sample  and  an  oil  droplet 
of  equal  volumes  were  co-injected  into  the  cell.  The  candidate 
agonists  tested  were  partially  dried  from  ethanol  stock  solutions, 
diluted  into  dimethyl  sulfoxide  (DMSO).  reconcentrated  under 
dry  N2  gas,  and  then  diluted  in  injection  buffer  (Ca2+-free  phos- 
phate buffered  saline;  1,  14).  To  reduce  the  possibility  that  the 
samples  would  oxidize  before  being  injected,  they  were  kept  in 
the  dark  and  under  dry  N2  gas  until  just  before  the  pipette  was 
loaded.  Given  the  high  degree  of  solubility  in  aqueous  media  of 
the  AA  metabolites  and  the  rapidity  of  the  microinjection  pro- 
cedure. I  assumed  that  the  majority  of  the  sample  remained  in 
the  aqueous  phase  before  injection.  Control  injections  of  injec- 
tion buffer,  DMSO  diluted  10-fold  in  injection  buffer,  deionized 
H2O.  and  vegetable  oil  evoked  no  increase  in  detectable  Ca2+- 
dependent  aequorin  luminescence.  Each  condition  was  tested 


at  least  four  times,  in  separate  cells,  to  assure  reproducability  of 
the  detected  response  (e.g..  1,2). 

Ca2+  release  from  endomembranes  was  elicited  in  vivo  by  LTB4 
injection.  No  release  of  Ca2+  was  evoked  from  isolated  endo- 
membrane stores  in  vivo  by:  AA,  prostaglandins  (G2,  H2,  E2, 
F2u).  thromboxane  A2,  leukotriene  A4  (LTA4),  leukotriene  C4, 
leukotriene  A5,  leukotriene  C5.  leukotriene  B5,  and  oxidized 
LTB4.  Ca2+  (as  calcium  chloride  solution)  and  Ca2+  released 
from  endomembrane  stores  by  divalent  ionophore  A23 1 87  also 
evoked  luminescence  in  the  aequorin  labeled  cells.  The  conserved 
PSTAIR  peptide  of  the  cell  cycle  related  kinase  p34  cdc2  was 
also  injected  to  test  an  earlier  report  of  its  ability  to  serve  as  an 
agonist  of  endomembrane  Ca2+  channels  (17).  PSTAIR  did  not 
evoke  the  release  of  Ca2+  from  endomembrane  stores;  the  positive 
effects  seen  earlier  could  have  been  due  to  impurities  attributable 
to  differences  in  preparation  of  the  synthetic  peptide. 

In  summary,  only  LTB4,  of  all  the  AA-derived  candidate  ag- 
onists, evoked  a  release  of  Ca2+  from  endomembrane  stores — 
seen  as  a  substantial  increase  in  the  number  and  density  of  cal- 
cium release  events  emitted  from  the  injected  cell  upon  injection 
of  the  candidate  agonist.  The  total  amount  of  Ca2+  released  was 
proportional  to  the  amount  of  LTB4,  1,4,5-IP3,  or  Ca2+  (CaCl2 
at  a  pipette  concentration  of  10~3A/)  injected.  Observations 
showed  that,  at  these  relatively  high  concentrations  of  LTB4. 
the  Ca2+  events  spread  radially  from  the  point  of  injection  at  a 
rate  of  about  5  micrometers  s~',  ending  at  the  inner  surface  of 
the  plasma  membrane;  Jaffe  has  reviewed  similar  native  Ca2+ 
waves  associated  with  fertilization  (18).  The  pattern  spread  of 
calcium  release  events  evoked  by  injection  of  unbuffered  Ca2+ 
was  highly  limited,  indicating  a  high  native  Ca2+-buffering  ca- 
pacity within  the  intracellular  compartment;  as  such,  the  dif- 
fusional  spread  of  Ca2+  injected  into  the  cell  would  be  highly 
restricted  in  space. 

Injection  of  LTB4  or  1,4,5-IP3  also  elevated  the  fertilization 
envelopes  of  sand  dollar  eggs  regardless  of  the  recipe  of  Ca2+- 
free  artificial  seawater  (MBL  or  Jamarin)  used;  each  seawater 
preparation  fully  supported  normal  embryonic  development  at 
least  to  the  pluteus  stage.  Eggs  injected  with  LTB4  typically 
showed  a  more  complete  and  native  fertilization  envelope  ele- 
vation than  those  injected  with  1,4.5-IP.,;  eggs  injected  with  CaCl2 
solutions  did  not  often  elevate  their  fertilization  envelopes,  most 
likely  due  to  the  high  intrinsic  intracellular  Ca2+  buffering  ca- 
pacity preventing  local  Ca,2+  concentration  from  reaching  suf- 
ficiently elevated  levels  to  induce  the  secretory  processes  nec- 
essary for  elevation  of  the  fertilization  envelope. 

LTB4  has  the  features  of  a  putative  agonist,  evoking  the  release 
of  Ca2+  from  endomembrane  stores  in  vivo  to  control  Ca,2+- 
dependent  processes  within  microdomains  in  eggs  and  mitotic 
second  cell  cycle  blastomeres.  Such  a  pulsed  release  of  Ca2+ 
is  consistent  with  the  calcium -dependent  luminescence  pat- 
terns observed  in  aequorin-loaded  eggs  and  mitotic  cells 
(C.K..  1.  5.  8). 


204 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


Grant  support  by  NSF  is  gratefully  acknowledged.  Aequorin 
preparations  ( 15)  were  generously  provided  by  Dr.  Shimomura 
(Marine  Biological  Laboratory)  and  his  colleagues  Drs.  Inouye, 
Musicki.  and  Kishi.  The  author  is  grateful  to  the  reviewers  for 
their  many  helpful  and  well  considered  suggestions  made  in  the 
final  preparation  of  this  manuscript. 


Literature  Cited 

1.  Silver,  R.  B.  1989.     Dcv  Biol  131:  1  1-26. 

2.  Silver,  R.  B.  1990.     Ann  N.  Y,  Actui.  Sci.  582:  207-221. 

3.  Llinas,  R.,  M.  Sugimori,  and  R.  B.  Silver.  1992.     Science  256:  677- 
679. 

4.  Berridge,  M.  J.,  and  R.  F.  Irvine.  1989.     Katitre  341:  197-205. 

5.  Silver,  R.  B.  1986.     J.  Cell  Biol.  103:  140a. 

6.  Silver,  R.  B.  1994.     Biol  Bull  187:  235-237. 

7.  Silver,  R.  B.,  A.  P.  Reeves,  M.  Whitman,  and  B.  Kelley.  1994.     Biol 
Bull.  187:  237-238. 


8.  Silver,  R.  B.,  J.  B.  Oblak,  G.  S.  Jeun,  J.  Sung,  and  T.  Dutta. 
1994.     Biol  Bull  187:  242-244. 

9.  Samuelsson,  B.  1983.     Science  220:  568-575. 

10.  Samuelsson,  B.,  S.-E.  Dahlen,  J.  A.  Lindgren.  C.  A.  Rouzer,  and 
C.  N.  Serhan.  1987.     Science  237:  1  171-1 176. 

11.  Buttner,  N.,  S.  A.  Siegelbaum,  and  A.  Volterra.  1989.     Mature 342: 
553-555. 

12.  Rich,  A.  M.,  G.  Weissmann,  C.  Anderson,  L.  Vosshall,  K.  A.  Haines, 
T.  Humphreys,  and  P.  Dunham.  1984.     Biochcm    Biophys.  Rex. 
Comm   121:  863-870. 

13.  Hayakawa.  M.,  N.  Ishida,  K.  Takeuchi,  S.  Shibamoto,  T.  Hori,  N. 
Oku,  F.  Ito,  and  M.  Tsujimoto.  1993.     J.  Biol.  C/icm  268:  1 1290- 
11295. 

14.  Silver,  R.  B.  1986.     Proc.  Nul.  Acad.  Sci.  U.S.A.  83:  4302-4306. 

15.  Shimomura,   O.,   S.   Inouye,   B.   Musicki,   and   Y.   Kishi.    1990. 
Biochcm  J  270:  309-322. 

16.  Hiramoto,  V.  1974.     Exp.  Cell  Res.  87:  403-406. 

1 7.  Picard,  A..  J.  C.  Cavadore,  P.  Lory,  J.  C.  Bernengo,  C.  Ojeda,  and 
M.  Doree.  1990.     Science  247:  327-329. 

18.  Jaffe,  L.  1993.     Cell  Calcium  14:  736-745. 


Reference:  Biol.  Bull  189:  204-205.  (October/November,  1995) 

Anaphase  Spindle  Dynamics  Under  D2O-enhanced  Microtubule  Polymerization 

Mira  Krendel  and  Shinya  I  none  (Marine  Biological  Laboratory) 


Heavy  water  (D:O)  promotes  microtubule  polymerization 
both  in  vitro  and  in  vivo  (1,2,  3).  Possible  mechanism  for  the 
enhancement  of  microtubule  polymerization  by  D2O  is  stabi- 
lization of  hydrophobic  interactions  between  tubulin  dimers  (2). 
Microtubules  are  the  primary  fibrous  components  of  the  mitotic 
and  meiotic  spindles,  and  treatment  of  dividing  cells  with  heavy 
water  increases  spindle  birefringence  which  reflects  enhanced 
microtubule  assembly.  Polymerization  and  depolymerization  of 
microtubules  are  thought  to  be  important  for  the  spindle  func- 
tions in  chromosome  separation  and  they  should  therefore  be 
precisely  regulated  during  cell  division.  High  concentrations  of 
D2O  (more  than  70%)  have  been  shown  to  block  mitosis  (4). 
We  have  investigated  the  effects  of  increased  polymerization  of 
microtubules  in  the  presence  of  D2O  on  the  first  meiotic  division 
in  Chaetopterus  pcrgamcntaceoux  oocytes. 

Birefringent  yolk  granules  present  in  oocytes  interfere  with 
the  observation  of  spindle  birefringence  under  polarized  light 
microscope.  Therefore,  clear  oocyte  fragments  were  prepared  by 
a  modification  of  the  centrifugation  method  described  in  (5). 
Oocytes  were  layered  onto  a  "cushion"  consisting  of  10  parts  of 
1.1  M  sucrose,  and  1  part  ASW  (Artificial  Sea  Water)  and  were 
centrifuged  in  a  microcentrifuge  at  7,000  rpm  for  8  min  and  at 
14,000  rpm  for  4  min.  Oocyte  fragments  were  collected  from 


the  sucrose-seawater  interface,  washed  with  seawater,  and 
mounted  for  observation  in  a  wedge-profiled  perfusion  chamber. 
After  preparation,  the  fragments  remained  arrested  in  the  meta- 
phase  of  the  first  meiotic  division  unless  they  were  induced  to 
proceed  through  the  cell-division  cycle  by  the  addition  of  sperm. 
Spindle  birefringence  was  observed  using  either  polarized  light 
microscopy  with  video  and  digital  contrast  enhancement  or  the 
new  pol-scope  (6). 

Addition  of  ASW  containing  40-50%  D;O  to  oocytes  in 
metaphase  or  anaphase  significantly  increased  spindle  birefrin- 
gence (Fig.  1  A,  C).  The  area  occupied  by  the  spindle  in  the  plane 
of  focus  also  increased,  on  average,  by  one-third  upon  addition 
of  D:O.  The  increased  spindle  birefringence  persisted  until  ana- 
phase,  when  the  spindle  was  rapidly  disassembled  (Fig.  IB.  D). 
Meiotic  division  and  first  polar  body  formation  in  the  presence 
of  D:O  proceeded  without  significant  delay.  No  decrease  in  bi- 
refringence with  time  was  observed  in  oocyte  fragments  that 
were  incubated  in  D:O  without  fertilization,  indicating  that  the 
rapid  fading  of  birefringence  in  cells  completing  meiosis  was  a 
function  of  the  cell  cycle  and  did  not  reflect  a  transient  effect  of 
D:O.  These  results  indicate  that  the  promotion  of  microtubule 
polymerization  by  up  to  50%  D:O  does  not  significantly  interfere 
with  the  mechanisms  responsible  for  shortening  and  disassembly 


CELL  CYCLE 


205 


Figure  1.  Effect  ofD2O  on  tin-  /ir\t  meiotic  division  in  Chaetopterus  pergamentaceous  oocytes.  (A).  (B)  Melaphasc  and  antiphase,  respectively,  of 
I  he  first  meiolic  division  in  control  oocytes.  fC).  (D)  First  meiotic  division  in  the  oocyte  treated  with  40%  £>,0  prior  hi  fertilization.  Note  the  increase 
in  spindle  size  and  hireringence  in  mctaphase  (C)  in  comparison  to  control  oocyles  ,-h  the  D:O-lrcalcd  oocyte  goes  through  anaphase.  the  spindle 
fibers  shorten  and  spindle  birefringence  decreases  (D) 


of  spindle  fibers  during  anaphase  chromosome  movement.  The 
inability  of  D:O  to  prevent  disassembly  of  anaphase  spindle  mi- 
crotubules  may  indicate  that  the  concentration  of  assembly- 
competent  tubulin  is  reduced  in  cells  completing  anaphase. 

We  thank  Rudolf  Oldenbourg  for  his  help  with  the  use  of  the 
pol-scope.  We  also  thank  Universal  Imaging  Corporation  for 
the  Physiology  post-course  fellowship  to  M.  K.  This  work  was 
supported  by  the  NIH  grant  R37  GM31617  awarded  to  S.  I. 


Literature  Cited 

1.  Olmsted,  J.  B.,  and  G.  G.  Borisy.  1973.     Biochemistry  12:  4282- 
4289. 

2.  Ito,  T.  J.,  and  H.Sato.  1984.     Biochnn .  Biop/ns  .lew  800:  21-27. 

3.  Inoue,  S.,  and  H.  Sato.  1967.     ./.  Gen.  Physiol.  Suppl.  50:  259-288. 

4.  Gross,  P.  R.,  and  \V.  Spindel.  1960.     Science  131:  37-39. 

5.  Inoue,  S.  1952.     E\p.  Cell.  Res  Suppl.  2:  305-318. 

6.  Oldenbourg,  R.,  and  G.  Mei.  1995.     ./  Microbe.,  in  press. 


206  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference: /;/.-/  Hull.  189:  206.  (October/November,  1995) 

Quantifying  Single  and  Bundled  Microtubules  with  the  Polarized  Light  Microscope 

Phong  Tran,  E.  D.  Salmon,  and  Rudolf  Oldenbourg  (Marine  Biological  Laboratory) 


Polarized  light  microscopy  has  been  an  important  tool  for 
noninvasive  imaging  of  fine  structures  directly  in  living  cells  ( 1 ). 
One  of  us  (R.O.)  has  improved  the  polarizing  microscope  by 
developing  a  precision  universal  compensator  made  of  electron- 
ically controlled  liquid  crystal  devices  and  circular  polarizers. 
Combined  with  special  processing  software,  the  new  "pol-scope" 
can  image  cellular  fine  structures  with  high  sensitivity  and  res- 
olution, irrespective  of  specimen  orientation  (2). 

We  have  used  the  pol-scope  to  image  and  quantify  the  inherent 
optical  properties  of  single  and  bundled  microtubules.  The  ability 
to  image  the  dynamics  of  a  single  microtubule  in  real  time  has 
been  demonstrated  with  light  microscopy  techniques  such  as 
darkfield,  differential  interference  contrast  (DIG),  and  fluores- 
cence. However,  each  of  these  techniques  has  limitations  that 
make  them  non-ideal  for  quantitative  measurements  of  micro- 
tubule  density  and  distribution.  For  instance,  darkfield  and  DIG 
microscopy,  while  noninvasive,  cannot  image  and  quantify  the 
number  of  microtubules  in  a  dense  region  of  microtubules  found 
in  the  mitotic  spindle;  and  fluorescence  microscopy,  while 
quantitative,  is  invasive  and  suffers  from  photobleaching.  The 
pol-scope  is  noninvasive,  does  not  suffer  from  photobleaching, 
and  is  quantitative  because  it  measures  the  inherent  optical 
properties  of  microtubules. 

Phosphocellulose-purified  bovine  brain  tubulin  was  allowed 
to  spontaneously  assemble  into  microtubules,  and  was  then  sta- 
bilized with  10  uAf  taxol.  The  stabilized  single  microtubules  were 
induced  to  form  bundles  of  various  numbers  by  the  addition  of 
inactive  KAR3,  a  kinesin-like  microtubule  motor  that  bundles 
microtubules.  The  microtubules  were  then  allowed  to  adhere  to 
the  coverslip  surface  of  a  slide  chamber  precoated  with  KAR3. 
Using  a  Nikon  PlanApo  60X/1.4NA  low-strain  objective  and  a 
matching  Nikon  Universal  1 .4NA  condenser  on  the  pol-scope, 
we  imaged  single  microtubules  with  polarized  light. 

Figure  1 A  shows  a  microtubule  bundle  imaged  with  DIG  mi- 
croscopy, which  cannot  easily  be  used  to  determine  the  exact 
number  of  microtubules  making  up  the  bundle.  For  comparison. 
Figure  1 B  shows  the  retardance  image  of  the  same  bundle  of 
microtubules  consisting  of  clearly  distinct  regions  of  one,  two. 
and  three  microtubules  observed  with  the  pol-scope.  The  re- 
tardance of  a  single  microtubule  was  measured  to  be  0.07 
±  0.02  nm  [n  =  30].  The  retardance  slow  axis  is  parallel  to  the 
long  axis  of  the  microtubule.  In  addition,  the  retardance  values 
were  found  to  be  quantized  and  increased  linearly  with  the 
number  of  microtubules  in  the  bundle  (Fig.  1G). 

We  will  use  the  new  pol-scope  to  noninvasively  quantify  the 
distribution  and  dynamics  of  spindle  microtubules  in  dividing 
cells. 


1 


Figure  I .  Spontaneously  assembled  microtubules  stabilized  with  Ia\ol 
and  imaged  with  differential-interference-contrast  (DIC)  microscopy  and 
the  new  pot-scope  using  a  Nikon  M icrophot-SA  microscope  equipped  with 
a  100-wattHg  arc  lamp  illuminator,  Nikon  PlanApo  60X/1.4NA  objective, 
and  Nikon  Universal  1.4NA  condenser.  (A)  A  small  bundle  of  microtubules 
observed  under  DIC.  and  with  digital  background  subtraction;  (B)  The 
same  microtubule  bundle  observed  mill  the  pol-scope.  and  with  back- 
ground subtraction  /horizontal  image  dimension  35  iiinl;  (C)  The  mea- 
sured retardance  ol  one.  two.  and  three  microtubules 


We  gratefully  acknowledge  the  support  of  members  of  the 
MBL  Architectural  Dynamics  in  the  Living  Cell  Program,  as 
well  as  NIH  grants  GM-49210  to  R.O.,  GM-24364  to  E.D.S., 
and  MBL  Eric  F.  Fries  Fellowship  to  P.T. 

Literature  Cited 

1.  Inoue,  S.  1953.     Chromosonm  5:  199-208. 

2.  Oldenbourg,  R.  and  G.  Mei.  1995.     /  Microsc.  in  press. 


CALCIUM 


207 


Reference:  Biol.  Bull  189:  207.  (October/November,  1995) 


Acetylcholine-Induced  Ca2f  Flux  across  the  Sarcolemma  of  an  Echinoderm  Smooth  Muscle 

C.  Leah  Devlin  (Department  of  Biology.  Penn  State  University  Ogont:  Campus, 
Abington,  PA  19001)  and  Peter  J.  S.  Smith 


Acetylcholine  (ACh)  causes  slow  contractions  of  the  longi- 
tudinal muscle  of  the  body  wall  (LMBW)  of  a  sea  cucumber. 
Sclerodactyla  briareus.  These  contractions  are  inhibited  by  Ca2+ 
channel  Mockers  diltiazem  and  verapamil  ( 1 ).  We  therefore  chose 
the  LMBW  as  a  model  system  and  the  vibrating  Ca2+-selective 
electrode  as  our  method  to  test  the  hypothesis  that  ACh  may  be 
stimulating  Ca24  influx  across  the  sarcolemma  of  smooth  muscle, 
thus  providing  a  source  of  Ca2+  during  excitation-contraction 
(E-C)  coupling. 

Technical  aspects  and  applications  of  the  vibrating  Ca2+-se- 
lective  electrode  technique  have  been  described  (2).  To  record 
Ca2+  flux  across  the  sarcolemma.  the  electrode  (placed  15  ^m 
from  the  tissue)  was  used  to  measure  voltage  differences  at  the 
two  extremes  of  vibration  10  ^m  apart.  Therefore,  measurements 
at  the  LMBW  were  recorded  at  a  distance  5  microns  or  less  from 
the  muscle  surface.  All  background  (control)  recordings  were 
conducted  at  an  electrode  distance  of  greater  than  450  microns 
from  the  muscle  surface.  PC-based  software  controlled  the  vi- 
bration of  the  electrode  and  calculated  the  voltage  (n\)  difference 
between  vibration  extremes  (2).  The  electrode  vibrated  at  a  fre- 
quency of  0.3  Hz  at  a  right  angle  to  the  long  axis  of  the  strap- 
like  LMBW.  This  low  vibration  frequency  minimized  mixing 
of  the  media  and  reduced  noise  in  the  system.  The  data  measured 
in  nV  was  converted  to  Ca2+  ion  flux  in  pmol  cm"2  s~'  using  a 
modification  of  the  Pick  equation  (2). 

A  wide  range  of  concentrations  of  ACh  ( 10""  M  to  10  '  Af) 
stimulated  a  Ca24  efflux  that  was  both  dose-  and  time-dependent. 
This  efflux  was  probably  the  result  of  recovery  from  a  preceding, 
rapid  Ca:*  influx  that  could  not  be  detected  by  the  relatively 
slow  time  constant  of  the  ionophore  in  the  electrode  tip.  Because 
10~6  At  ACh  lies  in  the  mid-range  of  effective  doses  and  was  the 
dose  used  in  an  earlier  mechanical  study  ( I ).  it  was  chosen  as 
the  concentration  to  be  challenged  by  the  Ca2+  channel  block- 
ing agents.  Treatment  with  10~6  M  ACh  caused  a  Ca2+  efflux  on 
the  order  of  3.39  pmol  cm"2  s~'  (S.D.  =  0.91  pmol  cm"2  s  ', 
N  =  6). 

We  then  tested  two  L-type  Ca24  channel  blockers.  diltiazem 
and  verapamil.  and  two  non-specific  Ca2+  blockers,  cobalt  chlo- 
ride and  lanthanum  chloride,  on  Ca2+  efflux  induced  by  10~6  M 
ACh.  10~5  M  diltiazem  or  verapamil  inhibited  efflux  caused  by 
10~6  M  ACh  by  52%  (N  =  6)  and  88%  (N  =  6),  respectively. 
10~4  Af  cobalt  chloride  or  lanthanum  chloride  inhibited  ACh- 
induced  Ca2+  efflux  by  62%  (N  =  6)  and  92%  (N  =  6).  respec- 
tively. These  data  suggest  that  the  entry  of  extracellular  Ca2+ 
through  voltage-gated  Ca2+  channels  is  source  of  the  ion  mo- 
bilized during  E-C  coupling. 

Alternative  sources  for  Ca2+  release  and  sequestration  in  the 
LMBW  may  be  a  series  of  elongated  subsarcolemmal  sacs  run- 
ning in  parallel  to  the  sarcolemma  in  the  LMBW  ofSclerodactyla 
briareus  (3).  Chen  (3)  suggests  that  excitation  of  the  membrane 
could  cause  a  release  of  Ca:+  from  these  internal  sacs  in  a  system 
similar  to  that  in  vertebrate  skeletal  muscle.  Suzuki  (4)  suggests 


that  calcium-containing  pyroantimonate  precipitates  found 
along  the  inner  membrane  surface  could  be  a  Ca2+  source  during 
E-C  coupling  as  well. 

To  elucidate  possible  mechanisms  of  Ca24  extrusion  we  chose 
agents  that  would  alter  the  normal  activity  of  the  Na-Ca  ex- 
changer or  the  Ca2+-ATPase.  In  the  first  series  of  experiments, 
Na+  ions  were  removed  from  the  bathing  saline  and  replaced 
with  the  same  concentration  (423  mAf)  of  lithium  (Li+);  the  aim 
was  to  test  the  hypothesis  that  Na+  may  enter  through  an  ACh 
receptor-complex  in  the  LMBW  similar  to  that  of  the  mam- 
malian nicotinic  ACh  receptor.  Li+  is  an  ion  that  can  pass 
through  Na+  channels  but  cannot  be  substituted  for  Na+  in  the 
Na-Ca  exchanger  (5).  When  we  replaced  Na+  in  the  bathing 
saline  with  Li+  and  then  applied  ACh,  Ca2+  efflux  was  inhibited 
by  about  50%  (N  =  6).  This  result  indicates  that  Na+  ions  are 
necessary,  first,  as  a  stimulus  for  Ca2+  mobilization  through 
voltage-gate  channels  or  from  an  intracellular  source,  or  perhaps 
during  Na+-induced  Ca2+-release  (5).  A  preliminary  experiment 
(N  =  1 )  showed  that  treatment  of  the  LMBW  with  a  Ca2+-ATPase 
inhibitor,  cyclopiazonic  acid  (10  5  Af)  blocked  ACh-induced 
Ca2+  efflux  by  only  22%.  The  experiments  above  suggest  that 
the  probable  mechanism  of  Ca2+  extrusion  in  the  LMBW  is  the 
Na-Ca  exchanger,  a  more  energy  efficient  mechanism  than  the 
Ca2+-ATPase. 

We  propose  that  the  ACh-induced  Ca2+  efflux  is  an  indirect 
measure  of  Ca2+  influx  through  voltage-gated  Ca2+  channels. 
Because  Ca2+  influx  (and  the  reciprocal  efflux)  was  inhibited  by 
the  L-type  channel  blockers  diltiazem  and  verapamil,  we  suggest 
that  L-type  Ca2+  channels  are  present  in  echinoderm  smooth 
muscle.  The  opening  of  these  voltage-gated  Ca2+  channels  was 
regulated  by  Na+  influx  as  revealed  by  Li+  substitution  experi- 
ments. The  process  of  Ca2+  extrusion  is  probably  the  result  of 
the  Na-Ca  exchanger  that  expels  excess  intracellular  Ca2+  while 
Na+  flows  in  across  the  membrane,  rather  than  the  activity  of  a 
sarcolemmal  Ca2+-ATPase.  Further  tests  with  pharmacological 
probes  and  metabolic  blockers  are  planned  to  distinguish  between 
these  alternatives. 

This  research  was  supported  by  an  MBL  Fellowship  and  Penn 
State  University  Research  Development  Grant  awarded  to 
C.  L.  Devlin  as  well  as  by  P41RROI395.  Thanks  are  extended 
to  Professor  C.  Ladd  Prosser  for  his  suggestions  on  lithium-sub- 
stitution experiments. 

Literature  Cited 

1.  Devlin,  C.  L.  1993.     Camp.  Biochcm.  Physiol.  160C:  573-577. 

2.  Smith,  P.  J.S.,Sanger,  R..  and  L.  F.Jaffe.  1994.     Pp.  115-134  in 
Methods  i  if  Cell  Biology:  A  Practical  Guide  to  the  Study  ol  Calcium 
in  the  Living  Cell.  I'ol-  40,  R.  Nuccitelli.  ed.  Academic  Press.  San 
Diego. 

3.  Chen,  C.  1983.     Ph.D.  Thesis.  University  of  Rhode  Island. 

4.  Suzuki,  S.  1982.     Cell  Tissue  Res   222:  1 1-24. 

5.  Lipp,  P.,  and  E.  Niggli.  1994.     J  Physiol  474:  439-446. 


208  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Biol  Bull.  189:  208-209.  (October/November.  1995) 

Retardation  of  the  Spread  of  Extracellular  Ca24  into  Transected,  Unsealed  Squid  Giant  Axons 

Harvey  M.  Fishman  (University  of  Texas  Medical  Branch).  Todd  L.  Krause, 
Andrew  L.  Miller,  and  George  D.  Bittner 


Survival  of  an  axon  after  injury  (e.g..  transection)  requires  a 
mechanism  that  prevents  internal  accumulation  of  extracellular 
Ca2+,  which  causes  degeneration  ( 1 ).  Repair  of  a  transected  axon 


10  mi 


GA 


J 


band 


13mm 


Figure  1.  :  rred  end  of  a  squid  giant  axon  10  nun  alter  Iran- 
section  in  artificial  •••••  .'.\,iler  I. IS  1C :  430  mM  NaCI.  10  mM  kCI,  10  mM 
CaCI:.  50  inM  \i  5  mM  Tri.sC/)  (B)  and  (C)  Luminous  images 

(reflecting  Ca2+  distribution}  tn»n  photons  ciniitcil  by  recombinanl  ae- 
iliKinn  inside  the  transected  axon  in  i  ti  timing  2-min  collection  interval* 
at  13  and 47  min post-severence.  I  mi\iiirco/ti5"c>  act/norm  (recombinant) 
and  15"i'  fluorescein-cenjugated  aeinionn  was  pressure-injected  with  a 
microelectrode  inl<>  the  axon  [oiicctcd  volume  /30  nl)  wa.s  <0.5"~r.  of  the 
entire  axoplasmic  volume]  The  infection  procedure  and  the  detection 


by  the  rapid  formation  ( <  1  h)  of  a  seal  (2)  is  one  such  mechanism; 
but.  some  axons  lack  the  ability  to  seal  rapidly,  yet  remain  func- 
tional for  hours  (2).  We  now  describe  the  spread  of  Ca2+-induced 
aequorin  luminescence  and  the  decay  of  injury  current  density 
(I,)  carried  by  Ca2+  (I,Ca)  in  transected,  unsealed  giant  axons  (GAs) 
excised  from  squid  (Loligo  pealei). 

We  assessed  Ca2+  movement  at  the  cut  end  of  unsealed  GAs 
(Fig.  1  A),  beginning  5  min  after  axonal  transection,  by  analyzing 
accumulated  photon  (luminous)  images  (Fig.  IB,  C)  emitted  by 


probe- 


neiai 

<\ 

1.0 

iCa 

fl 

Si                    °5 

\ 

^HHi 

1  0 


05 


0     10       30       50         0     10        30        50 
F         Time  (min)  G         Time  (min) 


system  tor  luminescence  and  fluorescence  were  as  described  previously 
(3).  Outlines  oj  /he  axon  shown  in  (A),  obtained  within  minutes  ol  each 
accumulated  photon  image,  were  drawn  in  while  on  luminous  images 
(B)-(D)  Horizontal  red  lines  mark  the  region  of  "spent "  aequorin.  (D). 
Fluorescence  offluorescein-conjugated  aequorin  at  50  min  post  severance 
showing  thai  aci/iionn  was  distributed  uniformly  in  the  GA.  IS  =  aeauunn 
mieciion  site  Scale  and  color  bar  =  1  mm  anil  Ca2*  concentration  /roin 
background  (black)  to  saturation  (red).  (E.  F.  G)  Ca:*  movement  into 
the  cut  end  ol  a  si/Hid  GA  measured  with  an  extracellular  Co?* -selective 
vibrating  probe  IF.)  Current-density  vector  (arrow)  determined  at  the  lip 
ol  a  Ca:t -\elecii\-e  vibrating  probe  at  t  he  cut  end  of  a  GA  rn.-l.Sir,  15  mm 
alter  traiiseeiion  The  one-dimensional  Ctf+-selective  vibrating  probe, 
tilled  with  a  calcium  i>  mophore  (Fluka  Chemika  #21048).  was  as  described 
previously  ( ~l  .  I  mm  length  =  32  ftA/cnr;  300  urn.  (F)  and(G)  Temporal 
decline  ol  m/i/n  current  density.  /,.  relative  to  the  initial  value  5  mm 
alter  transeclion  (closed  circles:  n  =  5)  and  of  lite  portion  of  injury  current 
density  carried  byCa2*.  /,<.•„,  relative  to  the  initial  value  ?  mm  alter  Iran- 
section  (closed  circles,  n  =  51  determined  in  the  511-inin  interval  following 
transeclion  ol  (i.  is  I'aned  axonal  segments,  obtained  from  the  transeclion 
ol  a  GA.  \rere  measured  simultaneously,  one  with  a  nonselective  probe 
I/J  and  the  other  with  a  Ccf* -selective  probe  (IlCJ.  All  determinations 
were  made  parallel  lo  the  longitudinal  axis  of  axons  and  with  the  lip  ol 
the  probe  placed  21r>  pm  Irom  the  cut  end. 


CALCIUM 


209 


aequorin-loaded  axons  (3).  Intact  GAs  in  artificial  seawater 
(ASW)  emitted  light  only  where  aequorin  had  been  injected  into 
an  axon  (IS.  Fig.  IB).  In  Ca2+-free  ASW.  the  GA  displayed  no 
luminescence  (n  =  4).  These  data  suggest  that  Mg2+  did  not 
contribute  to  luminescence  (4).  and  that  the  light  emitted  from 
GAs  severed  in  ASW  resulted  from  the  movement  of  Ca2+  into 
the  GAs  rather  than  from  an  injury-induced  release  of  intra- 
axonal  stores  of  Ca2+. 

After  transection  of  aequorin-loaded  GAs,  a  luminous  band 
moved  slowly  into  the  cut  end  (//  =  19).  At  13  min  after  tran- 
section of  a  representative  axon.  the  center  of  the  band  was 
364  ^m  from  the  cut  end  (Fig.  IB);  at  47  min  after  transection 
(Fig.  1C),  the  band  was  424  /*m  from  the  cut  end  (60  nm  from 
its  location  at  13  min).  When  the  rate  of  movement  of  the  lu- 
minous band  was  analyzed  at  2-min  intervals,  the  band  velocity 
was  0.35  nm/s  at  10  min  after  transection  and  0.25  nm/s  at 
35  min  after  transection.  After  axonal  shortening  (0.20  nm/s)  in 
the  region  of  the  luminous-band  movement  (within  1  mm  of 
the  cut  end)  was  taken  into  account  ( 5 ),  the  actual  rate  of  move- 
ment of  the  luminous  band  (reflecting  the  spread  of  extracellular 
Ca2+  into  the  transected  axon)  with  time  was  significantly  re- 
tarded (from  0.15  ^m/s  to  0.05  nm/s)  at  10  and  35  min,  re- 
spectively, following  transection. 

As  a  second  measure  of  retardation  of  the  spread  of  Ca2+  into 
the  cut  end  of  a  GA,  we  used,  extracellularly  (Fig.  IE),  either  a 
nonselective  vibrating  probe  that  measured  the  density  of  injury 
current  produced  by  all  ions  (I,),  or  an  ion-selective  probe  that 
measured  the  density  of  injury  current  produced  by  Ca2+  (IiCa) 
(6.  7).  To  compare  temporal  changes  in  I,  with  IiCa,  paired  seg- 
ments of  the  same  GA  (i.e..  the  two  axonal  segments  resulting 
from  a  transection)  were  always  measured  simultaneously,  one 
with  the  nonselective  probe  and  the  other  with  the  Ca2+-selective 
probe. 

In  the  50-min  interval  following  axonal  transection  in  four 
GAs,  inwardly  directed  I,  declined  75%  (Fig.  IF:  1 504.4  ±  108.2 
(SE)  MA/cm2  at  4  min,  to  341.4  ±  123.7  n\/cm2  at  50  min).  In 
the  first  10  min  following  transection,  I,  declined  22%.  The  large, 
persistent  I,  (341.4  ^A/cm2)  at  50  min  is  consistent  with  previous 
findings  (2)  that  GAs  do  not  seal  within  2.5  h.  Other  preliminary 


experiments  (data  not  shown),  with  an  Na+-selective  probe, 
demonstrated  that  most  of  I,  was  carried  by  Na+. 

In  the  50-min  interval  following  axonal  transection,  inwardly 
directed  IiCa  declined  97%  (Fig.  1G:  99.4  ±  23.6  /xA/cm2  at  4  min, 
to  3.5  ±  0.8  fiA/cnr  at  50  min).  In  the  first  10  min  following 
axonal  transection,  IlCa  declined  80%.  These  data  suggest  that 
Ca2+  movement  contributes  only  a  small  amount  (~1%)  to  I, 
at  post-severance  times  >20  min.  Furthermore,  in  separate  ex- 
periments (;;  ->.  when  the  ASW  was  replaced  with  Ca2+-free 
ASW.  at  post-sever^ i;L-e  times  >20  min.  I,  was  not  significantly 
different  from  the  cui  in  Fig.  IF. 

In  summary,  two  independent  measures — i.e..  the  decreased 
velocity  with  time  of  the  luminous  band  of  aequorin  and  the 
more  rapid  decay  of  IiCa  relative  to  the  decay  of  I, — both  suggest 
that  giant  axons  from  squid  possess  a  Ca2+-specific  mechanism 
capable  of  retarding  the  spread  of  Ca2+.  driven  by  the  high  ex- 
tracellular concentration  (10  mAf),  into  a  severed,  unsealed  cut 
end  minutes  after  transection. 

We  thank  Dr.  L.  F.  Jaffe  and  The  National  Vibrating  Probe 
Facility  for  discussions  and  the  use  of  facilities.  A.  Shipley  and 
E.  Karplus  for  technical  assistance,  and  Drs.  O.  Shimomura,  S. 
Inouye,  and  Prof.  Y.  Kishi  for  the  supply  of  aequorins.  Supported 
by  NIH  grant  NS31256  and  ATP  grant  003658-296. 


Literature  Cited 


1.  Schlaepfer,  \V.  W.,  and  R.  P.  Bunge.  1973.     /  Cell  Biol  59:  456- 
470. 

2.  Krause,  T.  L.,  H.  M.  Fishman,  M.  L.  Ballinger,  and  G.  D.  Bittncr. 
1994.     J.  Neurosci.  14(11)  part  1:6638-6651. 

3.  Miller,  A.  L.,  E.  Karplus,  and  I..  F.  Jaffe.  1994.     Methods  in  Cell 
Biology.  R.  Nuccitelli,  ed..  vol  40:  306-335,  Academic  Press,  NY. 

4.  Blinks,  J.  R.  1982.     Pp  1-38  Techniques  in  Cellular  Plmioloxy- 
Purl  II  Elsevier/North  Holland  Scientific  Publishers.  Amsterdam. 

5.  Todora,  M.  A.,  H.  M.  Fishman,  T.  L.  Krause,  and  G.  D.  Bittner. 
1994.     Neurti.ici.  l.llrs  179:  57-59. 

6.  Jaffe,  L.  F.,  and  R.  Nuccitelli.  1974.     J.  Cell  Biol  63:  6 14-628. 
Smith,  P.  J.  S.,  R.  H.  Sanger,  and  L.  F.  Jaffe.  1994.     Methods  in 
Cell  Biology.  R.  Nuccitelli.  ed.,  vol.  40:  1 15-134.  Academic  Press. 
NY. 


7 


Reference:  Biol.  Bull  189:  209-210.  (October/November.  1995) 

Effects  of  Exogenous  Heat  Shock  Protein  (hspTO)  on  Neuronai  Calcium  Flux 

Peter  J.  S.  Smith  (Marine  Biological  Laboratory),  Katherine  Hammar,  and  Michael  Tytell 


Understanding  the  molecular  and  cellular  responses  of  neural 
tissue  to  injury  has  been  a  driving  force  behind  a  large  body  of 
neuroscience  research.  Such  information  can  provide  the  bases 
for  strategies  designed  to  facilitate  repair  and  recovery  of  func- 
tion. One  promising  approach  involves  research  into  the  class 
of  proteins  known  as  heat  shock  proteins  (hsp).  This  study  uses 
an  hsp  belonging  to  a  family  of  characteristic  size:  70  kDaltons 
(hsp70).  These  molecules  appear  to  be  linked  to  cell  survival 
after  acute  metabolic  stress,  being  first  described  in  the  brain 


after  traumatic  injury  (1).  By  the  late  1980s  it  was  clear  that 
many  different  types  of  trauma  to  the  nervous  system  increase 
the  production  of  the  inducible  form  of  hsp70  (hsp,:  2).  Further 
evidence  suggested  that  elevated  hsp,  is  correlated  with  an  in- 
creased survival  of  neurons;  for  example,  transfection  mediated 
expression  of  human  hsp70i  protects  rat  dorsal  root  ganglion 
neurons  and  glia  from  severe  heat  stress  (3). 

Although  heat  shock  proteins  are  normally  intracellular  con- 
stituents, with  trauma  up-regulating  their  expression,  this  study 


210 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


has  taken  an  unconventional  approach — delivering  the  protein 
exogenously.  The  rationale  is  twofold.  First,  hsp  need  not  always 
be  synthesized  by  the  host  cell.  Glia.  for  example,  can  transfer 
hsp  across  the  axolemma  (4);  spermatozoa  are  provided  with 
hsp70  from  the  seminal  fluid  that  remains  associated  with  the 
outer  membrane  (5);  and  exogenous  application  of  hsp70  can 
have  such  profound  effects  as  protecting  the  retina  from  light 
damage  (6)  and  influencing  neuronal  survival  after  axotomy  (7). 
Secondly,  hsp  appears  to  insert  into  membranes,  inducing  chan- 
nels in  planar  bilayers  and  promoting  the  insertion  of  other  pro- 
tein molecules  by  altering  their  conformation  during,  as  well  as 
before  or  after  membrane  insertion  (8).  The  possibility  that  ex- 
ogenously applied  heat  shock  protein  70  might  alter  cellular  ion 
homeostasis  is  the  focus  of  this  study. 

We  have  used  two  methods  to  investigate  the  effect  that  ex- 
ogenous application  of  hsp70,  at  a  concentration  of  2.5  jtg-  ml~', 
might  have  on  cellular  ion  transport:  non-invasive  vibrating  ion- 
selective  probes  and  real-time  confocal  imaging.  The  emphasis 
has  been  on  calcium  ion  homeostasis.  Our  model  has  been  the 
aplysiid  bag  cell  neuron  cultured  by  methods  already  described 
(9).  Cells  were  grown  in  artificial  seawater  on  clean  glass  cov- 
erslips  (No.  2).  All  cells  were  examined  after  1  or  2  days  in 
culture.  Measurement  of  the  trans-membrane  calcium  flux  was 
done  as  previously  described  (10). 

Two  reporter  probes  for  studying  intracellular  free  calcium 
were  used,  Fluo-3-AM  or  Indo-1-AM  (Molecular  Probes,  Eugene, 
Oregon);  their  performance  was  indistinguishable,  and  Fluo-3- 
AM  results  will  be  discussed  here.  Cells  were  loaded  in  the  dark 
with  2  /^mol  •  1~'  Fluo-3-AM  dissolved  in  DMSO  for  30  min  at 
room  temperature.  Imaging  was  done  with  a  Nikon  RCM-8000 
confocal  microscope.  Laser  light  levels  were  minimized  to  avoid 
cellular  damage. 

All  experiments  were  conducted  in  artificial  seawater  (ASW) 
with  reduced  calcium  (50-100  jjmol  -1  ')  and  elevated  magne- 
sium (67  mmol-1  '). 

Resting  neurons,  cultured  in  ASW,  show  a  steady  state  mea- 
surable calcium  efflux  across  the  plasma  membrane  of  the  soma. 
as  measured  by  the  non-invasive  vibrating  calcium  selective 
probe.  This  flux  is  modulated  by  pharmacological  compounds, 
such  as  thapsigargin  (R.  ).  Knox.  unpub.)  as  well  as  phorbol 
esters  and  cGMP  (11).  The  range  of  efflux  values  recorded  is 
remarkably  uniform,  normally  being  between  30-40  ^V,  equiv- 
alent to  a  flux  of  2-3  pmol  -cm  2  -s~'.  The  variation  in  micro- 
volts for  8  neurons  from  this  study  is  38  n\  ±  7  (mean  ±  standard 
deviation).  Preincubation  of  the  neurons  in  hsp70  (StressGen) 
for  2  h  prior  to  measurement  causes  a  dramatic  elevation  of  the 
measured  efflux  (n  =  7:  1 10  nV  ±  43).  This  elevated  level  of 
activity  remains  even  24  h  after  the  hsp  has  been  removed  from 
the  bathing  medium.  Shorter  incubation  times  of  30-60  min. 
followed  by  washout,  also  show  an  elevated  transmembrane  ef- 
flux. In  all  the  above  experiments  hsp70  was  removed  from  the 
medium  prior  to  the  measurement  of  transmembrane  calcium 
flux. 


Attempts  to  measure  the  onset  of  this  elevated  efflux  during 
the  initial  application  of  hsp70  were  frustrated  by  the  interaction 
of  hsp70  with  the  calcium-selective  liquid  membrane  tipping 
the  ion  probe.  The  electrode  is  tipped  with  a  30  ^m  column  of 
calcium  ionophore  cocktail  A  (FLUKA)  and  the  hsp70  must 
either  insert  into  the  lipophilic  carrier  or  interfere  with  the  ion- 
ophore itself. 

Imaging  carried  out  during  hsp  exposure  revealed  little  change 
in  free  cytosolic  calcium  levels  even  over  periods  of  30  min  to 
1  h.  The  implication  of  this  surprising  observation  is  quite  pro- 
found, in  that  the  marked  increase  in  transmembrane  soma  efflux 
is  not  a  response  to  a  pronounced  increase  in  the  free  cytosolic 
calcium  level.  Some  other  explanation  must  be  sought. 

As  yet  we  can  only  speculate  about  the  mechanism  by  which 
hsp70  modulates  the  transmembrane  calcium  efflux.  The  lack 
of  any  noticeable  increase  in  the  free-calcium  level  within  the 
cell  implies  a  direct  action  on  transmembrane  calcium  regulation. 
Heat  shock  proteins  and  calcium  are  clearly  related  in  that  a 
rise  in  intracellular  calcium  or  an  increase  in  transmembrane 
calcium  current  causes  hsp  induction,  as  with  Epstein-Barr  virus 
infection  (12).  We  seem  to  be  observing  a  quite  different  series 
of  events.  Because  elevated  cellular  hsp  correlates  with  cellular 
protection,  our  results  suggest  that  one  feature  of  the  protection 
mechanism  of  hsp  involves  altered  calcium  ion  homeostasis. 

This  research  was  supported  by  NIH  grant  No.  P41  RR01395 
funding  the  National  Vibrating  Probe  Facility — National  Center 
for  Research  Resources  (PJSS).  The  authors  are  indebted  to  John 
Dow  of  Nikon  Instruments  for  technical  assistance. 


Literature  Cited 

1    Currie,  R.  \V.  and  F.  P.  White.  1981.     Science  214:  72-73. 

2.  Mayer,  J.  and  I.  Brown,  eds.  1994.     Heal  Shock  Proteins  in  the 
Nervous  System.  Academic  Press  NY. 

3.  Uney,  J.  B.,  J.  N.  C.  Kew,  K.  Staley,  P.  Tyers,  and  M.  V.  Sofroniew. 
1993.     FEBS  Letts.  334:  313-316. 

4.  Sheller,  R.  A.,  M.  Tytell,  M.  Smyers,  and  G.  D.  Bittner.  1995.     ./ 
Neunisci.  Res.  41:  324-334. 

5.  Miller,  D.,  S.  Brough,  and  O.  al-Harbi.  1992.     Hum   Reproil.  7: 
637-645. 

6.  Tytell,  M.,  M.  F.  Barbe,  and  I.  R.  Brown.  1994.     /  Neiirosci  Rex. 
38:  19-31. 

7.  Tytell,  M.,  L.  Li,  and  L.  J.  Houenou.  1994.     Trans  Anwr  Soc. 
Neurncliem.  25:  187. 

8.  Alder,  G.  M.,  B.  M.  Austen,  C.  L.  Bashford,  A.  Mehlerl,  and  C.  A. 
Pasternak.  1990.     Bioscience  Rep.  10:  509-518. 

9.  Knox,  R.  J.,  E.  A.  Quattrocki,  J.  A.  Connor,  and  I..  K.  kaczmarek. 
1992.     \enron  8:  883-889. 

10.  Smith,  P.  J.  S.,  R.  H.  Sanger,  and  L.  F.  Jaffe.  1994.     Methods  Cell 

Bio/.  40:  115-134. 

1  1.  Vatnoah,  K..  and  P.  J.  S.  Smith.  1994.     Biol  Bull  187:  1043. 
12.  Cheung,  R.  K.,  and  H.  M.  Dosch.  1993.      I'irology  193:  700-708. 


NEUROBIOLOGY 


211 


Reference:  Biol.  Bull  189:  21  1-212.  (October/November, 


Regional  Differences  in  Directional  Response  Properties  of  Afferents 
Along  the  Saccule  of  the  Toadfish,  Opsanus  tau 

Peggy  L.  Edds-Walton  and  Richard  R.  Fay  (Marine  Biological  Laboratory) 


The  inner  ears  of  fishes  consist  of  three  otolithic  endorgans: 
the  saccule.  lagena,  and  utricle.  In  most  fishes  the  saccule  is 
believed  to  be  the  primary  auditory  endorgan,  responding  to 
acoustic  particle  motion,  sound  pressure,  or  both,  depending  on 
species  ( 1 ).  Previously,  we  investigated  the  frequency  and  direc- 
tional response  characteristics  ofafferents  from  the  rostral  saccule 
to  determine  whether  the  direction  of  a  sound  source  could  be 
encoded.  We  found  that  most  afferents  are  highly  directional, 
with  widely  ranging  "best  directions"  (2).  We  have  extended  our 
study  to  determine  whether  variation  in  three-dimensional  di- 
rectional response  characteristics  can  be  related  to  the  morpho- 
logical orientation  of  the  sensory  hair  cells  along  the  saccule. 

The  toadfish  saccule  lies  along  the  longitudinal  axis  of  the  fish 
and  is  slanted  and  titled  slightly  away  from  a  parasagittal  plane. 
The  orientations  of  the  hair  cells  shift  gradually  along  the  rostro- 
caudal  axis  of  the  saccule  (Fig.  1A).  The  saccular  nerve  has  di- 
visions that  vary  in  size  and  location,  but  distinct  rostral  and 
caudal  bundles  are  always  present,  and  a  distinct  middle  bundle 
is  often  present.  We  recorded  extracellularly  from  afferents  near 
their  point  of  exit  from  the  saccule,  in  the  most  rostral  location 
of  the  saccular  nerve,  the  middle  bundle,  or  the  most  caudal 
location  of  the  saccular  nerve.  For  each  afferent,  we  measured 
sensitivity  and  directionality  in  three-dimensional  space. 

After  surgery  to  reveal  the  saccular  nerve,  the  lightly  anesthe- 
tized (aminobenzoic  acid,  methanesulfonate  salt,  Sigma)  and 
paralyzed  (pancuronium  bromide.  Sigma)  toadfish  was  placed 
in  a  specially  designed  head-holder  in  a  saltwater-filled  cylinder. 
We  made  extracellular  recordings  of  the  activity  of  saccular  af- 
ferents while  oscillating  the  toadfish  (=  particle  motion  stimu- 
lation). Linear  oscillations  were  produced  by  paired  orthogonal 
shakers  in  the  horizontal  plane  and  a  vertical  shaker.  Inputs  to 
the  three  shaker  channels  were  computer-controlled  to  produce 
sinusoidal  motion  along  six  axes  (0°,  30°,  60°,  90°.  120°,  150°) 
in  both  horizontal  and  mid-sagittal  planes. 

Afferents  in  the  rostral  bundle  (Fig.  IB)  tended  to  have  best 
elevations  below  45°  (mean  =  23°).  with  a  large  variety  of  best 
azimuths.  These  cells  probably  innervated  hair  cells  found  in 
the  most  rostral  saccule,  based  on  the  locations  of  our  electrodes 
and  their  directional  characteristics.  Afferents  in  the  middle 
bundle  (Fig.  1C)  tended  to  have  the  highest  best  elevations  (mean 
=  49°)  and  the  least  variable  best  azimuths,  which  is  consistent 
with  the  less  variable,  dorsal-ventral  hair  cell  orientations  in  the 
middle  saccule.  The  lowest  elevations  in  these  data  (0- 15°)  may 
represent  afferents  that  were  innervating  hair  cells  in  the  more 
rostral  region  of  the  middle  saccule  (large  arrow.  Fig.  1  A).  Lastly, 
the  responses  of  caudal  afferents  (Fig.  ID)  resembled  those  in 
the  rostral  bundle  (mean  =  35°),  which  is  consistent  with  the 
similaritv  in  hair  cell  orientations  in  the  two  areas. 


A. 


ROSTRAL 


Front 


DORS  A  L 


CAUDAL 

Figure  1.  (A)  The  saccule  «l  the  toadfish  (modified  from  5).  The 
arrows  represent  i/ie  hair  cell  oriental  inn  and  the  presumed  best  direction 
for  that  region  of  the  saccule.  huwil  on  what  is  known  about  the  electro- 
physiology  of  hair  cells.  The  large  arrow  indicates  the  edge  of  the  "middle" 
region  oj  the  saccule  (the  open  areas  were  lost  during  preparation  for 
electron  microscopy.)  (B-D)  Polar  plots  of  best  a:imnths  and  elevations 
for  individual  saccular  afferent  from  /he  rostral  (B).  middle  (C).  and 
caudal  (D)  bundles  of  the  saccular  nerve.  The  fish  cartoon  is  in  the  center 
<>l  the  thill  cned  "globe":  the  "North  Pole"  is  at  the  center  of  the  smallest 
circle,  and  I  he  perimeter  is  the  "equator.  "All  responses  were  arbitrarily 
plotted  in  the  "northern  hemisphere"  and  to  the  left  of  the  fish.  Open 
boxes  in  (A)  are  1994  data:  filled  boxes  are  current  data. 


The  most  sensitive  afferents  of  each  bundle  responded  to  dis- 
placements of  less  than  I  nm.  This  displacement  is  approxi- 
mately equal  to  that  of  the  mammalian  basilar  membrane  at 


212 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


the  threshold  of  hearing  (3).  The  neurally  coded  output  of  the 
toadfish  saccule  appears  sufficient  to  account  for  sound  detection 
thresholds  (4)  and  could  be  used  to  compute  the  axis  of  particle 
motion. 

Our  protocol  was  approved  by  the  Institutional  Animal 
Care  and  Use  Committee.  This  project  has  been  supported 
by  Program  Project  Grant  #1PO1DCO  18737  from  NIH. 
NIDCD.  Thanks  to  Philip  Ording  for  help  with  trigonom- 
etry. 


Literature  Cited 

1.  Fay,  R.  R.,  and  A.  N.  Popper.  1975.     J.  E\p.  Biol.  62:  379-388. 

2.  Fay,  R.  R.,  P.  L.  Edds-\Valton,  and  S.  M.  Highstein.  1994.     Biol. 
Bull.  187:  259-260. 

3.  Sellick,  P.  M.,  R.  B.  Paluzzi,  and  B.  M.  Johnstone.  1982.     /  Acoitsl. 
Sac.  Am.  72:  131-141. 

4.  Fish,  J.,  and  G.  Offutt.  1971.     J  Acoi/sl  Sac  Am  51:  1318-132' . 

5.  Edds-\\  alton,  P.  L.'and  A.  N.  Popper.  1995.     Ada  Zoo/.  76:  257- 
265. 


Reference:  Biol  Bull  189:  212-213.  (October/November,  1995) 


Optical  Imaging  of  Intrinsic  Signals  from  the  Limulus  Optic  Nerve 

Estela  ^'.  O'Brien  (The  Rockefeller  University)  and  Robert  B  Barlow 


We  report  here  visually  evoked  changes  in  the  reflectance 
properties  of  the  optic  nerve  of  the  horseshoe  crab  (Limulus 
polyplicmtis).  Because  this  signal  results  from  small  changes  in 
the  optical  properties  of  neural  tissue  and  can  be  imaged  optically 
without  the  application  of  exogenous  dyes,  it  is  called  an  intrinsic 
signal.  Such  signals  provide  a  minimally  invasive  means  of  sam- 
pling the  activity  of  many  neurons  simultaneously  (1).  First 
measured  by  Hill  and  Keynes  (2),  intrinsic  signals  are  small 
(fractional  changes  of  ~  1/10,000  of  reflected  light)  and  have 
multiple  sources  that  are  thought  to  include  light-scattering 
changes  in  response  to  stimulation,  metabolism-linked  changes 
associated  with  changes  in  the  transition  states  of  intrinsic  chro- 
mophores,  and  changes  in  blood  volume.  Past  studies  by  Hill 
and  Keynes  (2)  and  Cohen  el  al.  (3)  revealed  that  light-scattering 
changes  occur  during  and  after  electrical  stimulation  in  excised 
crab  and  squid  axons.  The  time  course  of  these  changes  in  in- 
vertebrate nerve  preparations  during  continuous  stimulation  is 
slow.  Their  initial  rise  above  baseline  lags  behind  the  stimulus 
by  300-500  ms  and  is  similar  to  the  time  course  of  the  visually 
evoked  signal  measured  in  mammalian  cortex  in  vivo,  which 
presumably  contains  multiple  components  ( 1 ).  After  the  offset 
of  electrical  stimulation,  however,  the  light-scattering  signals  of 
excised  invertebrate  preparations  decay  much  slower  than  those 
recorded  in  response  to  visual  stimuli  in  mammalian  cortex  in 
vivo. 

We  have  studied  the  spatial  extent  and  the  time  course  of 
both  electrically  and  visually  evoked  activity  in  the  excised  Lim- 
ulus optic  nerve.  Figure  1  illustrates  the  optical  imaging  of  in- 
trinsic signals  in  the  optic  nerve  of  the  lateral  eye  of  the  horseshoe 
crab.  The  excised  nerve  was  bathed  in  a  Limuliix  Ringers  solution 
(430  mAf  NaCl.  !0  mA/  KC1,  10  mM  CaCN.  10  m.W  MgCl:, 
20mA/MgSO4.  100  mMHEPES,  100  mA/TES)  and  stimulated 
with  a  Grass  stimulator  and  stimulus  isolation  unit  via  a  suction 
electrode  attached  to  one  end  of  the  nerve.  A  stabilized  DC 
tungsten  light  source  illuminated  the  nerve  with  600-nm  light. 


and  a  cooled  charge-coupled  device  (CCD)  camera  (Photomet- 
ries) acquired  images  of  the  nerve  that  were  synchronized  to  the 
stimulation  (10-Hz  train  of  1-ms  pulses).  In  most  cases,  a  re- 
cording suction  electrode  monitored  the  compound  action  po- 
tential at  the  other  end  of  the  excised  nerve.  Once  we  detected 
the  compound  action  potential,  we  crushed  the  nerve  midway 
between  the  two  electrodes  and  measured  the  spatial  extent  of 
optical  signals  generated  by  the  nerve. 

In  a  second  experiment,  we  excised  the  retina  together  with 
a  2-cm  length  of  nerve  and  placed  the  preparation  in  a  bath  with 
a  light-impermeable  shroud  that  optically  isolated  the  nerve  from 
the  retina.  Visual  stimulation  consisted  of  a  bright  white  light 
( 10  ms  flash  duration,  1-Hz  flash  repetition)  aimed  at  the  retina 
through  a  light  pipe  and  diffuser.  As  in  the  first  experiment,  we 
synchronized  the  collection  of  optical  data  to  the  stimulation 
and  analyzed  the  optical  data  with  extensions  of  Karhunen-Loeve 
principal  components  analysis  to  extract  spatial  maps  of  the 
intrinsic  signals  and  their  normalized  time  course  (4). 

Figure  I A  is  an  image  of  the  Limulus  optic  nerve  with  a  crush 
in  the  center  as  captured  by  the  CCD  camera.  Figure  1 B  is  the 
activity  map  of  the  same  nerve  in  response  to  electrical  stimu- 
lation. Note  that  the  activity  is  localized  to  the  region  proximal 
to  the  crush,  and  the  mean  intensity  per  pixel  proximal  to  the 
crush  is  a  factor  of  2  greater  than  that  distal  to  the  crush.  We 
found  that  visual  stimulation  also  evokes  intrinsic  signals  in  the 
optic  nerve,  and  we  measured  the  time  course  for  both  the  elec- 
trically and  visually  evoked  activity.  The  normalized  time  course 
of  the  signals  evoked  in  the  nerve  by  10  s  of  electrical  stimulation 
(Fig.  1C)  is  similar  to  that  of  the  electrically  evoked  light-scat- 
tering changes  measured  by  Cohen  and  Keynes  (5).  Figure  ID 
shows  the  normalized  time  course  of  intrinsic  signals  in  response 
to  visual  stimulation.  These  signals  can  be  fully  accounted  for 
by  light-scattering  changes.  The  time  course  of  the  return  to 
baseline  after  stimulus  offset  (Fig.  1C)  is  slower  than  that  mea- 
sured in  mammalian  cortex. 


NEUROBIOLOGY 


213 


Figure  I.  (A)  A  single  frame  of  optic  nerve  imaged  under  600-nm 
illumination.  The  region  of  the  crush  (demarcated  with  white  lines)  il- 
luminates poorlv  and  i.s  therefore  darker  than  the  rest  of  the  nerve.  (B) 
The  activity  mar  of  the  .same  nerve  during  electrical  stimulation.  Data 
sets  composed  of  20  frames,  each  with  a  2-s  exposure,  were  collected 
during  periods  chosen  randomly  in  which  the  nerve  was  either  stimulated 
or  unslimulated.  The  tissue  was  allowed  to  return  to  baseline  tor  30  s 
hetore  the  ncv.1  data  set  was  collected.  H7i«i  the  nerve  was  stimulated, 
the  reflectance  of  the  active  regions  decreased  Principal  components, 
which  represent  the  best  fit  to  the  data,  were  used  to  generate  the  activity 
map.  The  strength  of  activity  is  denoted  by  the  grey  level  in  the  image: 
a  bright  pixel  denotes  a  decrease  in  reflectance.  Activity  appears  to  be 
limited  to  the  region  proximal  to  the  crush.  (C)  The  time  course  of  elec- 
trically evoked  intrinsic  signal  activity  during  and  after  10  (solid  squares) 
and  100  iterations  (open  circles)  of  a  brief  (10s)  stimulation.  The  peak 
(normalized  to  - 1.0)  occurs  after  the  offset  of  stimulation,  and  the  return 
h  i  baseline  is  very  slow.  (D)  The  normalized  time  course  of  visually  evoked 
intrinsic  signal  activity 


o 
Z 


D 


0.0- 
-02- 
-0.4- 

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Electrically-Evoked  Response 


STIMULATION 


10 


20 


30 


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40 


Visually-Evoked  Response 


V— -*»*• 


STIMULATION 


0  10  20  30 

Time  (seconds) 


40 


Our  results  indicate  that  optical  intrinsic  signals  can  be  re- 
corded from  visual  pathways  in  the  invertebrate  nervous  system. 
These  intrinsic  signals  should  be  fruitful  for  studying  spatially 
localized  regions  of  activity  and  for  differentiating  the  variables 
which  contribute  to  the  intrinsic  signal. 

This  work  is  supported  by  NSF  grant  BNS9309539  and  NIH 
grants  EY06476,  EY00667.  and  MH49741.  E.  V.  O'Brien  is  a 
Grass  Fellow  at  the  Marine  Biological  Laboratory,  Woods  Hole. 


Literature  Cited 

1.  Grinvald,    A.,    R.    D.    Frostig,    E.    Lieke,    and    R.    Hildesheim. 
1988.     Physiol.  Rev  68:  1285-1366. 

2.  Hill,  D.  K.,  and  R.  D.  Keynes.  1949.     /  Physiol.  108:  278-281. 

3.  Cohen,  L.  B.,  R.  D.  Keynes,  and  B.  Hille.  1968.     Nature  218:  438- 
441. 

4.  Sirovich,  L.,  and  R.  M.  Everson.  1992.     Int  J  Supercomp.  App. 
6(1):  50-68. 

5.  Cohen,  L.  B.,  and  R.  D.  Keynes.  1971.     J.  Physiol.  212:  259-275. 


Reference:  Biol  Bull  189:  213-215.  (October/November,  1995) 

Limulus  Is  Tuned  into  Its  Visual  Environment 

C.  L.  Passaglia,  F.  A.  Dodge,  and  R.  B.  Barlow  (Syracuse  University.  Syracuse,     'ew  York  13244) 


Every'  spring  millions  of  horseshoe  crabs  invade  the  shallow 
waters  along  the  eastern  coast  of  North  America  in  search  of 
mates.  Behavioral  studies  show  that  male  crabs  use  vision  to 
find  mates,  whereas  female  crabs  use  vision  to  avoid  other  nesting 
crabs  ( 1,  2,  3).  Horseshoe  crabs  reliably  detect  one  another  under 
a  variety  of  environmental  lighting  conditions  despite  differences 
in  the  contrast  of  their  carapaces.  They  must  also  cope  with 
visual  interference  from  water  turbidity,  seaweed,  fish,  sandbars. 


etc.  Under  these  conditions,  it  is  remarkable  that  male  crabs 
can  detect  black  and  grey  cylindrical  targets  of  similar  size  and 
contrast  as  females  equally  .oil  day  and  night  (3).  Limulus  ap- 
pears to  achieve  such  visual  performance  by  tuning  into  the 
natural  fluctuations  of  light  in  its  environment. 

How  do  males  detect  low-contrast  objects  such  as  a  grey  target 
or  a  female  crab  of  light  carapace  (Fig.  1A)?  We  approach  this 
question  by  recording  the  spike  discharges  of  single  optic  nerve 


214 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


A. 


B. 


PH 


Time  (s) 


10     0 


Time  (s) 


10 


Environment 


> 

u 


0 


Frequency  (Hz) 


Eye 


10 


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Frequency  (Hz) 


Figure  1 .  (A)  Tup:  Images  of  a  hlack  target,  grey  large!,  and  female  horseshoe  crab  taken  with  an  underwater 
videocamera  mounted  on  a  crab  and  aligned  in  the  direction  ot  view  of  the  recorded  ommatidium.  Nonce 
the  bands  of  light  reflected  from  the  grey  target  and  female.  Bottom:  The  optic  nerve  responses  recorded  from 
single  ommalidia  as  the  crab  moved  past  the  above  targets.  The  average  instantaneous  firing  rales  (FR) 
when  the  ommalidnim  was  not  viewing  the  large!  were  — 14  and  —II  impulses  per  second  for  the  left  and 
right  experiments  respectively.  (B)  Top:  Power  spec/mm  of  the  light  signal  reflected  from  the  grey  target. 
Bottom:  Power  .spectrum  o/  the  train  ot  optic  nerve  impulses  recorded  from  a  single  ommatidium  viewing  the 
grey  target  Notice  that  the  eye  amplifies  the  environmental  light  signals  in  the  2-4  H:  range  while  attenuating 
I/lose  at  lower  frequencies  for  the  purpose  of  comparison,  the  power  spectra  of  the  light  signal  and  spike 
train  were  plotted  (bin  width  ot  0  3  Ilii  on  a  common  ordinale  scale  after  normalization  by  the  square  of 
their  respective  means. 


10 


fibers  while  a  crab  is  moving  freely  in  the  ocean  or  is  pulled 
along  a  track  (4).  We  mount  a  small  underwater  camera  above 
the  eye,  align  it  with  the  optic  axis  of  the  recorded  unit,  and 
record  what  the  crab  sees  as  it  passes  by  an  object  of  known 
contrast.  We  store  the  images  and  spikes  on  videotape  for  sub- 
sequent analysis  with  NIH-Image  and  other  software  installed 
in  a  660AV  Macintosh  computer. 

Black  and  grey  targets  modulate  the  firing  rate  of  optic  nerve 
fibers  as  they  move  past  the  eye  (Fig.  1  A).  Despite  their  difference 
in  contrast,  both  targets  evoke  sizeable  responses  consistent  with 
the  male's  ability  to  find  them  (2).  High-contrast  black  targets 
decrease  the  firing/ate  of  optic  nerve  fibers,  whereas  low-contrast 
grey  targets  generally  increase  the  firing  rate  in  a  quasi-periodic 


manner.  Such  peculiar  responses  to  grey  targets  in  the  ocean  are 
unlike  those  evoked  by  uniform  large-field  stimuli  in  the  labo- 
ratory. 

The  source  of  the  large  periodic  responses  to  low-contrast 
grey  targets  appears  to  be  the  natural  fluctuations  of  light  in  the 
animal's  underwater  environment.  As  waves  move  overhead, 
they  focus  light  onto  the  sandy  bottom  that  sweep  across  the 
scene  at  a  rate  of  2-4  Hz.  The  flickering  light  highlights  reflective 
surfaces  against  the  murky  background  that  scatters  light  and 
degrades  the  appearance  of  objects.  As  a  result,  grey  targets  and 
horseshoe  crabs  generate  bright,  quasi-periodic  signals  that  stim- 
ulate units  in  synchrony  with  the  traveling  overhead  waves  (Fig. 
1A).  Because  the  periodic  signals  also  move  through  space,  the 


NEUROBIOLOGY 


215 


activity  of  nearby  receptors  in  the  eye  that  see  an  object  becomes 
correlated  (5). 

The  lateral  eye  is  highly  sensitive  to  the  flickering  light  from 
the  overhead  waves.  A  large  component  of  the  light  reflected 
from  grey  targets  centers  at  ^2-4  Hz  which  is  the  peak  of  the 
temporal  transfer  function  of  the  eye  (Fig.  IB;  6,  7,  8).  Photo- 
transduction  mechanisms  set  the  underlying  shape  of  the  transfer 
function,  which  two  inhibitory  processes  then  sharpen  and  am- 
plify (6,  7).  As  a  result,  the  power  spectrum  of  the  spike  train 
recorded  from  receptors  viewing  the  grey  target  grows  consid- 
erably in  the  range  of  2-4  Hz  (Fig.  IB). 

In  this  paper  we  show  that  a  consideration  of  the  natural  en- 
vironment of  an  animal  can  lead  to  a  better  understanding  of 
its  visual  system.  The  Liinithts  eye  appears  to  be  adapted  to  a 
particular  feature  of  its  environment — the  flickering  light  re- 
flected off  the  carapace  of  a  potential  mate.  These  light  signals 
help  males  detect  a  female  irrespective  of  the  contrast  of  her 
carapace.  Frequency  tuning  of  vision  is  not  unique  to  horseshoe 
crabs  and  has  been  observed  in  many  animals,  such  as  cats  (9) 
and  humans  (10).  Perhaps  their  lighting  environment  also  de- 
serves a  closer  look. 


Supported   by   NSF  grant   BNS9309539   and   NIH   grants 
MH49741  and  EY00667. 

Literature  Cited 

1.  Barlow,  R.  B.,  L.  C.  Ireland,  and  L.  Kass.  1982.     Nature  296:  65- 
66. 

2.  Powers,  M.  K.,  R.  B.  Barlow,  and  I,.  Kass.  1991.     IV.v.  Neiirmci. 
1:  179-186. 

3.  Herzog,  E.  H.,  M.  K.  Powers,  and  R.  B.  Barlow.  1996.     I'is.  Neii- 
riKei.  in  press. 

4.  Herzog,  K.  H.,  C.  I,.  Passaglia,  S.  A.  Dodge,  N.  D.  I.evine,  and 
R.  B.  Barlow.  1993.     Biol.  Bull  185:  307-308. 

5.  Dodge,  F.  A.,  D.  M.  Porcello,  S.  A.  Dodge,  E.  Kaplan,  and  R.  B. 
Barlow.  1994.     Biol  Bull  187:  261-262. 

6.  Ratliff,    F.,    B.    \V.    Knight,    J.   Toyoda,    and    H.    K.    Hartline. 
1967.     Science  158:  392-393. 

7.  Knight,  B.  VV.,  J.  Toyoda,  and  F.  A.  Dodge.  1970.     ./  Gen.  Phy.tiol 
56:421-437. 

8.  Batra,  R.  and  R.  B.  Barlow.  1990.     ./.  Gen.  Plminl  95:  229-244. 

9.  Frishman,  L.  J.,  A.  \V.  Freeman,  J.  B.  Troy,  D.  E.  Schweitzer-Tong, 
and  C.  Enroth-Cugell.  1987.     J.  Gen.  Physiol  89:  599-628. 

10.  Robson,  J.  G.  1966.     J.  Opt  Soc  Am.  56:  1  141-1  142. 


Reference:  Bid.  Bull.  189:  215-216.  (October/November,  1995) 

Flutter-Like  Response  in  Visual  Cortex  of  the  Semi-Isolated  Turtle  Brain 

James  C.  Prcclitl  (Marine  Biology  Research  Div.,  Scripps  Instil ut ion  of  Oceanography, 
University  of  California  San  Diego,  La  Jolla,  California  92093-0202) 


There  is  increasing  evidence  that  high-frequency  synchroni- 
zation of  specific,  distributed  neuron  populations  is  a  reliable 
correlate  of  some  forms  of  sensory  processing!  1,  2)  and  of  some 
attentive  sensori-motor  behaviors  (3,  4,  5).  The  function  and 
fine  structure  of  such  responses  is,  however,  still  unclear.  In  the 
turtle,  every  salient  change  in  retinal  input,  whether  due  to  a 
stimulus  or  self-induced  by  visual  orienting,  is  correlated  with 
widely  distributed  20-Hz  field  potentials  in  its  visual  cortex  (6). 
The  oscillation  has  a  spindle  form  and  its  amplitude  and  fre- 
quency modulates  with  a  slow  potential  of  <4  Hz.  Although  this 
response  represents  increased  coherence  in  the  1 5-  to  25-Hz  band 
between  spatially  separated  loci,  recent  analyses  with  linear  elec- 
trode arrays  show  that,  along  the  rostro-caudal  axis,  systematic 
phase  lags  are  observed  between  most  waves  (7).  The  phase  lags 
change  from  cycle  to  cycle  and  represent  velocities  between  0.05 
and  0.3  m/s.  Synchronous  cycles  also  occur  in  each  response. 
Although  this  response  has  been  called  an  oscillation,  its  complex 
temporal  and  spatial  features  suggest  that  a  better  descriptor 
would  be  the  term  "flutter"  (i.e..  a  rapid,  nonstationary  undu- 
lation). Here  I  report  that  a  flutter-like  response  is  also  observed 


with  visual  stimulation  in  a  semi-isolated  brain  preparation 
(epipial  1 1 -electrode  linear  array,  250  ^m  spacing).  The  flutter- 
like  response  is  contrasted  with  a  more  regular  and  synchronous 
type  of  oscillation  that  was  induced  in  three  of  the  preparations 
with  DC  electrical  stimulation. 

The  semi-isolated  brain  is  prepared  by  sectioning  cranial 
nerves  V-XII  and  the  spinal  cord  under  anesthesia  (NIH  guide- 
lines), followed  by  intravascular  perfusion  with  oxygenated  ar- 
tificial cerebrospinal  fluid  (8).  Although  this  preparation  lacks 
the  tonic  somatic  and  visceral  afference  of  an  intact  animal, 
moving  stimuli  (black  bar,  20-cm  distant,  8.5  cm/s)  still  induce 
a  flutter  response. 

Figure  1  (upper  traces)  shows  a  high-amplitude  segment  of 
visually  induced  responses  recorded,  2  mm  apart,  from  the  rostral 
(solid  line)  and  caudal  (dotted)  poles  of  the  visual  cortex.  An- 
notated time  intervals  indicate  wave  lags;  intermediate  lags  are 
also  recorded  from  the  middle  seven  electrodes  (data  not  shown). 
The  lower  superimposed  oscillatory  responses  in  Figure  1,  re- 
corded with  the  same  electrodes,  were  induced  after  9  s  of  anodal 
DC  stimulation  with  a  blunt  surface  electrode  placed  on  the 


216 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


Visual 


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O 


sync. 


Electrical 


15  ms 


0 


0.25 
Time  (ms) 


0.5 


Figure  1 .  I  'isuallv  (upper  traces)  and  electrically  (lower)  induced  local 
field  potential  oscillations  recorded  /nun  rostral  (solid  trace)  and  caudal 
(dotted,  2-mm  span)  electrodes  in  turtle  visual  cortex.  Annotation  on 
upper  traces  indicates  synchronous  and  lagged  cycles.  Lower  traces  show 
the  highly  regular  oscillation  recorded  from  the  same  loci  during  DC 
electrical  stimulation  (digital  band pass-5-100  H:;  1024  pts/s). 


lateral  margin  of  the  visual  cortex.  The  responses  to  electrical 
stimulation  in  the  three  animals  studied  were  complex  and  varied 
over  time,  but  they  all  included  1-2-s  intervals  in  which  more 
than  95%  of  the  waves  in  the  15-25  Hz  band  appeared  syn- 
chronous (i.e.,  <3  ms  lag/mm;  Fig.  1  lower  traces).  In  contrast. 
1-s  samples  from  visually  induced  responses  in  the  same  animals 
never  included  more  than  4  waves  of  near  synchrony  (i.e..  <25%) 
(Fig.  1 ,  top  traces).  These  results  indicate  that  although  the  turtle's 
visual  cortex  is  capable  of  spatially  synchronizing  along  the  ros- 
tro-caudal  axis  in  the  20-Hz  band,  visual  stimulation  in  both 
the  intact  and  semi-isolated  brain  induces  a  flutter-like  response 
that  is  dominated  by  systematic  phase  differences.  In  the  awake 
and  attentive  animal,  flutter  occurs  during  changes  in  visual 
processing.  The  possible  role  of  this  response  in  cognitive  pro- 
cessing remains  to  be  discovered. 

This  work  was  inspired  by  discussions  with  M.  Hofmann  and 
T.  H.  Bullock,  and  supported  by  NINDS  grants  to  THB. 

Literature  Cited 

1.  Freeman,  VV.  J.  1959.     J.  Neurophysiol.  22:  644-666. 

2.  Gray,  C.  M.  199-4.     /  Comput.  Neurosci.  1:  1 1-38. 

3.  Bouyer,  J.  J.,  C.  Tilquin,  and  A.  Rougeul.  1983.     EEC  Clin.  Neu- 
ropkysiol  55:  180-187. 

4.  Murthy,  V.  N.,  and  E.  E.  Fetz.  1992.     Proc.  Nail.  Acad.  Sci.  U.S.A. 
89:  5670-5674. 

5    Nicolelis,  M.  A.  L.,  L.  A.  Baccala,  R.  C.  S.  Lin,  and  J.  K.  Chapin. 

1995.     Science  268:  1353-1358. 

6.  Prechtl,  J.  C.  1994.     Pm:  Natl.  Acad.  Sci.  U.S.A.  91:  12467-12471. 
7    Prechtl,  J.  C.,  and  T.  H.  Bullock.  1995.     Proc.  2nd  Joint  Symp. 

Neural  Comp,  CalTech-UCSD  Institute  for  Neural  Computation. 

UC  San  Diego  (in  press). 
8.  Mori,  K.,  M.  C.  Nowycky,  and  G.  M.  Shepherd.  1981 .     ./.  Physiol. 

(Land.)  314:  28 1-294. 


Reference:  Biol  Bull  189:  216-218.  (October/November,  1995) 

The  Neurofilamentous  Network-Smooth  Endoplasmic  Reticulum  Complex 
in  Transected  Squid  Giant  Axon 

J.  Metuzals  (University  of  Ottawa).  H.  M.  Fishman,  and  I.  A.  Robb 


At  the  cytoskeleton-membrane  interface,  Ca:+-activated  pro- 
teases (calpain  system)  influence  important  signal  pathways  that 
control  the  diverse  behavior  of  intracellular  proteins  and  organ- 
elles  ( I ).  Characteristic  changes  of  membranes  in  association 
with  cytoskeletal  assemblies  occur  in  nervous  tissue  after  injury 
and  in  neurodegenerative  diseases  (2).  The  squid  giant  nerve 
fiber  provides  a  simple,  easily  utilized  model  with  which  to  ex- 
plore the  basic  mechanisms  of  cytoskeletal  interactions  with 
membranes.  We  have  used  transmission  electron  microscopy  of 
squid  (Loligo  pealei)  giant  nerve  fibers  to  identify  the  factors 
that  initiate  the  degenerative  events  that  follow  transection  in 
Ca2+-containing  seawater. 

The  fibers  were  fixed  in  a  standard  glutaraldehyde- 
formaldehyde  mixture  (Figs.  1A-C),  and  some  were  also  treated 
with  osmium  tetroxide-potassium  ferrocyanide  (Fig.  ID)  which 


enabled  the  observation  of  Ca2+-containing  structures  (3). 
Electron  micrographs  of  transected  fibers  were  compared  with 
many  series  of  electron  micrographs  of  normal  fibers  prepared 
under  different  conditions  of  fixation  and  embedding.  The  dy- 
namic behavior  of  the  neurofilamentous  network  (NFN)  as- 
sociated with  the  axolemma  and  with  the  subaxolemmal  cis- 
ternae  of  the  smooth  endoplasmic  reticulum  (SER)  in  the  squid 
giant  axon  has  been  suggested,  and  Ca:+  has  been  localized  in 
the  subaxolemmal  cisternae  (4).  The  NFN  is  altered  by  Ca2+- 
activated  proteases,  and  the  cleaved  products  reassemble  into 
characteristic  aggregates  (5).  The  sinuous  form  of  the  tubules 
constituting  the  SER  and  their  association  with  neurofilaments 
have  been  observed  (6);  the  authors  concluded  that  the  tubules 
were  primarily  regulating  the  concentration  of  Ca:+  in  the 
axoplasm. 


NEUROB1OLOGY 


217 


A  .-•> 


«& 


Figure  1.  f.4J  NFN-SER  complex  localned  in  the  ti\(iplii\nt  c/»sc  /o  //ic  CU/VCA  7"/n-  A'FA'  \iimit<nil\  I  lie  profiles  of  the  SER  and  exicmh  in  the 
lubes  (arrows),  y.104.000.  (Bi  Paracrystalline  helical  hraids  consisting  of  intercoiled  sirumh,  liher\  and  filaments.  Asswiuin»n  with  SER  (arrows) 
X75.000.  (C)  Rectangular  densities  ol  debris  of  filaments  and  SER  surrounded  by  modified  neurofilamenls.  X  15,000.  (D)  Twisted,  osmiophilic 
prolusions  ofNFN-SER  complex  deep  into  the  a\oplasm:  contact  ol  the  complex  with  the  axolcmma  (arrow)-  axon  (A):  Schwann  cell  (S).  Osmium 
tetm.\idc-polasstitin  lerroi yanide  •  1/10,000.  All  fibers  were  transected  10  min  he/ore  fixation 


Five  hallmarks  of  pathologically  modified  NFN-SER  complex 
were  identified  in  the  transected  axons:  (a)  Characteristic  aggre- 
gates of  SER  tubes,  surrounded  by  NFN  can  be  identified  close 
to  the  cortex  of  the  axoplasm  (Fig.  1  A).  The  tubes  are  not  parallel 
to  each  other  and  often  show  a  sinuous  form.  The  NFN  also 
appears  to  be  attached  to  the  tubes  (Fig.  1  A,  arrows),  (b)  Braided 
structures  composed  of  filaments,  in  association  with  NFN  and 
SER.  can  be  seen  (Fig.  IB),  (c)  Paracrystalline  domains  (not 
shown)  of  modified  NFN  surrounded  by  modified  SER  were 
observed  close  to  the  transection  site,  (d)  Rectangular  dense 


's  (Fig.  1C)  of  modified  NFN  and  SER  are  also  found  close 
to  the  cut  axonal  end.  Finally  (e),  osmium  tetroxide-ferrocyanide 
stained  finger-like  protrusions  of  the  NFN-SER  complex  (Fig. 
1  D)  extend  from  the  axoplasmic  cortex  deep  into  the  axoplasm. 
The  staining  of  these  structures  indicates  the  presence  of  Ca2+. 
A  gradual  transition  from  normal  NFNs  to  modified  ones  can 
be  observed  in  micrographs,  indicating  that  the  modified  fila- 
ments originate  from  the  NFN. 

Our    observations    of  the    structural    modifications    in- 
duced by  injury  (transection)  support  our  hypothesis  that 


218 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


the  NFN-SER  complex  is  highly  dynamic.  The  origin  of  the 
different  structures  of  the  NFN-SER  complex  in  transected 
axons  (Fig.  1A-D)  can  be  explained  by  the  activation  of  dif- 
ferent calpains  which  are  dependent  on  intracellular  pH  and 
Ca:+  concentration  (I).  The  formation  of  braided  para- 
crystalline  assemblies  and  dense  bodies  may  reflect  similar 
basic  mechanisms  operating  in  neurodegenerative  dis- 
eases (7). 

In  summary,  transection  induces  segregation,  modification, 
and  reassembly  of  the  components  of  the  NFN-SER  complex. 
Consequently,  we  conclude  that  the  NFN  and  the  SER  are  com- 
plex and  interactive  structures  that  are  likely  to  have  an  impor- 
tant role  both  in  response  to  injury  and  in  the  pathogenesis  of 
neurodegenerative  diseases. 

Supported  by  NIH  grant  NS31256. 


Literature  Cited 

1.  Nivin,  R.  A.,  K.  I.  Sailo,  K.  Crynspan,  \V.  R.  Griffin,  S.  katayama, 
T.  Honda,  P.  S.  Mohan,  P.  S.  Shea,  and  M.  Beermann.  1994.     Pp. 

77-9 1  in  Calcium  Hypothesis  <>]  AXIIIK  unit  Dementia  J.  F.  Disterhoft, 
W.  H.  Gispen,  J.  Traber,  and  J.  Khachaturian,  eds.  Ann.  NY  Acad. 
Sci.  vol  747.  Plenum,  NY. 

2.  Fishman,  II.  M..  and  J.  Metuzals.  1993.     Bin/  Bull .185:  292-293. 

3.  Hayat,  M.  A.  1989.     Pnncii'les  and  Techniques  of  Electron  Mi- 
crosaipy.  CRC  Press.  Inc.,  Boca  Raton,  Florida. 

4    Metuzals,  J.,  I.Tasaki,  S.  Terakawa,  and  D.  F.  Clapin.  1981.     Cell 
Tissue  Res.  221:  1-15. 

5.  Metuzals,  J.,  H.  Pant,  H.  Gainer,  P.  A.  M.  Eagles,  N.  S.  White, 
and  S.  Houghton.  1988.     Cell  Tissue  Res  252:  249-262. 

6.  Burton,  P.  R.,  and  L.  A.  Laveri.  1985.     J.  Neumsci.  5:  3047-3060. 

7.  Hill,  \V.  D.,  V.  M.-Y.  Lee,  H.  I.  Hurtig,  J.  M.  Murray,  and  J.  Q. 
Trojanowski.  1991.     J.  Comp.  N  enrol.  309:  150-160. 


Reference:  S/o/  Bull.  189:  218-219.  (October/November,  1995) 


Fluorescent  Labeling  of  the  Glial  Sheath  of  Giant  Nerve  Fibers 

C.  S.  Eddleman  (University  of  Texas  Medical  Branch),  C.  M.  Godell, 
H.  M.  Fishman,  M.  Tytell,  and  G.  D.  Bittner 


Glial-axonal  interactions  are  essential  for  the  maintenance 
and  regulation  of  vital  axonal  processes,  including  the  response 
to  injury  and  heat  shock  (1,  2).  Interactions  between  adaxonal 
glia  and  axons  have  been  reported  for  intact  and  severed  axons 
from  squid  (3)  and  crayfish  (4).  But  many  putative  glial  functions 
have  not  been  documented  in  living  tissue  because  the  adaxonal 
glial  layer  is  only  2-6  ^m  thick  and  is  not  easily  localized  with 
certainty  in  conventional  light  microscopy.  We  now  report  that 
the  cell  membrane  permeable  fluorogenic  substrate  calcein  ace- 
toxymethyl  (AM)  ester,  which  is  converted  to  an  impermeable 
and  fluorescent  derivative  by  esterases,  differentially  labels  the 
adaxonal  glial  cells  and  other  structures  in  the  glial  sheath  sur- 
rounding the  squid  giant  axon  (GA)  and  the  crayfish  medial 
giant  axon  (MGA). 

Giant  axons  from  squid  (Loligo pealei)  and  crayfish  (Procam- 
baris  clarkii)  were  excised  and  isolated  as  described  previously 
(3,  5).  Isolated  GAs  were  placed  in  0.5  ml  of  artificial  seawater 
(ASW:  430mA/  NaCl.  5  mA/  KC1,  10mA/  CaCK.  50mA/ 
MgCl2,  Tris-CI  buffered  to  pH  7.4  at  room  temperature);  and 
isolated  MGAs  were  placed  in  van  Harreveld's  solution  (vanH: 
205  mA/  NaCl,  5.4  mA/  KC1.  1 3.5  mA/  CaCI:.  2.6  mA/  MgCl:. 
10  mA/  HEPES  buffered  at  pH  7.35  at  room  temperature)  con- 
tained in  a  chamber  constructed  of  wax  or  petroleum  jelly  on  a 
glass  slide.  GAs  and  MGAs  were  pulse-loaded  with  calcein  AM, 
which  is  pH-  and  temperature-insensitive  under  physiological 
conditions,  by  incubation  for  10  min  in  ASW  or  vanH  containing 
1  fiM  calcein  AM,  followed  by  replacement  with  ASW  or  vanH 
without  calcein  AM.  A  glass  coverslip  was  placed  over  the  prep- 
aration to  minimize  evaporation  and  changes  of  osmolality.  Ax- 
ons were  viewed  with  a  laser-scanning  confocal  microscope 
(Zeiss,  LSM-410)  at  wavelengths  of  488  and  568  nm.  After  cal- 
cein AM  was  removed  from  the  external  bath,  observations  were 
made  for  up  to  2  h  without  significant  change  in  the  distribution 
of  fluorescence  intensity.  The  intensity  of  the  stored  digital  image 
was  scanned  with  Zeiss  LSM  software  (version  3.8). 


Within  5  min  after  the  removal  of  calcein  AM  from  the  bath, 
both  differential  image  contrast  (DIC,  Fig.  1A,  B)  and  confocal 
fluorescent  images  (Fig.  1C,  D)  were  obtained.  Fluorescence  in- 
tensity scans  of  axonal  cross  sections  showed  a  pronounced  dif- 
ference in  fluorescence  intensity  (Fig.  1C,  D)  between  the  axo- 
plasm  and  sheath.  In  separate  experiments  (not  shown),  the  axo- 
plasm  was  loaded  with  hydrophilic  fluorescent  dye  (Texas  Red) 
before  exposing  the  giant  axons  to  calcein  AM  for  10  min.  Cal- 
cein fluorescence  was  observed  as  a  thin  (6-12  ^m)  band  sur- 
rounding a  central  core  of  Texas  Red  fluorescence,  indicating 
that  the  sheath  surrounding  the  axons  was  predominantly  labeled 
by  calcein,  and  the  axoplasm  by  Texas  Red.  When  calcein  AM 
remained  in  the  bath  for  up  to  I  h,  the  fluorescence  intensity  of 
the  axoplasm  uniformly  increased  with  time.  Nevertheless,  even 
after  1  h,  calcein  fluorescence  was  always  significantly  lower  in 
the  axon  than  in  the  sheath.  One  interpretation  of  these  data  is 
that  the  esterase  level  in  the  axoplasm  is  much  lower  than  that 
in  the  adaxonal  glia  and  other  sheath  structures.  Images  of  the 
sheath  at  higher  magnification  (Fig.  IE,  F)  showed  that  the 
thickness  of  the  calcein  fluorescence  band  (glial  layer  of  the 
sheath)  was  about  2.5  ^m  in  the  GA  and  about  5  ^m  in  the 
MGA.  in  agreement  with  previous  values  obtained  by  electron 
microscopy  from  fixed  GAs  (3,  6)  and  MGAs  (7).  To  ensure 
that  calcein  AM  filled  the  adaxonal  glial  cells,  acridine  orange, 
a  fluorescent  nucleic  acid  dye,  was  also  used  to  localize  the  nuclei 
in  the  glial  cytoplasm.  The  resulting  fluorescence  band  (data  not 
shown)  corresponded  well  to  the  glial  localization  made  with 
calcein  AM  (Fig.  IE.  F). 

In  summary,  our  data  from  GAs  and  MGAs  indicate  that 
adaxonal  glia  and  other  structures  in  the  glial  sheath  may  have 
a  greater  esterase  activity  than  does  axoplasm,  thereby  producing 
a  more  intense  fluorescence  band  of  calcein  in  the  sheath  com- 
pared to  the  axoplasm.  Furthermore,  these  observations  provide 
evidence  that  calcein  AM  can  be  used  to  preferentially  label  the 
sheath,  in  particular  the  glial  cell  cytosol,  in  contrast  to  previous 


NEUROBIOLOGY 


219 


markers  of  gliul  cell  nuclei,  providing  a  significant  new  method 
for  evaluating  glial-axonal  interactions  in  the  living  preparation. 
We  thank  Louis  Kerr  and  Carl  Zeiss  Inc.  for  use  of  equip- 
ment and  facilities.  Supported  by  NIH  (NS31256)  and  ATP 
(003658-296). 


Literature  Cited 


PIN 


Figure  1 .  (A.  B)  D1C  image*  ol  a  squid  giant  axon  (GA)  and  a  crayfish 
medial  giant  a.\on  (MGA)  in  artificial  seawater  (ASW)  and  van  Harre- 
veld's  solution  (van  H),  respectively.  (C.  D)  Conlocal.  fluorescence  (at 


1 .  Lieberman,  E.  M.,  P.  I .  I  largittai,  and  R.  M.  Grossfeld.  1994. 
44:  333-376. 

2.  Bittner,  G.  D.  1991.     TINS  14(5):  188-93. 

3.  Krause,  T.  L.,  H.  M.  Fishman,  M.  Ballinger,  and  G.  D.  Bittner. 

1994.  J.  NciiniM-i  14:6638-6651. 

4    Eddleman,  C.  S.,  C.  M.  Godell,  H.  M.  Fishman,  and  G.  D.  Bittner. 

1995.  J.  Neitmchem.  64:  S25D. 

5.  Sheller,  R.,  and  G.  D.  Bittner.  1992.     Brain  Res  580:  68-80. 

6.  Villegas,  G.  M.,  and  R.  Villegas.  1968.     J.  Gen  Phvsiol.  51:  44s- 
60s. 

7.  Ballinger,  M.,  and  G.  D.  Biltner.  1980.     Cell  Tiss.  Res  208:  123- 
133. 


4SS  nml  image'-  of  the  corresponding  a\on  segments  in  (A)  and  (B).  Five 
minutes  after  calcein  AM  removal  from  the  bath,  intensity  (I)  along  scan 
line(L)  across  the  fluorescence  images  shous  deviations  above  background 
(baseline)  in  the  axon  anil  suggests  the  presence  ofaxoplasmic  esterases. 
albeit  at  a  loner  concentration  than  that  <>/  the  sheath  (marked  by  the 
fluorescence  band  and  the  two  peaks  in  I).  Thin,  horizontal  lines  constitute 
the  intensity  scale:  II.  20.  40.  60.  and  80%  maximum  intensity,  with  0 
corresponding  to  the  lowest  line  IK.  Ft  Higher  magnification  conjocal. 
fluorescence  (at  4SS  nml  images  ol  GA  and  MGA  segments.  The  glial 
thickness  is  ~2.5  pm  in  the  GA  and  —5  urn  in  the  MGA.  The  presence 
ol  calcein  in  other  sheath  sinictures  surrounding  the  GA  (E)  can  be  seen. 
\otice  thai  ?  mm  alter  pulse-labeling  these  a\ons  with  calcein  AM.  flu- 
orescence in  ihc  iiMin  IA.\I  and  extracellular  fluid  (ECF)  is  undeteclable. 
bin  the  sheath  (Sht  Ihiorcsces  intensely.  All  eonlocal  images  are  optical 
sections  (<5  n>n  thick)  through  the  longitudinal  a.\is  of  an  axon. 


Reference:  Btol.  Bull  189:  219-220.  (October/November.  1995) 


Effect  of  pH  Buffers  on  Proton  Secretion  from  Gastric  Oxyntic  Cells  Measured 
with  Vibrating  Ion-Selective  Microelectrodes 

Jefferv  R.  Demurest  (Department  of  Biology,  Jitniata  College,  Huntingdon,  PA  16652) 

and  James  L.  M.  Morgan 


Assessing  proton  pump  (H+/K+-ATPase)  activity  in  isolated 
mammalian  gastric  oxyntic  cells  has  been  possible  only  with 
indirect  methods,  such  as  the  measurement  of  oxygen  con- 
sumption, or  the  accumulation  of  weak  bases  (e.g.,  l4C-ami- 
nopyrine)  in  acid-containing  compartments  formed  within  the 
cells  by  the  sealing  off  of  the  cannalicular-secretory  membrane 
(1).  In  contrast,  non-mammalian  gastric  oxyntic  cells  do  not 
form  an  intracellular  cannaliculus  or  other  acid-accumulating 
compartment.  Oxyntic  cells  enzymatically  isolated  from  Nec- 
ttirtis  gastric  mucosa  retain  apical  to  basolateral  membrane  po- 
larity and  exhibit  the  pronounced  increase  in  apical  membrane 
area  and  the  activation  of  Cl  channels  that  characterizes  their 
stimulation  in  the  intact  mucosa  (2).  But  the  absence  of  anv 


acid-accumulating  compartment  prevented  us  from  determining 
whether  these  cells  were  actually  secreting  protons  in  response 
to  stimulation.  We  have,  therefore,  turned  to  vibrating  ion-se- 
lective electrodes  to  measure  proton  secretion  by  discrete  regions 
of  the  membranes  of  individual  isolated  Nectitrus  oxyntic  cells. 
Proton  selective  liquid  ion-exchange  (Fluka  Hydrogen  lonophore 
Cocktail  B)  microelectrodes  with  tip  diameters  of  1-2  ^m  and 
90%  response  times  of  <300  mS  were  vibrated  at  0.3-0.5  Hz 
over  a  distance  of  10-1  5  /urn  at  a  distance  of  ~  1  pm  from  the 
apical  or  basolateral  surface  of  isolated  oxyntic  cells.  Proton 
fluxes  were  calculated  from  the  difference  in  [H+]  measured  at 
the  extremes  of  the  electrode  excursion  and  the  rate  of  diffusion 
of  H+  in  the  bathing  media.  In  order  to  maintain  the  bulk  so- 


220 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


lution  at  a  stable  pH  near  normal  for  these  animals  (7.9  at  22°C) 
and  a  bicarbonate  level  sufficient  to  support  acid  secretion,  pro- 
ton fluxes  were  measured  in  lightly  buffered  ([HEPES]  =  1  mM) 
bicarbonate-containing  media  ([HCO-f  ]  =  5  m.\f).  However, 
the  inclusion  of  buffers  in  the  media  attenuates  the  extracellular 
proton  gradient  by  dramatically  increasing  the  already  high  mo- 
bility of  protons  in  aqueous  solution. 

Proton  secretion  from  a  cell  produces  a  gradient  of  concen- 
tration that  decreases  from  the  cell  surface  outward  into  the 
bathing  medium.  In  an  unbuffered  medium  the  concentration 
profile  of  this  gradient,  as  a  function  of  distance  from  the  cell 
surface,  constantly  changes  with  time  as  protons  accumulate  in 
the  media  adjacent  to  the  cell.  By  facilitating  the  diffusion  of 
protons,  inclusion  of  pH  buffers  in  the  solution  tends  to  dissipate 
the  gradient  more  rapidly  than  simple  diffusion  alone.  In  addition 
to  providing  a  stable  pH  in  the  bulk  solution  for  reference,  buffers 
also  stabilize  the  concentration  profile,  thus  producing  a  standing 
gradient.  To  calculate  the  total  flux  of  protons  (TJH )  from  vi- 
brating electrode  measurements  in  the  presence  of  buffers,  the 
diffusional  (DJH )  and  facilitated  (FJH )  movement  of  protons  down 
the  gradient  must  be  taken  into  account. 

TJH  =  DJH  +  FJH  ( 1 ) 

The  diffusional  flux  of  protons  (DJH)  is  equal  to  the  product  of 
the  diffusion  coefficient  for  protons  (DH )  and  the  proton  gradient 
(d[H+]/dx). 

DJH  =  DH-(d[H  +  ]/dx)  (2) 

and  the  facilitated  flux  can  be  shown  to  be  (3), 
FJH  =  DBI-(d[Bl]/dx)  +  DB:-(d[B2]/dx) 

+  •  •  •  +  DBn-(d[Bn]/dx)      (3) 

where  DB,  is  the  diffusion  coefficient  of  a  buffer  species  and 
(d[Bl]/dx)  is  the  gradient  of  concentration  of  the  proton  acceptor 
form  of  the  buffer.  Dividing  equation  (3)  by  (2)  yields: 

FJH/DJH  =  (DBI/DH)-(d[Bl]/d[l-r])  +  (DB:/DH) 

•<d[B2]/d[H+])  +  •  •  •  +  (DBn/DH)-(d[Bn]/d[H+]).      (4) 

The  terms  (d[Bl]/d[H+J),  (d[B2]/d[H+](.  etc.,  are  the  buffering 
capacities  (j3B,,  0B2,  etc.)  of  the  buffer  species,  which  can  be 
calculated  from  their  respective  concentrations  and  dissociation 
constants  (4): 

0en  =  d(Bn)/d[H]  =  B/[rT]  |  f/(  1  +  f)2}  (4a) 

where  f  =  K<j/[H+]  and  K^  is  the  dissociation  constant. 


Substituting  the  buffering  capacities  into  equation  (4).  solving 
for  FJH  and  substituting  into  equation  (1)  yields: 

TJH  =  (DH  +  DB, -0Bi  +  DB2-/iB: 

+  •  •  •  +DBn-/iBn)-(d[H  +  ]/dx).      (5) 

The  buffers  used  in  the  present  experiments  effectively  en- 
hanced the  diffusion  of  protons  by  a  factor  of  2249  (i.e..  1374 
by  1  m.M  HEPES  and  875  by  5  mM  HCO3"). 

We  calculated  d[H+]/dx  from  the  probe  output  as  described 
previously  (5).  Using  literature  values  for  the  constants  (6)  and 
an  estimate  for  DHEPES  of  0.62  X  1CT5  cnr/s,  flux  density  in  pAl/ 
cm2 -sat  the  midpoint  of  the  probe  excursion  (i.e.,  ~5  nm  from 
the  cell  surface)  was  calculated  from  equation  (5). 

In  resting  cells,  before  activating  the  H+/K+-ATPase,  a  met- 
abolically  dependent,  basal  level  of  proton  flux  was  measured 
uniformly  from  all  surfaces  of  the  cells.  After  20-40  min  of 
stimulation  of  acid  secretion  with  dibutyryl  cyclic  adenosine 
monophosphate  ( 1  m.\f).  the  apical  proton  flux  alone  had  in- 
creased by  270  ±  47%  from  8.6  ±  1 .8  p.U/cnr  •  s  (mean  ±  SEM; 
n  =  6).  Stimulated,  but  not  basal,  apical  flux  was  inhibited  by 
SCH-28080  ( 10~7  M),  a  specific  inhibitor  of  the  gastric  H+/K+- 
ATPase. 

These  results  indicate  that  isolated  Necturits  oxyntic  cells  retain 
functional  secretory  polarity  as  well  as  morphological  polarity. 
This  technique  enables  the  determination  of  the  activity  and 
distribution  of  an  electroneutral  ion  pump,  thereby  character- 
izing net  transcellular  transport  in  single  isolated  secretory  epi- 
thelial cells. 

Supported  by  the  Howard  Hughes  Medical  Institute  Under- 
graduate Biological  Sciences  Education  Program  and  the  Na- 
tional Vibrating  Probe  Facility.  The  authors  thank  Kelley  A. 
Gebhardt  and  Stephen  J.  Eikenberry  for  technical  assistance. 

Literature  Cited 

1.  Soil,  A.  H.  1980.     Am.  J  Physwl  238:  G366-G375. 

2.  Demaresl,  J.  R.,  D.  D.  F.  Loo,  and  G.  Sachs.  1989.     Science  245: 
402-404. 

3.  Speksnijder,  J.  E.,  D.  \V.  Corson,  C.  Sardet,  and  L.  F.  Jaffe. 
1989.     DL'Y  Bn>/  135:  182-190. 

4.  I  .Kail.  J.  T.,  and  J.  VVyman.  1958.     Biophysical  Chemistry.  Vol. 
1 .  Academic  Press,  New  York. 

5.  Kuhtreiber,  VV .  M.,  and  L.  F.  Jaffe.  1990.     /  Cell  Biol  1 10:  1 565- 
1573. 

6.  Robinson,  R.  A.,  and  R.  H.  Stokes.  1959.     Electrolyte  Solutions. 
2nd  Ed.  Butterworths,  London. 


Reference:  Biol  Bull.  189:  220-222.  (October/November,  1995) 

Transmission  of  Polarized  Light  through  Sunfish  Double  Cones  Reveals  Minute  Optical  Anisotropies 

Inigo  Novales  Flamarique  (University  of  I  'ictoria),  Rudolf  Oldenbourg,  and  Ferenc  I.  Hdmsi 


Many  fish  retinas  possess  paired  photoreceptors  termed  double  tioning  membrane  along  the  minor  axis  of  the  ellipse  (Fig.  1A). 
cones.  At  the  level  of  the  inner  segment,  tangential  cross  sections  Furthermore,  the  cones  are  arranged  in  square  or  row  mosaic 
show  double  cones  to  be  approximately  elliptical,  with  a  parti-  patterns. 


BIOPHYSICS 


221 


650 


450    500    550    600 

Wavelength  (nm) 


-50  0  50 

Angle  of  ellipse  major  axis  wrt  E-vector  (  ) 

^^m^m^^^r-         ™^M^^M^^-^^^^^^«^"^^^^^^^— 

Figure  I.  lAi  Differential  interference  contrast  iinii.su'  of  snn/is/i  retinal  mosaic,  d,  double  cone,  v,  single  cone:  arrow  points  to  a  partitioning 
membrane.  (B)  NFS  birefringence  image:  arrow  indicates  slow  axis  of  propagation  near  the  partitioning  membrane,  relardance  scale  from  0  nm  (black) 
to  2  nm  (while).  (C)  Retardance  counts  for  a  double  cone  at  various  cone  orientations  using  MSP;  top  trace  mis  obtained  with  a  2-nm  quart:  standard. 
(D)  %  contrast  lor  double  cone  transmission  til  nirions  cone  orientations  (n  =  5)  for  4  wavelengths;  %  contrast  =  2  (!„,„  —  l.J/(lm,,  +  l.J,  where  lmm 
denotes  ihc  incident  E-vector  aligned  with  the  minor  axis  of  the  ellipse,  and  /,  is  a  variable  cone  orientation  In  1C)  and  (D),  0°  corresponds  to  the 
incident  E-veclor  parallel  to  the  major  axis  of  the  ellipse 


222 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


The  retina  of  the  green  sunfish  (Lc/'o/ws  cyane/lus)  is  primarily 
arranged  in  a  square  mosaic,  with  double  cones  forming  the 
sides  of  the  square  (1.2).  Behavioral  studies  show  that  this  fish 
can  detect  the  electric  field  vector  of  linearly  polarized  light, 
exhibiting  sensitivity  maxima  to  horizontal  and  vertical  E-vectors 
(2).  These  observations  led  to  the  hypothesis  that  polarization 
detection  in  this  fish  relies  on  the  "geometric  birefringence" 
waveguide  properties  of  double  cone  ellipsoids  (3).  According 
to  this  hypothesis,  photons  with  E-vectors  along  the  major  axis 
of  the  ellipse  would  be  guided  more  effectively  than  those  along 
the  minor  axis,  thus  constituting  a  mechanism  for  the  detection 
of  polarized  light. 

The  experiments  presented  here  were  designed  to  measure 
the  transmission  of  polarized  light  through  double  cones  in  the 
retinas  of  green  sunfish  and  pumpkinseed  (Lepomis  gibbosus). 
Ventro-temporal  retinal  quadrants  were  placed  in  slide  wells 
filled  with  Ringer  solution,  and  the  preparations  were  sealed 
with  cover  slips.  Rod-free  areas  comprising  end-on  oriented  cones 
were  examined  using  microspectrophotometry  (MSP)  and  the 
New  Polarized  Light  Microscope  (NPS)  (4).  We  used  condensors 
with  0.45-0.5  NA  to  approximate  physiological  illumination. 
Two  types  of  experiments  were  conducted.  In  one  type,  bire- 
fringence was  measured  as  retinal  retardance  between  crossed 
polarizers  for  various  cone  orientations.  In  the  second  type, 
transmission  of  linearly  polarized  light  fluxes  was  measured 
through  double  cones  for  various  cone  orientations. 

Results  from  both  instruments  show  that  the  retardance  for 
double  cone  ellipsoids  never  exceeded  2  nm  (Fig.  IB.  C),  with 
the  highest  values  found  along  the  partitioning  membranes.  NPS 


analysis  further  showed  that  the  orientation  of  the  slow  axis  in 
the  elliptical  cross  section  was  variable  for  different  locations. 
In  addition,  polarization  contrast  (3),  determined  from  trans- 
mitted light  fluxes  polarized  along  the  minor  and  major  axes, 
was  in  the  order  of  1-3%  and  showed  independence  of  wave- 
length (Fig.  ID). 

A  retardance  of  2  nm  corresponds  to  a  difference  in  refractive 
index  of  0.0002  between  the  slow-  and  fast-propagating  axes  of 
double  cones.  This  difference  is  about  100  times  smaller  than 
the  value  that  can  be  calculated  from  the  previous  waveguide 
model  (3). 

We  conclude  from  these  studies  that  the  optical  anisotropies 
at  the  inner  segment  of  cones  are  very  small,  and  that  birefrin- 
gence-based waveguiding  is  an  unlikely  mechanism  to  explain 
polarization  sensitivity  in  sunfish.  We  noticed,  however,  the 
possibility  that  the  partitioning  membrane  of  the  double  cones, 
which  appears  to  be  planar,  might  act  as  a  dielectric  mirror.  The 
polarization  anisotropy  caused  by  reflection  from  such  a  mirror 
is  consistent  with  these  results. 

This  work  was  sponsored  by  a  Grass  Fellowship  in  Neuro- 
physiology  to  INF. 


Literature  Cited 


1.  Cameron,  D.  A.,  and  S.  S.  Easter,  Jr.  1993.     I'M.  Neurosd.  10: 
375-384. 

2.  Cameron,  D.  A.,  and  E.  N.  Pugh,  Jr.  1991.     Nature  353:  161-164. 

3.  Rowe,  M.  P.,  N.  Engheta,  S.  S.  Easter,  Jr.,  and  E.  N.  Pugh,  Jr. 

1994.     J  Opt.  Soc  Am  A   11:  55-70. 
4    Oldenbourg,  R.,  and  G.  Mei.  1995.     J.  .\/«n>vo>/>r.  in  press. 


Reference:  Biol.  Bull  189:  222-223.  (October/November.  1995) 

Dogfish  (Mustelus  canis)  Lens  Catalase  Reduces  H2O2-Induced  Opacification 

Seymour  Zigman,  Nancy  S.  Rafierty,  and  Mark  Schult:  (University  of  Rochester  School  of  Medicine  & 
Dentistry.  Rochester,  New  York,  NY  14642,  and  Marine  Biological  Laboratory) 


Previous  studies  showed  that  LiVA  radiation  in  vitro  causes 
opalescence  in  dogfish  lenses  (1,2).  Protection  against  this  change 
is  afforded  by  n-tocopherol.  which  also  protects  cytoskeletal  actin 
and  catalase  from  damage  (2-5).  We  thus  hypothesize  that  UVA 
causes  lens  opalescence  by  reducing  the  activity  of  the  major 
antioxidative  enzyme  (6)  so  that  even  normal  levels  of  H^O: 
can  oxidize  lens  constituents  and  lead  to  cataracts. 

Our  purpose  was  (a)  to  determine  if  inhibition  of  catalase 
anti-oxidative  activity  by  photooxidation  in  vitro  causes  opal- 
escence in  dogfish  lens  and  (b)  to  observe  the  damage  produced 
by  UV  radiation  and  the  protection  afforded  by  deferoxamine 
to  catalase  activity  and  cytoskeletal  proteins. 

Dogfish  lenses  were  incubated  in  elasmobranch  Ringer's  me- 
dium under  95%  air  and  5%  CO:.  Lens  clarity  was  assessed  by 
viewing  a  pattern  of  computer-generated  fish  scales  through  the 
lens  and  by  slit-lamp  photography.  Catalase  activity,  measured 
as  O2  production,  was  determined  with  an  CK  meter  and  electrode 
(Microelectrodes.  Inc.).  Catalase  inhibition  and  cytoskeletal 


protein  degradation  were  measured  on  lenses  that  received  ~9  J/ 
crrr/h  of  UVA  (99.9%  UV-A,  0.1%  UV-B)  radiation.  Cytoskeletal 
immunocytochemistry  was  done  on  fixed  lens  epithelia  double- 
labeled  with  rabbit  antibodies  vs  «-tubulin-FITC  and  rhodamine- 
phalloidin.  Samples  were  photographed  with  a  Zeiss  Axiophot 
fluorescence  microscope. 

Our  results  showed  that  lenses  pre-exposed  to  90  J/cm2  of 
UVA  and  challenged  with  0. 1  to  1 .0  mM  [H2O2]  developed  dense 
cortical  opalescence  (Table  la).  Deferoxamine  (10  mM)  partially 
protected  lens  clarity  and  catalase  activity  (Table  Ib).  In  irradiated 
cells,  the  filamentous  nature  of  microtubules  had  degraded  nearly 
completely.  Fluorescence  appeared  to  be  associated  with  the 
plasma  membranes  and  microtubule  organizing  centers.  In 
unirradiated  cells,  brightly  displayed  microtubules  extended  from 
the  nuclei  to  the  periphery  of  the  cell.  Deferoxamine  partially 
protected  microtubules  against  the  effects  described  above. 

The  results  lead  us  to  two  conclusions.  ( 1 )  Catalase  activity 
in  lens  epithelium  is  inhibited  by  UVA  so  that  near-normal  levels 


COMPARATIVE  PHYSIOLOGY  AND  BIOCHEMISTRY 

Table  I 

ti  Ooij/M/i  Icin  opacitteatiim  by  II:O:  after  (T,  Irradiation 


223 


Results 


Lens 
treatment 


mM 


Verbal 


Photograph 


Unexposed 


UVA-exposed 


O.I 

1  0 

O.I 
1.0 


Opal  =  opalescent;  UVA-exposure  I  I  h.  H:O:  exposure  1  1  h. 
b.  Dejeroxiiminc  protection  of  lens  eatalase  activity 


Lens  treatment 


increase          Catalase 
per  min       activity  units 


Unexposed  4.25  ±  0.86  0.49 

Unexposed  plus  (10  m,l/)  deferoxamine  3.05  ±  0.87  0.40 

UVA-exposed  0.75  ±  0.37  0.11 

UVA-exposed  plus(10  mA/)  deferoxamine  1.78  ±0.50  0.24 

±  =  Standard  deviation. 
UVA-exposure  =  2  mW/cnr;  18  h;  20°C;  Lens  weight  =  400  ±  50  mg. 


of  H2O:  in  the  fluid  medium  (i.e..  aqueous  humor)  can  cause 
lens  opalescence.  (2)  Microtubules  are  damaged  hy  UVA  radia- 
tion and  are  partially  protected  by  deferoxamine. 

This  research  was  supported  by  the  National  Eye  Institute  Ey- 
00459  and  Research  to  Prevent  Blindness  Senior  Scientific  In- 
vestigator Award.  Thanks  to  B.  R.  Zigman  for  preparation  of 
the  manuscript. 

Literature  Cited 

I.  Zigman,  S.,  N.  S.  Rafferty,  and  R.  B.  Wheeler,  Jr.  1991.     Biol 

Bull  181:  341-342. 


1.  Zigman,  S.,  N.  S.  Rafferty,  D.  L.  Scholz,  and  K.  Lowe.  1992.     Exp. 
Eye  Res.  55:  193-201. 

3.  Zigman,  S.,  and  N.  S.  Rafferty.  1994.     Comp.  Biochem.  PhysioL 
109 A:  463-467. 

4    Zigman,  S.,  T.  McDaniel.  J.  B.  Schultz,  J.  Reddan,  and  M.  Meydani. 
1995.     Mol  Cell  Biochem.  143:  35-46. 

5.  Zigman,  S.,  T.  Yulo,  and  G.  A.  Greiss.  1976.     Mol  Cell  Biochem. 
11:  131-135. 

6.  Fuchs,  J.,  T.  Hufelel,  L.  M.  Rolhfuss,  D.  S.  Wilson,  G.  Carcamo, 
and  L.  Packer.  1989.     Photocliem  Photohiol  50:  739-744. 


Reference:  Biol.  Bull  189:  223-225.  (October/November.  1995) 


Sulfotransferase  Activities  in  the  Marine  Sponge  Microciona  prolifera: 

Correlation  with  Sulfated  Glycan  Adhesive  Structures 
William  ./.  Knhns,  Max  M.  Burger,  ami  Gradimir  Misevic  (Marine  Biological  Laboratorv) 


Sulfated  glycans  in  matrix  and  on  cell  membranes  mediate  a 
variety  of  cell  functions  (1.  2).  Their  highly  charged  nature  is 
known  to  influence  cell-cell  adhesion  and  cell  communication 
(3. 4).  Sultated  structures  have  been  demonstrated  in  the  adhesive 


proteoglycan  (AP)  of  Microciona  with  a  specific  aggregation- 
blocking  monoclonal  antibody  termed  Block  2  that  reacted  with 
purified  proteoglycan  oligosaccharides.  One  epitope  was  iden- 
tified as  a  sulfated  disaccharide,  3-SO,-A'-acetylglucosamine-j31- 


224 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


3-fucose.  A  -.xond  nonreactive  compound  was  identified  as  a 
sulfated  jjaUctose-linked  tetrasaccharide  (5).  On  the  basis  of  these 
structures,  we  predict  that  sponge  tissue  contains  specific  sul- 
fotransferases  (sulfo-T)  as  the  biosynthetic  enzymes  involved  in 
the  sulfation  process. 

To  explore  this  possibility,  we  performed  assays  for  sulfo-T 
using  Triton-extracted  sponge  microsomes  (7.5-12.5  mg/ml)  in 
the  presence  of  chemically  defined  monosaccharide  acceptor 
substrates  at  concentrations  of  20-320  nM.  The  co-substrate  and 
active  sulfate  donor  was  radiolabeled  3'-phosphoadenosine  5'- 
phosphosulfate  ( (35S)PAPS)  7  uM  (2145  dpm/pmol)  in  the 
presence  of  pyrophosphatase  and  sulfatase  inhibitors  and  10  mAf 
Mg2+  buffered  at  pH  6.3.  The  total  volume  of  the  reaction  mix- 
ture was  40  fi\  (6).  After  incubation  for  1  h  at  37°,  the  reaction 
was  terminated  by  adding  10 /jl  of  2%  sodium  borate/20  mM 
EDTA.  Separation  of  unreacted  PAPS  from  sulfated  product 
was  accomplished  by  thin-layer  chromatography  using  a  solvent 
system  containing  acetonitrile.  water  and  methanol  (4: 1 :0.2).  or 
high-voltage  electrophoresis  using  1%  sodium  borate  buffer  at 
pH  9.1.  The  figures  for  product  yield,  expressed  as  picomoles. 
were  the  average  of  duplicate  values  from  which  had  been  sub- 
tracted picomoles  present  in  endogenous  assays  in  which  acceptor 
had  been  omitted.  The  biosynthesized  product  was  expressed  as 
picomoles  per  hour  per  milligram  of  protein  (pmol/h/mg). 

Acceptor  substrates  were  monosaccharides  or  derivatized 
monosaccharides  representing  sugars  known  to  occur  in  Micro- 
dona  AP.  They  included  0-D-fucose,  a-L-fucose,  j3-r>glucuronide 
phenyl,  /J-phenyl-A'-acetyl-D-glucosamine(GlcNAc),  phenyl-/V 
acetyl-«-r>galactosamine(GalNAc),  phenyl-«-D-mannoside  and 


phenyl-^-D-galactoside(Gal).  Of  these.  phenyl-GlcNAc  and 
phenyl-Gal  gave  measurable  product  yields,  but  assays  using 
5  mM  phenyl  (phenol)  alone  were  negative.  In  the  case  of  phenyl- 
GlcNAc,  substrate-product  curves  generated  from  assays  with 
graded  quantities  of  acceptor  (20  to  320  nmol)  demonstrated 
I  mav  and  A",n  values  of  50  pmol/h/mg  and  1  mM,  respectively. 
The  proposed  reaction  is  visualized  as  follows: 


GlcNAc-0-phenyl 


("S)PAPS 


35SO,-0-GlcNAc-0-phenyl  +  PAP. 


Final  confirmation  of  the  position  of  the  sulfate  linkage  will 
require  additional  detailed  analyses  of  scaled-up  quantities  of 
product  (6). 

The  cellular  localization  of  sulfated  glycan  was  determined 
by  immunohistochemical  techniques  on  fixed  sections  of  washed 
Microcionu  cell  pellets  stained  with  Block  2  primary  antibody 
and  horseradish  peroxidase-labeled  secondary  antibody.  A  mixed 
pattern  of  stained  and  nonstained  cells  was  found,  with  the  most 
intense  staining  present  intracellularly  and  on  surface  membranes 
of  the  larger  cell  population,  including  cells  interpreted  as  ar- 
cheocytes  (Fig.  1 ).  This  finding  suggests  that  immunoseparation 
of  a  selected  cell  cohort  may  be  a  useful  preparative  technique 
when  preparing  purified  enzyme. 

In  summary,  the  enzyme  activities  defined  in  this  study  are 
consistent  with  earlier  findings  of  sulfated  glycan  structures  (5) 
and  suggest  that  quantitative  recoveries  of  pure  enzymes  will  be 
of  importance  in  studying  their  regulatory  role  in  sponge  cell 


100  Jim 


100  ^im 


Figure  1.  Comparison  ol  control  licit  I  and  treated  (right)  sections  of  washed  cell  pellet  Ironi  Microciona  prolifera.  The  cell  pellets  had  been  li\ed 
in  formalin-artificial  seawater,  sectional  (?  urn),  deparatlimied.  and  stained  with  Block  _  monoclonal  primary  antibody  (1-200)  followed  by  anti- 
mouse  horseradish  peroxidase  secondary  antibody  1 1-2500)  and  color  development  with  diaminobenzidine  (hematoxylin  was  the  counterstain).  A 
section  from  winch  primary  antibody  had  been  omitted  shows  only  counters/allied  cell',  licit)  In  contrast  to  the  control,  a  test  slide  shows  a  mixed 
population  ofperoxidasi  stained  and  unstained  cells  fright).  The  relative  magnifications  are  indicated  by  scale  bars  (bottom  left)  Many  ot  the  large 
stained  cells  resemble  an  lico,  ytcs  and  cslubit  internal  as  well  as  peripheral  membrane  reactivity  I'his  surface  reactivity  is  visualised  particularly 
well  as  a  /me  dark  brown  line  that  encircles  three  large  cells  in  triangular  configuration  near  the  middle  ol  the  section  toward  the  bottom  Other  large 
cells  in  this  section  are  sinnlarlv  encircled  Smaller  faintly  '-tamed  celts  may  he  ehoaiioeyles. 


COMPARATIVE  PHYSIOLOGY  AND  BIOCHEMISTRY 


225 


adhesion.  The  circumstances  under  which  sponge  cell  adhesion 
might  be  influenced  by  these  enzyme  activities  or  their  products 
are  unclear,  but  may  be  clarified  by  experiments  using  sulfo-T 
inhibitors  or  sulfatases,  or  calibrations  of  enzyme  product  re- 
sponses by  sponge  cells  after  the  manipulation  of  environmental 
sulfate(7). 

The  authors  thank  Dr.  Roxanna  Smolowitz  for  her  help  in 
the  preparation  of  the  photographs. 


Literature  Cited 

1.  Green,  E.,  el  al.  1986.     Mol  Cell  liioc/icm  71:81-100. 

2.  Cheng,  P.,  el  al.  1989.     ./.  Clin.  Invest.  84:  68-72. 

3.  Imai,  Y.,  el  al.  1992.     Nature  361:  555-557. 

4.  Brandley,  B.,  el  al.  1993.     UlycohtoloKy  3:  633-639. 

5.  Spillmann,  D.,  el  al.  1995.     ./  Biol.  Clwin.  270:  5089-5097. 

6.  Kuhns,  \\ '.,  el  al.     Glycobiology.  In  press. 

7.  Kuhns,  \\  .,  el  al.  1995.     ./  Cell.  Bioc/iem.  57:  71-89. 


Reference:  Biol.  Bull  189:  225-226.  (October/November.  1995) 


Preliminary  Investigations  on  the  Scavenger  Receptors  of  the  Amebocyte 
of  the  American  Horseshoe  Crab,  Limulus  polyphemus 

Ronald  T.  Ainies.  James  P.  Quiff  Icy.  Snehasikta  Swarnakar,  Dudley  K.  Strickland, 

and  Peter  B.  Armstrong  (Department  of  Molecular  and  Cellular  Biology. 

University  of  California,  Davis,  California  95616-8755) 


The  clearance  of  a  variety  of  ligands  from  the  tissue  fluids  of 
mammals  is  mediated  by  a  group  of  cell-surface  proteins  known 
as  the  scavenger  receptors.  These  receptors  bind  and  mediate 
the  endocytosis  of  target  molecules  including  low-density  lipo- 
protein  (LDL).  urokinase-type  plasminogen-activator-plasmin- 
ogen  activator  inhibitor- 1  complexes,  and  protease-reacted  «2- 
macroglobulin  (o:M)  (1).  The  LDL-receptor-related  protein/ 
«2M-receptor  (LRP/«2M-R)  is  a  member  of  the  LDL  receptor 
gene  family  consisting  of  at  least  six  cell-surface  receptors.  Three 
of  these  receptors — LRP.  the  LDL  receptor,  and  the  Heymann 
nephritis  antigen  (gp330) — are  known  to  bind  a  39  kDa  intra- 
cellular  receptor-associated  protein  (RAP)  (2).  RAP  binds  with 
high  affinity  in  a  calcium-dependent  manner  and  copurifies  with 
these  receptors  during  ligand  affinity  chromatography. 

Previous  work  from  our  laboratory  (3)  demonstrated  that  flu- 
orescently  labeled  protease-reacted  Limulus  polyphemus  «:M  is 
cleared  from  the  blood  of  Limulus  with  a  concomitant  associ- 
ation of  fluorescent  label  with  the  amebocytes  suggesting  the 
presence  of  a  specific  «2M  receptor  on  the  amebocyte.  We  have 
used  RAP-affinity  chromatography  (4)  to  search  for  this  and 
other  scavenger  receptors  in  the  blood  cells  (amebocytes)  of 
Limulus  polyphemus.  Amebocytes  were  collected  from  adult 
animals  (5);  washed  twice  with  sterile,  lipopolysaccharide-free 
3%  NaCl:  resuspended  in  the  same  buffer,  and  lysed  by  the  ad- 
dition of  an  equal  volume  of  2x  lysis  buffer  [0.1  A/  HEPES. 
0.3  M  NaCl.  0.02  MCaCl,,  2.0%  Triton  X-100  (TX-100),  0.1% 
Tween-20,  10  iiM  1.10-phenanthroline.  50  u.M  3,4-dichloroiso- 
coumarin.  2  fi.M  trans-epoxysuccinyl-L-leuc>lamino(4-guani- 
dino)-butane.  2  nM  pepstatin  A.  and  1  mM  phenylmethylsul- 
fonyl  fluoride,  pH  7.4].  The  samples  were  vortexed,  and  the 
insoluble  material  was  removed  by  centrifugation  at  1 4,000  rpm 
in  a  microcentrifuge.  Nonspecific  Sepharose-binding  proteins 
were  removed  (following  addition  of  calcium  to  make  a  con- 


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Figure  I.  RAP-ligand  hlol  <>/  a  representative  Limulus  amebocyte 
lysale  and  R.iP-Sepharose  purified  material  The  same  pattern  ofR.-lP- 
binding  proteins  has  been  found  in  eight  purifications  of  the  R.AP-binding 
proteins  from  five  different  amebocyte  lysates.  Samples  were  electropho- 
resed  on  a  standard  b'-'c  polyacrylamide-SDS gel  and  electrophoretically 
transferred  to  nitrocellulose  membranes  The  membranes  »  ere  processed 
ax  previously  described  (41,  using  recombinant  human  RAP  as  a  ligand. 
R.JP  binding  wa\  detected  with  a  polvclonal  anti-human  R.4P  antibody 
followed  by  goal  anti-rabbit-peroxidase  conjugated  \ccnnd  antibody  and 
development  \\ilh  SuperSignal  CL-HRP  Substrate  System  (Pierce.  Rock- 
Ion/.  IL)  Lane  I.  100  ng  purified  human  LRP:  lane  2.  5  M/  Sepharose 
4B-cleared  Limulus  umehocvte  twite,  lane  3.  7.5  pi  RAP-Sepharose  un- 
bound material.  lane\  -t-\  3?  M/  aliquots  of  column  wash:  lanes  6-10. 
30  pi  aliqnots  ofR.4P-Seplniro\e  pi  I  _  0  clutcil  material.  Arrows  indicate 
positions  of  RAP-binding  protein*,  found  in  Limulus  amebocyie  e.\trueis, 
large  arrow  indicates  the  Limulus  RAP-binding  protein  with  the  same 
eleclrophoreiic  position  as  human  LRP  Positions  of  molecular  mass 
standards  are  indicated  on  the  letl  in  kilodultons. 


226 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


centration  of  10  mM)  by  chromatography  on  a  Sepahrose  4B 
column  equilibrated  in  50  mM  Tris  (pH  8.0),  0.15  M  NaCl, 
0.01  M  CaC\2,  0.5%  TX-IOO,  0.05%  Tween-20.  The  unbound 
material  (4  ml)  was  collected,  applied  to  a  recombinant  human 
RAP-Sepharose  column  (3-ml  bed  volume),  and  washed  with 
30  ml  of  buffer.  The  bound  material  was  eluted  with  glycine  (pH 
2.0),  0.15  M  NaCl,  0.05%  TX-100,  0.05%  Tween-20;  1.5-ml 
fractions  were  collected  and  immediately  neutralized  with  1  M 
Tris  pH  8.0;  and  aliquots  were  analyzed  by  RAP  ligand  blotting 
(4).  Figure  1  shows  that  the  Limulus  amebocyte  contains  proteins 
capable  of  binding  to  human  recombinant  RAP,  and  that  these 
proteins  can  be  specifically  removed  from  the  cell  lysates  (lane 
3)  by  RAP-affinity.  At  least  one  protein  in  the  lysate  (lane  2) 
and  the  RAP-affinity  purified  material  (lane  6-10:  large  arrow) 
has  electrophoretic  mobility  similar  to  that  of  purified  human 
placental  LRP  (lane  1 ). 

We  have  shown  that  Limn/us  amebocytes  contain  a  RAP- 
binding  protein  with  similar  characteristics  to  human  LRP.  The 
fact  that  the  horseshoe  crab  contains  a  cell-associated  protein 
with  an  electrophoretic  mobility  similar  to  that  of  human  LRP 
and  that  is  capable  of  binding  human  RAP  suggests  a  high  degree 
of  conservation  in  scavenger  receptor  evolution.  This  potential 


receptor  for  molecules  targeted  for  clearance  from  the  circulation 
(e.g..  protease-reacted  oiM)  may  help  explain  the  rapid  clearance 
of  trypsin  from  the  circulation  in  experimental  animals  (3).  The 
ability  of  the  RAP-binding  proteins  from  Limulus  to  bind  pro- 
tease-reacted  «:A/  and  other  potential  ligands  has  not  been  dem- 
onstrated and  is  being  pursued. 

Supported  by  Grant  No.  MCB-92 1 8460  from  the  National 
Science  Foundation.  R.  Aimes  is  a  1995  Marine  Biological  Lab- 
oratory Bang  Fellow. 

Literature  Cited 

1.  Krieger,  M.,  and  J.  Herz.  1994.     Ann.  Rc\:  Biochem.  63:601-637. 

2.  Ashcom,  J.  D.,  S.  E.  Tiller,  K.  Dickerson,  J.  L.  Cravens,  W.  S. 
Argraves,  and  D.  K.  Strickland.  1990.     J  Cell.  Biol   110:  1041- 
1048. 

3.  Melchior,  R.,  J.  P.  Quigley,  and  P.  B.  Armstrong.  1995.     ./  Biol. 
Chein.  270:  13496-13502. 

4  Battey,  F.  D.,  M.  E.  Gafvels,  D.  J.  FitzGerald,  \V.  S.  Argraves, 
D.  A.  Chappell.  J.  F.  Strauss,  III,  and  D.  K.  Strickland.  1994.  ./ 
Biol.  Chen,  269:  23268-23273. 

5.  Armstrong,  P.  B.  1985.  Pp.  253-258  in  Blnnd  Cells  ol  Marine 
Invertebrates,  W.  D.  Cohen,  ed.  A.  R.  Liss.  New  York. 


Reference:  Biol.  Bull.  189:  226-227.  (October/November.  1995) 

Regulation  of  the  Plasma  Cytolytic  Pathway  of  Limulus  polyphemus  by  ai-Macroglobulin 

Snehasikta  Swarnakar.  Ralph  Melchior.  James  P.  Quigley.  and  Peter  B.  Armstrong  (Department  of 
Molecular  and  Cellular  Biology.  University  of  California,  Davis,  California  95616-8755) 


An  important  immune  defense  strategy  of  higher  animals  is 
to  kill  invading  pathogens  with  soluble  agents  in  the  plasma, 
usually  proteins,  that  provoke  the  cytolysis  of  foreign  cells.  These 
cytolytic  systems  are  typically  under  precise  regulation  to  prevent 
unintended  damage  to  the  cells  and  extracellular  matrix  of  the 
tissues  of  the  host.  The  plasma  cytolytic  system  of  the  horseshoe 
crab,  Limulus.  which  is  mediated  by  a  single  effector  protein — 
the  sialic  acid-binding  lectin  known  as  limulin  ( 1 ) — is  regulated 
specifically  by  the  protease-reacted  form  of  the  plasma  protease 
inhibitor  a2-macroglobulin  (LAM  for  Limulus  Alpha-2-Mac- 
roglobulin)  (Table  I,  line  4).  Limulin  is  a  member  of  the  pentraxin 
family  of  proteins  and  is  organized  as  a  homoduodecamer  of 
25  kDa  subunits  (1,  2),  and  LAM  is  a  soluble  protease-binding 
protein  organized  as  a  homodimer  of  185  kDa  subunits  (3).  Re- 
action of  LAM  with  the  small  primary  amine  methylamine  (MA) 
induces  an  activation  similar  to  that  produced  by  proteases  (3, 
4)  and  yields  a  conformation  that  is  similarly  inhibitory  to  limulin 
(Table  1,  line  3). 

Cytolysis  of  foreign  cells  was  detected  by  the  hemolysis  of 
sheep  erythrocytes  (5).  Protease-  and  MA-reacted  LAM,  but  not 
native  LAM,  depressed  the  hemolytic  action  of  purified  limulin 
at  a  ratio  of  30,-SO  moles  of  LAM  per  mole  of  limulin  (Table 
I).  The  addition  of  trypsin  or  MA  reduced  the  hemolytic  activity 
of  whole  plasma  (5):  presumably  they  react  with  endogenous 
LAM  in  the  plasma,  and  the  product  then  reduces  the  hemolytic 


Table  I 

ol  the  hemolytic  activity  of  purified  limulin  by  trypsin-  and 
methylamine  (MA)-reacted  Limulus  armacroglobulin  (LAM) 


Hemolysis  condition:  10  nM 
Limulin2  +  native  or  reacted  LAM 
No  addition 
860  nM  LAM' 
780  nM  MA-LAM4 
780  nM  trypsin-LAM5 
420  nM  trypsin/STI 
420  nM  trypsin/PMSF 


Hemolysis' 

(Mean  ±  standard  error) 
51.5  ±  5.2% 
51.1  ±  1.4% 
1 1.1  ±  1 .4% 
19.0  ±  9.8% 
46.3  ±  6.2% 
53.8  +  5.8% 


'  Fraction  of  erythrocytes  hemolyzed.  Hemolysis  was  conducted  as 
previously  described  (5)  (3  X  107  erythrocytes  per  sample,  final  sample 
volume  0.8  ml,  0.19  M  NaCl.  0.15  mA/CaCI;.  0.5  mMMgCl,,  2.5% 
glucose,  0.  \%  gelatin.  2.5  mM  barbital.  pH  7.3,  4  h  incubation  at  room 
T — after  which  the  intact  cells  were  removed  by  centrifugation  and  the 
released  hemoglobin  was  measured  spectrophotometncally  at  412  nm 
and  was  compared  with  full  hemolysis  produced  by  hypotonic  lysis  of 
an  aliquoit  of  erythrocytes). 

2  Limulin  was  purified  as  in  ( 1 ). 

3  LAM  was  purified  as  in  (3). 

4  LAM  was  treated  overnight  with  200  m.U  MA.  pH  8.0. 

5  LAM  was  treated  with  2-fold  excess  trypsin,  then  the  trypsin  was 
inactivated  with  phenylmethylsulfonyllluoride  (PMSF)  and  a  2-fold  excess 
soybean  trypsin  inhibitor  (STI). 


COMPARATIVE  PHYSIOLOGY  AND  BIOCHEMISTRY 


227 


activity  oflimulin.  The  inhibitory  activity  of  protease-  or  MA- 
reacted  LAM  was  seldom  complete  and  was  actually  absent  at 
molar  ratios  of  LAM:limulin  below  20-30  or  at  high  concen- 
trations oflimulin.  Limnliis  plasma  contains  1-5  fi.M  LAM  (6) 
and  about  30-50  nA/  limulin  (as  estimated  from  the  hemolytic 
activity  of  plasma).  Trypsin  inactivated  by  treatment  with  soy- 
bean trypsin  inhibitor  (STI)  or  phenylmethylsullbnylfluoride 
(PMSF)  failed  to  inhibit  hemolysis  by  purified  limulin  (Table  I, 
lines  5,  6)  or  whole  plasma.  Although  native  LAM  did  not  in- 
fluence the  lytic  activity  oflimulin.  it  did  reverse  the  inhibitory 
effects  of  protease-  or  MA-reacted  LAM  in  a  dose-dependent 
manner  (data  not  shown).  At  present  we  have  no  explanation 
for  this  effect. 

In  parallel  with  an  inhibition  of  hemolysis.  protease-  and  MA- 
reacted  LAM  inhibited  the  hemagglutinating  activity  oflimulin. 
One  hemagglutination  unit  of  limulin  for  sheep  erythrocytes 
was  2.5  nA/.  Inhibition  was  produced  by  300-400  nA/  MA-  or 
trypsin-LAM.  Inhibition  by  native  LAM  was  detected  at  700  nA/. 

The  apparent  target  of  protease-reacted  LAM  is  limulin,  rather 
than  the  erythrocytes.  Pretreatment  of  erythrocytes  with  MA- 
LAM  did  not  prevent  their  subsequent  hemolysis  by  limulin. 
MA-  and  protease-reacted  LAM  both  bound  limulin  more  avidly 
than  did  unreacted  LAM.  Immunoprecipitation  with  anti-li- 


mulin  antiserum  co-precipitated  MA-  and  trypsin-LAM.  but  not 
native  LAM;  substrate-immobilized  limulin  bound  MA-  and 
trypsin-activated  LAM  with  a  Kd  of  about  10  nA/,  but  failed  to 
bind  native,  unactivated  LAM. 

This  work  was  supported  by  Grant  No.  MCB-92 18460  from 
the  National  Science  Foundation. 

Literature  Cited 

1.  Armstrong,  P.  B.,  S.  Misquith,  S.  Srimal,  R.  Melchior,  and  J.  P. 
Quigley.  1994.     Biol  Bui/.  187:  227-228. 

2.  Tennent,  G.  A.,  P.  ,1.  G.  Buller,  I.  Mutton,  A.  R.  Woolfitt,  D.  J. 
Harvey,  T.  VV.  Rademacher,  and  M.  B.  Pepys.   1993.     Eur   ,1 
Biochem.  214:  91-97. 

3.  Armstrong,  P.  B.,  \V.  F.  Mangle,  J.  S.  Wall,  J.  F.  Hainfield,  K.  E. 
Van  Holde,  A.  Ikai,  and  J.  P.  Quigley.  1991.     J.  Biol.  Chem  266: 
2526-2530. 

4.  Quigley,  J.  P.,  A.  Ikai,  H.  Arakawa,  T.  Osada,  and  P.  B.  Armstrong. 
1991 .     J.  Biol.  Chem.  266:  1 9426- 1 943 1 . 

5.  Armstrong,  P.  B.,  M.  T.  Armstrong,  and  J.  P.  Quigley.  1993.     Mol. 
Immimol  30:  929-934. 

6.  Enghild,  J.  J.,  1.  B.  Thegersen,  G.  Salvesen,  G.  H.  Fey,  N.  L.  Figler, 
S.  L.  Gonias,  and  S.  V.  Pizzo.  1990.     Biochemistry  29:  10070- 
10080. 


Reference:  Biol.  Bull.  189:  227-228.  (October/November.  1995) 

Characterization  and  Use  of  Isolated  Toadfish  Hepatocytes 
for  Studies  of  Heme  Synthesis  and  Utilization 

Neal  W.  Cornell  (Marine  Biological  Laboratory).  Mark  E.  Hahn,  and  Holly  A.  Martin 


The  steady-state  level  of  hepatic  heme  is  the  net  result  of  bio- 
synthesis, degradation,  and  incorporation  of  heme  into  hemo- 
proteins  within  the  liver.  The  rate  of  synthesis  is  controlled  by 
5-aminolevulinate  synthase  (ALS).  the  first  enzyme  of  the  path- 
way: likewise,  heme  oxygenase  (HO),  the  first  enzyme  of  the 
degradative  pathway,  sets  the  rate  of  heme  catabolism.  Liver 
contains  many  different  hemoproteins,  but  the  turnover  of  cy- 
tochrome  P450  (P450)  quantitatively  accounts  for  the  major  frac- 
tion of  heme  utilization.  All  three  of  these  enzymes  are  known 
to  be  inducible  by  xenobiotics  and  toxicological  agents  (1-3), 
and  the  long-range  aim  of  our  studies  is  to  determine  the  effects 
of  environmental  contaminants  on  ALS.  HO,  and  P45n  in  the 
marine  fish  Opsunns  tau  (toadfish). 

The  lack  of  inbred,  regimen-raised  stocks  of  marine  fish  can 
present  serious  difficulties  in  studies  of  complex  hepatic  pro- 
cesses. To  minimize  such  difficulties,  we  are  conducting  our 
studies  with  isolated  liver  cells  prepared  by  the  collagenase  per- 
fusion  method  (4).  Some  characteristics  of  toadfish  hepatocytes 
have  been  determined  both  to  allow  results  obtained  with  the 
isolated  cells  to  be  referred  to  the  intact  liver  and  also  to  have 
criteria  for  evaluating  the  quality  of  each  cell  preparation.  From 
one  toadfish,  we  obtain  5-5.5  X  108  cells — enough  to  establish 
20-50  primary1  cultures,  thus  allowing  many  tests  to  be  run  with 
cells  from  the  same  fish.  Toadfish  hepatocytes  have  a  median 


diameter  of  15  urn.  and  there  are  3.4  X  108  cells  per  gram  wet 
wt.  Comparative  values  for  rat  hepatocytes  are  20-25  /*m  and 
1.2  X  10s  cells/g  wet  wt.  ATP  content  has  been  shown  to  be  an 
indicator  of  cell  integrity  and  viability  for  rat  hepatocytes  (5), 
and  the  same  is  assumed  to  be  true  for  toadfish  hepatocytes. 
ATP  in  freeze-clamped  toadfish  liver  (i.e..  in  vivo)  was  deter- 
mined to  be  1.84  f<mol/g  wet  wt,  and  freshly  isolated  hepatocytes 
had  a  similar  content  ( 1 .79  j/mol/g  wet  wt).  After  3  days  in  cul- 
ture, however,  the  cellular  ATP  content  had  risen  to  2. 1 5  ^mol/ 
g  wet  wt.  The  latter  approaches  the  value  (2.5  jumol/g  wet  wt) 
characteristic  of  rat  liver  in  vivo  and  of  isolated  rat  liver  cells. 
Protein  contents  were  also  measured  for  the  two  types  of  cells 
and  found  to  be  120-150  mg/g  wet  wt  (toadfish  cells)  and  180- 
200  mg/g  wet  wt  (rat  cells).  Finally,  consistent  with  fish  liver 
being  the  principal  site  of  both  lipid  biosynthesis  and  lipid  storage, 
initial  isolates  of  toadfish  cells  are  laden  with  lipid  droplets.  Those 
extracellular  lipid  droplets  can  be  eliminated  by  centrifuging 
toadfish  hepatocytes  through  a  layer  of  25%  sucrose;  this  minor 
modification  of  the  standard  procedure  for  preparing  hepatocytes 
permits  cells  of  good  quality  and  useful  quantity  to  be  obtained 
from  toadfish  liver. 

With  regard  to  heme  metabolism,  we  previously  reported  (6) 
that  toadfish  hepatic  ALS  is  inducible  by  several  agents,  most 
strongly  by  succinyl  acetone  (SA).  an  inactivator  of  aminolev- 


228 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


ulinate  dehydratase,  the  second  enzyme  of  the  heme  biosynthetic 
pathway.  In  land  vertebrates,  the  induction  of  ALS  by  SA  has 
been  suggested  to  result  from  a  decrease  in  hepatic  free  heme. 
a  feedback  regulator  of  ALS.  This  also  seems  to  be  the  case  with 
toadfish  liver,  since  the  15-  to  20-fold  induction  of  ALS  by 
0.5  mM  SA  is  completely  blocked  by  10  n.\I  heme  added  to  the 
culture  medium.  We  now  have  shown  that  ALS  is  induced  by 
a  polychlorinated  biphenyl  (PCB)  that  also  causes  a  strong  in- 
duction of  P450  in  toadfish  hepatocytes.  The  specific  PCB  con- 
gener chosen  for  initial  testing.  3,3',4,4',5-pentachlorobiphenyl 
(IUPAC  No.  126),  was  added  to  hepatocyte  culture  medium  at 
1,  10,  100,  and  1000  oA/.  Induction  of  P450  was  maximal  at 
100  nM;  at  that  dose,  ethoxyresorufin  0-deethylase  (enzyme  ac- 
tivity of  P4501 A 1  (2,  7)  )wasincreased  by  about  200-fold  relative 
to  untreated  cells.  The  simultaneous  presence  of  0.5  mJl/  SA 
had  no  significant  effect  on  the  induction  of  P450.  Induction  of 
ALS  was  also  maximal  at  100  n.M  PCB  and,  both  in  the  absence 
and  in  the  presence  of  0.5  mM  SA.  ALS  activity  was  increased 
by  about  50%  relative  to  the  appropriate  control  (i.e..  either 
untreated  cultures  or  those  with  SA  but  no  PCB).  These  results 
indicate  that  the  intracellular  pool  of  free  heme  is  large  enough 
to  support  enhanced  production  of  P4,0,  even  when  additional 
heme  svnthesis  is  blocked  bv  SA.  At  the  same  time,  the  dimi- 


nution in  free  heme  caused  by  SA  is  sufficient  to  permit  a  smaller 
but  significant  increase  in  ALS.  Because  HO  activity  is  very  low 
and.  consequently,  difficult  to  measure  in  fish  liver,  we  are  cur- 
rently developing  a  cDNA  probe  for  HO  mRNA  to  permit  as- 
sessment of  xenobiotic  effects  on  heme  degradation. 

This  work  was  supported  by  Endeavour  Foundation  (N.W.C.) 
and.  for  M.  E.  H.  of  the  Woods  Hole  Oceanographic  Institution, 
by  Sea  Grant  Project  No.  R/P-49  and  the  Penzance  Endowed 
Fund  in  Support  of  Scientific  Staff. 


Literature  Cited 


1 .  Marks,  G.  S.,  S.  A.  McCluskey,  J.  E.  Mackie,  D.  S.  Riddick,  and 
C.  A.  James.  1988.     FASEB  J.  2:  2772-2783. 

2.  llahn,  M.  E.,  and  J.  J.  Stegeman.  1994.     Toxicol.Appl  Pharmacol. 
127:  187-198. 

3.  Maines,  M.  D.  1988.     FASEB  J  2:  2557-2568. 

4.  Berry,  M.  N.,  and  D.  S.  Friend.  1969.     J  Cell  Biol.  43:  506-520. 

5.  Cornell,  N.W.  1983.     Pp.  [1-2Q  in  Isolation,  Characterization  and 
Use  of  Hepatocytes,  R.  A.  Harris  and  N.  W.  Cornell,  eds.  Elsevier, 
New  York. 

6.  Bruning,  G.,  M.  Ferkowicz,  and  N.  Cornell.  1993.     Biol.  Bull  185: 
327. 

7.  Klotz,  A.  V.,  J.  J.  Stegeman,  and  C.  Walsh.  1983.     Arch.  Biochcm 
Biopltyx.  226:  578-592. 


Reference:  Biol.  Bull-  189:  228-229.  (October/November,  1995) 

Suppression  of  Ca2  *  Flux  During  the  Transition  to  Anoxia  in  Turtle  Hepatocytes 
Revealed  by  a  Non-Invasive  Ca24 -Selective  Vibrating  Probe 

S.  C.  Land  and  P.  J.  S.  Smith  (National  Vibrating  Probe  Facility,  Marine  Biological  Laboratory) 


The  coordination  of  cellular  events  during  anoxic  metabolic 
suppression  has  been  studied  with  a  hepatocyte  preparation  iso- 
lated from  a  vertebrate  facultative  anaerobe,  the  western  painted 
turtle  [  (Chrysemys  picta  be/Hi)  ( 1 .  2)  ].  A  significant  fraction  of 
the  fall  in  total  ATP  demand  during  anoxic  metabolic  suppres- 
sion is  due  to  a  70%  reduction  in  rates  of  Na+/K+  ATPase  ac- 
tivity. Despite  these  changes,  the  cell  membrane  potential  re- 
mains at  —30  mV  throughout  anoxia  (3).  This  implies  that  an- 
oxia-induced metabolic  suppression  involves  a  cessation  of  flux 
through  ion  channels  that  is  coordinated  with  a  decrease  in  ion- 
pump  ATPase  activities  (4). 

The  present  study  examines  the  suppression  of  extracellular 
Ca2+  flux  (the  net  measure  of  Ca2+  ATPase,  transporter  and 
channel  activity)  during  the  transition  to  anoxia  in  turtle  he- 
patocytes. Cells  were  prepared  as  described  ( 1 )  and  cultured  on 
laminin-coated  glass  coverslips  ( <  103  cells  •  cm"2).  Extracellular 
Ca2+  flux  was  measured  with  a  Ca2+-selective  vibrating  probe 
(5)  from  cells  maintained  in  BSA-free,  Ca2+-depleted  medium 
(  [Ca2+]e  was  10-50  nM)  containing  10  mM  Mg2+.  Under  these 
conditions,  cells  demonstrated  a  steady-state  Ca2+  efflux  of -25.2 
±  8.6  ^V/cell  (mean  ±  SD)  over  1  week  of  culture  at  18°C.  In 
each  experiment,  anoxia  was  achieved  by  the  infusion  of  a  ni- 
trogen/CO:  atmosphere  into  a  chamber  surrounding  the  culture, 
and  dissolved  O2  concentrations  (  [O2]  )  were  monitored  si- 
multaneously with  a  polarographic  O2  microelectrode. 


A  representative  experiment  is  shown  in  Figure  1  A,  and  com- 
piled steady-state  data  are  shown  in  Figure  IB.  The  Ca2+  efflux 
was  diminished  by  75(v  during  anoxia,  and  it  recovered  towards 
control  values  on  re-oxygenation.  Notably,  this  suppression  of 
the  Ca2+  flux  began  early  in  the  transition  to  anoxia  and  had 
reached  a  new  steady-state  while  [O2]  was  above  lO^A/,  and 
therefore  still  theoretically  saturating  at  the  mitochondria  (6). 
In  previous  experiments  we  noted  that  aerobic  administration 
of  KCN  (inhibits  mitochondria!  oxidative  phosphorylation)  does 
not  diminish  net  Ca2+  efflux,  but  when  O2  is  depleted,  Ca2+ 
efflux  is  reversibly  suppressed  (data  not  shown).  Clearly,  the  re- 
duction in  Ca:+  efflux  in  the  transition  to  anoxia  is  an  O2-de- 
pendent  effect,  but  it  occurs  early  in  the  transition  to  anoxia, 
and  is  independent  of  the  role  of  O2  in  aerobic  metabolism. 
Therefore  we  tentatively  suggest  that  O2-receptive  mechanisms 
may  be  operative  in  the  modulation  of  Ca2+  efflux  in  anoxia  [as 
demonstrated  in  the  control  of  hypoxia-associated  protein 
expression  in  these  cells  (7)  ]. 

The  observed  hypoxia-induced  suppression  of  Ca2+  efflux  to 
a  new  steady  state  lends  support  to  the  concept  that  the  pres- 
ervation of  the  cell  membrane  potential  during  metabolic 
suppression  is  coordinated  among  multiple  processes.  We  are 
now  investigating  the  role  of  O2  in  the  control  of  membrane  and 
metabolic  events  during  the  transition  to  anoxia. 

This  work  was  conducted  with  the  generous  support  of  a  Lak- 


COMPARATIVE  PHYSIOLOGY  AND  BIOCHEMISTRY 


229 


A. 


B. 


[O,]  (MM) 


0.     -025- 


Bg    Al  Cell 


Or 


• 


>H" 


Ca;' 

Influx 


I  *I  Efflux 

1 


50  100 

Time  (mins) 


Bg  Nor         Anx       Re-oxy 

Figure  1 .  I. -I  i  Representative  experiment  demonstrating  the  suppres- 
sion nl  net  Cir*  etihi.x  during  ihe  transition  to  anoxia.  A  Cc?*-selective 
electrode  \\as  vibrated  at  0.3  H:  over  a  distance  ol  10  n>n  adjacent  to 
the  cell  membrane  (At  Cell).  Background  measures  were  taken  SO  tint 
twin  ike  cell  over  the  same  plane  of  vibration.  Measured  Orconceinralions 
are  in  »iM.  Each  plotted  value  represents  the  mean  ±  SD  of  10  independent 
dala  points  within  each  100  collected.  (B)  Compiled  steady-state  Ca~* 


ian  Postdoctoral  Fellowship  to  SCL.  The  National  Vibrating 
Probe  Facility  is  supported  by  NCRR. 

Literature  Cited 

1.  Buck,  I..  T.,  S.  C.  Land,  and  P.  \V.  Hochachka.  1993.     Am.  ./. 

Physiol  265(R34):  R49-R56. 

2.  Land,  S.  C.,  and  N.  J.  Bernier.  1995.     Pp.  379-410  in  Biochemistry 
and  Molecular  liiology  nl  Fishes.   \'ol.  5.  P.  W.  Hochachka  and 
T.  P.  Mommsen.  eds.  Elsevier.  Amsterdam. 

3.  Buck,  L.  T.,  and  P.  W.  Hochachka.  1993.     Am.  J  Physiol.  265(R34): 
R1014-R1019. 

4.  Hochachka,  P.  W.  1986.     Science  231:  234-241. 

5.  Smith,  P.  J.  S.,  R.  II.  Sanger.  and  L.  F.  Jaffe.  1994.     Melh.  Cell 
F  Biol.40:  115-134. 

6.  Jones,  D.  P.,  T.  V.  AH,  and  A.  H.  Sillau.  1990.     Expenenlia  46: 
1180-1185. 

7.  Land,  S.  C.,  and  P.  \V.  Hochachka.  1995.     Proe.  Xatl.  Acad.  Sci 
L'SA  92:  7505-7509. 


llux  dala.  I  'allies  are  means  ±  SD.  n  =  6  measurements  taken  on  cells 
Inini  independent  cultures  *P  =  0.01  (paired  Student's  \-test  relative  to 
control).  All  experiments  were  conducted  at  23-25° C.  Abbreviations:  Bg. 
background:  h'or,  normoxia:  Ax.  anoxia:  Re-oxy,  re-oxygenalion. 


Reference:  Biol.  Bull.  189:  229-230.  (October/November.  1995) 

Immunohistochemical  Localization  of  Saxitoxin  in  the  Siphon  Epithelium 
of  the  Butter  Clam,  Saxidomus  giganteus 

Roxanna  Smolowit:  (Laboratory  for  Marine  Animal  Health.  School  of  I  'eterinary  Medicine,  University  of 
Pennsylvania.  Marine  Biological  Laboratory)  and  Greg  Doucette 


Saxitoxin  (STX)  and  its  derivatives,  the  causes  of  a  lethal 
mammalian  neurotoxic  disease  called  paralytic  shellfish  poison- 
ing (PSP).  are  produced  by  several  dinoflagellate  genera  including 
Alexandrium,  Gymnodiniiim,  and  Pyrodinium  ( 1 ).  Butter  clams 
preferentially  accumulate  saxitoxin  in  their  siphon  tips  where 
the  toxin  can  remain  active  for  years:  this  is  a  defense  mechanism 
that  significantly  reduces  predation  of  the  clams  (2).  However, 
the  siphon  cell  type  in  which  toxin  accumulates  has  not  been 
identified.  A  recent  study  determined  that  nontoxic  butter  clams 
fed  Alexandrium  containing  gonyautoxin  (GTX)  and  neosaxi- 
toxin  (NEO).  but  no  STX.  accumulated  STX  in  the  siphon  and 
became  toxic.  The  authors  suggested  that  some  mechanism  other 
than  metabolic  conversion  of  GTX  and  NEO  to  STX  was  oc- 
curring since  GTX  and  NEO  were  depurated  from  the  clams 
before  STX  began  to  accumulate  in  the  siphon  (3). 

Kodama  (4)  suggested  that,  in  some  cases,  the  interaction  of 
bacteria  with  dinoflagellates  could  be  responsible  for  the  pro- 
duction of  PSP  toxin  by  the  dinoflagellates.  Indeed.  Alteromonas, 
a  bacterium,  was  recently  cultured  from  PSP-producing  dino- 
flagellates (G.  Doucette,  unpub.  data).  Kodama  (4)  further  sug- 
gested that  toxin-producing  bacteria  associated  with  the  dino- 


flagellates could  be  important  in  the  production  and  accumu- 
lation of  toxin  in  bivalves. 

Toxic  butter  clams  were  collected  from  a  restricted  area  in 
Washington  State  containing  toxic  clams.  Nontoxic  butter  clams 
were  collected  from  a  nearby  clean  area.  Sections  of  siphon  and 
other  organs  from  four  toxic  and  two  nontoxic  clams  were  fixed 
in  10%  buffered  formalin  in  seawater  and  processed  in  paraffin 
according  to  standard  methods.  Paraffin  sections  were  stained 
with  an  avidin-biotin  immunochemical  stain  and  a  peroxi- 
dase  tag. 

A  specific  antibody  (USA  rabbit  polyclonal  antibody  [USA 
PAB];  donated  by  Dr.  D.  Anderson,  Woods  Hole  Oceanographic 
Institution)  was  used  at  1/250  dilution  in  PBS.  Staining  controls 
consisted  of  either  normal  rabbit  serum  (NRS)  at  dilutions  of 
1/1000  and  1/2000  or  nondirected  rabbit  antibody  isotype  con- 
trol (control  AB)  (Zymed  08-6199)  substituted  for  the  specific 
antibody.  Sections  were  evaluated  with  an  Olympus  BH-2  pho- 
tomicroscope. 

Evaluation  of  immunohistochemically  stained  sections  of  toxic 
butter  clam  siphon  showed  multifocal  specific  staining  in  the 
columnar  epithelium  of  the  outer  (periostracial)  surface  of  the 


230 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


A 


B 


• 


siphon.  Staining  was  predominately  present  in  the  epithelium 
covering  the  tip  and  midportion  of  the  siphon.  Specific  staining 
was  intracellular  and  granular,  and  it  was  located  within  the 
apical  2/3  of  the  cells  (Fig.  1  A).  No  other  organs  or  cell  types  in 
the  animals  showed  specific  staining. 

Staining  was  not  present  in  control  sections  in  which  NRS  or 
control  AB  was  substituted  for  USA  PAB,  or  in  sections  of  siphon 
from  nontoxic  butter  clams  when  stained  with  USA  PAB  (Fig. 
IB)  or  control  solutions. 

Histologic  staining  of  siphon  sections  for  bacteria  adjacent  to 
or  within  epithelial  cells  was  negative.  Thus,  bacteria  seem  not 
to  be  responsible  for  the  occurrence  of  STX  in  these  cells.  Al- 
ternately, individual  or  small  groups  of  bacteria  could  be  in  res- 
idence in  or  between  the  epithelial  cells;  such  small  accumula- 
tions of  bacteria  are  not  easily  detected  with  a  histologic  stain. 

Now  that  we  have  identified  STX  in  the  columnar  epithelial 
cells  of  the  siphon's  outer  surface,  we  will  use  TEM  coupled 
with  immunocytochemical  staining  to  identify  the  organelles 
that  store  STX,  and  possibly,  associated  bacteria. 

This  work  was  supported  by  University  of  Pennsylvania  For- 
mula Funds. 

Literature  Cited 

1.  Anderson,  D.  M.  1990.     Pp.  41-51  in  Toxic  Marine  Phytoplankton, 
E.  Graneli,  B.  Sundsrom,  L.  Edler,  and  D.  M.  Anderson,  eds.  Elsevier, 
New  York. 

2.  Kvitek,  R.  G.  1993.     Pp.  407-41 1  in  Toxic  Phytoplanklon  Blooms 
in  ilic  Sen.  T.  J.  Smayda  and  Y.  Shimizu,  eds.  Elsevier,  New  York. 

3.  Beitler,  M.  K.,  and  J.  Listen.  1990.     Pp  257-262  in  Tn.\ic  Marine 
Phytoplankton,  E.  Graneli.  B.  Sundstrom,  L.  Edler,  and  D.  M.  An- 
derson, eds.  Elsevier,  New  York. 

4.  Kodama,  M.  1990.     Pp.  52-61  in  Toxic  Marine  Phytoplunkton,  E. 
Graneli,  B.  Sundsrom.  L.  Edler.  and  D.  M.  Anderson,  eds.  Elsevier. 
New  York. 


2 


Figure  1.  Photomicrographs  of  sections  ol  contracted  siphon  im- 
munohistochemically  stained  with  a  1/250  dilution  of  anii-ST\  i'SA 
rahhn  polydonal  antihody  and  counterstained  with  Mayers  hematoxylin. 
The  periostracum  uw.v  removed  from  the  outer  surface  of  the  siphon  he/ore 
processing  I.  II  (iramtlar  specific  staining  is  present  in  the  apical  2/3  of 
the  columnar  epithelial  cells  n/'ihe  outer  surface  of  a  toxic  clam 's  siphon 
(100X).  (B)  A'o  specific  sunning  is  present  in  the  columnar  epithelium  <>/ 
the  oulcr  surface  of  tins  nontoxic  clam's:  siphon  (100X).  1,  columnar 
epithelium  of  the  outer  (periostracial)  surface  of  the  siphon:  2.  subepithelial 
connective  tissue  of  the  siphon:  3,  specific  granular  staining:  4,  space 
representing  the  area  outside  of  the  animal's  body  (external  to  the  con- 
tracted siphonl. 


BEHAVIOR 


231 


Reference:  B/o/.  Bull  189:  231-232.  (October/November,  1995) 


Explorations  of  Turbulent  Odor  Plumes  with  an  Autonomous  Underwater  Robot 

T.  R.  Const  (MIT  Sea  Grant  Program),  F.  Grasso.  D.  Mountain,  and  J.  Atema 


Lobsters  extract  information  from  complex  signals  in  tur- 
bulent odor  plumes  and  it  guides  them  to  mates  or  food  sources. 
To  test  hypotheses  about  this  guidance  information,  we  have 
developed  a  robot  as  a  physical  model  of  a  lobster.  Here  we 
present  the  results  of  experiments  designed  to  test  the  efficacy 
of  amplitude  information — a  single  component  of  a  complex 
signal — in  guidance.  The  robot  used  a  bilateral  pair  of  conduc- 
tivity sensors  (sensor  surface  spacing  =  5-7  cm.  5  cm  separating 
two  1  -cm  wide  sensors)  to  sense  a  salt  plume  simulating  an  odor 
plume. 

The  experiments  were  performed  in  a  fresh-water  flume  with 
a  mean  flow  rate  of  0.6  cm/s.  A  0.76  vl/NaCl  solution  (containing 
crystal  violet  for  visualization  and  ethanol  to  adjust  buoyancy) 
was  injected  parallel  to  the  flow  from  a  2  mm  diameter  nozzle 
into  the  flume  at  a  rate  of  250  ml/min.  The  resulting  plume  had 
two  distinct  regions:  a  proximal  cone  originating  at  the  source, 
and  a  distal  patch  field  downstream  from  the  jet.  The  proximal 
jet  is  the  region  where  the  velocity  of  the  jet  exceeds  the  mean 
flow  in  the  flume.  The  distal  patch  field  corresponds  to  plume 
positions  downstream  from  the  proximal  cone  where  the  mean 
flow  is  the  major  source  of  plume  velocity  (relative  to  the  floor). 

Two  robot  control  algorithms  were  tested: 

1 .  The  robot  turns  toward  the  side  with  the  higher  salt  conduc- 
tance signal  or  goes  forward  if  the  difference  between  the 
right  and  left  sensor  signals  drop  below  9  j/S. 

2.  As  in  #  1 ,  with  the  added  feature  that  the  robot  goes  backward 
if  the  conductances  of  both  sensors  drop  below  a  threshold 
of7MS. 

The  robot  was  placed  in  the  center  of  the  flume.  90  cm  down- 
stream from  the  plume  source,  and  was  started  in  two  orienta- 
tions for  each  algorithm:  pointed  upstream  directly  into  the  on- 
coming plume,  and  pointed  45  degrees  to  the  right  of  the  plume 
axis.  Each  of  the  four  conditions  (2  orientations  and  2  algorithms) 
was  replicated  10  times.  The  robot's  trajectory  was  recorded  by 
a  video  camera.  Data  from  a  single  run  using  algorithm  #1  are 
presented  in  Figure  1. 

As  the  robot  moved  through  the  patch  field,  its  behavior  was 
characterized  by  sequences  of  abrupt,  brief  turns  that  occurred 
at  irregular  intervals.  When  it  entered  the  proximal  jet,  the  robot 
moved  with  more  regular  side-to-side  oscillations:  a  characteristic 
series  of  alternating  smooth  left  and  right  turns  (often  of  greater 
magnitude  than  those  seen  in  the  distal  patch  field).  Once  inside 
the  proximal  jet  the  robot  often  found  its  way  to  the  source  (50% 
algorithm  #1  and  72%  algorithm  #2). 

The  starting  orientation  had  a  substantial  effect  on  the  success 
of  the  algorithms.  Algorithm  #2  with  the  robot  pointing  into  the 
plume  had  a  higher  rate  of  direct  "hits"  onto  the  source  than 
algorithm  #1  with  the  same  orientation  (66%  vs  33%).  We  at- 
tribute the  greater  failure  rate  of  algorithm  #1  to  the  fact  that 
when  both  sensors  happen  to  exit  the  plume,  algorithm  #  1  moves 
the  robot  in  a  straight  line  away  from  the  point  of  exit.  The 


-  Right  Sensor 

•  -  -  Left  Sensor 
(inverted) 


100 


Closest  Approach 
to  Source 


Angular 
Velocity 

Right  -  Left 


Time  (sec) 


-20  L 


Source 


Distance  From  Origin  (cm) 


Figure  1 .  (A)  Right  and  left  conductance  measurements  during  an 
experimental  run  (algorithm  #1.  robin  start  pointed  up  the  central  plume 
a\isi  Left  sensor  data  were  inverted  for  visualization  purposes.  (B)  Con- 
ductance difference  inghl-lettl  and  turning  rate'-  Vertical  scale  is  in  ^s 
for  conductance  dillcrcncc  and  in  dcg/s  for  turning  rate.  Turning  events 
follow  sensor  signal  differences  by  0.6  s.  (C)  Robot  trajectory  during  the 
same  run  as  Figure  la.  b  Points  are  robot  positions  at  1-s  intervals.  The 
dolled  lines  denote  the  plume  til  the  end  of  the  experiment  and  show  the 
region  where  the  proximal  cone  nuikes  the  transition  into  the  distal  patch 
field. 


232 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


back-up  bi-h,  ior  of  algorithm  #2  corrects  for  plume  exit.  Ori- 
enting the  n.bot  at  45  degrees  reduced  the  probability  of  the 
robot  finding  the  cone  and  thus  reduced  the  probability  of  the 
robot  finding  the  source  (algorithm  #1:  33%  vs  0%,  algorithm 
#2:  66%  vs  10%). 

These  experiments  show  that  simple  bilateral  amplitude  com- 
parisons generally  suffice  to  guide  a  robot  within  the  proximal 
jet  of  a  chemical  plume.  Lobsters  may  use  such  an  algorithm 
for  guidance  in  the  proximal  cone.  This  simplest  algorithm  fails 


in  the  distal  patch  field  where  the  structure  of  the  chemical  signal 
is  less  regular.  Lobsters  show  different  behavioral  strategies  at 
different  distances  from  the  source  of  an  odor.  Consequently, 
additional  algorithms  will  be  required  for  successful  guidance 
of  the  robot  from  greater  distances  toward  the  proximal  cone. 
This  gradual  build-up  of  algorithm  complexity,  coupled  with 
lobster  behavior  analysis,  is  expected  to  lead  to  a  general  un- 
derstanding of  guidance  principles  in  odor  plumes. 
Supported  by  NSF  Grant  BES-93 15791  to  J.A. 


Reference:  Biol.  Bull.  189:  232-233.  (October/November.  1995) 

Effects  of  Varying  Plume  Turbulence  on  Temporal  Concentration  Signals  Available  to  Orienting  Lobsters 

Kevin  Dittmcr,  Frank  Grasso.  and  Jelle  Atema  (Boston  University  Marine  Program. 

Marine  Biological  Laboratory) 


Lobsters  locate  odor  sources  in  turbulent  plumes  (1.2).  Based 
on  the  speed  and  accuracy  with  which  lobsters  orient  to  such 
odor  sources.  Moore,  Scholz,  and  Atema  (2)  argued  that  these 
animals  are  guided  by  temporal  features  of  the  odor  concentra- 
tion profile  arriving  at  their  chemoreceptors  as  a  series  of  con- 
centration peaks.  Physical  investigations  (3,  4,  5)  of  the  temporal 
odor  signal  identified  a  number  of  peak  parameters  as  candidate 
guidance  cues.  These  physical  studies  and  the  behavioral  ones 
were  both  conducted  using  axisymmetrical  jet  plumes  generated 
in  a  single  turbulent  regime  (defined  by  mean  flow  through  the 
flume  and  jet  injection  rate).  Here  we  extend  the  physical  in- 
vestigations to  include  multiple  turbulent  regimes.  Specifically 
we  aim  to  address  the  question:  Do  the  candidate  peak  parameter 
gradients  identified  by  Moore  and  Atema  (5)  retain  their  guidance 
potential  for  the  lobster  in  plumes  generated  with  different  source 
injection  rates? 

To  facilitate  these  initial  measurements,  we  created  saline 
plumes  (source  0.76  M  NaCI)  containing  dye  and  ethanol  for 
neutral  buoyancy  in  a  fresh-water  flume  (366  X  90  X  36  cm, 
mean  flow  0.7  cm/s).  Conductivity  measurements  enabled  us  to 
estimate  salt  concentration  at  40  Hz  or  about  1 0  times  the  signal 
frequency  resolution  of  lobster  chemoreceptor  cells  (6).  The 
electrochemical  methods  used  in  the  previous  studies  (5)  were 
limited  to  10  Hz.  This  allowed  us  to  record  the  signal  amplitude 
while  minimizing  temporal  distortions  due  to  signal  masquer- 
ading in  the  bandwidth  of  lobster  chemoreceptor  cells  (6).  Our 
interest  here  is  in  turbulent  (inertial)  dispersal  where  flow  dom- 
inates molecular  transport.  Ethanol  is  not  measured  and  salt,  as 
well  as  ilopamine  which  was  used  in  some  of  our  earlier  studies, 
are  useful  tracers  for  turbulent  mixing  processes.  The  use  of  salt 
plumes  also  complements  our  studies  with  a  robot  that  orients 
with  conductivity  sensors.  Although  salt  and  ethanol  diffuse  dif- 
ferently than  food  odors  at  the  molecular  scale,  we  observed  no 
visual  differences  in  plume  structure  between  odor  plumes  and 
ethanol-salt  plumes.  Thus,  this  study  provides  a  reasonable 
physical  model  of  a  food  odor  plume  in  a  way  that  informs 
future  behavioral  and  robot  orientation  studies. 


We  estimated  the  salt  plume  concentration  time-course  (from 
measured  conductivity)  at  five  distances  along  the  Hume  mid- 
line  from  the  source  (0,  25,  50,  75.  and  100cm).  To  produce 
different  plumes  we  varied  the  rate  of  source  injection  [  (3  ml/ 
mm).  (40  ml/mm).  (80  ml/min).  (120ml/min)  and  (160ml/ 
min)  ].  Each  injection  was  delivered  continuously  through  a  2.2- 
mm  inner  diameter  glass  tube  located  at  the  flume  midline  9  cm 
from  the  Hume  floor.  Conductivity  measurements  were  taken 
with  a  pair  of  silver-tipped  electrodes  placed  9  cm  from  the  flume 
floor  (approximate  lobster  antennule  height).  The  electrodes  had 
a  1-mm  spatial  separation  (scale  of  a  lobster  sensillum).  Thus, 
we  sampled  a  horizontal  line  through  each  plume  at  an  elevation 
and  spatial  scale  that  matched  that  of  the  lobster  lateral  antennule 
receptors. 

We  converted  the  conductivity  profiles  to  concentration 
(moles/liter).  In  agreement  with  earlier  studies,  the  temporal 
profiles  that  we  examined  were  so  patchy  that  accurate  estimates 
of  the  concentration  gradient  would  require  greater  than  30  s  of 
sampling.  We  therefore  turned  to  an  analysis  of  the  temporal 
parameters  of  the  patches  themselves.  Patches  in  the  spatial  do- 
main are  seen  as  peaks  in  the  concentration  profile.  Peaks  were 
defined  (5)  as  the  profile  region  between  the  time  the  concen- 
tration exceeded  the  background  by  0.75  mM  and  the  time  the 
concentration  fell  below  30%  of  the  maximum  concentration  of 
the  peak.  We  examined  five  peak  parameters:  peak  height  (PH. 
maximum  concentration),  rise-time  (PR.  time  from  the  begin- 
ning of  a  peak  to  its  maximum),  peak  slope  (PS.  ratio  of  peak 
height  to  rise-time),  peak  duration  (PD  time  from  peak  start  to 
end),  and  interpeak  interval  (IPI,  time  between  consecutive  peak 
maxima). 

We  used  a  two-way  ANOVA  to  analyze  the  effects  of  distance 
and  injection  rate  on  each  of  the  five  peak  parameters.  These 
analyses  indicated  significant  effects  of  injection  rate  and  distance 
on  all  five  peak  parameters  (P  <  0.001  all  analyses).  These  results 
are  consistent  with  earlier  studies  (5).  They  also  revealed  signif- 
icant interactions  of  distance  and  injection  rate  on  all  five  peak 
parameters.  (P  <  0.001  all  analyses.) 


BEHAVIOR 


233 


However,  post  line  analyses  (GT2  test  for  unequal  sample 
sizes)  indicated  that  only  a  few  of  the  parameters  form  gradients 
that  could  be  used  as  cues  to  the  source.  The  effects  of  most 
injection  rates  were  only  distinguishable  nearest  the  source  (I3 
<  0.05.  all  distance  by  injection  comparisons  at  25  cm).  Only 
the  slowest  injection  rate  was  significant!)  different  from  all  other 
injection  rates  at  all  distances  (P  <  0.05.  for  all  distance  by  in- 
jection comparisons  with  the  injection  rate  of  3  ml/min).  Not 
all  comparisons  between  adjacent  distances  at  a  given  injection 
rate  were  significantly  different.  To  cite  one  example,  the  primary 
effect  of  distance  on  rise  time  could  be  traced  to  the  comparison 
between  25  and  100cm  (P  <  0.001).  Comparisons  of  PR  at 
intervening  distances  indicated  nonsignificant  differences.  Only 
PH  and  PS  showed  significant  differences  between  all  distances 
for  any  given  injection  rate  (P  <  0.05  for  all  comparisons)  except 
the  slowest  injection  rate  (3  ml/min).  In  PH  there  was  a  signif- 
icant distance  by  injection  interaction  at  an  injection  rate  of 
1 20  ml/min  (P  <  0.01 ).  Otherwise  PS  and  PH  decreased  mono- 
tonically  with  distance  from  the  source. 

The  results  allow  us  to  draw  four  conclusions  (that  pertain  to 
the  orientation  of  lobsters  and  robots):  1.  A  strategy  of  climbing 
PH  or  PS  gradients  within  a  single  plume  will  eventually  lead 
to  the  source,  except  for  plumes  generated  by  our  slowest  injec- 
tion rate.  2.  The  parameters  IPI.  PR.  and  PD  are  not  reliable 
cues  to  the  source  when  the  plume  is  sampled  at  25-cm  intervals 
along  the  midline  regardless  of  injection  rate.  3.  The  slowest 
injection  rate  (a  single  ribbon-like  filament)  does  not  afford  a 


significant  gradient  of  any  of  the  five  peak  parameters.  4.  PH 
and  PS  not  only  provide  good  spatial  gradients  over  100  cm. 
they  also  allow  us  to  differentiate  between  source  injection  rates 
at  a  distance  of  25  cm  from  the  source.  We  infer  that  lobsters 
or  robots  could  use  spatial  gradients  of  PH  and  PS  to  locate 
odor  sources  up  to  100  cm  away.  Information  about  the  strength 
of  the  jet  source  gradually  improves  until  it  becomes  clear  at 
25  cm  from  the  source. 

We  thank  Dr.  Jennifer  Basil,  Dr.  Rainer  Voigt,  and  Dr.  Rich- 
ard Hill  for  help  and  practical  advice.  This  work  was  supported 
by  NSF  grant  BES-93 1 579 1  to  J.  A.  and  awards  from  the  Woods 
Hole  Marine  Science  Consortium  Fund,  Michigan  State  Uni- 
versity Honors  College,  and  the  Jeffrey  Boetcher  Travel  Fund 
to  K..  D. 


Literature  Cited 

1.  Basil,  J.,  and  J.  Atema.  1994.     Biol  Bull  187:  272-273. 

2.  Moore,  P.,  N.  Scholz  and  J.  Alcma.  1991.     /  Chem.  Ecol.  17: 
1293-1307. 

3.  Moore,  P..  N.  Scholz,  L.  I.acomis  and  J.  Atema.  1991.     Biol.  Bull. 
Ill:  329. 

4.  Moore,  P.,  G.  Gerhardt,  and  J.  Atema.  1989.     Chem.  Senses  14: 
829-845. 

5.  Moore,  P.,  and  J.  Atema.  1991.     Biol.  Bull.  181:  408-418. 

6.  Gomez,  G.,  R.  Voigt,  and  J.  Atema.  1994.     J.  Comp.  Physio/.  174: 
803-811. 


Reference:  Biol  Bull  189:  233-234.  (October/November.  199?) 

Accumulation  and  Retention  of  Dimethylsulfoniopropionate  by  Bivalve  Molluscs: 

High  and  Nonnormal  Variation 

Richard  \V.  Hill  (Department  of  Zoology,  Michigan  State  University.  East  Lansing,  MI  48824), 
John  W.  H.  Dacev,  David  K.  D.  Hill,  Judith  E.  McDowell,  and  Dale  F.  Leavitt 


Dimethylsulfoniopropionate  (DMSP)  is  a  principal  sulfur 
compound  of  many  bivalve  molluscs  (I,  2,  3).  Many  phyto- 
plankters  synthesize  DMSP  (4).  and  bivalves  are  assumed  to 
accumulate  DMSP  from  phytoplankton  in  their  diet. 

DMSP  is  of  current  interest  in  bivalve  biology  for  two  major 
reasons,  both  linked  to  the  production  of  volatile  dimethyl  sulfide 
(DMS)  from  breakdown  of  DMSP  (nonvolatile).  First.  DMS  is 
evidently  a  critical  component  of  the  normal  taste  and  odor  of 
many  bivalves  (2,  5).  Second.  DMS  that  enters  the  atmosphere 
can  affect  climate  (6.  7).  Estimates  of  the  biomass  of  MytHm 
edulis  in  the  Baltic  Sea  (8),  together  with  known  filtering  rates, 
suggest  that  water  equivalent  to  half  the  Baltic  is  filtered  by  M. 
edulis  each  year.  Much  faster  filtration  of  bodies  of  water  by 
communities  of  bivalves  can  occur  (9).  Thus,  on  local — albeit 
not  global — scales,  major  fractions  of  phytoplanktonically  pro- 
duced DMSP  may  be  processed  by  bivalves,  and  the  molluscs 
might  thereby  significantly  affect  transfer  of  biogenic  sulfur  to 
the  atmosphere. 


Prior  reports  on  DMSP  in  bivalves  (1,  2.  3.  5)  typically  rest 
on  small  sample  sizes  and  stress  average  results.  Our  purpose  is 
to  bring  to  light  remarkable  features  of  individual  variation  in 
the  extent  of  accumulation  of  DMSP.  We  illustrate  using  data 
from  two  ongoing  studies  of  DSMP  in  M  edulis,  one  on  field 
levels,  the  second  on  retention. 

Tissues  were  prepared  for  analysis  by  incubation  in  cold  1-2 
N  K.OH  within  sealed  serum  bottles,  converting  DMSP  to  DMS 
(10).  Headspace  gas  from  the  bottles  was  analyzed  by  gas  chro- 
matography  (Chromosil  330  column,  Sievers  350B  chemilu- 
minescence  detector). 

The  July  data  in  Figure  1  depict  whole-body  DMSP/g  for  15 
mussels  collected  at  Sandwich,  Massachusetts,  in  July  1992.  Even 
though  all  animals  were  from  a  single  clump  and  were  within  a 
restricted  size  range  (6.2-8.1  cm  shell  length),  their  accumula- 
tions of  DMSP/g  varied  enormously  (standard  deviation  =  54% 
of  mean).  This  cannot  be  attributed  merely  to  differences  in 
feeding  history  just  before  collection  because  we  know  (see  below) 


234 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


f  4000 

A 
O 

—            o 

Whole  body 
Digestive  gland  (DG) 
Tissues  besides  DG 

0) 

A 

* 

S 

A 
A 

A 

fll 

3   3000 

A 

M 

A 

*-• 

A 

1 

A 

A 

•                                              0 

A 

o 

o)  2000 

<D 

4A 

• 

0. 

AA 

^^^ 

0° 

A 

O 

A 

A 

••                               o 

E 

A 

• 

c,  1000 

A 

• 

A 

A 

•• 

D. 

A 

A 

•       o                         o 

CO 

A 

i 

O                                    ° 

Q 

A 

A 

• 

••      n               *t^Q°° 

0  L 

July 

Aug          Start        End 

Field 
Levels 


Retention 
Study 


Figure  1.  Distribution  <>/  DMSP/g  in  ttnir  groups  <>/  Mytilus  edulis. 
Each  point  represents  one  mussel.  Lateral  displacement  of  points  is  for 
clarity.  Field  levels  are  for  whole  animals  freshly  collected  during  2  months. 
Mussels  in  the  retention  study  were  deprived  oj  DMSP  in  food  for  5  weeks. 
They  were  divided  into  digestive  glands  and  other  tissues  for  analysis. 


that  variation  in  tissues  outside  the  digestive  gland  is  as  great  as 
that  in  the  digestive  gland,  and  tissue  burdens  do  not  change 
rapidly  in  bivalves  (1).  The  August  data  (Fig.  1)  represent  15 
mussels  from  a  single  clump  collected  at  Sandwich  in  August 
1992.  These  results  show  nonnormality  of  distribution  (skewed, 
P  <  0.03;  1 1)  as  well  as  high  variation.  We  find  that,  among  M. 
edulis  living  as  neighbors,  high  variation  in  DMSP/g  is  the  rule, 
and  nonnormality  is  common.  High  variation  is  seen  in  pub- 
lished data  for  Afya  arenaria  (2). 

Each  M.  edit/is  was  subdivided  into  two  parts — the  digestive 
gland  and  the  rest  of  the  body — for  the  retention  study  in  Figure 
1.  The  mussels  (6.0-7.5  cm  length)  were  collected  from  1  m2  at 
Sandwich.  The  start  sample  shows  10  animals  soon  after  collec- 


tion. The  end  sample  depicts  15  mussels  after  5  weeks  of  living 
in  filtered  water  and  being  given  only  DMSP-free  food.  The 
distributions  in  the  latter  sample  are  the  most  strongly  nonnor- 
mal  we  have  encountered  (skewed,  P  <  0.002  for  digestive  gland, 
P  <  0.01  for  other  tissues).  Evidently,  some  individuals  voided 
DMSP  during  the  5  weeks  of  nonexposure,  but  others  retained 
it  tightly.  Slow  turnover  has  been  reported  previously  ( 1 ). 

The  features  of  the  statistical  distribution  of  DMSP  accu- 
mulation in  M.  edulis  emphasized  here  raise  both  biological 
questions  and  practical  concerns.  Biologically,  we  can  now  only 
wonder  why  neighbors  often  differ  so  strongly  in  accumulation; 
DMSP/g  is  not  correlated  with  shell  length  or  other  parameters 
we  have  examined.  The  tight  retention  of  DMSP  by  some  in- 
dividuals is  remarkable  and  raises  the  possibility  that  DMSP 
may  have  a  functional  role  in  bivalves.  At  a  practical  level,  the 
contribution  of  high  variation  in  DMSP/g  to  variation  in  taste 
needs  investigation.  Further,  the  distributional  features  we  report 
often  create  havoc  in  experimental  designs.  When  mussels  are 
fed  DMSP-containing  food,  we  believe  the  short-term  fate  of 
most  DMSP  is  accumulation  in  tissues,  but  testing  this  hypothesis 
is  challenging  because  the  high  intrinsic  variation  in  DMSP/g 
makes  statistical  discrimination  of  fed  and  control  groups  diffi- 
cult (12). 

S.  Hill.  D.  Franks,  B.  Lancaster,  F.  Nichy.  D.  Radosh,  and 
E.  Enos  made  necessary  contributions.  Supported  in  part  by 
NSFOCE9 1-02532. 

Literature  Cited 

1 .  Ackman,  R.  G.,  and  H.  J.  Hingley.  1968.     J  Fish.  Res  Board  Can. 
25:  267-284. 

2.  Brooke,  R.  O.,  J.  M.  Mendelsohn,  and  F.  J.  King.  1968.     J.  Fish. 
Res  Board  Can.  25:  2453-2460. 

3.  lida,  H.,  and  T.  Tokunaga.  1986.     Bull.  Jap.  Soc.  Sri.  Fish.  52: 
557-563. 

4.  Keller,  M.  D.,  \V.  K.  Bellows,  and  R.  R.  L.  Guillard.  1989.     Pp. 
167-182  in  Biogenic  Sit/lur  in  the  Environment.  E.  S.  Saltzman  and 
W.  J.  Cooper,  eds.  Amer.  Chem.  Soc..  Washington.  DC. 

5.  Ronald,  A.  P.,  and  W.  A.  B.  Thomson.  1964.     J  Fish.  Res  Board 
Can  21:  I4XI-1487. 

6.  Shaw,  G.  1983.     Chin.  Change  5:  297-303. 

7.  Charlson,  R.  J.,  J.  E.  Lovelock,  M.  O.  Andreae,  and  S.  G.  Warren. 
1987.     Nature  326:  655-661. 

8.  Kautsky,  N.,  and  I.  \\allenlinus.  1980.     Ophelia.  Suppl.  I:  17-30. 

9.  Smaal,  A.  C.,  and  T.  C.  Prins.  1993.     Pp.  271-298  in  Bivalve  Filter 
Feeders  in  Estuarine  and  Coastal  Ecosystem  Processes.  R.  F.  Dame, 
ed.  Springer- Verlag,  New  York. 

10.  Dacey,  J.  W.  H.,  and  N.  V.  Blough.  1987.     Geophys  Res  Lett.  14: 

1246-1249. 
1  I     D'Agostino,  R.  B.,  and  G.  L.  Tietjen.  1973.     Biomelnka  60:  169- 

173. 
12    Hill,  R.  W.,  J.  \V.  H.  Dacey,  J.  E.  McDowell,  and  D.  F.  Leavitt. 

1993.     Biol  Bull  185:  322-323. 


PHYSIOLOGICAL  ECOLOGY  AND  BEHAVIOR 
Reference:  Biol.  Bull.  189:  235-236.  (October/November.  1995) 


235 


Accumulation  of  Dimethylsulfoniopropionate  in  Geukensia  demissa  Depends  on  Trophic  Interactions 

Bradley  A.  White  (Department  of'/.oology,  Michigan  State  University,  East  Lansing,  MI  48824), 

Richard  IT.  /////.  and  John  \V.  H.  Dacev 


Dimethyl  sulfide  (DMS)  constitutes  up  to  half  of  the  atmo- 
spheric sulfur  produced  biogenically  (1,2)  and  may  affect  global 
climate  (3).  A  major  source  of  atmospheric  DMS  is  the  enzymatic 
cleavage  of  dimethylsulfoniopropionate  (DMSP),  which  is  syn- 
thesized by  many  phytoplankters  (4.  5)  and  a  few  vascular  plants, 
including  Sptinimi  aherniflora  (5).  Most  DMSP  is  released  fol- 
lowing rupture  of  cell  walls  (6.  7)  and  is  then  subject  to  microbial 
degradation  to  DMS  (8,  9).  Little  attention  has  been  given  to 
salt  marsh  DMSP  fluxes  outside  the  autotrophic  and  microbial 
components  of  the  food  web. 

The  purpose  of  this  study  was  to  explore  DMSP  pathways 
within  the  Great  Sippewissett  Marsh  (Falmouth.  Massachusetts), 
determining  in  particular  whether  tissue  concentrations  of  DMSP 
in  Genkciisui  demissa  vary  with  food  resources.  G  demissa,  the 
ribbed  mussel,  is  the  dominant  animal  in  salt  marshes  in  the 
eastern  United  States  (10).  Ribbed  mussels  in  the  Great  Sippe- 
wissett Marsh  filter  most  of  the  marsh  water  during  each  tidal 
cycle  (11)  and  thus  are  likely  to  play  a  key  role  in  marsh  DMSP 
fluxes.  (J  demissa  can  directly  consume  S.  aherniflora  detritus 
in  addition  to  plankton  and  bacteria  ( 1 2).  Peterson  el  al.  showed 
that  the  isotopic  composition  of  G.  demissa  in  the  Great  Sip- 
pewissett Marsh  follows  a  horizontal  gradient  reflecting  a  shift 
in  available  food  resources,  from  a  diet  high  in  phytoplankton 
(up  to  70%)  near  the  bay.  to  mostly  Spartina  detritus  (80%)  in 
the  marsh  interior  ( 1 3).  Delta  values  for  34S  isotope  indicate  that 
much  of  this  shift  occurs  in  a  relatively  short  region  along  the 
tidal  channel  between  sites  3  and  4  (Fig.  1 ). 

We  hypothesized  that  mussels  near  the  bay  would  have  rel- 
atively high  levels  of  DMSP  due  to  a  diet  rich  in  live  phyto- 
plankton. Conversely,  mussels  in  the  interior  of  the  marsh  were 
expected  to  have  lower  levels  of  DMSP.  Their  diet  is  dominated 
by  Spui'iina  detritus,  which,  if  directly  consumed,  would  probably 
be  depleted  of  DMSP  by  leaching,  and  if  indirectly  consumed 
via  bacteria  and  nanozooplankton  filtration,  would  likely  be  de- 
pleted of  DMSP  by  microbial  decomposition.  We  expected  this 
shift  in  DMSP  concentration  to  be  most  dramatic  between  site 
4  and  all  others,  following  the  shift  in  "S  delta  values  (Fig.  1 ). 

The  mussel  collection  sites  in  Figure  1  were  chosen  to  cor- 
respond to  isotope  study  sites  (13).  Five  mussels  (5-7.5  cm  long) 
were  collected  from  each  site  in  August  1994,  and  the  digestive 
glands  were  analyzed  separately  from  the  rest  of  the  body.  The 
samples  were  incubated  in  2 A'  KOH  in  sealed  vials  at  25°C  for 
24  h.  allowing  DMS  from  hydroxide  decomposition  of  DMSP 
to  partition  to  equilibrium.  Mussel  DMSP  content  was  calculated 
from  determinations  of  DMS  in  headspace  samples  by  gaschro- 
matography  (Chromosil  330  column.  Sievers  350B  Sulfur  Che- 
miluminescence  Detector). 

To  test  for  differences  among  collection  sites,  the  Kruskal- 
Wallis  nonparametric  procedure  was  used  (SPSS/PC+).  Vari- 
ances for  DMSP/g  in  mussels  are  often  high  and  nonnormal 
( 14).  In  our  study,  they  were  also  nonhomogeneous.  The  Krus- 
kal-Wallis  test  eliminates  dependency  on  normal  distributions, 
homogeneity  of  variances,  and  other  parametric  test  assump- 


tions. In  spite  of  small  sample  sizes  and  high  variation  within 
sites — both  decreasing  the  likelihood  of  detecting  differences — 
the  DMSP  concentration  in  digestive  glands  (P  =  0.01 1)  and 


100 

Buzzards  Bay 


DMSP  concentration  (nmol/g)         Delta 


Rest  of  body       isotoperatio 


Y 

99.4 
154.7 
98.1 
412 

SD. 

36.7 
126.0 
46.1 
17.7 

+12.143 
b 

+  8.55 
+  0.54C 

Site   Digestive  gland 
~T~       SD. 

1  449.9      128.4 

2  569.0     4212 

3  590.6      319.0 

4  121.7       33.1 


Figure  I.  Aerial  new  of  Geukensia  demissa  sampling  sues  in  Great 
Sippewisseti  Marsh  adapted  from  Peterson  et  al.  (13).  The  table  reports 
means  and  standard  deviations  lor  DMSP  concentration  (nmol/g)  in 
digestive  gland  and  the  rest  of  the  body  tissue,  and  delta  values  for  i4S 
isotope  at  each  of  the  four  si/es  on  the  map.  Only  larger  tidal  channels 
are  shown.  At  sile  2.  mussels  were  collected  Irani  a  panne  on  the  marsh 
surface.  Mussels  at  other  sites  were  collected  from  the  creek  bank.  Site  4 
is  a  mosquito  ditch,  a  =  adjacent  creek  bank,  b  =  no  value  reported,  c 
=  neighboring  mosi/iiila  ditch 


236 


REPORTS  FROM  THE  MBL  GENERAL   SCIENTIFIC  MEETINGS 


the  rest  of  the  body  tissue  (P  =  0.05 1 )  proved  to  be  lower  at  site 
4  than  for  other  sites. 

As  hypothesized,  from  the  main  tidal  channel  to  the  marsh 
interior.  DMSP  concentration  undergoes  a  shift  that  reflects  the 
isotopic  gradient.  This  evidence  suggests  spatial  heterogeneity 
in  DMSP  fluxes  through  G  demissa  concordant  with  trophic 
differences  in  the  marsh.  The  shift  is  greatest  for  the  digestive 
gland  tissue,  which  contained  relatively  high  levels  of  DMSP. 

The  dynamics  and  functional  significance  of  DMSP  in  indi- 
vidual mussels,  the  extent  of  direct  versus  indirect  consumption 
ofSpartina  detritus,  and  the  rate  of  detrital  DMSP  loss  remain 
undetermined.  Measurement  of  DMSP  in  the  water  column  at 
various  sites  in  the  marsh  will  further  elucidate  these  relation- 
ships. We  are  in  the  process  of  expanding  the  data  set  reported 
here  to  clarify  Geitkensia  DMSP  gradients  in  the  marsh. 

This  study  was  conducted  as  part  of  a  Woods  Hole  Marine 
Science  Consortium  internship  to  B.  A.  W.,  and  was  supported 
in  part  by  NSF  OCE  9102532.  We  thank  Ivan  Valiela  for  his 
assistance. 

Literature  Cited 

1.  Andreae,  M.  O.,  and  H.  Raemdonck.  1983.     Science  221:  744-747. 

2.  Bates,  T.  S.,  J.  D.  Cline,  R.  H.  Gammon,  and  S.  R.  Kelly-Hansen. 

1987.     J.  C/V,y>/in   Re-,  92:  2930-2938. 


3.  Charleston,  R.  J.,  J.  E.  Lovelock,  M.  O.  Andreae,  and  S.  G.  Warren. 
1987.     Nature  326:  655-661. 

4.  Andreae,  M.  O.  1986.     Pp.  331-362  in  The  Role  of  Air-Sea  Ex- 
change in  Geochemical  Cycling.  P.  Buat-Menard.  ed.  D.  Reidel. 
Dordrecht.  The  Netherlands. 

5.  Charlston,  R.  J.  1995.     Pp.  251-262  in  Biotic  Feedbacks  in  the 
Global  Climatic  System.  G.  M.  Woodwell  and  F.  T.  Mackenzie, 
eds.  Oxford  University  Press,  New  York. 

6.  Dacey,  J.  \V.  H.,  G.  M.  King,  and  S.  G.  Wakeham.  1987.     Nature 
330:  643-645. 

7.  Kiene,  R.  P.  1988.     FEMS  Microbiol.  Ecol.  53:  71-78. 

8.  Krouse,  H.  R.,  and  R.  G.  L.  McCready.  1979.     Pp.  401-431  in 
Biogeochemical  Cycling  of  Mineral  Forming  Elements,  P.  A.  Tru- 
dinger  and  D.  J.  Swaine.  eds.  Elsevier.  Amsterdam. 

9    Ledyard,  K.  M.,  E.  F.  DeLong,  and  J.  \V.  H.  Dacey.  1993.     Arch. 
\lurohiol.  160:  312-318. 

10.  Bertness,  M.  D.  1984.     Ecology  65:  1794-1807. 

11.  Jordan,  T.  E.,  and  I.  Valiela.  1982.     Limnol.  Oceanogr.  27:  75-90. 


Mar.  Ecol.  Prog.  Ser. 


12.  Langdon,  C.  J.,  and  R.  I.  E.  Newell.  1990. 

58:  299-310. 

1 3.  Peterson,  B.  J.,  R.  \V.  Howarth,  and  R.  H.  Garritt.  1985.     Science 
227:  1361-1363. 

14    Hill,  R.  \V.,  J.  \V.  H.  Dacey,  D.  K.  D.  Hill,  J.  E.  McDowell,  and 
D.  F.  Leautt.  1995.     Bin.  Bull  189:  000-000. 


Reference:  Biol.  Bull.  189:  236-237.  (October/November,  1995) 

Localization  of  Digestion  Activities  in  the  Sea  Anemone  Haliplanella  luciae 

Dirk  Bumann  (Marine  Biological  Laboratory) 


Among  Cnidaria,  the  Anthozoa  have  many  radial  septa  called 
mesenteries,  which  project  from  the  body  wall  into  the  gastric 
cavity.  Anthozoa  are  typically  larger  than  other  cnidarians  and 
therefore  might  have  needed  to  increase  their  inner  surface/body 
weight  ratios.  Therefore,  a  generally  proposed  function  of  the 
mesenteries  is  to  enhance  the  gastrodermal  surface,  and  thereby 
the  digestive  capacity  ( 1 ).  Cells  that  secrete  enzymes  for  extra- 
cellular digestion  are  highly  localized  in  small  areas  at  the  edges 
of  the  mesenteries  called  filaments  (2).  Hence,  any  enhancement 
of  the  digestive  capabilities  would  be  only  due  to  an  increase  in 
the  number  of  cells  absorbing  food  fragments.  Indeed,  the  whole 
gastrodermis  is  capable  of  endocytosis  in  sea  anemones  (2.  3). 

In  the  studies  discussed  above,  however,  artificial  food  was 
used,  and  the  results  contradict  those  obtained  many  decades 
ago,  also  with  artificial  food,  indicating  that  the  septa  were  mainly 
active  in  absorption  (4,  5).  It  is  possible  that  only  parts  of  the 
gastrodermis  are  active  when  little  food  is  available,  whereas 
major  fractions  become  active  when  food  is  abundant  (as  ob- 
served for  the  scvphozoan  Attrclia  aurita.  D.  B.  and  G.  Jarms. 
unpub.  obs.).  In,the  early  experiments,  the  digestive  capacity  of 
the  sea  anemones  may  not  have  been  saturated.  Moreover,  the 
relevance  of  experiments  with  artificial  food  for  the  functional 
significance  of  the  mesenteries  is  doubtful.  Hence  experiments 


with  natural  food  and  feeding  to  satiation  are  needed  to  critically 
test  the  hypothesis. 

The  anthozoan  Haliplanella  luciae.  obtained  from  Mill  Pond. 
Woods  Hole,  Massachusetts,  was  fed  to  satiation  with  protists 
and  crustaceans,  which  are  major  constituents  of  their  natural 
diet  (2.  6).  To  trace  ingestion,  digestion,  and  absorption,  the 
food  was  labeled  with  fluorescent  dyes.  Digestion  in  living  ane- 
mones (10  per  food  type)  was  observed  with  fluorescence  mi- 
croscopy for  2-4  h  after  feeding.  Additionally,  some  anemones 
were  anesthetized  and  fixed  at  given  time  intervals  up  to  6  h 
after  feeding.  After  bisection,  they  were  examined  with  a  dis- 
secting scope.  For  intracellular  localization  of  dyes,  dissected 
pieces  of  the  anemones  were  examined  with  a  fluorescence  mi- 
croscope. 

For  microphagous  feeding  (2),  yeast  cells  were  labeled  with 
the  dye  Evans  blue.  Haliplanella  ingested  yeast  cells  and  mixed 
them  with  mucus  in  the  basal  part  of  the  pharynx.  The  yeast- 
containing  mucus  was  transported  to  the  edges  of  the  mesenteries 
where,  in  all  anemones  tested,  absorption  almost  exclusively 
occurred.  Little  absorption  could  be  detected  in  the  tentacle  gas- 
trodermis and  none  at  all  was  seen  in  the  rest  of  the  mesenteries 
or  in  the  column  wall,  even  after  6  h  in  the  presence  of  5  X  106 
yeast  cells  ml~'.  However,  isolated  column  wall  fragments  can 


PHYSIOLOGICAL  ECOLOGY  AND  BEHAVIOR 


237 


Figure  1 .  Longitudinal  section  of  the  sea  anemone  Haliplanella  luciae 
fixed  4  h  after  feeding  to  satiation  with  Artemia  salina  nauplii  labeled 
with  Evans  blue.  The  arrowheads  indicate  the  me.senterial  regions  adjacent 
it  i  lite  tilameius  where  endocytosis  almost  exclusively  occurred.  The  scale 
bar  represents  I  mm 

absorb  yeast  cells  or  other  particles  in  Haliplanella.  Apparently 
in  intact  Haliplanella,  all  yeast  cells  ingested  are  trapped  in  the 
pharyngal  mucus  and  therefore  might  not  be  available  to  the 
column  wall  cells.  On  the  other  hand,  a  small  fraction  of  the 
artificial  food  India  ink  passed  the  pharynx  without  being  trapped 
and  was  then  absorbed  in  the  whole  gastrodermis  as  previously 
observed  in  other  species  (2).  The  differential  affinities  of  yeast 
and  India  ink  for  mucus  are  currently  under  investigation. 

For  macrophagous  feeding.  Anemia  salina  nauplii  were  co- 
valently  labeled  with  the  dyes  Texas  red  sulfonyl  chloride  and 
fluorescein  isothiocyanate,  or  non-covalently  labeled  with  Evans 
blue  (7).  The  flexible  mesenteries  formed  tight  sacs  around  all 
ingested  nauplii  as  observed  previously  (8.  9).  These  food  sacs 


were  further  wrapped  with  mucus  as  shown  by  mucicarmine 
staining.  During  extracellular  digestion  of  the  wrapped  food, 
very  little  of  the  three  dyes  could  be  detected  in  the  gastric  cavity 
in  all  ten  tested  anemones,  which  indicated  that  paniculate  food 
fragments  as  well  as  soluble  constituents  (e.g..  the  water-soluble 
dye  Evans  blue)  were  almost  completely  trapped  in  the  mes- 
enterial  sacs.  Hence,  no  mixing  of  the  digestive  juices  occurred 
within  the  gastric  cavity.  This  explains  why  very  little  digestive 
enzymes  were  previously  detected  in  the  gastric  cavity  during 
digestion  (9,  10).  Endocytosis  took  place  almost  exclusively  in 
the  mesenterial  regions  directly  adjacent  to  the  filaments,  even 
when  the  animals  were  fed  to  satiation  (Fig.  1).  No  endocytosis 
was  detected  in  the  rest  of  the  mesenteries  or  column  walls. 

In  summary,  the  results  show  that  in  Haliplanella  only  a  very 
small  part  of  the  gastrodermal  surface  area  is  used  for  digestion 
of  natural  food.  This  is  not  due  to  a  general  lack  of  uptake  ca- 
pabilities in  the  other  areas,  but  is  due  to  a  highly  localized 
digestion  during  which  even  soluble  food  constituents  remain 
trapped  in  the  sacks  formed  by  the  mesenteries.  Only  small  frac- 
tions of  artificial  food  were  absorbed  in  the  column  walls.  En- 
hancement of  the  gastrodermal  surface  area  as  the  main  function 
of  the  mesenteries  ( I )  is  not  likely,  at  least  in  this  species.  Instead, 
mesenteries  serve  mainly  biomechanical  functions  (11). 

I  thank  the  Deutsche  Forschungsgemeinschaft  for  financial 
support  in  the  form  of  a  postdoctoral  fellowship  (Bu  971/-1) 
and  Alan  Kuzinan.  Eugene  Tassinari.  Hemant  Chikarmane,  and 
Gerhard  Jarms  for  helpful  discussions  and  support. 


Literature  Cited 


10 


11 


Schuchert,  P.  1993.     Z  Zoo/.  Syst.  Evol.-forsch.  31:  161-173. 

Yan  Praet,  M.  1985.     Adv  Mar.  Binl.  22:  65-99. 

Yan  Praet,  M.  1980.     Reprod  \mr  Dev.  20:  1393-1399. 

Metschnikoff,  E.  1880.     Zoo/.  An:.  3:  261-263. 

Mesnil,  F.  1901.     Ann  Insl.  Past  15:  352-397. 

6.  Chintiroglou,  C.  1992.     Helxol.  Meereswiterx.  46:  53-62. 

7.  Dunne,  J.  F.,  and  l.ittlefield,  C.  L.  1983.     Pp.  131-140  in  Hydra: 
Research  Methods.  H.  M.  Lenhoff.  ed.  Plenum  Press.  New  York. 

8.  MacGinitie,  G.  E.,  and  MacGinitie,  N.  1949.     Natural  History  of 
Marine  Animals,  p.  126.  McGraw-Hill.  New  York. 

9.  Nicol,  J.  A.  C.  1959.     J.  Mar.  Binl.  Assoc  L'.K  38:  469-476. 
Krijgsam,  B.  J.,  and  Talbot,  F.  H.  1953.     Arch.  Inl   Physio/.  61: 
277-291. 

Batham,  E.  J.,  and  Pantin,  C.  F.  A.  1950.     ./  K.\p.  Biol  27:  264- 
289. 


Reference:  Biol.  Bull  189:  237-238.  (October/November.  1995) 


Natural  Diets  for  Hermissenda  crassicornis  Mariculture 

Conxita  Avila  and  Alan  M.  Kniirian  (Marine  Biological  Laboratory) 


A  main  goal  at  the  Hermissenda  Resource  Facility  is  to  max- 
imize maintenance  conditions  for  survival,  fecundity,  and  de- 
velopment rates  on  a  steady,  year-round  basis  ( 1 ).  At  times,  the 
main  laboratory  food,  the  hydroid  Tubularia  cmcea.  is  difficult 


to  obtain.  For  that  reason,  some  alternative  artificial  diets,  such 
as  crab  meat  and  fish  food  pellets,  have  been  used  to  feed  Her- 
missenda juveniles  (2.  3).  Of  the  diets  tested,  soft  fish  food  pellets 
gave  optimal  results,  although  water  fouling  could  not  be  com- 


238 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


Predator 
No  predator 


Tubularia  Ciona  Mytilus  Metridium  Haliplanella 


M 

600 
500 
400 

;       i 

0) 
CO 

•c 

C 

300 
200 

H 

W 

100 

•  ^         m 

<u 

-100 

w  w 

r1 

-200 

</%/ 

ncrease  i 

-300 
-400 
-500 

.. 

Figure  I .  Hermissenda  crassicornis  feeding  experiment  with  five  dij- 
lerenl  diets :  the  hydnrid Tubularia  crocea,  the  liinieale Ciona  intestinalis, 
//)(•  mussel  Mytilus  edulis.  ami  I  he  anemones  Metridium  senile  and  Hal- 
iplanella luciae.  (A)  Decrease  in  the  //mil  weight  (ing)  in  the  presence  and 
absence  <>l  H.  crassicornis.  (B)  Increase  in  weight  (mg)  in  H.  crassicornis 
with  di/lerenl  diets.  I  'allies  are  means  <>1  three  replicates  ±SE 


pletely  avoided  (2).  Therefore,  we  found  it  useful  to  try  additional 
natural  diets  that  could  not  only  provide  a  temporary  food 
source,  but  also  allow  adult  Hermissenda  to  grow  and  produce 
egg  masses. 

We  tested  the  tunicate  Ciona  intestinalis.  the  mussel  Mytilus 
edulis,  the  anemones  Metridium  senile  and  Haliplanella  luciae. 
with  the  hydroid  T.  crocea  as  a  control.  Adult  Hermissenda 
(three  replicates/treatment)  were  maintained  in  individual  200- 
ml  containers  at  12°C  and  fed  ad  libitum,  except  those  on  the 
Haliplanella  diet.  The  cnidarians  were  offered  live,  while  the 
tunicates  (cut)  and  mussels  (opened)  were  offered  fresh.  The 


seawater  was  changed  every  other  day,  and  food  was  added  as 
necessary.  Food  items  and  the  Hermissenda  specimens  were 
weighed  before  and  after  the  experiment,  which  lasted  1  week. 
Eggs  laid  were  also  weighed.  In  addition,  food  items  were  kept 
in  containers  without  predators  to  evaluate  changes  in  weight 
other  than  those  accounted  for  by  consumption.  Data  were  sta- 
tistically analyzed  by  using  ANOVA  and  the  Student-Newman- 
Keuls  test. 

Our  data  indicate  that  all  the  food  items  were  eaten  by  Her- 
missenda (Fig.  1A),  although  the  amounts  consumed  yielded 
different  growth  rates.  Changes  in  food  weight  from  causes  other 
than  predation  were  minimal  (Fig.  1A).  Most  specimens  laid  at 
least  one  egg  mass,  with  the  exception  of  those  on  the  tunicate 
diet  and  two  animals  on  the  mussel  diet.  As  in  other  studies 
with  opisthobranch  molluscs  (4),  egg  mass  and  adult  weight  were 
considered  together  as  the  cumulative  increase  in  weight  (Fig. 
1  B).  Statistical  analysis  indicated  that  the  treatments  produced 
significantly  different  weight  gains  (ANOVA.  p  =  0.03.  d.f.  =  4). 
The  results  of  the  Student-Newman-Keuls  test  indicated  signif- 
icant differences  between  the  following  pairs  of  diets:  Tubularia/ 
Ciona  (p  =  0.04),  Tubularia/Mytilus  (p  =  0.04),  Ciona/Metri- 
dium  (p  =  0.019),  Mytilus/Metridium  (p  =  0.017).  and  Metri- 
dium/Haliplanella  (p  =  0.039).  Therefore,  for  the  growth  of 
Hermisscnilu  in  the  laboratory,  the  Tubularia  diet  is  better  than 
the  Ciona  or  Mvliltis  diets.  Furthermore,  the  Metridium  diet  is 
better  than  the  Ciona.  Mytilus,  or  Haliplanella  diets.  The  Ciona 
and  Myliliis  diets,  although  eaten  by  Hermissenda,  resulted  in 
significant  weight  loss  and  a  rapid  increase  in  water  fouling.  All 
the  cnidarian  foods  produced  a  weight  increase  in  Hermissenda. 
with  no  effects  on  water  quality.  The  weight  gain  was  most  sig- 
nificant for  animals  fed  with  M.  senile,  followed  by  T  crocea. 
and  then  //  luciae  (Fig.  IB).  //.  luciae.  which  was  confirmed  as 
a  suitable  prey  in  two  earlier  experiments  (not  reported  here), 
was  rapidly  eaten  by  Hermissenda,  but  could  not  be  used  ad 
libitum  due  to  limited  availability.  Further  experiments  should 
analyze  the  long-term  effects  of  these  different  diets,  particularly 
Metridium  and  Haliplanella.  both  on  adult  growth  and  on  re- 
productive effort. 

A  Spanish  Government  postdoctoral  fellowship  to  C.A.  is 
gratefully  acknowledged.  This  research  was  supported  by  an 
NCRR-N1H  grant  (P40-RR03820)  to  A.M.K.  //.  luciae  was 
kindly  provided  by  C.  M.  Chester  of  the  University  of  New 
Hampshire. 

Literature  Cited 

I     Kuzirian,  A.  M.,  C.  T.  Tamse,  and  E.  Yamoah.  1989.     Am.  Zool. 
29:  333. 

2.  Yamoah,  E.,  A.  M.  Kuzirian,  D.  Phie,  and  L.  Matzel.  1988.     Biol. 
Bull.  175:  309. 

3.  Harrigan,  J.  E.,  and  D.  L.  Alkon.  1978.     Biol.  Bull.  154:  430-439. 

4.  Havcnhand,  J.  N.,  and  C.  D.  Todd.  1988.     ./  Kxp  Mar  Biol.  Ecol. 
118:  173-189. 


PHYSIOLOGICAL  ECOLOGY  AND  BEHAVIOR 
Reference:  Biol.  Bull  189:  239.  (October/November. 


239 


The  Effect  of  Temperature  on  the  Relationship  Between  a  Ciliated  Protozoan,  Trichodina  cottidarum, 
and  the  Longhorn  Sculpin,  Myoxocephalus  octodecemspinosus 

David  C.  Bra:ik  and  Robert  A.  Bitllis  (School  of  Veterinary  Medicine, 
University  of  Pennsylvania,  Marine  Biological  Laboratory) 


The  longhorn  sculpin  (Myoxocephalus  octodecemspinosus)  is 
the  host  of  Trichodina  cottidarum,  a  frequent  inhabitant  of  the 
gills.  Natural  repellents  in  the  mucus  of  the  fish's  surface  help 
limit  the  number  of  Trichodina  present  (1).  The  antimicrobial 
properties  of  this  protective  layer  either  diminish  or  change  when 
an  animal  becomes  stressed,  allowing  Trichodina  to  increase  in 
numbers,  which  causes  damage  to  the  gills.  Sculpins  captured 
and  held  at  colder  temperatures  exhibited  little  mortality,  but 
as  the  ambient  temperature  rose  in  the  spring,  sculpin  mortality 
associated  with  protozoal  branchitis  increased  (Bullis,  unpub. 
data).  Thus,  increased  water  temperature  may  play  a  deleterious 
role  in  the  relationship  between  these  organisms. 

Forty  sculpin  were  obtained  from  the  Marine  Resources  Cen- 
ter of  the  Marine  Biological  Laboratory.  Four  20-gallon  tanks 
were  set  up  with  running  ambient  seawater  and  each  had  an 
undergravel  filter  of  native  sand.  Ten  fish  were  placed  in  each 
aquarium.  Heaters  were  placed  in  three  tanks  to  slowly  raise  the 
temperature  of  the  water  by  2°C  per  day  for  5  days.  One  tank 
was  heated  and  held  at  10°C,  two  at  15°C,  and  the  fourth  tank 
remained  at  ambient  (~5°C)  for  the  remainder  of  the  experi- 
ment (4  weeks).  One  of  the  15°  tanks  was  used  as  a  treatment 
tank.  After  ~50%  of  this  group  had  died  and  been  confirmed 
as  being  Trichodina  positive,  the  temperature  was  allowed  to 
return  to  ambient  in  an  attempt  to  retard  protozoal  growth. 
Water  quality  was  tested  weekly  to  ensure  that  there  was  no 
build-up  of  ammonia  or  nitrite  that  could  damage  the  gills  of 
the  animals.  The  fish  were  not  fed  during  the  experiment. 

Tanks  were  observed  daily  for  mortality.  When  a  fish  died, 
its  gills  were  examined  for  signs  of  petechiation,  and  the  second 
gill  arch  was  removed  and  examined  microscopically  for  the 
presence  of  Trichodina.  Infections  were  rated  as  mild  (<1  par- 
asite/lOOX  field),  moderate  (1-10/field),  or  severe  (>10/field). 

Survival  patterns  indicate  that  fish  lived  longer  at  the  lower 
temperatures  (Fig.  1).  The  fish  population  decreased  fastest  in 
the  15°C  tank  and  slowest  in  the  ambient-water  tank.  Lowering 
the  water  temperature  only  prolonged  the  time  until  mortality. 

Because  Trichodina  is  normally  found  in  wild  populations  of 
sculpins.  these  fish  must  enter  the  captive  environment  already 
harboring  this  protozoan  (2).  In  the  wild,  only  mild  (80%)  and 
moderate  (20%)  infections  were  seen.  After  only  10  days  in  cap- 
tivity, a  relatively  even  distribution  of  infection  (36%  mild,  21% 
mod.,  43%  severe)  was  noted  in  captive  fish.  Clearly,  the  captive 
environment  is  playing  a  role  in  the  development  of  this  disease. 


of  Fish 


Date 


Figure  1 .  Shows  the  siirnvtil  in  the  captive  environment  oj  longhorn 
sculpins  held  at  different  temperatures.  Survival  was  greatest  in  animals 
that  were  held  at  ambient  temperature  (5"C).  The  onset  of  mortality  was 
faster  in  fish  held  at  higher  temperatures.  Allowing  the  temperature  to 
return  to  ambient  in  the  I5°C  (T)  prolonged  the  survival  offish  as  com- 
pared to  the  group  that  remained  at  15°C. 


Fluctuation  in  temperature  and  high  stocking  density  were  en- 
vironmental stresses  that  should  have  been  avoided.  Better  tem- 
perature control  and  an  understanding  of  the  role  that  anemia 
plays  are  essential  to  further  elucidating  the  problem.  But  cur- 
rently, low  temperatures  and  short  holding  times  are  the  most 
effective  means  of  maintaining  the  health  of  sculpins  in  captivity. 
David  Brazik  is  a  student  in  the  Marine  Program  at  Bowling 
Green  State  University.  The  authors  thank  the  Boston  University 
Marine  Program  for  supplying  us  with  laboratory  space  and 
supplies.  Dr.  Rainer  Voigt  for  assisting  with  the  experimental 
setup,  and  Mr.  Ed  Enos  of  the  MBL  Marine  Resources  Depart- 
ment for  generously  donating  the  fish  used  in  this  project. 


Literature  Cited 

1 .  Lorn,  J.,  and  I.  Dykova.  1992.     Pp.  269-279  in  Protozoan  Parasites 
of  Fishes.  Elsevier.  Amsterdam. 

2.  Lorn,  J..  and  M.  I.aird.  l%9.     Can.  J.  Zoo/.  47:  1367-1380. 


240  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Bi, </  Rail  189:  240-241.  (October/November.  1995) 

A  Settlement  Bioassay  Assessing  the  Response  of  Soft  Shell  Clam  Larvae  to  Sediments 
from  Various  Sites  in  Massachusetts  Bay 

M.  Wintermyer,  D.  Leavitt.  and  J.  McDowell  (Woods  Hole  Oceanographic  Institution. 

Woods  Hole,  Massachusetts  02543) 


Many  environmental  influences  including  pollution,  preda- 
tion,  water  currents,  and  food  availability  can  impact  the  survival 
and  settlement  of  bivalve  larvae  ( 1 ).  Larval  survival  and  settle- 
ment are  key  factors  in  structuring  populations  of  the  soft  shell 
clam  (Alya  arenaria)  (2).  If  larval  survival  and  settlement  are 
impacted  by  environmental  contaminants,  one  could  expect  to 
observe  changes  in  population  structure  of  soft  shell  clams  at 
the  experimental  sites.  A  settlement  bioassay  was  performed  us- 
ing soft  shell  clam  larvae  to  determine  the  impacts  on  survival 
and  settlement  of  the  larvae  on  various  levels  of  contaminated 
sediments  collected  from  Massachusetts. 

Hatchery-reared  larvae  of  the  soft  shell  clam  were  exposed  to 
sediment  collected  from  five  sites  within  Massachusetts  Bay: 
Wellfleet  Harbor,  Barnstable  Harbor,  Saugus  River,  Quincy/ 
Neponset  River,  and  Fort  Point  Channel.  The  five  test  sediments 
are  known  to  represent  a  range  of  polycyclic  aromatic  hydro- 
carbon levels  based  on  analyses  by  Moore  cl  al.  (3).  Several  of 
the  sites  (Fort  Point  Channel,  Saugus,  and  Quincy/Neponset) 
are  within  the  city  limits  of  areas  highly  industrialized  and  whose 
marine  ecosystems  are  influenced  by  many  nonpoint  sources  of 
pollution.  The  contaminant  status  of  the  five  sites  is  as  follows: 
Wellfleet  Harbor  and  Barnstable  Harbor  are  minimally  impacted, 
Quincy/Neponset  River  and  Saugus  River  are  moderately  con- 
taminated, and  Fort  Point  Channel  is  highly  contaminated.  Two 
controls,  filtered  seawater  (no  sediment)  and  an  artificial  sedi- 
ment composed  of  microbeads.  were  also  used.  The  sediments 
were  wet-sieved  through  a  96-^m-mesh  screen  and  centrifuged 
to  remove  all  sediment  from  the  water  column.  The  %-h  assay 
was  performed  using  24-well  tissue  culture  plates.  Each  of  the 
sediment  treatments  consisted  of  1 0  replicates.  Each  well  received 
2  ml  of  filtered  (0.45  um)  seawater;  controls  had  either  seawater 
alone  or  seawater  plus  1  ml  of  microbeads.  Competent  larvae 
were  added  at  a  density  of  30  larvae  per  1  ml  of  the  test  sediment 
as  described  by  Phelps  ct  al.  (4).  Survival  and  settlement  were 
assessed  visually  after  the  96  h.  Larvae  were  considered  to  be  in 
the  veliger  stage  if  they  were  swimming  in  the  water  column: 
settled  larvae  were  identified  by  the  appearance  of  a  foot  and  a 
dorsoventrally  flattened  shell.  Dead  larvae  were  opaque  and  often 
infected  with  protozoans.  Although  neutral  red  dye  is  sometimes 
used  to  aid  in  distinguishing  settled  larvae  from  dead  larvae,  this 
was  not  necessary  with  M.  arenaria. 

The  results  of  this  bioassay  are  depicted  in  Figure  1.  The  fil- 
tered seawater  control  had  significantly  less  settlement  than  any 
other  treatment:  33.0%  of  the  living  larvae  settled  in  the  filtered 
seawater  compared  to  98.2%  in  the  control  microbead  sediment, 
indicating  that  the  larvae  required  a  sediment  substrate  for  set- 
tlement. Survival  was  20%-30%  higher  for  larvae  exposed  to 
control  treatments  than  for  larvae  exposed  to  test  sediments. 
The  difference  between  the  controls  and  test  treatments  was  sig- 


30 


Veliger 
Settled 
Dead 


T 

n 


20 


01 
to 


E 

3 

z 

c 

(0 

OJ 


10 


FSW     CB     WH     BH     QN      SR     FPC 

Treatment 

Figure  1.  Menu  number  of  swimming  veligers,  settled  larvae,  and 
dead  larvae  from  the  settlement  bioassay  assessed  after  96  h.  Filtered 
seawater  (WS)  mid  control  microbead  sediment  (CB)  are  control  treat- 
ment-, l\cllllccl  Harbor  (\VH)  and  Barnstable  Harbor  (BH)  are  minimally 
impacted  sites.  Suuxus  River  (SR)  and  Quincy/Neponset  (QN)  are  mod- 
eratelv  contaminated,  and  l-'orl  Point  Channel  (FPC)  is  highly  contam- 
inated 


nificant  for  sediment  from  Wellfleet  Harbor  (a  site  with  low 
contamination),  possibly  because  it  was  anoxic.  The  control  mi- 
crobead sediment  had  the  highest  number  of  settled  larvae  (225), 
but  percent  settlement  was  not  significantly  different  among  all 
six  test  sediments  (range  of  89%- 100%  of  living  larvae  settled). 
This  work  demonstrates  that  each  of  the  six  sediments,  re- 
gardless of  contaminant  loading,  was  able  to  induce  settlement 
of  the  soft  shell  clam  larvae.  Larval  mortality  was  not  different 
between  sites,  with  the  exception  of  an  apparent  anoxic  condition 
in  Wellfleet  sediment.  It  is  unlikely,  on  the  basis  of  these  exper- 
imental results,  that  the  mortality  and  settlement  of  soft  shell 


PHYSIOLOGICAL  ECOLOGY  AND  BEHAVIOR 


241 


clam  larvae  could  affect  the  structure  of  the  population.  Differ- 
ences in  population  structure  are  more  likely  to  be  caused  by 
other  factors,  including  the  availability  of  larvae  and  post-set- 
tlement mortality. 

Literature  Cited 

I .  \\ilbur,  K.  1983.     Pp.  299-336  in  The .\follitxeu.  vol.  3  N  H.  Ver- 


donk.  J.  A.  M.  Van  Den  Biggelaar.  and  A.  S.  Tompa.  eds.  Academic- 
Press.  New  York. 

2.  Brousseau,  D.  J.  1976.     P   151.  Ph.D.  Dissertation.  Univ.  of  Mas- 
sachusetts. Amherst,  MA. 

3.  Moore,  M.,  R.  Smolowitz.  D.  I.eavitt,  B.  Jensen,  B.  \\oodin,  and 
J.  Stegeman.  1995.     National  Extuarine  Program  Coastal  Tech- 
nology Transfer  Conference.  New  Orleans.  Feb.  13-16. 

4.  Phelps,  H.,  and  K.  Warner.  1990.     Bull  Environ.  Contain.  Toxicol. 
44:  197-204. 


Reference:  Biol.  Bull  189:  241-242.  (October/November.  1995) 

Invertebrate  Response  to  Nutrient-Induced  Changes  in  Macrophyte  Assemblages  in  Waquoit  Bay 
Jenny  Ahern,  Julie  Lyons,  James  McClelland,  and  Ivan  Valiela  (Boston  University  Marine  Program, 

Marine  Biological  Laboratory) 


Coastal  marine  systems  such  as  Waquoit  Bay,  Massachusetts. 
have  experienced  a  major  increase  in  nutrient  loads  through 
groundwater  in  recent  decades  ( 1 ).  Five  estuaries  of  Waquoit 
Bay  (Childs  River,  Hamblin  Pond,  Jehu  Pond,  Quashnet  River, 
and  Sage  Lot  Pond)  are  subject  to  different  nutrient  loads  due 
to  different  land  uses  in  their  watersheds  ( 1 ).  Nutrient  loading 
prompts  replacement  of  eelgrass  (Zostera  marina)  by  macroal- 
gae-dominated  communities  (2).  In  this  paper  we  address 
whether  the  vegetation  changes  caused  by  nutrient  loading  result 
in  parallel  changes  in  the  benthic  fauna  of  the  affected  areas. 

In  each  of  the  five  estuaries,  the  macrophyte  and  invertebrate 
communities  were  sampled  at  10  sites  (randomly  selected  within 
representative  depth  strata)  with  an  Eckman  dredge  (0.15  m2), 
June-August  1995.  Samples  were  rinsed  through  a  1-mm  sieve. 
Macrophytes  were  sorted  by  species,  dried,  and  weighed  (above- 
ground  biomass  only  for  eelgrass).  Macroinvertebrates  were  cat- 
egorized into  taxonomic  groupings  and  counted. 

The  estuaries  varied  widely  in  eelgrass  and  macroalgal  biomass 
(Fig.  1).  Benthic  invertebrate  density  did  not  change  markedly 
with  the  decrease  in  eelgrass  biomass  from  Sage  Lot  Pond  and 
Jehu  Pond  to  the  other  estuaries  (Fig.  1,  top).  In  contrast,  in- 
vertebrate density  showed  a  clear  inverse  relationship  with  ma- 
croalgal biomass  (r  =  0.5,  P  =  0.003)  (Fig.  1.  bottom).  Childs 
River,  the  most  nutrient-loaded  site,  had  a  consistently  higher 
macroalgal  biomass  and  a  lower  invertebrate  density  (Fig.  1, 
bottom).  The  other  sites  (shown  without  initials  in  Fig.  1)  had 
much  lower  macroalgal  biomass  than  Childs  River,  but  still  show 
a  strong  negative  relationship  between  invertebrate  density  and 
macroalgal  biomass  (r  =  0.3.  P  =  0.07)  when  Childs  River  points 
are  excluded.  The  other  estuaries  did  not,  however,  have  con- 
sistently different  macroalgal  biomass  from  each  other. 

Although  the  decrease  in  eelgrass  biomass  did  not  alter  in- 
vertebrate density,  it  did  change  faunal  composition.  In  eelgrass- 
dominated  communities  (Jehu  Pond.  Sage  Lot  Pond),  poly- 
chaeles  composed  26rc  of  the  invertebrate  abundance,  whereas 
in  macroalgae-dominated  communities  polychaetes  increased 
to  41%  of  benthic  invertebrates.  Changes  in  the  abundance  of  a 
single  species  in  response  to  eelgrass  loss  have  been  documented 
for  shellfish  species  of  Waquoit  Bay  ( 1 ). 


Many  factors  may  influence  invertebrate  abundance  in  es- 
tuaries. Low-oxygen  conditions  associated  with  accumulations 
of  macroalgae  (3)  may  be  responsible  for  the  decrease  in  inver- 
tebrate density  with  increasing  macroalgae  biomass;  this  oxygen 


6000-, 


5000- 


4000- 


3000- 


c 

OJ 

•o 
o 

S 

.0 


EELGRASS 


QR          HP 


,  CR 


_      1000 

>,         o 


10 


SOI  III-, 


6000- 


4000- 


2000- 


20  30  40  50 


MACROALGAE 


60 


0  100  200  300 

Biomass  (g  m-2) 

Figure  1.  Invertebrate  density  versus  biomass  oj 'celgraxs  (top)  and 
macroalgae  (bottom)  in  live  estuaries  <>/  H'utiimit  Bay.  Samples  were 
taken  June-August  1995.  Points  are  summer  average  ±  SE  fn  =  30) 
(lop)  and  monthly  average  ±  SE  (n  =  10)  (bottom! 


242 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


depletion  is  caused  by  an  imbalance  between  respiration  and 
photosynthesis  during  periods  of  low  irradiance.  Childs  River, 
in  particular,  has  more  frequent  anoxic  events  than  other  Wa- 
qiaoit  Bay  estuaries  (4).  possibly  accounting  for  the  lower  densities 
of  invertebrates  in  this  estuary.  Salinity  was  25-30%o  near  the 
bottom  at  all  sites,  suggesting  that  this  variable  was  not  a  sig- 
nificant factor.  Differential  predation.  sediment  composition, 
and  depth  may  also  influence  invertebrate  density  and  taxonomic 
composition,  thus  contributing  to  the  variability  of  the  data. 

Nutrient  loading  from  watersheds  has  induced  replacement 
of  eelgrass  by  macroalgae-dominated  communities  in  many 
shallow  estuaries  (2,  5).  Our  study  demonstrates  that  these  shifts 
in  macrophyte  assemblages  may  play  a  key  role  in  macrobenthic 
invertebrate  community  changes.  Both  eelgrass-dominated  and 
macroalgae-dominated  sites  can  support  comparable  invertebrate 


densities;  however,  as  macroalgae  increase,  benthic  invertebrate 
numbers  decrease.  The  switch  in  dominance  from  eelgrass  to 
macroalgae  is  also  accompanied  by  changes  in  taxonomic  com- 
position. 

This  work  was  supported  by  REU-NSF  (OCE  9300490), 
NOAA  (NA170R21 101),  and  the  Waquoit  Bay  Fellowship. 

Literature  Cited 

1.  Valiela,  I.,  el  al.  1992.     Esluark-s  15:  443-457. 

2.  Duarte,  C.  M.  1995.     Ophelia  41:  87-1  12. 

3.  llcip,  C.  1995.     Ophelia  41:  1  13-136. 

4.  D'Avanzo,  C.,  and  J.  N.  Kremer.  199-4.     Estuaries  17:  131-139. 

5.  Lyons,  J.  A.,  J.  Ahern,  J.  McClelland,  and  I.  Valiela.  1995.     Bin/. 
Bull.  189:  255-256. 


Reference:  Biol.  Bull  189:  242-243.  (October/November,  1995) 


Effect  of  Changing  Plant  Morphology  on  Invertebrate  Susceptibility  to  Predation  in  Eelgrass  Beds 

Matthew  C.  Prcisser  and  Linda  A.  Deegan  (Marine  Biological  Laboratory) 


Long-term  coastal  eutrophication  leads  to  changes  in  the 
macrophyte  community  in  eelgrass  beds  by  allowing  macroalgae 
to  outcompete  the  eelgrass  ( 1 ).  Changes  in  plant  species  may 
affect  predation  on  the  invertebrate  community  by  altering  hab- 
itat morphology.  This  study  analyzed  predation  rates,  in  habitats 
of  contrasting  plant  morphology,  on  two  invertebrate  species 
that  exhibit  different  strategies  for  avoiding  predation. 

Rates  of  predation  by  Fundulus  lu'tcmclilits  on  the  isopod 
Erichsonella  filiformis  and  the  amphipod  Lysianopsis  alba  were 
measured  in  the  laboratory  under  conditions  simulating  three 
habitats:  no  vegetation,  eelgrass  (Zostera  marina),  and  the  ma- 
croalga  Cladophora  spp.  Twelve  trials  of  each  experimental 
treatment  were  run,  each  with  80  g  (wet  biomass)  of  vegetation 
in  a  20-1  aquarium  flushed  with  running  ambient  seawater.  The 
same  aquaria  and  vegetation  were  used  for  the  1 2  replications 
of  each  treatment;  new  prey  and  predators  were  introduced  at 
the  beginning  of  each  of  these  trials.  Macrophyte  densities  were 
comparable  to  those  encountered  in  the  field. 

Twenty  individuals  of  a  single  prey  species  were  placed  in 
each  tank  and  allowed  to  acclimate  for  45  min.  One  Fundulus 
(starved  for  24  h)  was  then  introduced.  This  prey  density,  higher 
than  the  natural  field  density,  was  used  to  allow  the  predator  to 
forage  for  the  full  experiment.  The  fish  was  removed  alter  1  h 
and  the  remaining  prey  were  counted.  Missing  prey  were  as- 
sumed to  have  been  eaten;  no  invertebrates  were  found  partially 
eaten  or  dead.  Predation  rate  was  calculated  as  the  percentage 
of  prey  eaten  during  the  1-h  interval. 

Predation  on  Erichwnella  was  lowest  (30.4%)  in  eelgrass  (Fig. 
1 ,  top).  With  its  king  thin  body  and  green  coloration,  this  isopod 
resembles  a  blade  of  eelgrass.  The  strategy  of  "background 
matching"  may  make  detection  by  visual  predators  difficult  in 
eelgrass  habitats.  Predation  was  moderate  (47. 1%)  in  the  aquaria 


with  Cladiiphnra,  possibly  because  the  algae  provided  some 
structure  and  a  green  background.  Predation  was  highest  (94.2%) 
in  the  'no  vegetation'  treatment  because  the  isopod  is  not  a  strong 
swimmer  and  had  no  refuge. 


100% 


O 
"O 


80% 
60% 
40% 
20% 


L  alba 


no  vegetation  Cladophora       eelgrass 

Figure  1.  f'redalion  rule  (mean  %  ±  standard  error)  of  Fundulus 
heteroclitus  on  Erichsonella  nliformis  (lop)  and  Lysianopsis  alba  (holtoin) 
in  hahituts  nl  dittoing  morphology. 


ECOLOGY:  FISH  AND  INVERTEBRATES 


243 


Predation  on  Lysianopsis  was  lowest  (15.4%)  in  Cladophora 
(Fig.  1,  bottom),  which  forms  a  thick  intertwining  mat.  The 
amphipod's  strategy  for  avoiding  predation  was  to  burrow  deep 
into  the  algae,  putting  itself  out  of  the  reach  of  the  predator.  In 
the  field,  however,  such  behavior  might  be  hindered  by  the  anoxic 
conditions  often  found  in  the  mat.  The  experimental  method- 
ology of  this  study  allowed  for  a  fairly  complete  exchange  of  the 
interstitial  mat  water,  which  resulted  in  aerobic  conditions 
throughout  the  mat.  Predation  was  high  (73.8%)  in  the  eelgrass. 
probably  because  crawling  and  burrowing  amphipods  cannot 
take  advantage  of  the  vertical  structure  of  the  plant.  Predation 
was  highest  (93.3%)  in  the  'no  vegetation'  treatment  because 
there  was  no  substrate  available  to  provide  protection. 

Predation  rate  was  analyzed  in  a  one-factor  ANOVA  (P 
=  0.05).  Differences  among  treatment  means  were  evaluated 
using  Fisher's  PLSD  (P  =  0.05).  There  were  strong  differences 


in  predation  rates  between  prey  in  different  vegetation  types  (F 
=  227.615).  All  predation  rates  were  different  from  each  other 
except  in  the  'no  vegetation'  treatment. 

Many  invertebrate  species  are  highly  dependent  upon  mac- 
rophyte  structure  and  color  for  protection  against  predation  (2). 
Alteration  of  the  primary  producer  community  due  to  eutro- 
phication  may  change  the  structure  of  the  habitat  and  may  po- 
tentially have  second-order  effects  on  the  invertebrate  com- 
munity. 

This  study  was  supported  by  the  Boston  University  Marine 
Program  and  the  Cox  Foundation. 

Literature  Cited 

1.  Valiela,  I.,  el  al.  1992.     Estuaries  15:  443-457. 

2.  Nelson,  \V.  G.  1979.     J  Exp.  Mar,  Biol.  Ecol.  38:  225-245. 


Reference:  Biol  BuIL  189:  243-244.  (October/November.  1995) 

Effect  of  Algal  Cover  on  Size-Selective  Predation  of  Gammarus  mucronatus 
by  the  Striped  killifish,  Fundulus  majalis 

Chaka  Drake,  Peter  J.  Bc/ir.  and  Ivan  I  'aliela  (Boston  University  Marine  Program, 

Marine  Biological  Laboratory) 


The  relative  sizes  of  predators  and  prey  influence  top-down 
control  of  prey  as  well  as  success  of  predation  ( I ).  The  striped 
killihsh.  Fundulus  majalis.  is  an  abundant  predator  (2)  in  es- 
tuaries of  Waquoit  Bay  and  often  feeds  on  the  amphipod  Gam- 
marus mucronatus.  one  of  the  most  common  benthic  species  in 
the  Bay. 

Predation  by  fish  is  also  affected  by  the  physical  structure  of 
the  habitat  in  which  predation  takes  place  (3.  4).  In  Waquoit 
Bay,  one  major  aspect  of  habitat  structure  is  the  presence  or 
absence  of  macroalgae.  To  determine  the  effects  of  macroalgal 
cover  and  its  possible  interactions  with  size-selective  predation, 
we  tested  predation  by  different  sizes  of  F.  majalis  on  different 
sizes  of  G  mucronatus  in  the  presence  and  absence  of  the  green 
alga  Cladophora  vagabunda.  C.  vagabunda  is  the  dominant  ma- 
croalgal species  in  the  Bay. 

Fish,  amphipods.  and  algae  were  collected  from  estuaries  of 
Waquoit  Bay.  Massachusetts.  The  specimens  were  held  in  flow- 
through  seawater  tanks  for  2-5  days  before  use  in  experiments. 
C '  vagabunda  was  cleaned  of  all  debris  and  organisms  before 
use  in  experiments.  Individuals  of  6'.  miicrtmatus  were  separated 
into  three  size  categories,  small  (0.3-0.8  mm),  medium  (0.8- 
1.3  mm),  and  large  (1.4-2. Omm).  by  measuring  from  eye  to 


Figure  1.  Consumption  (amphipods  ealen  ±  SE)  o/Gammarus  mu- 
cronatus ol  different  size  by  Fundulus  majalis  ol  different  ,si:e  »'itli  (top), 
and  without  ihoitomi  cover  ol'  the  alga  Cladophora  vagabunda.  Con- 
sumption was  over  a  2-h  period. 


WITH  ALGAL  COVER 


C 

<u 

—  ' 

cd 


D- 


D  Small  fish  (4.2  ±0.1  cm) 
B  Medium  fish  (6.0  ±0.1  cm) 
•  Large  fish  (8.6  ±  0.2  cm) 


Prey  size  (mm) 


244 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


second  abdominal  segment.  F.  majalis  specimens  were  also 
placed  into  three  total-length  size  categories:  small  (2.9-4.9  cm), 
medium  (5.0-6.9  cm),  and  large  (7.0-12.4  cm). 

Five  small,  medium,  and  large  G  inucronatus  were  placed  in 
3-1-capacity  jars  with  a  1.7-1  volume  of  loosely  packed  C.  va- 
gabitnda.  The  amphipods  were  allowed  to  acclimate  to  the  jars 
for  1  h  before  addition  of  F.  majalis.  Fish  were  fed  2  h  prior  to 
experimentation  and  then  placed  individually  within  the  3-1  jars 
to  feed  for  2  h.  After  the  feeding  period,  the  fish  were  removed 
and  measured.  The  amphipods  remaining  in  the  jars  were  cat- 
egorized by  size  class  and  counted.  The  trial  runs  were  made 
with  algae  (58  replicates)  and  without  algae  (60  leplicates).  A 
three-way  ANOVA  was  used  for  comparison  of  cover,  prey  size, 
and  predator  size. 

Consumption  was  affected  by  algal  cover  (F  =  62.4;  P  <  0.00 1 ) 
and  an  interaction  between  sizes  of  prey  and  predators  (F  =  4.8; 
P  <  0.001).  Prey  consumption  was  about  twice  as  high  in  the 
absence  of  algae  (Fig.  1 ).  In  jars  containing  algae,  the  large  fish 
consumed  fewer  small  prey  than  smaller  fish  (Fig.  1,  top  left). 
Additionally,  small  fish  consumed  fewer  large  prey  than  larger 


fish  (Fig.  1,  right  top  and  bottom).  Intermediate-sized  prey  were 
fed  upon  at  about  the  same  amount,  regardless  of  size  of  fish 
(Fig.  1,  middle  top  and  bottom).  The  presence  or  absence  of 
macroalgal  cover  did  not  affect  size-selective  feeding  (the  F  value 
for  the  3-way  interaction  was  0.2;  P  =  0.960). 

The  data  suggest  that  as  fish  grow,  their  food  preferences 
change  from  small  to  large  prey,  so  that  predation  is  exerted  on 
different  parts  of  benthic  invertebrate  populations.  Macroalgal 
cover  reduced  predation  success,  but  did  not  change  size  selec- 
tivity. 

This  work  was  supported  by  a  Waquoit  Bay  Land  Margin 
Ecosystems  Research  REU  internship. 


Literature  Cited 


1.  Ryer,  C.  1988.     Mw 

2.  Werme,  C.  E.  1981. 
PP. 

3.  Vince,  S.,  et  al.  1976. 

4.  Jones,  G.,  et  al.  1991. 


/•:«•/  Prog.  Scr.  48:  37-45. 

Ph.D.  Dissertation.  Boston  University.  126 

J.  E.\p.  Mar  Biol.  Ecol.  23:  255-266. 
Pp.  1 7 1-1 72  in  The  Ecology  of  Fishes  on 


Coral  Reefy.  P.  Sale.  ed.  Academic  Press,  San  Diego. 


Reference:  Biol.  Bull  189:  244-245.  (October/November,  1995) 

Effect  of  Macroalgal  Species  and  Nitrogen-Loading  Rates  on  Colonization 
of  Macroalgae  by  Herbivorous  Amphipods 

Nicole  Martinez,  Jennifer  Hauxwell.  and  Ivan  I  'aliela  (Boston  University  Marine  Program, 

Marine  Biological  Laboratory) 


The  macroalgae  Cladophura  vagabunda  (green,  filamentous) 
and  Gracilaria  tikvahiae  (red,  branching)  are  common  in  es- 
tuanes  of  Waquoit  Bay  subject  to  different  nitrogen-loading  rates 
(1).  The  total  biomass  and  relative  abundances  of  the  two  ma- 
croalgal species  may  depend  not  only  on  nitrogen-loading  rates 
(1,2)  but  also  on  significant  consumption  by  herbivorous  am- 
phipods (Hauxwell  et  til.,  unpub. ).  Because  food  preference  of 
an  abundant  grazer  can  significantly  decrease  the  abundance  of 
targeted  macroalgae  and  alter  competitive  interactions  among 
producers  (3),  we  conducted  a  colonization  experiment  to  de- 
termine ( 1 )  whether  grazers  prefer  C'  vagabunda  or  G.  tikvahiae 
and  (2)  whether  preferences  for  the  two  macroalgal  species  differ 
in  estuaries  subject  to  different  nitrogen-loading  rates. 

Because  relatively  small  amphipod  herbivores  hide  within  the 
macroalgae  they  consume,  preference  may  be  linked  not  only 
to  food  quality  (nitrogen  content,  digestibility)  but  also  to  the 
amount  of  shelter  an  alga  affords  from  predation.  To  assess  grazer 
macroalgal  substrate  preference,  we  deployed  three  (30  X  16 
X  10  cm)  cages  containing  equal  volumes  of  either  C.  vagabunilu 
(34  g  ww)  or  (;  nkvahiae  (60  g  ww)  in  estuaries  with  high  (Childs 
River)  and  low  (Sage  Lot  Pond)  nitrogen  loadings  ( 1 )  and  mea- 
sured colonization  by  amphipods.  To  assess  whether  shelter  was 
important  as  a  cue  in  selection  by  grazers,  we  eliminated  food 
cues  from  a  third  treatment  by  replacing  the  macroalgae  with 
an  equivalent  volume,  of  plastic  mesh. 


C  vagabunda  and  6"  tikvahiae  were  collected  and  cleaned  of 
grazers  before  being  placed  in  cages,  and  were  indigenous  to  the 
estuary  in  which  they  were  used.  The  cages,' constructed  with  2- 
mm  mesh  to  allow  entry  of  grazers  while  retaining  algae,  were 
secured  to  the  algal  mat  by  attaching  both  ends  to  reinforcing 
rods  driven  into  the  sediment.  Swivels  were  used  to  connect 
each  end  to  a  string  loop  around  the  reinforcing  rod,  which 
enabled  a  snorkeler  to  detach  the  cage  without  disturbing  its 
contents.  Cages  were  retrieved  after  1  week;  while  one  snorkeler 
detached  an  end,  another  placed  a  bag  around  the  entire  cage 
to  retain  all  grazers.  Grazer  density  and  species  composition 
were  recorded  for  each  cage.  Three  replicates  of  each  of  the  algal 
species  were  installed  in  each  estuary.  Six  one-week  trials  were 
run  during  midsummer. 

There  were  seasonal  patterns  in  the  data  (ANOVA,  P  <  0.05 
in  all  cases),  showing  peak  abundances  in  midsummer.  This 
trend  parallels  survey  data  obtained  from  our  these  estuaries  (J. 
McClelland,  unpub.).  However,  where  preferences  were  found, 
the  proportion  of  grazers  found  in  C.  vagabunda  relative  to  G. 
tikvuliiae  was  similar  across  trials.  We  therefore  present  averages 
pooled  across  trials. 

Provision  of  cover  alone  did  not  seem  to  influence  grazer 
substrate  preference,  because  amphipods  preferred  algae  over 
artificial  cover  in  all  cases  (Table  I).  The  preference  of  amphipods 
between  the  two  algal  species  differed  between  estuaries  (Table 


ECOLOGY:  FISH  AND  INVERTEBRATES 
Table  I 


245 


.\umher  (mean  ±  standard  error)  of  amphiriods  of  di/lerein  v/xr;o  found  in  experimental  cagc\  containing  either  Cladophora  vagabunda. 
Gracilaria  tikvahiae.  or  artificial  cover  al  e\lnane\  »nh  /i/itli  It  'hildx  River)  and  low  (Sage  Lot  Pond)  nitrogen-loading  rale-.  Because  these  species 
are  not  readily  distinguishable,  dala  lor  Ampithoc  longimana  and  Cymadusa  compta  were  combined 


Grazers 

Childs  River 

Sage  Lot  Pond 

Cladophora 
vagabunda 

Gracilaria 
tikvahiae 

Cover 
Treatment 

Cladophora 
vagabunda 

Gracilaria 
tikvahiae 

Cover 
Treatment 

Microdeutopus  gryllotalpa 

Ainpnhov  hngimana/ 
Cymadusa  cumpia 

147  ±  33 
149  ±  54 

28  ±    5 
1  56  ±  34 

18  ±    6 
66  ±  13 

128  ±  32 
15  ±    8 

184  ±65 
29  ±11 

44  ±  10 
9±    3 

Total  Grazers 

304  ±  66 

188  ±  38 

85  ±  17 

157  ±  40 

244  ±63 

59  ±    9 

I).  In  Childs  River.  Microdeutopus  gryllotalpa  clearly  preferred 
C.  vagahunda.  while  the  other  amphipods  (Ampithoc  longimana 
and  Cymadusa  compta)  showed  no  preference.  Total  grazers 
(also  including  Lysianopsis  alha,  Gammarus  mitcnmattis.  and 
Corophium  sp.)  were  found  in  higher  densities  in  C  vagabunda 
cages  than  in  G.  tikvahiae  cages.  In  Sage  Lot  Pond,  no  significant 
preferences  were  apparent. 

Our  results  show  (1)  that  grazers  select  algae  principally  on 
the  basis  of  food  value,  rather  than  cover,  (2)  that  C.  vagabunda 
is  preferred  over  G.  tikvahiae  by  M.  gryllotalpa  and  total  grazers 
in  estuaries  subject  to  high  nutrient  loading,  and  (3)  that  grazers 
showed  no  significant  preference  at  the  site  with  low  nitrogen 
loading.  These  shifts  in  preference  between  sites  may  be  attrib- 
uted to  differences  in  food  quality  [as.  for  example,  by  increased 


nitrogen  content  in  more  loaded  estuaries  (2)]  as  perceived  by 
two  amphiod  populations.  The  potential  impact  of  herbivores 
in  controlling  macroalgal  biomass  is  therefore  directed  toward 
different  macroalgal  species  in  different  estuaries,  and  the  role 
of  nitrogen  loading  mediates  the  colonization  by  grazers  of  dif- 
ferent macroalgal  species. 

This  work  was  supported  by  NSF-Research  Experiences  for 
Undergraduates. 

Literature  Cited 

1.  Valiela,  I.,  el  al.  1992.     Exliiarie'i  15:  443-57. 

2.  Peckol,  P.,  el  al.  1994.     Mar  Bio/  121:  175-185. 

3.  Lubchenco,  J.  1978.     Am.  Nat.  112:  23-39. 


Reference:  Biol.  Bull  189:  245-246.  (October/November.  1995) 

Differences  in  Benthic  Invertebrate  Assemblages  in  Two  Estuaries  of  Waquoit  Bay 

Receiving  Disparate  Nutrient  Loads 

Rafael  Sardd  (Centra  de  Estitdios  Avanzados  de  Blanes.  CSIC,  17300-Blanes  [Girona],  Spain), 

Kenneth  Foreman,  and  Ivan  I  'aliela 


We  evaluated  the  effects  of  nutrient  loading  on  benthic  com- 
munities by  measuring  the  abundance  and  composition  of  ma- 
croinfaunal  assemblages  inhabiting  similar  habitats  within  two 
Waquoit  Bay  estuaries  subject  to  different  rates  of  nutrient  load- 
ing. On  the  basis  of  water  recharge  rates  and  dissolved  inorganic 
nitrogen  concentrations  in  groundwater  at  the  shore,  we  esti- 
mated that  each  year  about  8500  kg  of  nitrogen  enter  the  Childs 
River  estuary  (4.6  moles  nT2  year"1)  and  roughly  3500  kg 
(1.5  moles  irT2  year~')  enter  the  Quashnet  River.  The  intensity 
of  nutrient  loading  to  these  estuaries  differs  primarily  because 
ot  differences  in  the  density  of  houses  and  septic  systems  within 
their  surrounding  watersheds  (2). 

In  both  estuaries,  the  benthic  assemblages  differed  depending 
on  location  within  the  gradient  of  fresh  to  saltwater.  The  poly- 


chaete  Amphicteis  gtinneri  and  the  amphipod  Leptocheirus  phi- 
mulosus  dominated  faunal  abundance  and  biomass  in  low-sa- 
linity areas  of  the  Quashnet  River.  In  the  Childs  River,  low- 
salinity  areas  were  dominated  by  insect  larvae.  Abundance  and 
diversity  were  highest  in  the  medium-salinity  areas  in  both  rivers. 
In  the  Quashnet  River,  the  polychaetes  Streblospio  benedict i 
and  Amphicteis  gunneri.  a  tubificid  oligochaete  that  we  believe 
to  be  a  species  of  Limnodrillus,  and  the  anemone  Nematostella 
vectensis  were  the  most  abundant  benthic  organisms,  and  the 
polychaete  Heteromastus  fi/i/onni*  was  the  main  contributor  to 
biomass.  In  the  Childs  River,  the  oligochaete  was  by  far  the  most 
abundant  species,  accounting  for  70%  of  the  total  abundance, 
and  the  carnivorous  polychaete  Neanthes  vircns  was  the  main 
contributor  to  biomass.  In  high-salinity  areas,  the  polychaete 


246 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


100  -i 


100  - 

Upper 

estuary    so  -< 
< 

25  - 


Middle 
estuary    so  -{i 


Lower 
estuary    50 


10  1 

Species  rank 

Figure  1.     K-dominance  curves  for  abundance  (upcn  circles)  ami  hin- 

mass  (black  ciirle.\l  nl  macrolauna  in  the  upper,  middle,  and  lower  parts 
of  the  Qiiaslmel  Riwr  (/ell)  and  Childs  River  fright). 


salinity  range,  eutrophication  seemed  to  be  responsible  for  dif- 
ferences in  the  composition  and  abundance  of  macrofauna  be- 
tween estuaries.  The  abundance  of  oligochaetes  and  insect  larvae 
and  the  occurrence  of  a  large  carnivore  increased  with  nutrient 
loading,  whereas  the  number  and  biomass  of  other  typically  res- 
ident species  decreased.  Most  significantly,  the  ratio  of  poly- 
chaetes  to  oligochaetes  was  clearly  lower  in  the  Childs  River 
(0.36)  than  in  the  Quashnet  River  (3.58). 

We  contrasted  the  benthic  assemblages  within  the  heavily  nu- 
trient-loaded Childs  River  estuary  to  those  of  the  more  mod- 
erately loaded  Quashnet  River  estuary  by  constructing  plots  of 
ranked  species  dominance  (3).  The  biomass  and  abundance  of 
each  species  were  ranked  from  highest  to  lowest  and  plotted 
against  the  cumulative  percent  biomass  or  percent  abundance 
(Fig.  1 ).  Plots  in  which  the  k-dominance  curve  for  biomass  falls 
above  the  curve  for  abundance  are  indicative  of  communities 
in  which  the  most  abundant  fauna  are  large,  slower  growing 
species.  These  communities  are  indicative  of  relatively  unstressed 
conditions.  Plots  in  which  the  curve  for  abundance  falls  above 
the  curve  for  biomass  represent  communities  in  which  the  most 
abundant  species  are  small  and  rapidly  growing.  These  are  more 
typical  of  disturbed  or  eutrophied  communities  ( 1 ).  No  large 
differences  were  apparent  between  the  k-dominance  curves  of 
abundance  and  biomass  in  the  upper  portion  of  either  estuary. 
In  both  the  middle  and  lower  portions  of  the  estuaries,  the  eval- 
uation suggests  that  Childs  River,  with  its  more  urbanized  wa- 
tershed, is  more  impacted  by  nutrient  loading  than  the  Quashnet 
River. 

We  acknowledge  the  MBL  Associates  Fellowship  that  sup- 
ported Rafael  Sarda  during  this  work. 


\laren:ellena  viriilis  dominated  the  benthos  of  the  Quashnet 
River  in  both  abundance  and  biomass.  In  the  Childs  River, 
Lumhricilliis  spp.  and  Neanthes  virens  were  again  the  main  con- 
tributors. 

Salinity  appeared  to  be  the  primary  factor  controlling  the  dis- 
tribution of  the  macroinfaunal  species;  but  within  any  single 


Literature  Cited 

1.  Valiela,  I.,  et  al..  1992.     l-:\tiuirie\  15(4:)  443-457. 

2.  Warwick,  R.  M.  1986.     Mar  Bioi  92:  557-562. 

3.  Pearson,  T.  H.,  and  R.  Rosenberg.  1978.     Oceanngr   Mar.  Binl. 
Ann.  Re\:  16:229-311. 


Reference:  Biol  Bull  189:  246-247.  (October/November,  1995) 


Impact  on  Marine  Species  of  New  England  Recreational  Fishing  Policies 

Jonathan  S.  O'Ncil  (University  of  Rhode  Island)  and  Ilene  M.  Kaplan 


This  study  examines  the  status  of  the  Massachusetts  Atlantic 
cod  (Gadus  morhua)  recreational  fishery  and  the  potential  impact 
of  proposed  governmental  marine  regulations.  Data  from  the 
1993  National  Marine  Fisheries  Service's  catch  and  fish  surveys 
along  the  eastern  coastal  LInited  States  (1)  and  the  1994  Add- 
on Marine  Recreational  Economics  Survey  of  the  Marine  Rec- 


reational Fishing  Statistical  Survey  were  examined.  Data  collec- 
tion methodology  consisted  of  interviews  with  fishermen  at  on- 
site  fishing  locations  and  follow-up  telephone  interviews.  Survey 
sampling  sites  were  randomly  selected  from  lists  of  fishing  access 
sites  weighted  by  expected  fishing  activity. 

Reported  commercial  and  recreational  landings  of  Atlantic 


ECOLOGY:  FISH  AND  INVERTEBRATES 


247 


cod  strongly  indicate  a  decline  in  this  fishery,  with  the  recreational 
fishery  experiencing  a  greater  decline  proportionally  (see  Table 
I).  The  potential  decline  in  recreational  stocks  is  even  greater, 
because  statistics  on  catch  and  release  mortality  from  recreational 
fishing  are  not  available. 

Fishery  managers  seeking  to  reduce  the  harvest  of  cod  are 
now  targeting  the  recreational  fishing  industry.  The  data  collected 
from  the  recreational  fishermen  indicate  that  they  favor  the  con- 
servation measures  that  fishery  managers  are  proposing,  although 
a  sizeable  minority  are  fearful  of  restrictions  in  certain  geographic 
areas  and  during  vacation  or  leisure  time  periods  (see  Table  I). 

The  data  strongly  suggest  that  the  economic  hardships  pre- 
dicted by  operators  and  owners  of  businesses  related  to  recre- 
ational fishing,  particularly  in  the  charter,  party,  and  rental  boat 
sectors,  will  not  be  sustained,  because  recreational  fishermen 
will  still  pursue  this  activity.  Additional  data  from  this  survey, 
indicating  that  the  motivation  to  fish  stems  from  experiential 
variables  that  are  independent  of  policy  limitations  (other  than 
a  complete  moratorium),  also  support  this  conclusion.  This  is 
a  particularly  important  finding,  for  it  shows  how  contact  with 
fishermen  can  actually  be  used  to  support  ecological  measures 
without  impeding  economic  pursuits.  We  suggest,  however,  that 
interviews  be  conducted  within  the  business  sector. 

The  data  collected  in  the  Add-on  Marine  Recreational  Eco- 
nomics Survey  are  significant,  illustrating  that  fishermen  must 
be  incorporated  into  policy  decisions.  We  also  suggest  that,  in 
addition  to  overfishing,  other  sources  of  stock  depletion,  such 
as  pollution  of  the  habitat,  be  investigated.  A  better  understand- 
ing of  the  conflicts  between  ecological  and  economic  interest 
groups,  as  well  as  among  different  types  of  fishermen,  is  essential 
if  fisheries  management  is  to  be  effective  (2,  3,  4,  5). 

The  authors  gratefully  acknowledge  the  assistance  of  the  Na- 
tional Marine  Fisheries  Service,  the  Woods  Hole  Oceanographic 
Institution,  and  the  Marine  Biological  Laboratory  in  Woods 
Hole.  Massachusetts. 


I  ..Mr  I 

Decline  in  cm/  win 'As  ami  \ta\\achu\cll\  recreational  lishermen's 
reactions  to  proposed  rexulalions 


US.  Commercial  and  Recreational  Landings  (in 
metric  tons)* 


1990 


1992 


U.S.  Recreational  Landings** 

5.200 

1,300 

U.S.  Commercial  Landings** 

43,400 

27,700 

Party  and  Charter  Boat  Fishermen  (n  =  60)*** 

Support 

Oppose 

Limits  on  the  minimum  size  of  fish  that  can  he 

kept 

95% 

3% 

Limits  on  the  number  offish  that  can  be  kept 

93% 

7% 

Limits  on  the  time  of  year  when  fish  can  be  kept 

83% 

17% 

Limits  on  area  where  fishing  can  occur 

88% 

12% 

Private  and  Rental  Boat  Fishermen  (n  =  414)*** 

Support 

Oppose 

Limits  on  the  minimum  size  offish  that  can  be 

kept 

95% 

5% 

Limits  on  the  number  offish  that  can  he  kept 

92% 

8% 

Limits  on  the  time  of  year  when  fish  can  be  kept 

79% 

21% 

Limits  on  area  where  fishing  can  occur 

67% 

33% 

NOAA  Tech.  Mem  NMFS-F/NEC-101.  1993. 
**     Landings  for  Gulf  of  Maine.  Georges  Bank  and  areas  south. 
***  Compiled  from  NMFS  Add-On  Marine  Economics  Survey  1994. 

Literature  Cited 

1.  NOAA  1993.     Tech.  Mem  NMFS-F/NEC-101:  45. 

2.  Kaplan,  I.  M.,  and  B.  C.  Boyer.  1992.     Biol.  Bull-  183:  379-380. 

3.  Kaplan,  I.  M.,  B.  C.  Boyer,  and  D.  E.  Hoffman.  1990     Biol.  Bull. 
179:  227. 

4.  Kaplan.  I.  M..  B.  C.  Boyer,  and  D.  E.  Hoffman.  1989.     Biol.  Bull. 
Ill:  327. 

5    Kaplan,  I.  M.,  B.  C.  Boyer,  and  K.  A.  Santos.  1988.     Biol  Bull 
175:  312. 


Reference:  Biol  Bull  189:  247-248.  (October/November,  1995) 

The  Effect  of  Residential  and  Forested  Watershed  Land  Cover  on  Nutrient  Loading  to  Hambiin 

and  Jehu  Ponds,  VVaquoit  Bay,  Massachusetts 

Sue  Ann  Chaplin,  Catherine  Hunter  MacGregor,  Ivan  Valiela,  Kenneth  Foreman,  and  Lori  Soucv 
(Boston  University  Marine  Program,  Marine  Biological  Laboratory) 


Atmospheric  deposition,  fertilizer  use.  and  wastewater  are 
major  sources  of  nitrogen  to  watersheds  of  Waquoit  Bay  estu- 
aries. Groundwater  transports  nitrogen  from  all  land  covers 
within  Waquoit  Bay  because  precipitation  quickly  percolates 
into  sands  underlying  the  watershed  surface  ( 1 ).  The  major  land 
covers  in  Waquoit  Bay  watershed  are  forest  and  residential.  The 
major  external  input  of  nitrogen  to  forests  is  atmospheric  de- 
position. The  major  inputs  to  residential  land  parcels  are  at- 
mospheric deposition  as  well  as  fertilizer  and  wastewater  nitro- 
gen. The  fate  of  nitrogen  under  forested  or  residential  land  cover 


differs  (Valiela  el  a!.,  unpub.  data).  We  asked  whether  water 
catchment  areas  with  different  proportions  of  forested  and  res- 
idential land  lead  to  different  nitrogen  concentrations  in 
groundwater  heading  to  receiving  estuaries.  To  address  that 
question,  we  measured  concentrations  of  nitrate  (NO3~),  am- 
monium (NH4+),  and  dissolved  organic  nitrogen  (DON)  in 
groundwater  draining  from  areas  with  different  proportions  of 
forest  and  residential  land  cover  types. 

Jehu  and  Hambiin  Ponds,  two  estuaries  of  Waquoit  Bay,  have 
both  forested  and  residential  areas  within  their  watersheds.  We 


248 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


FORESTS 


RESIDENTIAL 


o     in     o     in     o 
CM     m     r-     o 


%    of   Watershed  Area 

Figure  1.  Concentrations  of  inorganic  and  organic  nitrogen  in 
groundwaler  as  a  function  oj  the  percentage  n/  the  watershed  covered  by 
forests  (left  panels)  and  residential  areas  (right  panels)  as  measured  in 
seven  catchment  areas  o/Hainh/in  and  Jehu  Ponds.  Values  are  means 
(±standard  error)  of  samples  for  DON  (dissolved  organic  nitrogen),  nitrate, 
ammonium,  and  TON  (total  dissolved  nitrogen)  All  regression  lines  are 
significant  at  the  0.05  level. 

divided  the  watersheds  into  seven  catchment  areas,  four  on  Jehu 
and  three  on  Hamblin.  Samples  of  groundwater  were  taken  at 
!00-m  intervals  around  each  estuary  by  placing  a  drive-point 
piezometer  into  the  soil  above  the  high-water  mark.  Groundwater 
was  vacuum  filtered  and  acidified  with  1  n\  of  5  N  HCI  for  even. 
1  ml  of  sample,  and  concentrations  of  NO3  ,  NH4+,  and  dissolved 
organic  nitrogen  (DON)  were  determined  using  a  LACHAT  au- 
toanalyzer. 


We  obtained  estimates  of  forest  and  residential  areas  from  a 
geographical  information  systems  map  of  the  Waquoit  Bay  area. 
We  subdivided  the  samples  of  groundwater  according  to  the 
seven  catchment  basins,  and  could  therefore  plot  average  nitro- 
gen concentration  in  groundwater  in  relation  to  land  use  in 
catchment  basins. 

The  increased  proportion  of  forested  and  residential  areas 
within  a  catchment  basin  was  related  to  the  concentration  of 
nitrogen  in  groundwater  about  to  leave  the  aquifer  (Fig.  1 ).  The 
more  forested  the  catchment  basin,  the  lower  the  concentration 
of  nitrogen  (Fig.  1,  left  panel).  This  observation  is  consistent 
with  Keeney's  conclusion  that  forests  intercept  (by  storing  in 
the  soil  and  tree  biomass  and  converting  nitrate  to  nitrogen  gas) 
atmospherically  derived  nitrogen  (2). 

In  contrast,  the  more  residential  a  catchment  basin,  the  higher 
the  concentration  of  nitrogen  (Fig.  1 .  right  panels).  This  is  prob- 
ably the  result  of  lower  interception  of  atmospheric  nitrogen, 
combined  with  the  addition  of  nitrogen  from  wastewater  and 
lawn  fertilizer.  This  suggests  that  nitrogen  loading  in  Waquoit 
Bay  depends  largely  on  nitrogen  release  associated  with  residen- 
tial land  cover  (Valiela  et  ai.  unpub.  data).  As  a  landscape  be- 
comes more  urbanized,  there  will  be  a  progressive  increase  in 
nitrogen  loads  to  receiving  estuaries,  both  because  of  reduction 
of  forest  interception  and  because  of  increased  delivery  of  waste- 
water  and  lawn  fertilizer. 

The  major  type  of  nitrogen  delivered  to  estuaries  from  ur- 
banized watersheds  is  dissolved  organic  nitrogen  (DON),  with 
considerable  NH4+  and  smaller  concentrations  of  NO3~.  These 
relative  concentrations  suggest  that  it  is  the  nitrogen  from  dwell- 
ings near  the  shore  that  are  making  the  larger  contribution  to 
the  load.  For  example,  if  wastewater  were  to  travel  for  any  dis- 
tance in  the  aquifer,  we  would  expect  more  nitrification  and 
thus  a  higher  concentration  of  nitrate  (3)  than  we  observed. 

Work  supported  by  the  WBLMER  Research  Experience  for 
Undergraduates  grant. 


Literature  Cited 


1.  Valiela,  I.,  et  al.  1992.     Estuaries  15:  443-457. 

2.  Lajitha,  K.,  et  al.  1995.     Btogeochemistry  28:  33-54. 

3.  Keeney,  D.,  1986.     Critical  Reviews  in  Environmental  Control  16: 
257-304. 


Reference:  Biol  Bull  189:  248-249.  (October/November.  1995) 


Land  Cover  Effects  on  Inorganic  Nutrients  in  Groundwater  and  the  Role  of  Salt  Marshes  in  Interception 
of  Land-Derived  Nutrients  Entering  Estuaries  of  Waquoit  Bay,  Massachusetts 

Catherine  Hunter  MacGregor,  Site  Ann  Chaplin,  and  Ivan  Valiela  (Boston  University  Marine  Program, 

Marine  Biological  Laboratory) 


Nutrients  from  atmospheric  deposition,  fertilizer  use,  and 
wastewater  are  delivered  to  coastal  watersheds  and  have  different 
fates  as  they  are  transported  through  different  land  covers  (1). 


In  an  unconsolidated  sandy  watershed  such  as  that  of  Waquoit 
Bay,  Massachusetts,  hydraulic  conductivity  is  high,  and  ground- 
water  is  the  estuary's  primary  source  of  fresh  water  and  inorganic 


ECOLOGY:  BIOGEOCHEMISTRY  AND  NUTRIENT  CYCLING 


249 


nutrients  ( 1 ).  We  assessed  the  effect  of  forest  area  and  number 
of  houses  on  inorganic  nutrient  concentrations  in  groundwater 
leaving  Waquoit  Bay  watersheds  with  different  proportions  of 
these  land  covers. 

Salt  marshes  grow  between  land  and  estuaries,  and  ground- 
water-borne  nutrients  usually  have  to  pass  through  salt  marshes 
to  reach  estuaries.  Salt  marshes  are  known  to  support  substantial 
rates  of  denitrification  (2).  We  hypothesized  that  only  a  fraction 
of  the  nutrients  passing  from  land  through  salt  marshes  make 
it  into  estuaries. 

To  measure  the  nutrient  content  of  groundwater  delivered  to 
Waquoit  Bay,  samples  were  collected  at  the  back  of  the  salt 
marsh  fringe  all  along  the  periphery  of  Sage  Lot  Pond  (3,  4). 
Quashnet  River  (3,  4),  Hamblin  Pond,  Jehu  Pond,  and  Childs 
River  (3,  4).  To  find  the  nutrient  content  of  water  that  passed 
through  salt  marsh,  samples  of  water  were  taken  from  outflowing 
tidal  creeks  and  springs  in  the  salt  marsh  of  Sage  Lot  Pond  (Mar- 
tin, unpub.  data),  Hamblin  Pond,  and  Jehu  Pond.  The  Quashnet 
and  Childs  Rivers  have  very  little  fringing  marsh.  In  all  samples, 
nitrate  (NO,")  and  ammonium  (NH4f)  were  measured  with  a 
Lachat  Autoanalyzer,  and  phosphate  (PO43+)  was  measured  by 
a  method  adapted  from  Strickland  and  Parsons  (5). 

We  first  focused  on  the  effect  of  the  two  principal  land  covers, 
forest  and  residential  land  (6).  on  groundwater  nutrient  content. 
To  do  this,  we  plotted  (Fig.  1,  top)  mean  DIN  (NO,  +  NH4+) 
and  PO43+  concentrations  in  groundwater  about  to  leave  the 
aquifer  against  the  percentage  of  forested  area,  and  against  the 
number  of  houses  per  watershed  (unpub.  data). 

We  found  that  the  greater  the  proportion  of  forested  land  in 
a  watershed,  the  lower  the  concentration  of  DIN  and  PO43+  in 
groundwater  entering  the  salt  marsh  (Fig.  1,  upper  and  lower 
left,  black  circles).  This  may  be  the  result  of  uptake  and  use  of 
atmospherically  delivered  DIN  and  PO43+  by  forests  (7). 

Houses  contribute  lawn  fertilizer  and  nutrient-rich  septic  sys- 
tem wastewater  to  the  watershed  (8),  so  it  was  not  surprising  to 
find  that  DIN  in  groundwater  entering  the  salt  marsh  was  pos- 
itively related  to  the  number  of  houses  in  the  watersheds  (Fig. 
1,  upper  right).  There  was  no  significant  relationship  between 
the  number  of  houses  in  the  watersheds  and  PO43+  concentra- 
tions in  groundwater  entering  the  marsh  (Fig.  1,  lower  right). 

Second,  to  examine  possible  interception  of  nutrients  during 
passage  through  the  salt  marsh  (Fig.  1.  white  circles),  we  looked 
at  concentrations  in  the  samples  from  tidal  creeks  and  springs 
in  each  of  the  three  estuaries  that  had  a  marsh  fringe.  The  DIN 
concentrations  in  groundwater  leaving  the  salt  marsh  were  lower 
than  in  groundwater  entering  the  salt  marsh  (compare  black  to 
white  circles.  Fig.  I,  upper  left  and  right)  by  47%  at  Jehu.  61% 
at  Hamblin,  and  43%  at  Sage  Lot  (mean  =  50%)  (Fig.  I.  lower 
left  and  right).  PO4'+  concentrations  in  groundwater  leaving  the 
salt  marsh  were  lower  than  in  groundwater  entering  the  salt 
marsh  by  98%  at  Jehu  and  40%  at  Hamblin  (mean  =  69%)  (Fig. 
1.  bottom  panels).  This  suggests  that  plants  and  sediments  of 
the  salt  marsh  remove,  use,  and  transform  half  the  inorganic 
nitrogen  and  69%  of  the  phosphorus  brought  by  groundwater 
as  it  passes  from  watershed  to  estuary. 

Forests  intercept  atmospherically  delivered  inorganic  nitrogen 
and  phosphorus,  and  houses  increase  nitrogen  and  phosphorus 


120-, 


80- 


40- 


I          0. 

co 


DIN 


03 

o 

c 
o 
O 


3- 


2- 


1- 


PO4 


o 
o 
o 


%  forest 


No.  houses 


Figure  1.  Mt'an  concentrations  of  dissolved  inorganic  nitrogen  and 
phosphate  in  groundwater  (black  circles)  from  watersheds  of  ll'aqiioit 
Bay.  both  plollt'd  in  relation  in  the  percentage  afforest  area  in  the  wa- 
tersheds (left  panels)  and  to  the  number  of  houses  on  the  watersheds  (right 
panels).  Concentrations  of  nutrients  in  groundwater  leaving  salt  marsh 
growing  along  the  border  of  the  watersheds  and  estuaries  are  shown  as 
unfilled  circles.  Statistically  significant  relationships  between  %  forest  or 
No.  houses  and  nutrient  concentrations  are  indicated  by  the  regression 
line:  where  the  regression  was  not  significant,  no  line  was  included. 
Regression  lines  and  correlations  were  y  =  -2.15X  +  162.27.  r  =  0.54 
for  upper  left:  y  =  O.OSX  -  2.38.  r  =  0.94  for  upper  right:  y  =  -0.11 
+  7.42.  T  =  0.90  for  lower  right. 


loads  from  land  to  estuary.  Salt  marshes  growing  between  land 
and  estuary  intercept  considerable  amounts  of  land-derived  in- 
organic nitrogen  and  phosphorus.  These  results  argue  the  im- 
portance of  conservation  of  green  land  cover  and  coastal  wetlands 
to  maintain  estuarine  water  quality. 

We  thank  Lori  Soucy  for  her  assistance  in  the  laboratory. 
This  research  was  supported  by  internships  from  the  Woods 
Hole  Marine  Sciences  Consortium  and  Research  Experience  for 
Undergraduates  through  Waquoit  Bay  Land  Margin  Ecosystems 
Research,  and  by  a  grant  from  NOAA  Coastal  Oceans  Program. 


Literature  Cited 

I     Valiela,  I.,  el  al.  1990.     Biogeochemis/ry  10:  177-197. 

2.  Johnson,  R.,  et  al.  1994.     liiol  Hull  187:289-290. 

3.  McDonnell,  K.,  et  al.  1994.     Biol.  Bull.  187:  276-277. 
4    Rudy,  M.,«fl/.  1990.     Buil  Bull  187:278-279. 

5.  Strickland,  J.,  and  T.  Parsons.  I960.     Bull.  Fish.  Res  Board  Can 
125:  1-185. 

6.  Valiela,  I.,  et  al.  1995.     Ecol  .•!/>/>/.  In  Press. 

7.  I  ajlha.  K.,  et  al.  1995.     Biogeochemistry  28:  33-54. 

8.  Valiela,  I.,  et  al.  1992.     I'Muunes  15:  443-457. 


250  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Bun  Bull.  189:  250-251.  (October/November,  1995) 

Pelagic  Metabolism  in  the  Parker  River/Plum  Island  Sound  Estuarine  System 

Derrick  1C.  M.  Alderman  (Bowdoin  College),  Brian  R.  Balsis.  I  sin  D.  Biiffam, 
Robert  H.  Gairitt,  Charles  S.  Hopkinson  Jr.,  and  Joseph  J.  \'allino 


In  this  experiment  we  quantified  pelagic  metabolism  in  the 
Plum  Island  Sound  estuary,  Massachusetts.  Pelagic  metabolism 
is  an  important  process  in  estuarine  ecosystems,  with  //;  situ 
primary  production  usually  being  the  primary  source  of  organic 
carbon  supporting  the  trophic  web. 

Production  and  respiration  were  determined  by  incubating 
water  in  bottles  for  14  and  24  h  every  other  day  lor  three  con- 
secutive weeks  in  June  and  July  1995.  Water  was  collected  at 
three  stations  along  the  estuary  at  sunrise  and  incubated  in  MIU 
at  light  levels  corresponding  to  0%,  54%.  90%,  and  100%  light 
extinction.  Net  daytime  production  (NDP),  dark  respiration 
(NR).  gross  production  (GP),  and  net  community  production 
(NCP)  were  calculated  from  changes  in  dissolved  oxygen  ( 1 ) 
measured  by  automated  Winkler  titration.  Chlorophyll-^  con- 
centrations were  measured  concurrently. 

Spatial  patterns  were  evident  throughout  the  estuary.  NDP 
and  NCP  are  highest  in  the  upper  water  column  and  decrease 
with  depth.  Surface  productivity  decreases  from  greater  than 
100  mmol  O2  m~3  d~'  in  the  upper  estuary  to  less  than  20  mmol 
O2irr3d~'  in  the  lower  Sound  (Fig.  I  A).  Turbidity,  as  measured 
by  light  extinction  (Fig.  IB),  and  chlorophyll  levels  are  highest 
in  the  upper  estuary  (Fig.  1C).  Respiration  remains  relatively 
constant  (averaging  —55  mmol  Oi  rrT:  d  ')  through  much  of 
the  estuary,  but  rises  to  an  average  —83  mmol  CK  irT:  d  '  in 
the  Sound  portion  of  the  estuary  (conductivities  greater  than 
45  mS  cm"1).  NCP  levels  indicate  that  the  water  column  is  net 
autotrophic  until  conductivities  exceed  46.5  mS  cm"1  in  the 
Sound,  at  which  point  it  becomes  net  heterotrophic  (Fig.  ID). 
This  spatial  pattern  of  autotrophy  and  heterotrophy  may  reflect 
the  utilization  of  watershed  inputs  of  inorganic  nutrients  in  the 
upper  estuary  and  the  remineralization  of  autochthonous  organic 
matter  transported  downstream  to  the  lower  estuary. 


Patterns  of  turbidity  and  production  per  unit  chlorophyll  pro- 
vide some  insight  into  the  controls  of  primary  production  in  the 
estuary.  The  pattern  of  increasing  production  per  unit  chloro- 
phyll down  the  estuary  suggests  that  production  is  light-limited. 
Although  the  upper  estuary  contains  a  larger  amount  of  phy- 
toplankton,  as  indicated  by  chlorophyll  concentrations,  turbidity 
of  the  water  reduces  light  availability  and  hence  production  per 
unit  chlorophyll.  In  the  Sound,  clarity  of  the  water  column  allows 
light  penetration  to  all  levels,  increasing  the  specific  production 
rate. 

Bottle  methods  for  studying  metabolism  often  underestimate 
total  system  metabolism  because  they  fail  to  measure  the  sub- 
stantial metabolic  contributions  by  benthic  and  nektonic  com- 
munities (I).  Estimated  benthic  respiration  in  the  estuary  is 
—46  mmol  m  ~  d~'  (Hopkinson,  unpub.).  In  comparison,  pelagic 
metabolism  (-55  and  -83  mmol  O:  rrT-  d  ')  contributes  54% 
to  64%  of  total  system  respiration  (i.e.,  sum  of  benthic  and  pelagic 
metabolism).  This  proportion  is  similar  to  what  has  been  ob- 
served in  comparable  estuaries  (2).  However,  in  contrast  to  mea- 
surements of  whole  system  respiration  (3),  respiration  estimated 
from  the  sum  of  the  benthic  and  pelagic  components  is  much 
lower.  This  difference  may  be  due  to  the  resuspension  and  mixing 
of  labile  organic  matter  between  benthos  and  water  column;  this 
mixing  does  not  occur  in  bottles  or  in  core  tube  measures  of 
benthic  respiration. 

This  research  was  supported  by  an  LMER  grant  (#OCE- 
92144M). 

Literature  Cited 

1.   Odum,  H.  T.  1956.     l.imnol  Oivamw  1:  102-117. 

2    Hopkinson,  C.  S.  1985.     Mar  Bin/.  87:  19-32. 

3.  Balsis,  B.  R.,  e I  al.  1995.     Biol.  Bull  189:  252-254. 


ECOLOGY:  BIOGEOCHEMISTRY  AND  NUTRIENT  CYCLING 


251 


§      30 


20 

_     15 
B      '0 


-20 
•85 


Ipswich  Bay  Yacht  Club 


>        20        25        30        35        40 

conductivity  (mS/cm) 


15        20       25       30       35       40 
Conductivity  (mS/cm) 


200 


-200 


10 


15          20          25          30 
Conductivity  (mS/cm) 


35 


45      45.5      46      46.5      47      47.5      48      48.5      49      49.5      50 
Conductivity  (mS/cm) 


- 

NR 

-B- 

NCP 

-— 

NDP 

-+- 

GP 

D 


Figure  1.  (A)  Depth  profiles  for  sampling  sites,  averaged  over  the  three-week  study  period.  Points  represent  depths  in  water  column  for  0%,  54%, 
90%.  and  99.8%  (dark)  light  extinction;  the  vertical  lines  are  the  average  depths  at  each  site.  For  daily  profiles,  the  integrated  area  under  the  NCP 
curve  represents  net  community  production  (imnol  in  ~  d~'):  the  area  under  the  NDP  curve  represents  net  daytime  production  (inmol  in~2  f).  (B) 
Extinction  coefficients  (given  as  positive  values)  along  the  length  of  the  estuary.  (C)  Spatial  pattern  ii/'ehl-a  (ng  l~')  and  production  per  unit  chl-& 
(nunol  O2  m'2  d~'  per  unit  chl-n.  (D)  Metabolism  calculated  through  changes  in  BOD  bottle  oxygen,  across  a  range  of  conductivities  in  the  estuary 
(note  change  in  scale  on  x-axis). 


252  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Biol.  Bull  189:  252-254.  (October/November,  1995) 


Total  System  Metabolism  of  the  Plum  Island  Sound  Estuarine  System 

Brian  R.  Balsis  (Dartmouth  College),  Derrick  W.  M.  Alderman,  Ishi  D.  Buffam,  Robert  H.  Garritt, 

Charles  S.  Hopkinson  Jr.,  and  Joseph  J.  1'allino 


In  early  summer  1995,  we  surveyed  total  system  metabolism 
in  the  Plum  Island  estuary.  Objectives  included  ( 1 )  estimation 
of  ecosystem  metabolism  via  open-water  oxygen  measurements, 
(2)  determination  of  the  autotrophic  and  heterotrophic  regions 
of  the  estuary,  and  (3)  comparison  of  the  loading  of  organic 
carbon  from  the  watershed  to  measurements  of  autochthonous 
production. 

Metabolism  was  calculated  using  two  techniques:  (1)  24-h 
Lagrangian  surveys  at  three  points  in  the  riverine  portion  of  the 
estuary  at  conductivities  2.7  mS/cm.  20.1  mS/cm,  and  38.1  mS/ 
cm,  and  (2)  eight  dissolved  oxygen  (DO)  transects  along  the 
entire  length  of  the  estuary,  including  Plum  Island  Sound,  from 


27  June  through  29  June.  Respiration,  gross  daytime  production 
(GDP),  net  daytime  production  (NDP),  and  net  community 
production  (NCP)  were  measured  according  to  the  procedure 
of  Odum  (1). 

Lagrangian  surveys  demonstrated  a  dynamic  pattern  of  DO 
change  over  the  day.  For  example,  in  the  mid-estuary  at  20. 1  mS/ 
cm,  the  mass  of  DO  varied  over  the  day  from  360  to  450  mmol/ 
m:  (Fig.  la).  Rates  of  DO  change  corrected  for  flux  across  the 
air-sea  interface  (2)  indicated  that  primary  production  was  high- 
est in  late  morning  and  respiration  was  nearly  constant  through- 
out the  night  (Fig.  Ib).  Rates  of  metabolism  were  comparable 
at  the  three  conductivities  surveyed  (Fig.  Ic).  Gross  production 


Lagrangian  Surveys 


330 
320 
310 


E     290- 

E 


270- 
260' 


250 


06 


1  12  14 

Time  (Fraction  of  Day) 


1  8 


1  1.2  14 

Time  (Fraction  of  Day) 


250- 

200- 

150- 

100- 

OJ 

m 

^ 

50- 

fiih 

1 

11    f 

0 

: 

"o 

E 

•50- 

: 

E 

; 

-100- 

: 

•150- 

; 

-200- 

: 

: 

NDP 

GO 

3           24HrRes          NCP 
38.1  mS/cm 

ft: 

^ 

20.1  mS/cm 

S 

2.7   mS/cm 

•  Uncorrected 


Corrected 


Figure  1 .  (a)  Dii'l  pattern  of  dissolved  oxygen  (DO)  measured  during 
a  24-h  Lagrangian  survev.  The  best-fit  curve  overlies  actual  data  (b) 
Diffusion  correction  per  15-min  interval  of  6/22-3  Lagrangian  shown 
along  with  the  uncorrected  and  corrected  rate  of  change  of  DO.  (c)  Daily 
measures  of  net  daytime  production  (NDP).  gross  daytime  production 
(GDP).  24-h  respiration  and  net  community  production  (NCP)  from  three 
regions  within  the  Parker  River  estuary  calculated  by  Lagrangian  surveys, 
(di  Patterns  ol  dissolved  oxygen  concentration  measured  over  the  course 
of  a  day  along  the  entire  length  of  the  Plum  Island  Sound  estuary,  (e) 
Spatial  pal  terns  oj  GDP.  NDP.  and  24-h  respiration  obtained  with  the 
transect  approach,  if)  Pattern  of  NCP  along  the  length  ol  Plum  Island 
Sound  estuary,  (g)  Calculated  inputs  of  organic  carbon  loading  to  the 
estuary  from  the  watershed  during  summer  and  annual  periods  compared 
to  transect  measures  of  summer  metabolism  convened  to  carbon,  assum- 
ing an  eanimolar  O::CO:  equivalency. 


ECOLOGY:  BIOGEOCHEMISTRY  AND  NUTRIENT  CYCLING 


253 


ranged  from  1 70  to  2 10  and  respiration  from  200  to  250  mmol 
O:/m:/day.  Overall.  NCP  was  less  than  zero  at  all  sites  and  in- 
dicated that  the  system  was  net  heterotrophic  and  dependent  on 
allochthonous  inputs  of  organic  matter. 

The  transect  surveys  covered  the  entire  length  of  the  estuary 
and  showed  the  clearest  spatial  patterns  of  metabolism  (Fig.  If- 
g).  Daily  DO  levels  along  the  length  of  the  estuary  ranged  from 
5.2  to  10.9  mg/1  (Fig.  Id).  Almost  all  waters  were  undersaturated 
with  oxygen  throughout  the  day.  GDP  ranged  from  180  to 
300  mmol  O:/m:/day  and  NDP  ranged  from  20  to  150  mmol 
O:/m:/day  (Fig.  le).  Spatial  patterns  of  GDP  and  NDP  were 
similar,  being  highest  in  the  mid-riverine  portion  of  the  estuary 
(20  mS/cm)  and  lowest  in  the  lower  riverine  portion  (35  mS/ 
cm).  Daily  respiration  ranged  from  200  to  310  mmol  OI/ITT/ 
day.  It  was  lowest  in  the  upper  estuary  and  highest  in  the  mid- 
estuary  ( 1 5-20  mS/cm)  (Fig.  le).  Patterns  of  NCP  indicated  that 
only  the  extreme  upper  estuary  was  net  autotrophic  (60  mmol 


OI/ITT  per  day)  (Fig.  If).  Although  the  patterns  illustrated  with 
the  two  techniques  were  similar,  the  transect  approach  provided 
much  greater  spatial  information  at  lower  cost  and  is  the  rec- 
ommended method  for  future  studies. 

Spatial  patterns  of  metabolism  provide  clues  about  the  im- 
portance of  allochthonous  inputs  of  organic  matter  and  inorganic 
nutrients  to  the  estuary.  This  system  is  clearly  dependent  on 
allochthonous  inputs  of  organic  matter  as  almost  the  entire  es- 
tuary is  net  heterotrophic  (Fig.  If.g).  The  regions  of  highest  GDP 
may  reflect  the  importance  of  inorganic  nutrient  inputs  from 
the  watershed  (in  the  upper  estuary)  and  the  utilization  of  nu- 
tnents  remineralized  from  organic  matter  that  has  been  trans- 
ported downstream  to  the  mid-estuary  (Fig.  le).  Average  daily 
watershed  inputs  of  organic  matter  during  the  summer  are  in- 
sufficient to  sustain  estuarine  metabolism.  NCP  and  24-h  res- 
piration exceed  watershed  inputs  by  more  than  an  order  of  mag- 
nitude (Fig.  Ig).  Two  possible  explanations  could  account  for 


Transect  Surveys 


11 


10- 

9- 
Ij" 
B) 
§      8- 

O 
o 

7 
6 


10          15          20          25          30          35          40          45 
Conductivity  (mS/cm) 


15          20          25          30 
Conductivity  (mS/cm) 


12  00pm      M     4  00pm 


•  8  00pm 


NCP  June  29 


•  NCP  June  29 


200 


|    100- 

c3          0 
O 

o     -100 

E     -200 

-300 


15          20         25         30 
Conductivity  (mS/cm) 


35 


45 


GDP  June  28 
•  NDP  June  29 


NDP  June  28 
•  24Hr  Resp 


GDP  June  29 


>-    ~S 
I    1 


300- 
200- 
100 


Bfe 


-100 
-200 

-300 


Sum  Load         Ann  Load        Sum  Metab 
Key  For  Riverine  Summer  Metab 


I  NCP 


|  24Hr  Resp  f_H]  NDP  j^  GDP 


254 


this  seasonal  pattern:  (1)  metabolic  needs  during  the  summer 
are  met  by  watershed  inputs  during  the  rest  of  the  year,  or  (2) 
summer  needs  are  met  by  inputs  from  adjacent  intertidal 
marshes. 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Literature  Cited 


1.  Odum,  H.  T.  1956.     l.imnol.  Oceanogr.  1:  102-1 17. 

2.  Marino,  R.,  and  R.  \V.  llowarth.  1993.     Estuaries  16:  433-445. 


Reference:  Binl.  Bull.  189:  254-255.  (October/November.  1995) 

Effects  of  Nitrogen  Loading  and  Salt  Marsh  Habitat  on  Gross  Primary  Production  and 
Chlorophyll  a  in  Estuaries  of  VVaquoit  Bay 

David  H'.  Callaway.  Ivan  l'a/ie/a,  Kenneth  Foreman,  and  Lori  A.  Soucy 
(Boston  University  Marine  Program,  Marine  Biological  Laboratory) 


Nixon  ( 1 )  showed,  using  comparative  data  from  different  sys- 
tems, that  increased  nitrogen  load  to  shallow  coastal  estuaries 
increased  production  of  phytoplankton.  Furthermore,  it  has  been 
well  established  that  the  growth  of  coastal  producers  is  nitrogen 
limited  (2).  In  Waquoit  Bay,  we  have  a  complex  of  separate 
estuaries  that  are  subject  to  different  nitrogen  loading  rates  (3). 
This  variation  in  loading  rate  provides  the  opportunity  to  test. 
in  one  system,  whether  increased  nitrogen  loads  result  in  in- 
creased production. 

The  range  of  nitrogen  loading  to  the  estuaries  extended  from 
a  high  rate  of  8. 1  X  103  kg  N  y~'  in  Childs  River  to  approximately 
0.05 1  kg  N  y"1  in  Sage  Lot  Pond.  Because  phytoplankton  growth 
in  shallow  estuaries  is  nitrogen  limited  (2),  increased  loading 
rates  are  likely  to  affect  activity  and  abundance  of  these  primary 
producers. 

Salt  marsh  habitats  are  active  sites  of  denitriftcation  and  nu- 
trient uptake  (2).  A  strip  of  salt  marsh  located  between  the  wa- 
tershed and  the  estuary  could,  therefore,  intercept  incoming  ni- 
trogen and  significantly  reduce  estuarine  nitrogen  loading.  The 
estuaries  of  Waquoit  Bay  are  surrounded  by  different  areas  of 
salt  marsh.  We  could,  consequently,  also  evaluate  the  effects  of 
salt  marsh  on  interception  of  nitrogen  by  comparing  phyto- 
plankton abundance  and  activity  in  estuaries  with  different  ex- 
tents of  fringing  salt  marsh. 

In  this  paper  we  ask,  first,  whether  there  is  a  relationship  be- 
tween nitrogen  loading  rate  and  phytoplankton  abundance  and 
productivity;  and  second,  whether  the  presence  of  a  salt  marsh 
fringe  decreases  the  nitrogen  loading  rate  and,  accordingly,  lowers 
phytoplankton  abundance  and  productivity. 

We  measured  gross  primary  production  (GPP)  and  chlorophyll 
a  concentration  at  two  stations  in  each  of  five  estuaries  of  Wa- 
quoit Buy  (Childs  River,  Quashnet  River,  Jehu  Pond,  Hamblin 
Pond,  and  Sage  Lot  Pond).  We  used  standard  light/dark  bottle 
technique  v.ith  5-h  in  situ  incubation  period,  and  the  Winkler 
titration  method  to  determine  primary  production  of  the  estu- 
aries. Chlorophyll  a  concentration  was  measured  by  the  Lor- 
enzen  method  (4).  The  nitrogen  loading  rate  was  calibrated  based 
on  total  dissolved  nitrogen  (DIN)  at  shore  edge,  rate  of  water 
recharge,  and  total  area  of  the  estuary. 


GPP  and  chlorophyll  a  increased  significantly  with  higher  ni- 
trogen loads  (Fig.  1,  top  panels).  For  the  regression  of  phyto- 
plankton and  loading,  P  <  0.003  for  both  GPP  and  Chi  a.  In 
Childs  River,  for  example,  the  average  chlorophyll  a  concentra- 
tion and  GPP  levels  were  about  three  times  as  high  as  those  in 
Sage  Lot  Pond. 

Both  GPP  rates  and  chlorophyll  a  concentration  decreased 
in  estuaries  with  larger  areas  of  fringing  salt  marsh  (Fig.  1,  middle 
panels).  The  cause  of  this  decrease  is  not  well  established.  The 
salt  marshes  could  be  physically  removing  phytoplankton  from 
the  flooding  estuarine  water  during  high  tide  and.  thus,  lowering 


1! 


0         25        50        75 
N-  Load  (10=  Kg  Ny  '  ) 


100  0         25        50        75-     100 

N- Load  (10=  Kg  N  y1  ) 


d 

5 

M 

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0    3- 

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i 

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*                     *            D* 

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i 

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—      0          10        20        30 

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Q_       Salt  marsh  area!  104  nr) 
PH 

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O 

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25 


50 


75        100 


0 


25 


50 


75 


100 


<7c  Salt  marsh  around  shore 


%  Salt  marsh  around  shore 


Figure  1.  Gross  primary  production  (left  panels)  and  chlorophyll  con- 
centration (right  panels)  plotted  versus  nitrogen  load  I  upper  panels),  salt 
marsh  area  (middle  panels),  and  percentage  of  the  periphery  of  each 
estuary  that  is  made  up  />/  sail  mar.s/i.  Data  from  estuaries  ol  H'aQtioit 
Hay,  including  Childs  River  (Mack  squares).  Quashnet  River  (black  dia- 
monds), Jehu  Pond  (hlaek  circles).  Hamhlin  Pond  ihlack  triangles),  and 
Sage  Lot  Pond  (open  squares). 


ECOLOGY:  BIOGEOCHEMISTRY  AND  NUTRIENT  CYCLING 


255 


GPP  and  chlorophyll  a.  Because  tidal  ranges  reach,  at  most,  0.5 
m  in  Waquoit  Bay.  and  coverage  of  vegetation  occurs  only  during 
a  few  days  of  spring  tides  each  month,  this  mechanism  does  not 
seem  convincing.  More  likely,  denitritication  and  storage  of  wa- 
tershed-derived nitrogen  in  salt  marshes  could  he  responsible 
for  a  sufficient  reduction  of  nitrogen  supplies  to  lower  phyto- 
plunkton  production.  Similar  results  were  obtained  when  we 
compared  GPP  or  chlorophyll  a  in  relation  to  the  percent  of  the 
periphery  of  each  estuary  that  is  composed  of  salt  marsh  fringes 
(Fig.  1.  bottom  panels). 

We  therefore  conclude  that  the  increase  in  producer  activity 
due  to  nitrogen  loading  from  watersheds  may  be  mediated  by 
the  extent  of  salt  marsh  interposed  between  land  and  estuary. 


Further,  salt  marshes  can  decrease  the  nutrient  supply  to  the 
estuaries  and  limit  phytoplankton  production  and  chlorophyll 
a  levels. 

This  work  was  supported  by  the  WBMLER  Research  Expe- 
rience for  LIndergraduates  Grant  and  by  a  grant  from  NOAA 
Coastal  Ocean  Studies  Program. 

Literature  Cited 

1.  Nixon,  S.  1988.     l.inniol  Oceunot;.  33:  1005-1025. 

2.  Howarth,  R.  VV.  1992.     .-Inn  Rev  Ecol  S.v.v/.  19:  89-1 10. 

3.  Valiela,  I.,  et  al.  1992.     E\tmiries  15:  443-457. 

4.  Lorenzen,  C.  J.  1979.     I.imnol.  Oceanog.  24:  1117-1 120. 


Reference:  Bio/.  Bull  189:  255-256.  (October/November,  1995) 

Macrophyte  Abundances  in  Waquoit  Bay  Estuaries  Subject  to  Different  Nutrient  Loads  and  the  Potential 
Role  of  Fringing  Salt  Marsh  in  Groundwater  Nitrogen  Interception 

Julie  Lyons.  Jenny  Ahern,  James  McClelland,  and  Ivan  ralie/a  (Boston  University  Marine  Program, 

Marine  Biological  Laboratory) 


Increased  nutrient  loading  has  shifted  seagrass-dominated 
habitats  to  macroalgae-dominated  habitats  in  many  coastal  re- 
gions ( 1 ).  In  Waquoit  Bay,  Massachusetts,  eelgrass  (/.usicni  ma- 
rina) has  diminished  markedly  in  abundance  over  the  last  four 
decades  and  been  replaced  by  macroalgae  (2).  In  most  estuaries, 
nitrogen  loads  are  furnished  by  the  adjoining  watershed  (3.  4, 
5),  and  the  major  form  of  nitrogen  is  nitrate.  Many  of  these 
estuaries  have  fringes  of  salt  marsh  interposed  between  land  and 
water.  Salt  marshes  have  high  rates  of  denitrification  (6),  and 
may  therefore  act  as  a  buffer  against  eutrophication.  uncoupling 
the  link  between  the  load  of  nutrients  from  land  and  the  estuarine 
benthic  vegetation.  In  this  study  we  investigate  the  relationship 
between  nutrient  loading  and  macrophyte  abundance  in  estuaries 
of  Waquoit  Bay  and  address  the  potential  role  of  fringing  salt 
marsh  in  reducing  loadings  to  estuaries. 

Macrophyte  abundance  was  measured  from  June  to  August 
1995  in  five  estuaries  of  Waquoit  Bay  (Childs  River,  Quashnet 
River,  Jehu  Pond.  Hamblin  Pond,  and  Sage  Lot  Pond).  These 
estuaries  receive  different  nutrient  loadings  from  their  watersheds 


Figure  1 .  Macrophyte  biomass  versus  nitrogen  loading  (top)  and  salt 
marsh  area  (hotlom)  at  Childs  River  (CR),  Quashnel  River  (QR).  Jehu 
Pond  UP),  Hamhlin  Pond  (HP),  and  Sage  Lot  Pond  ISLP).  esluarwi  ol 
Wai/noil  Bay  sampled  June-August  7995.  Eelgra\i  point*  are  mean  ± 
SE.  and  macroalgae  points  are  medians  ±  SEmeit  (n  =  30  lor  SLP,  QR, 
and  CR,  and  n  =  50  lor  HP  and  JP). 


EELGRASS 


MACROALGAE 


E 

o 


02468      10       02468       10 

Nitrogen   load   (103  Kg  N  yr1) 


60-1 
50- 
40- 
30- 
20- 
10- 


150- 


100- 


50- 


0 


ISLP 


0   100  200  300  400   0   100  200  300  400 

Salt  marsh  area  (103  m2) 


256 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


(Valiela  el  al..  unpub.  data)  and  possess  different  amounts  of 
surrounding  salt  marsh.  Ten  sites  at  each  estuary  were  sampled 
with  an  Eckman  dredge  (0. 1 5  m2)  at  the  beginning  of  each  month 
in  all  estuaries  and  at  intervening  2-week  intervals  in  Jehu  and 
Hamblin  Ponds.  Samples  were  sorted  by  species,  dried,  and 
weighed.  Nitrogen  loading  and  salt  marsh  area  showed  no  re- 
lationship (/Tor  regression  >0.05).  We  therefore  regressed  nu- 
trient loading  versus  biomass,  and  salt  marsh  area  versus  biomass. 
Eelgrass  biomass  was  inversely  related  to  nitrogen  loading  (Fig. 
1,  top  left;  P  =  0.0001 ),  in  contrast  to  macroalgal  biomass,  which 
increased  with  nutrient  loading  (top  right;  P  =  0.0041).  The 
shapes  of  the  best-fit  curves  suggest  that  eelgrass  is  particularly 
sensitive  to  even  low  rates  of  additional  nutrient  loading  (Fig. 
1,  top  left),  but  macroalgae  respond  linearly  over  a  broader  range 
of  nutrient  loadings.  The  correlations  (Fig.  1,  top),  however, 
have  considerable  variation;  for  instance,  Jehu  Pond  has  sub- 
stantially more  eelgrass  and  less  algae  than  would  be  expected 
based  on  nutrient  loadings  alone.  Part  of  this  variability  may  be 
associated  with  the  amount  of  salt  marsh  in  the  estuaries.  As 
salt  marsh  area  increases,  eelgrass  biomass  increases  (Fig.  1 ,  bot- 
tom left;  P  =  0.0001 )  and  macroalgal  biomass  decreases  (Fig.  1, 
bottom  right;  P  =  0.001 1).  Correlation  coefficients  for  best-fit 
lines  improved  for  both  eelgrass  and  macroalgal  biomass  when 
plotted  against  salt  marsh  area  (compare  r  values  in  top  and 
bottom  panels.  Fig.  1).  This  result  could  be  caused  by  lowered 
nitrogen  loading  owing  to  denitrification  in  salt  marshes. 


Seagrass  growth  is  inhibited  under  increased  nitrogen  loading 
and  macroalgal  growth  is  enhanced.  Eelgrass  apparently  under- 
goes rapid,  near-exponential  reduction  in  biomass  over  a  narrow 
range  of  nutrient  loadings,  whereas  macroalgae  respond  in  a 
linear  fashion  over  a  broader  range.  The  relationship  between 
nutrient  loading  to  coastal  watersheds  and  estuarine  macrophytes 
may,  however,  be  strongly  influenced  by  salt  marsh  fringing  the 
estuaries.  Fringing  salt  marsh  might  act  as  a  buffer  against  nu- 
trient loading,  and  thus  might  allow  eelgrass  beds  to  be  main- 
tained even  when  they  are  surrounded  by  relatively  urbanized 
watersheds. 

This  work  was  supported  by  REU-NSF  (OCE  9300490), 
NOAA  (NA170R21 101),  and  The  Waquoit  Bay  Fellowship 
awarded  to  J.  McClelland. 


Literature  Cited 


1.  Duarle,  C.  M.  1995.     Ophelia  41:  87-1 12. 

2.  Valiela,  I.,  et  al.  1992.     Exliianc*  15:  443-457. 

3.  Giblin,  A.  E.,  and  A.  Gaines.  1990.     Biogeochemislry  10:  309-328. 

4.  Simmons,  G.  M.,  Jr.  1992.     Mar  Ecol,  Prog.  Ser  84:  173-184. 

5.  Millham,  N.  P.,  and  B.  L.  Howes.  1994.     Limnnl.  Oceanogr.  19: 
1928-1944. 

6.  Johnson,  R.,  et  al.  1994.     Binl  Bull  187:289-290. 


Reference:  Biol.  Bull  189:  256-257.  (October/November.  1995) 


Effects  of  Land  L'se  on  the  Degradability  of  Dissolved  Organic  Matter 
in  Three  VV  atersheds  of  the  Plum  Island  Sound  Estuary 

AmyG.  Uhlenhopp,  John  E.  Hobble,  and  Joseph  J.  } 'a/lino 
(The  Ecosystems  Center,  Marine  Biological  Laboratory) 


Different  types  of  land  use  influence  many  of  the  fundamental 
processes  of  terrestrial  ecosystems  and  affect  the  materials  moving 
from  land  to  estuaries  and  coastal  marine  systems.  One  input 
to  estuaries  is  dissolved  organic  carbon  (DOC);  changes  in  wa- 
tershed land  use  have  increased  the  levels  of  total  organic  carbon 
transported  to  the  ocean  by  3-5  times  those  of  natural  levels 
( I ).  Yet  it  is  not  clear  how  much  of  the  DOC  entering  estuaries 
is  actually  used  by  microbes  and  contributes  to  the  food  web 
and  how  much  is  resistant  to  degradation  and  moves  out  to  the 
oceans.  In  our  experiment,  we  determined  the  concentration 
and  degradability  of  the  DOC  entering  a  northern  Massachusetts 
estuary  from  three  areas  with  different  land  uses:  agriculture, 
forest,  and  urban. 

Stream  water  collected  9  August  1994  from  agricultural,  ur- 
banized, and  forested  watersheds  was  filtered  (0.2  ^m)  to  remove 
most  of  the  bacteria  and  reinoculated  with  estuarine  bacteria 
(20:1  dilution)  from  the  oligohaline  portion  of  the  Parker  River 
in  the  Plum  Island  Sound  estuary.  Inoculated  water  samples 
were  incubated  in  15-1  Mylar  bags,  and  changes  in  bacterial 


numbers,  dissolved  organic  and  inorganic  carbon  (DOC.  DIC), 
and  dissolved  oxygen  (DO)  were  analyzed  at  several  time  points 
over  a  2-week  period. 

Bacteria  in  the  agricultural  samples  consumed  a  greater 
amount  of  oxygen,  and  did  so  at  a  faster  rate,  than  bacteria 
growing  in  the  urban  or  forest  samples  (Fig.  1 ).  The  greatest  total 
change  of  oxygen  occurred  in  the  agricultural  water  samples,  a 
90.8  nM  (±0.8  nM}  decrease.  Total  changes  in  the  urban  and 
forest  bags  were,  respectively,  41.2  jiA/(±1.5  ^\/)  and  34.5  nM 
(±6.8  tiAf).  The  DOC  in  the  agricultural  samples  showed  a  90.3 
jiA/(±6.3  nAI)  decrease;  the  largest  decrease  of  the  three  samples 
(Fig.  1 ).  There  was  a  48.2  ^M  (±5. 1  nM)  decrease  of  DOC  in 
the  forest  samples,  and  a  33.5  nM  (±4.3  v-M)  decrease  in  the 
urban  samples.  Most  of  the  DOC  consumed  was  completely 
oxidized,  as  DIC  concentrations  increased  by  104  pM,  49  pM, 
and  33  nM  in  the  agricultural,  forest,  and  urban  samples,  re- 
spectively (Fig.  1 ). 

The  actual  concentrations  of  DOC  were  more  than  twice  as 
high  in  the  agricultural  samples  as  in  either  the  forest  or  urban 


ECOLOGY:  BIOGEOCHEMISTRY  AND  NUTRIENT  CYCLING 


257 


260 
240 


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200 

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•  Q 

O 
240  0 

180 

- 

•  • 

-im 

i  ....  i  ....  i  .... 

pnn 

5          10 
Time  (d) 


15 


samples.  The  percentage  of  the  total  DOC  pool  utilized  was 
similar  among  all  three  water  samples:  15.7%  in  the  forest,  10.3% 
in  the  agricultural,  and  1 1.0%  in  the  urban  samples. 

The  organic  matter  from  the  agricultural  watershed  clearly 
had  the  greatest  potential  impact  on  the  estuary.  The  unexpected 
conclusion  from  the  experiment,  however,  is  that  the  total 
quantity  of  organic  matter  is  most  important;  the  usable  DOC 
made  up  a  similar  percentage  of  the  total  in  water  from  all  three 
watersheds. 

The  research  was  supported  by  an  LMER  grant  (NSF-OCE- 
9214461).  We  thank  Ishi  Buftam,  David  Giehtbrock,  Charles 
Hopkinson,  and  Eileen  Monaghan  for  advice  and  assistance  with 
sample  analysis. 

Literature  Cited 
1.  Maybeck,  M.  1982.     Am.  J  Sci  282:  401-450. 


Figure  1.  The  average  concentrations  (i*M)  of  dissolved  organic  carbon 
(DOC),  dissolved  inorganic  carbon  (DIC).  and  dissolved  oxygen  (DO)  in 
replicate  15-1  Mylar  bags  during  a  13-day  incubation.  DIC  was  measured 
by  high-temperature  catalytic  oxidation.  DO  hr  automated  ll'inkler  ti- 
tralion.  and  DIC  by  precision  coulometric  CO:  analyzer. 


Reference:  Biol  Bull  189:  257-258.  (October/November,  1995) 

Nutrient  Limitation  of  Phytoplankton  Growth  in  Waquoit  Bay,  Massachusetts 

Gabrit'l/c  Tomasky  and  Ivan  I  'aliela  (Boston  I'nivcrsily  Marine  Program.  Marine  Biological  Laboratory) 


Studies  of  nutrient  limitation  in  fresh  water  and  seawater  ( 1 , 
2,  3,  4,  5,  6)  show  that  phosphorus  limits  phytoplankton  growth 
in  fresh  water  and  nitrogen  does  in  saline  water.  Estuaries  are 
situated  between  a  fresh-water  source  and  the  sea  and  are  char- 
acterized by  a  gradient  of  salinities;  hence,  each  estuary  must 
have  a  point  at  which  the  limiting  nutrient  switches  from  phos- 
phorus to  nitrogen.  In  this  study  we  asked  whether  the  effect  of 
N  or  P  varies  along  a  fresh  to  salty  gradient  within  the  Childs 
River  estuary  of  Waquoit  Bay.  Massachusetts,  and  whether  the 
limiting  roles  of  N  and  P  vary  seasonally. 

Bottle  enrichment  experiments  were  carried  out  using  water 
from  sites  of  low  (0-9%»),  intermediate  ( 10-19%»).  and  high  (20- 
28%»)  salinities.  Water  was  filtered  through  a  253-^m-mesh  net 
to  remove  large  zooplankton.  Enrichment  treatments  consisted 
of  additions  that  furnished  100  fiM  of  NOJ  or  PO~4;  controls 
received  no  additions.  The  treatments  were  applied  to  two  rep- 
licate bottles.  All  bottles  were  incubated  near  the  surface  in  the 
Bay,  and  then  collected  at  1-  or  2-day  intervals  to  furnish  a  time 
sequence  of  phytoplankton  growth,  as  measured  by  chlorophyll 
concentration.  The  shallow  incubation  reduced  the  probability 


of  light  limitation.  The  containers  were  incubated  in  the  Bay  by 
attaching  them  to  a  floating  rack  that  was  anchored  to  the  bottom 
of  the  estuary. 

Phytoplankton  grew  in  virtually  all  of  the  containers  over  the 
incubation  periods.  We  calculated  chlorophyll-specific  growth 
rates  for  each  month  by  normalizing  growth  rates,  relative  to 
controls,  to  compensate  for  the  marked  seasonal  change  in  chlo- 
rophyll standing  crop  in  Childs  River.  We  calculated  the  nor- 
malized growth  rate  as  G  =  (Pt.  -  Ic)/d  -  (Pc  -  Ic)/d,  where  Pe 
and  Ie  are  the  peak  and  initial  concentrations  in  the  enriched 
treatments,  Pc  and  Ic  are  the  peak  and  initial  chlorophyll  con- 
centrations in  the  control  treatment,  and  d  is  the  number  of 
days  of  the  incubation. 

The  normalized  phytoplankton  growth  rates  peaked  in  July- 
August  in  all  treatments  (Fig.  1 ).  The  magnitude  of  the  peak 
depended  on  the  supply  of  nitrogen  or  phosphorus  and  the 
provenance  of  the  water.  In  water  from  the  upper  reaches  of  the 
river,  with  salinity  <10%<>,  phosphate  enrichment  prompted  a 
modest  increase  in  phytoplankton  growth  during  summer  (Fig. 
1,  top);  note  that  the  standard  error  of  means  do  not  overlap 


258 


REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 


Q. 
O 


-C 
O 

O) 
0) 

s 

-C 
-t— « 

O 

O 


40-i 
30- 
20- 
10- 
0- 

-10- 

-20 


40-, 
30- 
20- 
10- 
0- 

-10- 

-20 


40-, 
30- 
20- 
10- 
0- 


0-9  %0 


PO, 


10-19  %0 


20-28  %o 


A      M      J      J      A    S 


Figure  1.  Normalized  phytoplankton  growth  rates  imcan  ±  propa- 
gated SE),  relative  to  controls  in  each  enrichment  treatment,  shown  over 
the  rear  for  the  three  reaches  of  Child*  River,  identified  as  different  ranges 
of  salinity. 


during  summer.  In  water  from  the  lower  reaches  of  Childs  River, 
with  salinity  >10%».  nitrate  stimulated  growth  during  the  three 
summer  months.  Phosphate  stimulated  growth  for  1  summer 
month  (July)  (Fig.  1.  middle  and  bottom):  this  experiment  was 
run  with  water  of  10%o,  it  was,  therefore,  the  fresher  end  of  the 


10-19%0  range.  Nutrient  additions  stimulated  phytoplankton 
growth  during  2  months  in  water  from  the  upper  reaches,  3 
months  in  water  from  the  middle  reaches,  and  4  months  in 
water  from  the  lower  reaches  (Fig.  1.  top,  middle,  and  bottom). 
This  suggests  that  phytoplankton  found  near  the  mouth  of  Childs 
River  were  more  nutrient-limited  for  more  of  the  year  than  were 
cells  at  the  upper  reach  of  the  estuary. 

Our  results  show  that  phytoplankton  growth  can  be  nutrient- 
limited,  even  in  an  estuary  that  receives  reasonably  high  nitrate 
loads,  2.4  moles  N  irT2y~L  (Valiela  el  al.  unpub.),  from  its  wa- 
tershed, and  where  ambient  concentrations  are  reasonably  high 
compared  to  other  seawaters.  The  range  of  ambient  nitrate  con- 
centration was  18-42  /jM  in  the  upper  reaches  of  the  estuary 
and  1-16  nM  in  the  intermediate  to  lower  reaches  of  the  estuary, 
and  the  range  of  ambient  phosphate  concentrations  was  0.7- 
1.9  nM  in  the  upper  reaches  of  the  estuary  and  0.1-1.5  in  the 
intermediate  to  lower  reaches  of  the  estuary  (7).  In  the  fresher 
end  of  the  estuary  there  is  a  suggestion  of  modest  limitation  of 
phytoplankton  growth,  but  because  nutrient  concentrations  are 
relatively  high  (7),  the  growth  response,  even  to  P,  is  modest. 

Nitrogen  supply  controls  phytoplankton  growth  over  the  warm 
months  in  saltier  water.  There  was  an  apparent  switch  from  P 
to  N  limitation  within  the  estuary  in  water  of  around  10%»,  and 
there  were  no  evident  seasonal  shifts  in  N  or  P  limitation. 

The  work  of  the  Waquoit  Bay  Land-Margin  Ecosystem  Re- 
search (LMER)  project  was  supported  by  grants  from  NSF  (OCE 
891479),  REU  (OCE  9300490),  NOAA  (NA  170R21101),  and 
NSF/EPA(OCE  8914729). 

Literature  Cited 

1.  Caraco,  N.,  A.  Tamse,  O.  Boutros,  and  I.  Valiela.  1987.     Can.  ./ 
Fish.  Aaual.  Set.  44:  473-476. 

2.  Fisher,  T.  R.,  E.  R.  Peele,  J.  \V.  Ammerman,  and  I..  \\ .  Harding. 
1992.     Mar.  Ecol.  Prog.  Ser  82:  51-63. 

3.  Howarth,  R.  \V.  1988.     Ann.  Rev  Ecol.  Sysl.  19:  89-1  10. 

4.  Howarth,  R.  \\ '.,  and  J.  J.  Cole.  1985.     Science  229:  653-655. 

5.  Nixon,  S.  \V .,  C.  A.  Oviatt,  J.  Frithsen,  and  B.  Sullivan.  1986.     ./ 
Umnol.  Soc  South.  Air  12:  43-71. 

6.  Vince,  S.,  and  I.  Valiela.  1973.     Mar.  Biol  19:  69-73. 

7.  Valiela,  I.,  K.  Foreman,  M.  LaMontagne,  D.  Hersh,  J.  Costa,  P. 
Peckol,  B.  DeMeo-Anderson.  C.  D'Avanzo,  M.  Babione,  C.  H.  Sham, 
J.  Brawley,  and  K.  l.ajlha.  1992.     Estuaries  15:  443-457. 


Reference:  Biol.  Bull  189:  258-259.  (October/November,  1995) 


Effect  of  Nutrient  Enrichment  on  Phytoplankton  Growth  in  Waquoit  Bay,  Massachusetts 

Cecelia  C.  Sheridan,  Ivan  I'aliela,  Kenneth  Foreman,  and  Lori  A.  Soucy 
(Boston  University  Marine  Program,  Marine  Biological  Laboratory) 


The  relative  importance  of  nitrogen  and  phosphorus  limitation 
on  growth  of  coastal  phytoplankton  has  been  much  discussed 
( 1,  2,  3,  4,  5).  In  addition,  there  may  be  an  interaction  between 
nitrogen  and  phosphorus  limitation  and  the  overall  rate  of  nu- 
trient loading  in  the  estuaries.  To  investigate  this  interaction, 
we  conduced  a  set  of  enrichment  experiments  in  which  NO-T 


+  PO4'~,  NO3~,  or  PO43  was  added  to  water  from  estuaries  of 
Waquoit  Bay  that  are  exposed  to  different  rates  of  nutrient  load- 
ing (6). 

Experiments  were  conducted  during  June,  July,  and  August, 
1995.  Filtered  water  samples  from  22-31  ppt  salinity  sites  in 
Childs  River,  Quashnet  River,  and  Sage  Lot  Pond  were  collected 


ECOLOGY:  BIOGF.OCHEMISTRY  AND  NUTRIENT  CYCLING 


259 


'- 

20- 

Sage  Lot  Pond 

on 

15- 

3r 

10- 

rentration 

5- 

0- 

20- 

S         ' 

Quashnel  River 

^p 

c 
o 
U 

15- 
10- 

.^ 

o. 
o 

5- 

0- 

_c 

20- 

Childs  River    ^_^* 

-= 
U 

15- 

•^^ 

."§ 

10- 

'5 

— 

5- 
n- 

O 

O 


June     July    August 


80- 
60- 
40- 
20- 


Sage  Lot  Pond 


o. 

o 

o 


60- 
40- 
20- 


80- 
60- 
40- 
20- 
0 


Quashnet   River 


Childs   River 


June      July    August 


• —     NO3  +  POJ  enriched 

Summer    1995    — »—  N°3  enrkhed 

— • —     PO4  enriched 

Figure  1 .  Left  panels:  Initial  ambient  chlorophyll  concentrations  for 
Sage  Lot  Pond.  Quashnel  River,  and  Childs  River,  three  estuaries  of 
llut/uoir  Bay.  Massachusetts.  Right  panels:  Phytoplankton  growth  rates 
with  nitrate  +  phosphate,  nitrate,  and  phosphate  enrichment  in  water 
samples  from  Sage  Lot  Pond.  Quashnet  River,  and  Childs  River.  Nutrient 
enrichment  experiments  were  completed  in  June.  July,  and  August.  1995. 


and  initial  concentrations  in  the  enriched  treatments,  and  d  is 
the  number  of  days  of  incubation).  The  calculation  normalizes 
growth,  compensating  for  the  difference  in  chlorophyll  that  oc- 
curs over  time  in  the  estuary  (Fig.  1,  left  panel). 

Phytoplankton  growth  rates  for  water  samples  enriched  with 
NO3^  and  NOr  +  PO43  peaked  during  mid-July  in  the  three 
estuaries  (Fig.  1,  right  panel).  The  phytoplankton  was  primarily 
nitrogen-limited:  nitrogen  addition  stimulated  growth  (in  both 
NOr  and  NO3~  +  PO43~  enrichments),  but  PO43~  addition  alone 
did  not  (Fig.  1,  right  panel).  There  was  some  degree  of  secondary 
limitation  by  PO43~  when  NOr  was  available,  as  suggested  by 
the  moderate  increase  of  chlorophyll  growth  in  NO3~  +  PO43 
treatment  compared  to  NO,"  treatment  (Fig.  1,  right  panel). 

Surprisingly,  the  growth  response  (largely  due  to  nitrogen 
supply)  was  most  pronounced  where  the  algal  cells  were  chron- 
ically exposed  to  greater  nitrogen  loading.  Compare,  for  example, 
the  response  in  Childs  River  (Fig.  1,  bottom  right)  to  that  in 
Sage  Lot  Pond  (Fig.  1.  top  right).  The  differences  in  growth  re- 
sponse may  be  attributed  to  the  threefold  higher  initial  ambient 
concentration  of  chlorophyll  in  Childs  River  than  in  Sage  Lot 
Pond  (Fig.  1,  top  and  bottom,  left  panel). 

The  enriched  samples  from  different  estuaries  differ  in  phy- 
toplankton species  composition.  A  bloom  of  the  chain-forming 
diatom  Rhizosolenia  delicatula  comprised  35.7%  of  the  cells  in 
the  NO3~-enriched  treatments,  and  42.5%  of  the  cells  in  NO3~ 
+  PO43~  enriched  samples  from  Childs  River  in  July.  The  same 
diatom  made  up  only  25.5%  of  the  cells  in  the  NOr  +  PO43~ 
enriched  samples  from  Sage  Lot  Pond  in  July. 

Nitrogen  is  the  primary  limiting  nutrient  in  Waquoit  Bay. 
Phosphate  plays  a  secondary'  role.  The  different  growth  responses 
in  estuaries  of  Waquoit  Bay  can  be  attributed  to  differences  in 
the  composition  and  initial  biomass  of  the  phytoplankton. 

Work  supported  by  the  Waquoit  Bay  Land  Margin  Ecosystems 
Research  project  and  a  NSF-REU  grant. 


in  2-1  polyethylene  bottles.  After  the  addition  of  100  nM  NO3~. 
100  tiM  PO43-,  or  50  »M  NO3750  nM  PCX,'-,  control  and  en- 
riched samples  were  incubated  on  site  in  Waquoit  Bay.  Water 
samples  harvested  between  days  0  and  4  of  each  experiment 
were  analyzed  to  determine  nutrient  (ammonium,  nitrate,  and 
phosphate)  concentrations  and  phytoplankton  biomass  (7).  Wa- 
ter samples  preserved  with  Lugol's  solution  were  allowed  to  settle, 
and  then  observed  by  inverted  microscopy  (8).  Growth  of  phy- 
toplankton was  calculated  by  the  equation  G  =  [(Pc  -  Ie)/d]  - 
[(Pc  ~  U/d]  (where  Pc  and  Ic  are  the  peak  and  initial  chlorophyll 
concentrations  in  the  control  treatment.  Pc  and  Ic  are  the  peak 


Literature  Cited 

1.  Ryther,  J.  H.,  and  \V.  M.  Dunslan.  1971.     Science  171:  1008-1013. 

2.  Vince,  S.,  and  I.  Valiela.  1973.     Mar  Biol  19:  69-73. 

3.  Caraco,  N.,  et  al.  1987.     Can  ./  l-'tsh  Aquat.  Sa.  44:  473-476. 

4.  D'Elia,  C.  F.,  et  al.  1986.     Can  ./.  Fish  Aquat.  Sci.  43:  401-403. 

5.  Fisher,  T.  R.,  et  al.  1992.     Mar.  Ecol  Prog.  Ser.  82:  5 1-63. 

6.  Valiela,  I.,  et  al.  1992.     Estuaries  15:  443-457. 

7.  Wetzel,  R.  G.,  and  G.  E.  Likens.  1991 .     Pp.  S 1  - 1 65  in  Limnological 
Analyses.  Springer-Verlag,  New  York. 

8.  lltermohl,  H.  1958.     Milt.  Int    \'er  Theor  Angew.  Limnoi  9:  1- 
38. 


260  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Kiol.  Bull.  189:  260.  (October/November,  1995) 

Effect  of  Epiphyte  Biomass  on  Growth  Rate  of  Zostera  marina 
in  Estuaries  Subject  to  Different  Nutrient  Loading 

Travis  Bohrer,  Amos  Wright,  Jennifer Hauxwell,  and  Ivan  ]'aliela 
(Boston  University  Marine  Program,  Marine  Biological  Laboratory) 


Nutrient  enrichment  has  been  claimed  to  increase  epiphyte 
biomass  growing  on  eelgrass  (Zostera  marina)  leaves  (1),  and 
growth  of  epiphytes  on  eelgrass  leaves  can  restrict  light  enough 
to  eventually  decrease  productivity  of  eelgrass  (2).  We  examined 
the  effect  of  nutrient  loading  to  estuaries  on  growth  of  eelgrass 
in  three  estuaries  of  Waquoit  Bay,  Massachusetts,  subject  to 
different  nutrient-loading  rates  (3).  The  estuaries  and  their  nu- 
trient-loading rates  are  Sage  Lot  Pond  (0.16  g  N  rrf-y'1),  Ham- 
blin  Pond  (5.3  gN  m  V  ')•  and  Jehu  Pond  (6.4  gN  m  :y  '). 
We  also  assessed  the  possible  inhibition  of  eelgrass  growth  due 
to  the  presence  of  epiphytes  by  comparing  growth  rates  of  cleaned 
and  uncleaned  leaves  at  each  site. 

To  measure  growth  rates  of  eelgrass  leaves,  20  plants  from 
natural  populations  were  marked  in  each  estuary  in  July  and 
August  1995.  Marking  was  accomplished  by  making  a  needle 
hole  in  the  sheath.  Because  the  hole  moves  with  the  growing 
leaf,  the  distance  between  the  hole  and  sheath  scar  is  a  measure 
of  growth  rate.  Ten  of  the  marked  plants  were  entirely  cleaned 
of  epiphytes  in  tint  to  determine  whether  epiphytes  impaired 
growth  of  eelgrass.  Plants  were  harvested  4-6  days  after  marking. 
Because  leaves  of  different  ages  grow  at  different  rates  (4),  com- 
parisons among  estuaries  were  confined  to  first  (youngest)  or 
second  leaves. 

In  July,  growth  rates  of  both  the  first  and  second  cleaned  and 
uncleaned  leaves  increased  with  nutrient  load  (regression.  P 
<  0.05  in  each  case)  (Fig.  1,  top  and  middle)  but  were  largely 
unaffected  by  epiphyte  removal  (2-factor  ANOVA:  first  leaf,  P 
=  0.49;  second  leaf,  P  =  0.07)  (Fig.  1.  top  and  middle).  These 
results  suggest  that,  under  these  nutrient-loading  rates,  growth 
of  eelgrass  is  nutrient-limited,  rather  than  light-limited.  In  Au- 
gust, average  growth  rates  of  the  first  leaves  were  significantly 
slower  than  in  July,  indicating  seasonal  decreases  in  eelgrass 
growth  (Student's  /-test,  P  <  0.05  in  all  cases)  (Fig.  1.  bottom 
vs.  top),  and  cleaned  and  uncleaned  leaves  grew  slower  as  nutrient 
loading  increased  (negative  slopes  in  regression)  (Fig.  1,  bottom). 
Growth  rate  did  not  vary  between  cleaned  and  uncleaned  leaves 
(ANOVA:  first  leaf,  P  =  0.23). 

Despite  an  increase  in  epiphyte  biomass  with  nutrient  load 
(5),  growth  rate  of  eelgrass  was  unaffected  by  epiphytes  in  this 
experiment.  Contrary  to  what  we  expected,  the  July  results  sug- 
gest that  nutrient  loading  increased  growth.  Other  studies  have 
shown  eelgrass  production  increases  with  nitrogen  availability 
(6.  7).  Eelgrass,  however,  was  found  only  in  the  three  estuaries 
studied  and  was  absent  from  other  Waquoit  Bay  estuaries  that 
have  higher  nutrient  loads  (Quashnet  River,  39.3  gN  m~2y~' 
and  Childs  River,  45.5  g  N  irT2y~'),  and  whose  benthic  primary 
production  has  shifted  from  eelgrass-  to  macroalgal-dominated 
habitats.  This  suggests  that  nutrient  loading  may  not  negatively 
affect  eelgrass  production  within  the  range  of  loading  rates  of 
Sage  Lot  Pond,  Hamblin  Pond,  or  Jehu  Pond,  but  does  at  the 
higher  nutrient-loading  rates. 


Epiphytes  absent 
Epiphytes  present 


CD    3-, 


2- 


0 


August 
1st  Leaf 


-S— 3 


8 


0246 

Nutrient 
loading  rate  (gNm-2y-i) 

Figure  1.  Eelgrass  (Zostera  marina)  gnmlli  rate  (mean  ±  SE,  cm 
d~')  vs.  inilnent-loading  rates  (g  N  m~2  y~')  of  Sage  Lot  Pond.  Hamblin 
Pond,  and  Jehu  Pond  for  leaves  \viih  epiphytes  absent  and  present.  Leaves 
were  collected  in  July  (1st  leaf:  top,  2nd  /eat:  middle)  and  August  (1st 
leal:  bottom). 

Nutrient  loading  seems  most  important  for  eelgrass  growth 
during  the  peak  growth  season  (July).  Later  in  the  growing  season 
(August)  growth  rates  may  be  determined  by  internal  physio- 
logical controls,  changes  in  light  or  temperature,  and  competition 
for  nutrients  with  phytoplankton  or  macroalgae. 

This  work  was  made  possible  by  WBLMER  Research  Expe- 
rience for  Undergraduates  program  and  a  grant  from  the  NOAA 
Coastal  Oceans  Program. 

Literature  Cited 

1.  Borum,  J.  1985.     Mai  :  Biol  87:  21  1-218. 

2  Orth.  R.  J.,  and  K.  J.  Moore.  1983.     Science  222:  5  1  -53. 

3.  Valicla,  I.,  el  at.  1992.     Estuaries  15:  443-457. 

4.  Costa,  J.   1988.     Ph.D.  Dissertation,  Boston  University  Marine 
Program. 

5.  Wright,  A.,  el  al.  1995.     Biol  Bull  .189:  261. 

6.  Murray,  I...  el  al.  1992.     Auitat.  Bol.  44:  83-100. 

7.  Short,  F.  T.  1987.     Auiial  Boi  27:41-57. 


H'OHXiY     HKKilOCHl  MISIKY    AND   Nl'IRHN]    CYC)  INC. 
Reference:  Biol.  Bull  189:  261.  (October/November,  1995) 


261 


Growth  of  Epiphytes  on  Zostera  marina  in  Estuaries  Subject  to  Different  Nutrient  Loading 

Amos  ll'riglu.  Travis  Bohrer.  Jennifer  Ihinxwell.  ami  Ivan  I  'aliela  (Boston  University  Marine  Program, 

Marine  Biological  Laboratory) 


Epiphytes  may  limit  distribution  and  productivity  of  seagrasses 
by  reducing  the  light  available  to  eelgrass  blades  ( 1 ).  Primary 
production  of  eelgrass  (Zostera  marina)  epiphytes  increases  with 
nutrients  in  the  water  column  (2).  In  Waquoit  Bay  different 
estuaries  are  subject  to  different  N  loading  rates  from  their  wa- 
tersheds. This  offers  the  possibility  of  testing  whether  the  accu- 
mulation of  epiphytes  increases  as  N  supply  increases.  We  in- 
vestigated the  growth  of  epiphytes  on  Z.  marina  in  Sage  Lot 
Pond  (N  load  =  0.16  g  m-2y-').  Hamblin  Pond  (N  load  =  5.3 


g  m  -y  '),  and  Jehu  Pond  (N  load  =  6.4  g  m  2y 

Epiphyte  biomass,  including  primary  producers,  animals,  and 
detritus,  was  quantified  by  measurement  of  chlorophyll  a  and 
epiphyte  dry  weight.  Twenty  leaves  were  collected  from  each 
estuary  in  June  and  again  in  July.  1995.  To  collect  leaves  of 
about  the  same  age,  the  third  oldest  leaf  was  always  sampled 
(3).  Each  leaf  was  cut  into  10-cm  sections  starting  at  the  youngest 
end,  and  the  epiphytes  were  gently  scraped  off  and  filtered  onto 
either  Whatman  glass  fiber  filters  (chl.  a  measurements)  or  a 
200-j<m-mesh  screen  (biomass  measurements).  Chlorophyll  a 
was  measured  spectrophotometrically  after  extraction  with  90% 
acetone  for  2  days  (4). 

Epiphyte  biomass  was  much  greater  on  the  tops  of  older  leaves 
than  on  the  bottom  of  older  leaves  or  on  newer  leaves  (data  not 
shown).  This  accumulation  of  epiphytes  on  older  leaf  segments 
suggests  that  epiphyte  biomass  depends  on  the  duration  of  un- 
disturbed colonization  and  growth.  Therefore,  to  assess  the  rates 
of  accumulation  of  epiphyte  biomass  on  eelgrass  growing  in  dif- 
ferent estuaries,  the  age  of  different  leaf  segments  had  to  be  de- 
termined. We  calculated  the  age  of  each  10-cm  segment  from 
measured  rates  of  blade  elongation  from  each  estuary  (3).  These 
calculated  ages  allowed  us  to  define  the  interval  of  time  over 
which  epiphytic  biomass  had  accumulated. 

The  accumulation  of  epiphyte  biomass  and  of  chlorophyll 
was  highest  in  the  estuary  with  the  largest  N  load  (JP)  and  lowest 
in  the  estuary  with  the  lowest  load  (SLP)  (Fig.  1  top).  Thus,  the 
algae  that  make  up  part  of  the  epiphytic  biomass,  as  well  as  the 
animals  and  detntal  material,  all  increased  faster  in  the  estuary 
with  the  highest  N  load.  We  know  that  nutrient  concentrations 
in  the  water  in  the  three  estuaries  were  in  proportion  to  N  loads 
(unpub.  data). 

When  we  plotted  the  slopes  of  the  regression  lines  from  Figure 
1  (top  panels)  against  N  loading  rates  (unpub.  data),  we  found 
that  the  accumulation  rate  of  both  epiphyte  biomass  and  chlo- 
rophyll increased  as  nitrogen  loads  to  the  estuaries  increased 
(Fig.  1.  bottom  panels). 

These  results  suggest  that  external  N  loading  controls  the 
growth  of  epiphytic  matter  on  eelgrass  leaves.  The  more  epiphyte 
biomass,  the  more  light  is  intercepted  before  it  reaches  the  leaves. 
We  suggest  that  nitrogen  loading  acts  through  this  indirect 
mechanism  to  decrease  the  growth  of  eelgrass. 

Our  results  clearly  show  an  effect  of  N  load  on  epiphyte  bio- 
mass. The  further  supposed  effect  on  eelgrass  growth,  however, 
may  be  an  oversimplification  for  at  least  two  reasons.  First,  the 


Biomass 


JP:  r  =  0.858 
HP  r  =  0.654 
SLP:  r  =  0.196 


Chlorophyll 


JP:  r  =  0.938 
HP:  r  =  0.921 
SLP  r  =  0.697 


0        10      20      30      40      50  0       10 

Time  (days) 


20      30      40      50 


JP 


HP 

SLP 

/ 

y  =  0.004*  +  0.017.  r  =  0.805 


468  024 

Nutrient  load  (g  m"2  y-i) 


8 


Figure  1.  Biomass  flop  /ell)  and  chlorophyll  a  (top  right)  in  relation 
to  the  duration  of  growth  on  celgras.i  leaves  in  each  of  the  three  estuaries 
(JP  =  Jehu  Pond.  HP  =  Hamblin  Pond.  SLP  =  Sage  Lot  Pond).  Rale 
of  biomass  and  chlorophyll  a  increases  (obtained  from  slopes  of  the 
regressions  of  the  upper  two  panels)  in  relation  to  nitrogen  loading  rates 
./«/•  the  watershed  (bottom  left  and  right,  respectively). 

effect  of  loading  on  growth  rate  of  eelgrass  depends  on  dose,  and 
inhibition  of  growth  is  likely  only  at  the  highest  loading  rates 
within  Waquoit  Bay  estuaries  (5).  Second,  since  eelgrass  leaves 
grow  basally,  and  more  epiphytic  biomass  occurs  on  the  older, 
apical  portions  of  leaves,  epiphytes  may  have  less  of  an  effect 
on  total  leaf  growth  than  we  might  suppose.  Accumulation  of 
unattached  macroalgae  around  the  bases  of  eelgrass  plants  may 
have  shading  effects  on  the  leaves  as  important  as  the  effects  of 
does  epiphytic  biomass. 

This  work  was  supported  by  an  internship  from  the  Woods 
Hole  Marine  Sciences  Consortium  and  a  grant  from  NOAA 
Coastal  Oceans  Program. 

Literature  Cited 

1    Orlh,  R.  J.,  and  J.  V.  Montfrans.  1984.     Aunal.  Bui  18:43-69. 

2.  Borum,  J.  1985.     Mar.  Biol  87:  211-218. 

3.  Costa,  J.   1988.     Ph.D.  Dissertation,  Boston  University  Marine 
Program. 

4.  Lorenzen,  C.  J.  1967.     l.unnol  Oceanogr  12:  343-346. 

5.  Bohrer,  T.,  et  al.  1995.     Biol  Bull.  189:  260. 


262  REPORTS  FROM  THE  MBL  GENERAL  SCIENTIFIC  MEETINGS 

Reference:  Bid.  Bull  189:  262.  (October/November.  1995) 

Foliar  Release  of  Ammonium  and  Dissolved  Organic  Nitrogen  by  Spartina  alterniflora 

Cheryl  Ann  Wolfe,  Carol  Rietsma,  and  Ivan  Valiela  (Boston  University  Marine  Program, 

Marine  Biological  Laboratory) 


To  cope  with  osmotic  stress  associated  with  saline  environ- 
ments, salt  marsh  plants  secrete  salt  and  use  amino  compounds 
to  balance  osmotic  pressure  (1,2,3).  It  seems  likely  that  some 
nitrogen  compounds  are  excreted  along  with  salts  (3).  but  little 
is  known  about  the  magnitude  of  ammonium  (NH4)  and  dis- 
solved organic  nitrogen  (DON)  release  from  salt  marsh  plants. 
and  even  less  about  this  release  to  the  water  relative  to  nitrogen 
loads  received  from  adjoining  watersheds.  To  investigate  rate  of 
release  and  rate  of  release  per  square  meter  by  salt  marsh  plants, 
we  measured  foliar  release  rate  of  NH4  and  DON  by  Spariina 
alterniflora,  and  compared  these  rates  to  nitrogen  entering  es- 
tuaries from  watersheds. 

We  hypothesized  that  high  nitrogen  loading  to  an  estuary  leads 
to  higher  rates  of  foliar  nitrogen  release.  We  tested  this  by  mea- 
suring NH4  and  DON  foliar  exudates  from  S  u/icrni/loru  in 
three  estuaries  of  Waquoit  Bay  that  receive  different  nitrogen 
loads  (Table  I).  To  measure  foliar  release,  the  uppermost,  fully 
extended  leaves  of  5.  aherniflora  were  placed,  during  low  tide, 
in  25-ml  test  tubes  of  filtered  seawater  and  incubated  for  3  h. 
The  NH4  released  was  measured  using  a  LACHAT  autoanalyzer 
and  DON  was  measured  using  a  modified  dissolved  nitrogen 
protocol  (4). 

Rain,  dew,  and  tidal  submergence  affect  leaching  from  leaves 
(3).  Results  of  a  timecourse  experiment  suggest  that  the  nitrogen 
collected  in  the  tubes  is  that  which  had  accumulated  on  the 
leaves  since  the  last  time  water  removed  exuded  nitrogen.  Tidal 
height  measurements  showed  that  plants  were  not  submerged, 
even  during  spring  high  tides.  We  also  concluded  from  prelim- 
inary experiments  that  dew  was  not  as  effective  as  rain  in  re- 
moving accumulated  NH4  and  DON  from  leaves.  Thus  we  cal- 
culated rates  of  leaching  as  the  amount  of  NH4  and  DON  re- 


ceived in  the  tubes  divided  by  the  number  of  hours  since  the 
last  rainfall.  Assuming  all  leached  nitrogen  reached  estuarine 
waters,  we  calculated  the  amount  of  nitrogen  released  by  S.  al- 
icrnijlora leaves  into  each  estuary  during  the  growing  season. 

The  weight-specific  release  rates  of  both  NH4  and  DON  were 
not  significantly  different  at  each  location  (Table  I,  cols.  1  and 
2).  Nitrogen  loading  from  watersheds  therefore  does  not  seem 
to  affect  rates  of  foliar  release.  Increased  loading  does,  however, 
increase  biomass  of  S  alterniflora  (Table  I,  col.  4).  Multiplying 
release  rates  by  standing  crop  yields  the  area-specific  release  of 
nitrogen.  Because  of  the  differences  in  biomass,  area-specific  re- 
lease does  seem  related  to  nitrogen  load  (Table  I). 

In  estuaries  subject  to  larger  nitrogen  load,  the  amounts  of  N 
released  by  salt  marsh  plants  per  square  meter  are  greater,  but 
the  overall  release  per  square  meter  of  marsh  is  small  compared 
to  the  external  nitrogen  load.  Sage  Lot  Pond,  the  estuary  exposed 
to  the  lowest  nitrogen  load,  released  about  75%  of  the  external 
inputs.  This  suggests  that  in  pristine  systems,  in  which  atmo- 
spheric nitrogen  is  the  largest  nitrogen  influence,  foliar  release 
by  salt  marsh  vegetation  can  lead  to  significant  release  of  nitrogen. 
This  is  a  previously  unsuspected  mechanism  that  appears  to 
convey  considerable  nitrogen  to  estuaries. 

This  research  was  supported  by  LMER  Research  Experience 
for  Undergraduates  program,  and  by  a  grant  from  NOAA  Coastal 
Ocean  Program. 

Literature  Cited 


Pakulski,  J.  D.  1986.     Em  Coast.  Shelf  Sci.  22:  385-394. 
Jefferies,  R.  L.  1981.     BioScience  31:  42-46. 
Turner,  R.  E.  1978.     l.imiwl.  Oecanogr.  23:  442-451. 
D'Klia,  C,  el  al.  1977.     Lininol  Oecanogr.  22:  760-764. 


Table  I 

Weight-specific  nitrogen  release  rales,  area  <>/  marsh  surrounding  the  esluarr.  nitrogen-loading  rale  to  estuary  (II'BLAIER  unpuh.  data),  and  area- 
specific  nitrogen  release:  last  lm>  columns  com/Hire  foliar  release  to  loading  rates  from  watershed 


%  of  N  load 

released  to 

NH4  mean 

DON  mean 

Area  of 

N  loading  rate 

5.  (i/lenii/lora 

NH4 

DON 

estuary 

release  ±  SE 

release  ±  SE 

marsh 

(kg  N/ha/y) 

biomass 

released 

released 

Estuary 

(nmoles/g-h) 

(/imoles/g-h) 

(nr) 

from  watersheds 

(g/ITT) 

(kg)a 

(kg)a 

NH4        DON 

Jehu  Pond 

0.03  ±  0.018 

0.02  ±  0.010 

1.2  x  105 

2606 

892 

231 

154 

9              6 

Humblin 

Pond 

0.0  1  ±  0.003 

0.03  ±  0.004 

9.5  x  10" 

1615 

445 

30 

91 

2              6 

Sage  Lot  Pond 

0.03  ±  0.009 

0.03  ±  0.012 

1.4  '.  105 

259 

307 

93 

93 

36            36 

a  During  the  entiie  growing  season,  while  there  was  significant  hiomass  of  grass  present. 


CONTENTS 


RESEARCH  NOTES 


Renninger,  G.  H.,  L.  Kass,  R.  A.  Gleeson,  C.  L.  Van 
Dover,  B.-A.  Battelle,  R.  N.  Jinks,  E.  D.  Herzog,  and 
S.  C.  Chamberlain 

Sulfide  as  a  chemical  stimulus  for  deep-sea  hydro- 
thermal  vent  shrimp     69 

Rodhouse,  Paul  G.,  and  Martin  G.  White 

Cephalopods  occupy  the  ecological  niche  of  epipe- 
lagic  fish  in  the  Antarctic  polar  frontal  zone    ....         77 


ECOLOGY  AND  EVOLUTION 


Katayama,  Tomoe,  Hiroshi  Wada,  Hidetaka  Furuya, 
Noriyuki  Satoh,  and  Masamichi  Yamamoto 

Phylogenetic  position  of  the  dicyemid  mesozoa  in- 
ferred from  18S  rDNA  sequences 81 

Kelly,  Maeve  S.,  M.  F.  Barker,  J.  Douglas  McKenzie, 

and  Jan  Powell 

The  incidence  and  morphology  of  subcuticular 
bacteria  in  the  echinoderm  fauna  of  New  Zealand  9 1 

Pancer,  Zeev,  Harriet  Gershon,  and  Baruch  Rin- 

kevich 

Coexistence  and  possible  parasitism  of  somatic  and 
germ  cell  lines  in  chimeras  of  the  colonial  urochor- 
date  Bntn'llm  srhlosseri  106 

Voight,  Janet 

Sexual  dimorphism  and  niche  divergence  in  a  mid- 
water  octopod  (Cephalopoda:  Bolitaenidae)  ....  113 


BEHAVIOR 

fillers,  Olaf 

Behavioral  control  of  swash-riding  in  the  clam  Donax 
variahilis 120 

Ellers,  Olaf 

Discrimination  among  wave-generated  sounds  by  a 
swash-riding  clam  128 

Ellers,  Olaf 

Form  and  motion  of  Donax  rariribilis  in  flow    ....       138 

NEUROBIOLOGY 

Niida,  Akiyoshi,  Yoshiko  Takatsuki,  and  Tsuneo 
Yamaguchi 

Morphology  and  physiology  of  the  thoracic  and  ab- 
dominal stretch  receptors  of  the  isopod  crustacean 
Ligui  exotica  1 48 

PHYSIOLOGY 

Jaeckle,  William  B. 

Transport  and  metabolism  of  alanine  and  palmitic 
acid  by  field-collected  larvae  of  Tedania  ignis  (Pori- 
fera,  Demospongiae):  estimated  consequences  of 
limited  label  translocation  159 

Rosas,  Carlos,  Andrea  Bolongaro-Crevenna,  Adolfo 

Sanchez,   Gabriela   Gaxiola,   Luis  Soto,   and   Elva 

Escobar 

Role  of  digestive  gland  in  the  energetic  metabolism 

of  Penaeus  setiferus    168 

Saigusa,  Masayuki 

Bioassay  and  preliminary  characterization  of  ovi- 
gerous-hair  stripping  substance  (OHSS)  in  hatch 
water  of  crab  larvae 175 

Short  Reports  from  the  Marine  Biological  Labora- 
tory's General  Scientific  Meetings     185 


Volume  189 


THE 


Number  3 


BIOLOGICAL 
BULLETIN 


DECEMBER,  1995 


Published  by  the  Marine  Biological  Laboratory 


THE 


BIOLOGICAL  BULLETIN 


PUBLISHED  BY 
THE  MARINE  BIOLOGICAL  LABORATORY 


Associate  Editors 


1  A 


PETER  A.  V.  ANDERSON,  The  Whitney  Laboratory,  University  of  Florida 

WILLIAM  D.  COHEN,  Hunter  College,  City  University  of  New  York 

DAVID  EPEL,  Hopkins  Marine  Station,  Stanford  University 

J.  MALCOLM  SHICK,  University  of  Maine.  Orono 


Editorial  Board 


PETER  B.  ARMSTRONG,  University  of  California,  Davis 
THOMAS  H.  DIETZ,  Louisiana  State  University 
DAPHNE  GAIL  FAUTIN,  University  of  Kansas 

Wn  LIAM  F.  GILLY,  Hopkins  Marine  Station.  Stanford 

University 

ROGER  T.  HANLON,  Marine  Biological  Laboratory 
MICHAEL  LABARBERA.  University  of  Chicago 
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BARUCH     RINKEVICH,     Israel    Oceanographic    & 
Limnological  Research  Ltd. 

RICHARD  STRATHMANN.  Friday  Harbor  Laboratories. 
University  of  Washington 

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SARAH  ANN  WOODIN,  University  of  South  Carolina 

RICHARD   K.   ZIMMER-FALIST,    University   of  South 

Carolina 


Editor:  MICHAEL  J.  GREENBERG.  The  Whitney  Laboratory,  University  of  Florida 
.\tana.vnif;  i.dilur  PAMELA  L.  CLAPP.  Marine  Biological  Laboratory 


DECEMBER,  1995 


Printed  and  Issued  by 
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CONTENTS 


No.  1,  AUGUST  1995 


HISTORICAL  REVIEW 


ECOLOGY  AND  EVOLUTION 


Shimomura,  Osamu 

A  short  story  of  aequorin 


DEVELOPMENT  AND  REPRODUCTION 

Morisawa,  Sachiko 

Fine  structure  of  spermatozoa  of  the  hagfish  EpUt- 

tretus  burgeri  (Agnatha)    6 

Glas,  Patricia  S.,  Jeffrey  D.  Green,  and  John  W.  Lynn 
Oxidase  activity  associated  with  the  elevation  of  the 
penaeoid  shrimp  hatching  envelope  13 

PHYSIOLOGY 

Scholnick,  David  A. 

Sensitivity  of  metabolic  rate,  growth,  and  fecundity 
of  tadpole  shrimp  Tiinj^  longicaudatus  to  environ- 
mental variation  22 

IMMUNOLOGY 

Hirose,  Euichi,  and  Teruhisa  Ishii 

Microfilament  contraction  promotes  rounding  of 
tunic  slides:  an  integumentary  defense  system  in  the 
colonial  ascidian  Aplidiiim  \iinnr./i  29 


Chadwick-Furman,    Nanette    E.,    and    Irving    L. 
Weissman 

Life  histories  and  senescence  of  Botryllus  schlosseri 
(Chordata,  Ascidiacea)  in  Monterey  Bay    36 

Hairston,  Nelson  G.,  Jr.,  and  Colleen  M.  Kearns 
The  interaction  of  photoperiod  and  temperature  in 
diapause  timing:  a  copepod  example    42 

Woodin,  Sarah  A.,  Sara  M.  Lindsay,  and  David  S. 

Wethey 

Process-specific  recruitment  cues  in  marine  sedi- 
mentarv  systems  49 


FUNCTIONAL  MORPHOLOGY 

Carefoot,  Thomas  H.,  and  Deborah  A.  Donovan 

Functional  significance  of  varices  in  the  muricid 

gastropod  Ci'ml/ntmim  f/iliiitiim    59 

Annual  Report  of  the  Marine  Biological  Laboratory       Rl 


No.  2,  OCTOBER/NOVEMBFR  1995 


RESEARCH  NOTES 

Renninger,  G.  H.,  L.  Kass,  R.  A.  Gleeson,  C.  L.  Van 
Dover,  B.-A.  Battelle,  R.  N.  Jinks,  E.  D.  Herzog,  and 
S.  C.  Chamberlain 

Sulfide  as  a  chemical  stimulus  for  deep-sea  hydro- 
thermal  vent  shrimp    69 

Rodhouse,  Paul  G.,  and  Martin  G.  White 

Cephalopods  occupy  the  ecological  niche  of  epipe- 
lagic  fish  in  the  Antarctic  polar  frontal  zone    ....         77 


ECOLOGY  AND  EVOLUTION 

Katayama,  Tomoe,  Hiroshi  Wada,  Hidetaka  Furuya, 
Noriyuki  Satoh,  and  Masamichi  Yamamoto 

1'hvlogenetk  position  of  the  dkyemid  mesozoa  in- 
ferred from  18S  rDNA  sequences SI 


Kelly,  Maeve  S.,  M.  F.  Barker,  J.  Douglas  McKenzie, 
and  Jan  Powell 

The   incidence   and   morphology   of  subcuticular 
bacteria  in  the  echmoderrn  fauna  of  New  Zealand         91 

Pancer,  Zeev,  Harriet  Gershon,  and  Baruch  Rin- 

kevich 

Coexistence  and  possible  parasitism  of  somatic  and 
germ  cell  lines  in  chimeras  of  the  colonial  urochor- 

date  Bntr\Iln.'i  .«7i/o.wn    106 

Voight,  Janet 

Sexual  dimorphism  and  niche  divergence  in  a  mid- 
water  octopod  (Cephalopoda:  Bolitaenidae)     ....       113 

BEHAVIOR 

Filers,  Olaf 

Behavioral  control  of  swash-riding  in  the  clam  Duniix 
I'tiniihili*  .  120 


CONTENTS 


Ellers,  Olaf 

Disci  iinm.Hion  among  wave-generated  sounds  l>\  .1 

swash-riding  dam     1  'JcS 

Ellers,  Olaf 

Form  and  motion  of  Di»iu\  vnriabili*  in  How  138 


NEUROBIOLOGY 

Niida,  Akiyoshi,  Yoshiko  Takatsuki,  and  Tsunco 

Yamaguchi 

Morphology  and  plivsiologv  of  the  thoracic  and  ab- 
dominal stretch  receptors  of  the  isopod  crustacean 


Rosas,  Carlos,  Andrea  Bolongaro-Crevenna,  Adolfo 
Sanchez,  Gabriela  Gaxiola,  Luis  Soto,  and  Elva 
Escobar 

Role  of  digestive  gland  in  the  energetic  metabolism 

of  Penaeus  wti/rriH    168 

Saigusa,  Masayuki 

Bioassay  and  preliminary  characterization  of  ovi- 
gerous-liair  stripping  substance  (OHSS)  in  hatch 
water  of  crab  larvae 175 

Short  Reports  from  the  Marine  Biological  Labora- 
tory's General  Scientific  Meetings  185 


148 


PHYSIOLOGY 

Jaeckle,  William  B. 

Transport  and  metabolism  of  alanine  and  palmitic 
acid  by  field-collected  larvae  of  Ti'd/iniii  ignis  (Poi  i- 
fera,  Demospongiae):  estimated  consequences  of 
limited  label  ti anslocation  .  159 


No.  3,  DECEMBKR  1995 


NEUROBIOLOGY  AND  BEHAVIOR 

Fleischer,  KellieJ.,  and  James  F.  Case 

Cephalopod  predation  facilitated  by  dinoflagellate 
luminescence 263 

Rodriguez,  Sebastian  R.,  Carlos  Riquelme,  Eliseo  O. 

Campos,  Pamela  Chavez,  Enrique  Brandan,  and  Ni- 

baldo  C.  Inestrosa 

Behavioral  responses  of  Cotifluilcpas  itni(h<ili'jni\ 
(Bruguiere,  1789)  larvae  to  natural  and  artificial 
settlement  cues  and  microbial  films 272 

Westfall,  Jane  A.,  Kelley  L.  Sayyar,  Carol  F.  Elliott, 

and  Cornells  J.  P.  Grimmelikhuijzen 

Ultrastructural  localization  of  Antho-RWamides  I 
and  11  at  neuromuscular  synapses  in  the  gastro- 
dermisand  oral  sphincter  muscle  of  the  sea  anemone 
Calli(irli<i  fmraaiticn  280 

PHYSIOLOGY 

Dove,   Sophie   G.,   Misaki   Takabayashi,   and   Ove 
Hoegh-Guldberg 

Isolation  and  partial  characterization  of  the-  pink 
and  blue  pigments  of  pocilloporid  and  acroporid 

corals 288 

Fitt,  W.  K.,  and  M.  E.  Warner 

Bleaching  patterns  of  four  species  of  Caribbean  reef 
corals  .  .  298 


Silverman,  H.,  E.  C.  Achberger,  J.  W.  Lynn,  and 
T.  H.  Dietz 

Filtration  and  utili/ation  of  laboratory-cultured 
bacteria  by  Dreiaacttn  polymorpha,  C<n~l>n  ula  fluminea. 
and  Carunculinn  ti'\<i\i:>i.\L\  308 

DEVELOPMENT  AND  REPRODUCTION 

Fong,  Peter  P.,  Keiichiro  Kyozuka,  Jill  Duncan, 
Stacy  Rynkowski,  Daniel  Mekasha,  and  Jeffrey  L. 
Ram 

The  effect  of  salinity  and  temperature  on  spawning 
and  fertilization  in  the  zebra  mussel  Dreisseiw  pol\- 
iiinr/iliii  (Pallas)  from  North  America  320 

Togo,  Tatsuru,  Kenzi  Osanai,  and  Masaaki  Mori- 

sawa 

Existence  of  three  mechanisms  for  blocking  poly- 
spermy  in  oocytes  of  the  mussel  MMilu*  edulis  .  .  .  330 

Sarojini,   Rachakonda,    Rachakonda    Nagabhusha- 

M. mi.  and  Milton  Fingerman 

In  I'ii'ii  effects  of  dopamine  and  dopaminergic  an- 
tagonists on  testicular  maturation  in  the  red  swamp 
crayfish.  Pnntiiiilxini-,  iltirkii  340 

SYMBIOSIS 

Doino,  Judith  A.,  and  Margaret  J.  McFall-Ngai 

A  transient  exposure  to  symbiosis-competent  bac- 
teria induces  light  organ  morphogenesis  in  the  host 
squid  347 


CONTENTS 
ECOLOGY  AND  EVOLUTION  CELL  BIOLOGY 

Haddock,  Steven  H.  D.,  and  James  F.  Case  Sequeira,    Teresa,    Manuel    Vilanova,    Alexandre 

Not  all  ctenophores  are  bioluminescent:  Pli'itrolirti-  Lobo-da-Cunha,   Luis   Baldaia,   and   Mario   Arala- 

fliui     356         Chaves 

Ilan,  Micha,  and  Avigdor  Abelson  Flow  cytometric  analysis  of  molt-related  changes  in 

The  life  of  a  sponge  in  a  sandy  lagoon     363  hemocyte  type  in  male  and  female  Penaeus  japtmicus      376 

Inoue,  Koji,  J.  Herbert  Waite,  Makoto  Matsuoka, 

Satoshi  Odo,  and  Shigeaki  Harayama  Index  for  Volume  189    381 

Interspecific  variations  in  adhesive  protein  se- 
quences of  i\l\tilu\  IV/H/M.  M.  gallopravincialis,  and 
M.  trmsulii'i  37(1 


Reference:  Biol.  Bull  189:  263-271.  (December.  1995) 


Cephalopod  Predation  Facilitated  by 
Dinoflagellate  Luminescence 

KELLIE  J.  FLEISHER  AND  JAMES  F.  CASE* 

Marine  Science  Institute.  University  of  California  at  Santa  Barbara, 
Santa  Barbara,  California  93106 


Abstract.  Predation  by  nocturnal  cephalopods  on  non- 
luminous  prey  was  examined  in  the  presence  of  dinofla- 
gellate  bioluminescence.  Sepia  qfficinalis  Linnaeus  and 
Euprymna  scolopes  Berry  were  tested  for  predation  effi- 
ciency in  darkness  illuminated  by  the  luminescent  dino- 
flagellate  Pyrocystis fusiformis  Murry.  Prey  were  rnysids, 
Holmesimysis  sculpta  (Tattersall);  grass  shrimp,  Palae- 
monetes  pugio  Holthuis;  and  mosquito  fish,  Gambusia 
qffinis  Baird  and  Girard.  Tests  were  conducted  in  aquaria 
containing  0-20  cells  ml"1  of  P.  fusiformis.  Predation  in- 
creased as  numbers  of  luminescent  dinoflagellates  in- 
creased. Controls  were  predation  tests  in  the  presence  of 
P.  fusiformis  during  nonluminescent  photophase  or  in 
the  absence  of  dinoflagellates.  Movements  of  squid  and 
prey  readily  stimulated  luminescence.  Behavior  and  cor- 
related luminescence  in  infrared-illuminated  aquaria  were 
recorded  by  image-intensified  and  infrared  video  cameras. 
Sepia  strikes  on  prey  were  common  under  luminescent 
conditions — 85%  occurred  in  less  than  10  min;  but  strikes 
in  darkness  were  rare.  E.  scolopes  attacked  more  fre- 
quently than  Sepia,  and  almost  90%  obtained  prey  under 
luminescent  conditions.  This  study  demonstrates  the 
ability  of  squid  to  use  dinoflagellate  bioluminescence  to 
locate  and  capture  nonluminous  prey.  The  burglar  alarm 
theory  of  the  adaptive  significance  of  dinoflagellate  bio- 
luminescence is  supported. 

Introduction 

At  least  20  functions  of  bioluminescence  have  been 
advanced  (Tett  and  Kelly,  1973;  Buck,  1978).  One  of 
these,  the  burglar  alarm  theory,  holds  that  light  produced 
by  luminescent  prey  upon  attack  by  a  predator  might  at- 

Received  27  April  1995;  accepted  21  September  1995. 
'Author  to  whom  correspondence  should  be  addressed. 


tract  its  own  predators,  thereby  reducing  predation  pres- 
sure on  the  bioluminescent  organism.  The  result  would 
be  of  little  use  to  the  prey  unless  it  survived  the  attack, 
for  which  there  is  some  experimental  evidence  in  dino- 
flagellates (Buskey  et  a!.,  1985).  However,  even  with  prey 
mortality,  benefit  could  accrue  to  the  species  as  a  whole 
by  such  a  process.  This  is  particularly  true  in  dinoflagel- 
lates, which  tend  to  exist  in  localized  clones,  so  that  the 
sacrifice  of  some  members  of  the  clone  would  directly 
favor  survival  of  the  luminescent  genotype  (Burkenroad, 
1943).  The  theory  is  supported  by  demonstration  that  or- 
ganisms apt  to  graze  on  luminescent  dinoflagellates  are 
induced  by  luminescence  to  undertake  evasive  behavior 
that  would  tend  to  reduce  grazing  (Esaias  and  Curl,  1972; 
White,  1979;  Buskey  and  Swift,  1983).  Until  recently, 
however,  there  has  been  little  evidence  for  the  second  crit- 
ical element  of  the  theory,  namely  that  higher  level  pred- 
ators are  able  to  hunt  animals  efficiently  by  the  light  these 
latter  trigger  from  bioluminescent  organisms,  either  by 
feeding  on  or  by  moving  among  them. 

Mensinger  and  Case  (1992)  showed  that  juvenile  mid- 
shipman fish.  Porichthys  not  at  us  Girard.  midwater  am- 
bush predators,  feed  efficiently  on  nonluminescent  prey 
by  dinoflagellate  light.  Here  we  extend  these  observations 
to  the  Cephalopoda,  predators  with  superb  vision  (Young, 
1991)  and  remarkably  developed  hunting  behavior. 
Demonstration  that  these  invertebrate  predators  are  able 
to  hunt  effectively  with  the  aid  of  bioluminescence 
strongly  reinforces  the  burglar  alarm  theory.  The  work 
also  has  implications  for  interpretation  of  the  role  of 
luminescence  in  the  population  dynamics  of  marine 
organisms. 

As  predators  we  used  Euprymna  scolopes  Berry,  a  shal- 
low benthic  squid  indigenous  to  the  Hawaiian  archipelago 
(Singley,  1983).  and  Sepia  officinalis  Linnaeus,  a  benthic- 
to-midwater  cuttlefish  found  in  the  Eastern  Atlantic  Ocean 


263 


264 


K.  J.  FLE1SHER  AND  J.  F.  CASE 


and  the  Mediterranean  Sea  (Boletzky,  1983).  E.  scolopes 
tends  to  approximate  the  ambush  attack  of  the  midship- 
man fish,  hut  from  a  position  on  the  bottom.  S.  officinalis 
differs  markedly  from  the  midshipman  fish  in  hunting 
behavior  by  roving  actively  in  the  midwaters. 

E.  scolopes  adults  eat  primarily  mysid  shrimp;  in 
aquaria,  the  young  also  take  Anemia  (Singley,  1983). 
Members  of  this  species  are  active  only  at  night,  when 
they  are  able  to  produce  bioluminescence  from  a  light 
organ  populated  by  luminescent  bacteria  (Singley,  1983; 
McFall-Ngai  and  Montgomery,  1990).  They  camouflage 
themselves  in  the  sand  during  daylight.  A  feeding  strategy 
consisting  of  approach,  tracking,  and  capture  phases, 
similar  to  that  of  Sepia,  has  been  reported  in  other  squid 
(Foyle  and  O'Dor,  1988).  However,  our  laboratory  ob- 
servations show  that  E.  scolopes  actually  tends  to  wait  for 
the  approach  of  prey. 

S.  officinalis  adults  are  roving  nocturnal  predators  that 
feed  on  a  variety  of  prey  including  small  crustaceans,  fish, 
or  even  smaller  Sepia  (Boletzky.  1983).  The  young  eat 
mainly  small  crustaceans.  The  day  is  spent  in  the  sand 
and  they  rise  into  the  water  column  at  night  to  hunt, 
aided  by  a  diurnal  cycle  of  buoyancy  change  (Denton  and 
Gilpin-Brown,  1961).  Their  vision  is  excellent  and  they 
use  both  binocular  and  monocular  fixation  to  locate  prey 
(Messenger,  1968).  Attack  is  by  one  of  two  strategies,  de- 
pending on  prey  size  and  potential  risk  to  the  attacker: 
(1)  rapid  extension  of  the  two  prehensile  tentacles,  or  (2) 
envelopment  of  the  prey  (Duval  el  a/.,  1984).  The  tentacle 
extension  process  has  three  phases — attention,  position- 
ing, and  seizure.  The  first  two  are  visually  controlled, 
whereas  the  last  is  so  rapid  that  there  is  no  time  for  visual 
feedback.  Accuracy  consequently  depends  on  reducing 
the  visual  error  to  near  zero  (Messenger,  1968). 

Materials  and  Methods 

Co/lection  and  maintenance  of  experimental  animals 

Juvenile  and  adult  Eitprymna  scolopes  were  generously 
provided  by  Professor  M.  McFall-Ngai,  who  periodically 
collected  specimens  from  Kaneohe  and  Niu  Bays  on  the 
coast  of  Oahu,  Hawaii.  Animals  were  kept  in  a  40-gallon 
aquarium  with  single-pass,  heated  seawater  (20°  — 24°C) 
and  a  1.0-cm-deep  sand  bottom.  Experimental  animals 
were  kept  on  a  12;  12  light-dark  (LD)  cycle,  the  same  LD 
cycle  as  the  rest  of  the  animals  in  this  study.  Food  consisted 
of  brackish-water  grass  shrimp  (Palaemonetes  pugio  Hol- 
thuis).  All  experiments  reported  here  were  done  with 
adults. 

Juvenile  cuttlefish.  Sepia  officinalis.  were  purchased 
from  the  University  of  Texas  Marine  Biomedical  Institute, 
Galveston,  Texas  (Boletzky  and  Hanlon.  1983;  DeRusha 
et  at..  1989).  They  were  kept  in  60-gallon  aquaria  with 
single-pass  seawater  ( 14°  -  18°C)  and  2.5-to-3.8-cm-deep 


sand  bottoms.  All  animals  in  this  study  were  maintained 
on  the  same  12:12  LD  cycle.  Mortality  was  low,  with  good 
survival  to  reproductive  age.  Animals  used  in  these  ex- 
periments were  about  2  months  old  and  averaged  25  mm 
in  length.  Food  consisted  of  kelp-canopy  mysids  (Hol- 
mesimysis  sculpta  [Tattersall]);  top  smelt  (Atharinops  af- 
fmis  Aries),  both  live  and  frozen);  striped  shore  crabs 
(Pachygrapsus  crassipes  Randall);  and  mosquito  fish 
(Gambusia  affinis  Baird  and  Girard).  Prey  varied  accord- 
ing to  cuttlefish  size  and  food  requirements. 

The  various  food  and  prey  animals  were  obtained  and 
handled  as  follows.  Mysids  were  collected  weekly  by  dip 
netting  from  kelp  canopies  along  the  Santa  Barbara  coast; 
maintained  in  aerated,  free-flowing  aquaria;  and  used 
within  10  days  of  capture.  Mosquito  fish  were  obtained 
every  2  weeks  from  a  local  aquarium  store;  fed  daily;  and 
maintained  in  a  50-gallon  aerated,  fresh-water  tank.  Grass 
shrimp  were  obtained  periodically  from  a  local  supplier; 
maintained  in  brackish  water  at  room  temperature;  fed 
weekly;  and  used  within  15  days.  All  prey  animals  ap- 
peared to  remain  in  excellent  condition  during  the  spec- 
ified holding  periods. 

Dinoflagellate  culture  ami  luminescence  cycle 

Unialgal  cultures  of  the  dinoflagellate  Pyrocystis  fusi- 
formis  Murry  were  originally  supplied  by  the  late  B.  M. 
Sweeney  and  maintained  using  the  techniques  of  Widder 
and  Case  ( 1982).  Cells  were  maintained  on  the  same  12: 
12  LD  cycle  as  the  squid  at  between  18°  and  20°C.  in 
sterilized  filtered  seawater  enriched  with  f/2  formula 
(Guillard  and  Ryther.  1962)  and  soil  extract,  omitting 
silicate.  During  the  day-phase,  cells  were  illuminated  from 
above  with  cool-white  fluorescent  bulbs  at  500  /uW  cm"2 
as  measured  by  a  United  Detector  Technology  Model  40x 
photometer.  Two  populations  were  maintained  on  op- 
posite LD  cycles  for  simultaneous  use  of  day-  and  night- 
phase  cells.  On  experimental  days,  cell  concentrations 
were  determined  with  a  cell-counting  chamber  (Hausser). 
Under  these  conditions,  maximum  scotophase  biolumi- 
nescence intensity  was  10"'  photons -cell  '  s  '. 

Optimal  controls  for  this  study  would  involve  use  of 
completely  nonluminescent  photophase  dinoflagellates. 
However,  although  the  cells  used  in  control  experiments 
were  at  least  3  h  into  photophase,  as  soon  as  they  were 
placed  in  darkness  at  the  beginning  of  the  experiment 
they  rapidly  recovered  enough  luminescent  capacity  to 
aid  vision  of  the  squid.  To  assess  the  magnitude  of  recovery 
as  a  function  of  time  in  the  dark,  tests  were  conducted  to 
quantify  mechanically  excitable  bioluminescence.  Cells 
used  for  this  test  were  at  least  5  h  into  scotophase.  Quan- 
tum emission  was  measured  in  a  10-in-diameter  inte- 
grating sphere  collector  (Labsphere,  Inc.),  with  an  RCA 
model  8850  photon-counting  photomultiplier  operating 


D1NOFLAGELLATE-AIDED  PREDATION 


265 


IR  PASS  FILTER 


IR  BLOCKER 


ISIT  =  BL 


IR  =  PREDATOR 
PREY 

LOCATION 


MIXER  RECORDER 


Figure  1.     Split-screen  video  camera  arrangement  for  monitoring 
predator/prey  interactions. 


at  -1680  V  (Latz  et  a!..  1987).  Cell  samples  were  stim- 
ulated to  exhaustion  with  a  stirring  rod  consisting  of  a 
stainless  steel  shaft  with  three  cross  tines,  coupled  to  a 
DC  motor  (Latz  el  a/.,  1 990)  operating  at  a  standard  speed. 
Motor  speed  was  measured  with  a  magnetic  pick-up 
mounted  on  the  motor  shaft  and  displayed  on  a  Visi-tach 
digital  ratemeter.  Light  emission  was  monitored  for 
250  ms  by  ACE-MCS  software  operating  with  a  channel 
dwell  time  of  5  ms.  Previously  unstimulated  cells  were 
run  every  1 5  min  for  3  h. 

Quantitative  predation  experiments  with  Sepia 
officinalis 

Twelve  tests  with  single  animals  in  12-liter  glass  tanks 
were  run  concurrently  in  a  darkroom.  Six  were  controls, 
either  with  dinoflagellates  absent  or  in  photophase;  and 
six  were  experimental  tanks  with  dinoflagellates  in  sco- 
tophase.  Tanks  were  separated  by  opaque  dividers.  Water 
temperature  was  maintained  at  15°C.  S.  officinalis  (av- 
erage mantle  length  =  23.3  mm  ±  0.39;  n  =  50)  were 
placed  in  individual  tanks  no  later  than  1  h  before  onset 
of  the  dark  cycle  to  allow  recovery  after  transfer.  Dino- 
flagellates in  final  concentrations  of  1.  2,  5.  10,  15.  and 
20  cells/ml  were  added  2  h  after  onset  of  scotophase.  Be- 
cause cells  tend  to  settle  over  time,  concentrations  indi- 
cated are  for  initial  conditions.  With  care  taken  to  min- 
imize bioluminescence.  10  mysids  (carapace  length: 
1.9mm  to  3.6mm)  were  added  simultaneously  to  all 
tanks.  Preliminary  experiments  of  up  to  6  h  were  con- 
ducted to  determine  optimal  time  span  and  prey  density. 
Results  showed  that  Sepia  of  the  ages  used  (2  to  4  months) 
were  satiated  after  3  h  and  never  consumed  more  than 
10  mysids  during  that  time.  To  minimize  disturbance  and 
maintain  dark  adaptation,  the  Sepia  were  handled  with 


the  aid  of  an  IR-light  and  IR-image  converter.  At  the  end 
of  an  experiment,  the  surviving  mysids  were  counted  after 
the  cephalopods  had  been  returned  to  their  home  tanks. 
Experimental  tanks  were  emptied  and  the  sand  was 
washed  free  of  dinoflagellates  every  night  and  refilled  with 
filtered  seawater  the  next  morning.  The  laboratory  filtering 
system  ensured  that  the  seawater  was  free  of  other  visibly 
bioluminescent  organisms. 

Predator/prey  interactions 

Behavior  of  S.  officinalis  and  E.  scolopes  was  monitored 
with  DAGE  MTI  image-intensified  (ISIT-66LX)  and  in- 
frared (IR)  (SC-66LX)  video  cameras  during  predator/ 
prey  interactions.  The  aquarium  was  illuminated  from 
above  by  a  25-W  incandescent  lamp  screened  by  a  Kodak 
IR  filter  (Wratten  No.  87),  eliminating  wavelengths  shorter 
than  700  nm.  A  Panasonic  special-effects  generator  (WJ- 
4600 A)  produced  a  horizontal  split-screen  image  of  the 
aquarium.  Half  of  the  screen  displayed  the  animals  as 
viewed  under  IR  light,  and  the  other  half  displayed  di- 
noflagellate  luminescence  as  viewed  by  the  ISIT.  The  ISIT 
was  fitted  with  a  red-absorbing  blue-green  glass  filter 
(Melles-Griot  BG  18)  to  block  wavelengths  longer  than 
650  nm.  Data  were  stored  on  a  Sony  Hi-8  EV  C100  video 
recorder  and  transferred  to  a  Power  Macintosh  8100/80 
AV  computer  for  detailed  analysis.  The  experimental  ar- 
rangements are  shown  in  Figure  1 . 

In  work  with  5.  officinalis,  2  h  after  onset  of  the  di- 
noflagellate  scotophase  a  single  cuttlefish  was  placed  in  a 
10-1  aquarium  containing  40  cells  mL1  of  dinoflagellates. 
Tank  size  was  determined  by  limitations  of  camera  res- 
olution. A  single  mosquito  fish  (length  =  23-36.4  mm) 


Photophase  (non-luminescent) 
Scotophase  (luminescent) 


TIME  (min) 

Figure  2.  Luminescence  produced  by  photophase  dinoflagellates 
(Pyrocystis  fusiformis)  over  a  3-h  period  after  transfer  to  darkness  as 
compared  with  cells  in  scotophase.  Error  bars  represent  standard  errors. 


266 


K.  J.  FLEISHER  AND  J.  F.  CASE 


was  added  1 5  mm  later.  Optimal  prey  size  was  determined 
by  the  aggressiveness  of  Sepia,  which  ignored  small  targets. 
Events  were  monitored  for  a  subsequent  30  min  with  the 
ISIT/IR  video  recording  system.  Four  trials  were  con- 
ducted on  a  given  day,  for  a  total  number  of  20  runs  over 
a  2-month  period. 

With  E.  scolopes,  procedures  differed  slightly  owing  to 
its  smaller  size.  An  hour  prior  to  the  night  cycle,  each  test 
animal  (average  mantle  length  =  14.15  ±  .34  mm;  n=  10) 
was  moved  to  an  individual  3.5-1  experimental  aquarium 
and  allowed  to  acclimate  for  3  h.  Tank  size  was  small  to 
ensure  that  strikes  could  be  recorded  with  high  resolution. 
Each  tank  was  aerated  and  kept  at  the  same  temperature 
as  the  holding  aquarium  (~23°C).  Dinoflagellates  in  final 
concentrations  of  0,  5,  10,  20,  and  40  cells/ml  were  added 
slowly  from  a  wide-mouth  container  into  each  tank  to 
minimize  premature  stimulation.  A  single  grass  shrimp 
(carapace  length  =  8.2 — 1 1 .7  mm)  was  added  1 5  min  after 
the  dinotlagellates  to  allow  calming  time  for  the  squid. 
E.  scolopes  are  significantly  harder  to  feed  in  captivity 
than  Sepia.  The  prey  chosen  for  this  experiment  was  both 
familiar  to  them  and  large  enough  to  attract  their  atten- 
tion. Monitoring  continued  for  a  subsequent  30  min. 
Trials  (n  =  5)  were  conducted  daily,  for  a  total  of  90  runs 
over  a  3-month  period.  Interactions  of  predators  and  prey 
were  monitored  and  analyzed  with  the  same  split-screen 
apparatus  used  for  Sepia  (Fig.  1 ). 

Results 

Dinoflagellate  luminescence  recovery  upon  light  to  dark 
transfer 

P.  fusiformis  in  photophase  proved  difficult  to  use  as  a 
control  because  cells  became  luminescent  relatively 
quickly  after  being  placed  in  the  dark.  A  similar  phenom- 
enon has  been  observed  in  Pyrodinium  baharnense  (Bigg- 
ley  et  ai.  1969)  and  Pyrocystis  hmula  (Co\zv\co\o,  1992). 
Our  results  showed  increasing  luminescence  with  passage 
of  time  in  darkness  (Fig.  2).  After  3  h  in  darkness  the  light 
produced  by  20  cells/ml  of  photophase  P  fusiformis  is 
comparable  to  that  produced  by  1  cell/ml  in  full  scoto- 
phase  (Fig.  2).  This  intensity  is  sufficient  to  improve  the 
feeding  accuracy  of  Sepia.  Therefore,  to  ensure  complete 
darkness,  subsequent  controls  in  our  experiments  con- 
tained no  dinoflagellates.  This  would  appear  reasonable 
because  no  adverse  effects  on  the  squid  or  prey  were  ever 
seen  for  the  concentrations  used;  mortality  was  quite  low 
for  both  species  of  cephalopods  over  the  19-month  ex- 
perimental period. 

Predation  experiments 

These  experiments  were  conducted  exclusively  on  5. 
officinalis.  After  an  acclimation  time  of  3  h,  all  animals 


I I        Riotophase 
Scotophase 


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0) 

3 
U> 
C 
O 

o 


o 
£ 

o 

3 


I 


Dinoflagellate  Concentration  (cells/ml) 

Figure  3.  Average  number  of  mysids  (Holmesimysis  costala)  con- 
sumed by  cuttlefish  (Sepia  officinalis)  as  a  function  of  concentration  of 
scotophase  and  photophase  dinoflagellates  (Pyrocystis  fiisiformis).  Pho- 
tophase cells  become  luminescent  as  time  in  darkness  progresses  (see 
text).  Error  bars  represent  standard  errors. 


were  behaving  normally,  hovering  above  the  sand  and 
feeding.  Tanks  containing  P.  fusiformis.  both  scotophase 
(test)  and  photophase  (control),  had  fewer  mysids  present 
at  the  end  of  the  experiment  than  did  tanks  without  di- 
noflagellates. In  control  tanks  containing  photophase  di- 
noflagellates, the  average  number  of  mysids  consumed 
was  from  0.88  to  4.75,  increasing  with  dinoflagellate  con- 
centration (Student's  /-test;  no  significant  difference  at  any 
concentration,  P  >  0.078  -  0.662;  Fig.  3).  This  effect  is 
attributed  to  recovery  of  luminescent  capacity  in  the 
course  of  the  experiment.  Confirmation  comes  from  the 
fact  that  the  average  number  of  mysids  consumed  in  tanks 
containing  no  dinoflagellates  was  0.2  (Fig.  4).  By  contrast, 
in  the  tanks  containing  scotophase,  fully  luminescent  di- 
noflagellates, the  number  eaten  varied  from  4.2  to  8.0, 
increasing  with  dinoflagellate  concentration  ( ANOVA  and 
Dunnett  one-sided  test;  P  <  0.015;  Fig.  4).  Thus  predation 
of  cuttlefish  on  mysids  was  correlated  with  the  presence 
of  scotophase  dinoflagellates  (Pyrocystis  fusiformis).  Un- 
like the  situation  reported  for  the  midshipman  fish,  Por- 
ichtliys  notatus  (Mensinger  and  Case,  1992),  no  significant 
inhibition  of  predation  was  observed  at  high  dinoflagellate 
concentrations. 

Observations  of  predator-prey  interactions 

The  dual  camera  system  allowed  simultaneous  viewing 
of  predator-prey  interactions  and  the  resultant  lumines- 


DINOFI.AGELLATE-AIDED  PREDATION 


267 


c 

Q) 

15 

UJ 
W 
• 


O 

S 
S3 


<u 

> 


1  5 


20 


Dinoflagellate  Concentration  (cells/ml) 

Figure  4.  Average  number  of  mysids  (Holmesimysis  cosiata)  con- 
sumed by  cuttlefish  (Sepia  offidnalis)  as  a  function  of  concentration  of 
luminescent  dmoflagellates  (Pyrocystis  fusiformis).  Error  bars  represent 
standard  errors. 


cence.  No  behavioral  change  was  noted  between  organ- 
isms in  holding  or  experimental  tanks;  thus  it  was  assumed 
that  any  direct  effect  of  dinoflagellates  (exclusive  of  bio- 
luminescence)  at  all  concentrations  was  insufficient  to  bias 
the  experiments.  In  experimental  runs  the  dinoflagellate 
concentration  was  40  cells  ml"1.  Controls  for  this  exper- 
iment were  conducted  without  dinoflagellates. 

Strikes  by  Sepia  and  E.  scolopes  were  easily  discernible 
using  both  the  IR  and  ISIT  camera  (Figs.  5  and  6).  Details 
as  fine  as  eye  movements  tracking  the  luminescence  were 
visible  with  the  IR  camera.  Mosquito  fish  were  observed 
to  trigger  luminescence  with  each  tail  stroke,  which  Sepia 
monitored  closely.  Grass  shrimp  appendages  triggered 
ample  luminescence  to  attract  the  attention  of  E.  scolopes. 

Cuttlefish  strikes  were  all  or  none,  and  misses  were 
never  observed  in  a  total  of  20  attacks.  A  strike  or  other 
rapid  movement  elicited  a  large  cloud  of  luminescence 
that  was  easily  observed  with  the  ISIT  camera,  but  the 
normal  rise  and  hover  movements  of  Sepia  triggered  no 
luminescence.  Due  to  acclimation  time  (15  min).  Sepia 
feeding  behavior  was  not  affected  by  the  confines  of  the 
aquaria,  and  strikes  were  primarily  away  from  aquarium 
walls.  Mosquito  fish  appeared  to  swim  normally  under 
the  experimental  conditions.  Sixty-five  percent  of  the  Se- 
pia in  the  presence  of  scotophase  dinoflagellates  were  suc- 
cessful in  prey  capture,  whereas  only  5%  of  the  animals 
in  the  control  tank  (no  luminescence)  obtained  prey  (Chi- 
square  test;  P  <  0.000 1 ).  Eleven  individuals  in  the  presence 


of  luminescence  took  less  than  10  min  to  capture  prey, 
and  all  strikes  occurred  in  under  20  min.  In  the  control 
tanks,  only  one  strike  occurred  out  of  20  tests,  and  this 
occurred  after  almost  30  min  (Fig.  7). 

Messenger  (1968)  defined  the  attack  of  S.  offidnalis  as 
including  three  components:  attention,  positioning,  and 
seizure.  Attention,  the  interval  between  the  time  when 
the  prey  enters  the  field  of  view  and  when  the  cuttlefish 
and  prey  are  on  the  same  axis,  can  take  less  than  1  s  or  it 
may  last  for  up  to  10  s  (Messenger,  1968).  In  this  study, 
the  average  duration  of  attention  was  10.9  s  (SE  =  ±2;  n 
=  10).  Positioning,  which  begins  when  the  cuttlefish  faces 
the  prey  and  ends  with  the  strike,  can  last  from  less  than 
1  to  more  than  10  s  (Messenger,  1968).  During  our  ex- 
periments. Sepia  averaged  7.3  s  (SE  =  ±1.1;  n  =  10)  for 
this  component  of  the  attack  sequence.  The  final  act,  sei- 
zure, is  marked  by  the  extension  of  the  tentacles  and  ends 
with  the  prey  held  by  all  arms,  taking  about  2  s  (Messenger. 
1968).  Our  specimens  accomplished  this  in  an  average  of 
0.83  s(SE  =  ±0.05;  n  =  10). 

E.  scolopes  has  a  different  attack  mode.  Instead  of  the 
hover  and  strike  method  of  the  cuttlefish,  E.  scolopes  re- 
mains poised  on  the  bottom,  frequently  in  a  depression 
deliberately  made  by  blowing  sand  with  the  siphon,  where 
it  waits  for  prey  to  move  within  its  strike  zone.  The  size 
of  the  strike  zone  varies  with  each  animal  but  is  typically 
a  circle,  with  the  squid  at  its  center,  whose  radius  is  about 
twice  the  body  length  of  the  animal.  Once  a  target  is  in 
that  strike  zone,  the  squid  rapidly  turns,  points  all  arms 
in  the  direction  of  the  prey,  and  strikes  by  launching  its 
two  tentacles,  as  with  Sepia.  Our  video  analysis  shows  no 
evidence,  by  body  movement  or  other  sign,  of  the  atten- 
tion component  noted  in  the  cuttlefish.  The  actions  off. 
sco/opes  are  similar  to  those  of  an  ambush  predator,  going 
from  sedentary  directly  and  rapidly  to  positioning  and 
seizure.  Unlike  Sepia,  E.  sco/opes  does  not  adjust  its  dis- 
tance to  the  prey  during  positioning.  Were  it  not  for  the 
launching  of  the  tentacles,  positioning  and  seizure  by  this 
squid  would  be  considered  one  step.  The  average  time 
taken  by  E.  sco/opes  for  positioning  was  l.ls  (SE  =  ±0.09; 
n  =  10)  and  for  seizure.  0.63  s  (SE  =  ±0.03;  n  =  10). 
When  a  miss  occurred,  the  squid  did  not  pursue  the  prey 
and  continue  the  attack  immediately,  even  though  the 
prey's  luminescent  track  was  distinct.  All  movement  was 
easily  discernible  on  the  monitor  with  the  ISIT  camera, 
including  luminescence  induced  by  siphon  exhaust  as  the 
squid  excavated  a  resting  place  in  the  sand.  Motion  by 
the  grass  shrimp  prey,  both  "walking"  along  the  bottom 
and  swimming,  stimulated  dinoflagellate  luminescence. 
No  noticeable  attention  was  given  to  prey  outside  the 
strike  zone. 

There  was  a  significant  increase  in  frequency  of  pre- 
dation  in  aquaria  containing  luminescent  dinoflagellates 
(Fig.  8).  In  the  absence  of  luminescence,  E.  scolopes  struck 


268 


K.  J.  FLEISHER  AND  J.  F.  CASE 


Figure  5.  Split-screen  video  image  of  Sepia  qffidnalis  feeding  on  ghost  shnmp  (Palaemoneles  pugio). 
Image-intensifying  camera,  top  view.  Infrared  camera,  bottom  view.  (A)  Attention;  (B)  Positioning;  (C) 
Seizure.  Luminescence  is  produced  by  Pyrocystis  fusiformis  upon  being  stimulated  by  ghost  shrimp  move- 
ments. Bar  scale  =  2  cm. 


in  only  37%  of  the  total  tests  (Chi-square  test;  P  <  0.008). 
Under  luminescent  conditions  the  frequency  was  higher: 
79%  with  20  cells/ml  and  63%  at  a  concentration  of 
40  cells/ml.  Comparisons  made  between  concentrations 
showed  no  significant  differences  (Chi-square  test;  P 
=  0.76),  nor  did  a  comparison  of  strike  rale  among  all 
concentrations  (ANOVA  and  Dunnett  one-sided  test:  P 
=  0.46). 

Discussion 

Cephalopods  employ  many  sophisticated  sensory  or- 
gans during  prey  capture,  namely  eyes,  statocysts  (Bu- 
delmann,  1979),  and  lateral  line  analog  (Budelmann  el 
ul.,  1991).  Stimuli  that  induce  attacks  appear  to  be  pri- 
marily visual  since  prey  in  an  adjacent  aquarium  are  just 
as  likely  to  be  attacked  as  those  swimming  in  the  same 
aquarium  with  the  cephalopods  (Wells.  1958).  Both  S. 
officinalis  and  E.  scolopes  are  nocturnal  predators  living 
in  waters  where  bioluminescent  dinoflagellates  are  present 


in  notable  quantities:  1 1  dinoflagellate  cells  1  '  in  the 
Northeastern  Atlantic  and  >1  cell  I"1  for  tropical  waters, 
to  a  depth  up  to  1 50  m  or  more  depending  on  clarity  and 
mixing  (D.  Lapota,  pers.  comm.).  Dinoflagellate  concen- 
trations used  in  these  experiments  exceed  those  that  occur 
naturally  but  are  lower  than  concentrations  used  in  pre- 
vious burglar  alarm  studies  (Esaias  and  Curl,  1972;  White, 
1979;  Buskey  et  a/.,  1983).  Some  of  the  lower  concentra- 
tions used  in  our  study  are  not  unusual  in  dinoflagellate 
bloom  conditions. 

Locomotion  of  mysid  (Holmesimysis  sculpta),  mos- 
quito fish  (Gambusia  affinis),  and  grass  shrimp  (Palae- 
monetes pugio)  readily  stimulated  dinoflagellate  (Pyro- 
cystis fusiformis)  luminescence  at  all  concentrations,  il- 
luminating the  prey  and  thereby  increasing  their 
susceptibility  to  squid  predation.  Their  swimming  hydro- 
dynamic  forces  approximate  the  1.0  dyne  cm"2  required 
to  excite  luminescence  by  couette  flow  (Latz  et  al .  1994). 
Luminescence  appeared  to  be  the  primary  factor  in  in- 
ducing predation,  as  the  absence  of  dinoflagellates  resulted 


DINOFLAGELLATE-AIDED  PREDATION 


269 


Figure  6.  Split-screen  video  image  ofEuprymna  scolopes  feeding  on  ghost  shrimp  (Palaemonetes pugio). 
Image-intensifying  camera,  top  view.  Infrared  camera,  bottom  view.  (A)  Pre-attack  position;  (B)  Positioning; 
(C)  Seizure.  Luminescence  is  produced  by  Pyrocystis  fusiformis  stimulated  by  ghost  shrimp  movements. 
Bar  scale  =  1  cm. 


1  - 

X    with  luminescence 

O    no  luminescence 

tr 

In        10  - 

X   (0:07) 

1 

x                                                                   xx 

55       100  - 
o 

x         X 

X 

p 

X 

~       1000  - 

X    (19:36)                                                                      X 

O 

no  strike    - 

OOBOBOBOBBOOOBO            OBOO 

2  4  6  8  10         12         14         16         18         20 

Test  Number 

Figure  7.     Time  required  by  Sepia  nffidnalis  to  strike  mosquito  fish  (Gamhusia  qffinis)  in  the  presence 
of  luminous  and  nonluminous  dinoflagellates  (Pyrocystis  fusiformis). 


270 


R    J.   FLEISHER  AND  J.   F.  CASE 


I 
.1 

M 

'5 

I" 


Dinoflagellate  concentration  (cells/ml) 

Figure  8.  The  frequency  with  which  Euprymna  sailapea  attack  ghost 
shnmp  (Palaemonetes  pugio)  as  a  function  of  concentration  of  lumi- 
nescent dinotlagellates  (Pyrocystis  fusiformis).  Error  bars  represent  stan- 
dard errors. 


in  markedly  lower  predation.  At  the  same  time,  the  pres- 
ence of  dinoflagellates  had  no  obvious  direct  detrimental 
or  behavioral  effects  on  prey  within  the  time  scale  of  the 
experiments. 

S.  officinalis  enters  the  water  column  at  night  to  feed. 
Hovering  just  off  the  sand  bottom,  the  cuttlefish  either 
wait  for  or  swim  in  search  of  prey.  Luminescent  dinofla- 
gellates occur  naturally  in  waters  off  the  British  coast  of 
France,  the  Mediterranean,  and  Great  Britain  where  S. 
officinalis  are  found.  Predation  experiments  showed  that 
cuttlefish  have  the  ability  to  use  light  provided  by  dino- 
flagellates to  locate  prey.  Without  this  light  there  is  little 
predation  success.  The  higher  the  dinoflagellate  concen- 
tration the  more  prey  S.  officinalis  obtained  (Fig.  4).  We 
suspect  that  the  ability  to  regulate  bouyancy  improves 
concealment  of  the  cuttlefish  from  its  prey  or  possible 
predators  by  reducing  the  necessity  for  locomotor  activity. 

A  difficulty  with  these  experiments  was  the  recovery  of 
luminescence  by  photophase  dinoflagellates  in  the  control 
tanks  (Fig.  2).  As  bioluminescence  competence  increased 
over  the  3-hour  test.  Sepia  hunted  more  effectively.  Also 
unexpected  was  the  fact  that  in  total  darkness,  few  prey 
were  attacked.  This  is  contrary  to  the  observations  of  Bu- 
delmann  et  al.  ( 1991 ),  who  found  that  S.  officinalis  uses 
a  lateral  line  system  similar  to  the  mechanoreceptive  lat- 
eral lines  of  fish  and  aquatic  amphibians  to  find  about 
50%  of  available  prey.  In  complete  darkness.  Sepia  in  this 
experiment  consumed  significantly  less  than  50%  of 
available  prey  (Fig.  4). 


Observation  of  predator/prey  interactions  with  mos- 
quito fish  and  ISIT/IR  video  showed  that  luminescence 
from  dinoflagellates  aids  Sepia  to  visually  locate  and 
strike  prey.  Prey  size  and  type  in  these  tanks  were  such 
that  Sepia  always  attacked  by  discharging  its  two  pre- 
hensile tentacles.  Video  analysis  of  the  predator-prey 
interactions  and  correlated  bioluminescence  clearly 
showed  the  eye  movements,  body  orientation,  and  sub- 
sequent strike  of  individual  S.  officinalis  as  they  fol- 
lowed mosquito  fish  through  luminescent  water  (Fig. 
5).  In  water  populated  with  scotophase  dinoflagellates, 
1 3  out  of  the  20  Sepia  successfully  struck  the  prey,  and 
1 1  strikes  took  place  in  less  than  10  min.  Without 
luminescent  cells  only  one  strike  took  place,  and  this 
at  greater  than  29  min.  (Fig.  7). 

E.  scolopes  emerges  from  the  sand  at  night  to  await 
prey.  Once  prey  is  in  an  individual's  strike  zone,  the  squid 
orients  and  strikes — remaining  off  the  bottom  for  a  brief 
period  and  then  returning  to  the  sand  to  complete  feeding. 
E.  scolopes  has  a  slightly  different  attack  mode  than  Sepia. 
This  cephalopod  will  only  strike  prey  within  a  defined 
strike  zone  and  spends  little  or  no  time  adjusting  distance 
to  the  prey  along  the  prey  axis  to  ensure  seizure  (Fig.  6). 
This,  coupled  with  the  highly  variable  movements  of  the 
grass  shrimp,  may  serve  to  explain  the  high  variance  of 
strike  rates.  Nonetheless,  the  frequency  with  which  E. 
scolopes  struck  was  much  greater  in  luminescent  water 
(79%)  than  in  dinoflagellate-free  control  tanks  (37%;  Fig. 
8).  Successful  strikes  in  darkness  are  unexplainable,  but 
may  well  involve  mechanoreception  or  near-field  acoustic 
sensitivity.  Comparing  strikes  alone,  the  rates  show  no 
significant  differences,  indicating  no  increase  or  decrease 
in  predation  success,  due  to  specific  concentration  of  di- 
noflagellates. One  possible  explanation  is  that  the  lumi- 
nescence assisted  the  squid  in  locating  prey  but  not  nec- 
essarily in  attack  success.  Luminescent  dinoflagellates  oc- 
cur in  measurable  quantities  on  the  coast  of  Hawaii  where 
E.  scolopes  is  found. 

These  experiments,  along  with  those  of  Mensinger  and 
Case  (1992),  clearly  establish  on  an  experimental  basis 
that  predators  as  widely  disparate  as  fish  and  cephalopods 
are  able  to  use  the  light  of  dinoflagellates  as  an  effective 
aid  in  hunting  nonluminescent  prey.  The  work  also  sup- 
ports the  concept  of  a  more  general  role  for  biolumines- 
cence in  which  detection  of  bioluminescence,  by  increas- 
ing the  sensory  domain  of  nocturnal  and  deep-sea  animals, 
contributes  to  their  estimation  of  the  carrying  capacity  of 
the  local  environment  (Case  et  al.,  1994).  Bioluminescent 
events,  typically  representing  predator/prey  interactions, 
can  be  seen  at  several  meters  distance  in  clear  oceanic 
waters,  and  thereby  allow  animals  with  good  vision  to 
census  local  populations  in  a  way  well  beyond  the  range 
of  sensory  modalities  other  than  acoustic. 


DINOFLAGELLATE-AIDED  PREDATION 


271 


Acknowledgments 

This  work  was  supported  by  the  Office  of  Naval  Re- 
search (NOO 14-94- 1-0751)  and  the  FBN  fund.  We  would 
particularly  like  to  thank  M.  McFall-Ngai  for  assistance 
in  obtaining  and  maintaining  E.  scolopes  and  J.  Forsythe 
for  the  same  with  S.  officinalis.  We  are  indebted  to  S. 
Haddock,  J.  Moeller,  D.  Cook,  D.  Neilson,  and  Mark 
Ussini  for  discussions  and  assistance  in  the  laboratory.  S. 
Anderson  and  J.  McCullagh.  as  usual,  provided  invaluable 
support  in  animal  maintenance. 

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Reference:  Biol.  Bull  189:  272-279.  (December,  1995) 


Behavioral  Responses  of  Concholepas  concholepas 

(Bruguiere,  1789)  Larvae  to  Natural  and  Artificial 

Settlement  Cues  and  Microbial  Films 


SEBASTIAN  R.  RODRIGUEZ1,  CARLOS  RIQUELME2,  ELISEO  O.  CAMPOS', 
PAMELA  CHAVEZ2,  ENRIQUE  BRANDAN1,  AND  NIBALDO  C.  INESTROSA1 

' Departamento  cle  Biologia  Cehtlar  y  Molecular.  Facultad  de  Ciencias  Biologicas,  Pontijicia 

Universidad  Catolica  de  Chile,  and 2 Departamento  de  Aciiiculinra. 

Facultad  de  Recursos  del  Mar.  Universidad  de  Antofagasta 


Abstract.  The  behavioral  responses  of  veliger  larvae  of 
the  gastropod  Concholepas  concholepas  were  studied  in 
the  presence  of  different  natural  and  artificial  settlement 
cues  and  microbial  films.  Early  pre-competent  larvae 
stopped  swimming,  sank  (due  to  ciliary  arrests,  retraction 
of  the  velum  into  the  shell,  or  both),  and  remained  inactive 
on  the  substratum  when  exposed  to  conspecific  mucus 
and  hemolymph.  In  both  cases  the  effect  was  time-de- 
pendent and  the  number  of  larvae  showing  these  behaviors 
decreased  over  time.  Larvae  exposed  to  NH4C1  (ammo- 
nium ion)  showed  a  similar  time-  and  dose-dependent 
response.  A  positive  and  time-dependent  response  was 
also  observed  when  larvae  were  exposed  to  different  ex- 
tracellular matrix  (ECM)  components  (i.e..  collagen,  gel- 
atin, and  nbronectin)  and  sulfated  polysaccharides  (i.e.. 
carrageenan,  heparin,  and  chondroitin  sulfate).  In  this  case 
the  larvae  remained  attached  to  the  substratum.  However, 
the  effect  of  sulfated  polysaccharides  on  C  concholepas 
larval  behavior  was  faster  than  that  observed  with  other 
ECM  molecules.  We  also  studied  the  responses  of  pre- 
metamorphic  C.  concholepas  larvae  exposed  to  different 
microbial  films.  In  chemotaxis  experiments  with  different 
films,  with  glass  as  the  substratum,  larvae  showed  a  sig- 
nificant preference  for  multispecific  and  diatoms  films. 
When  shells  of  C.  concholepas  were  used  as  the  substra- 
tum, the  preference  for  multispecific  films  was  clear  and 
significant.  Likewise,  larvae  showed  velar  contractions  in 
the  presence  of  all  the  films  tested.  Larvae  exposed  to 


Received  1  1  November  1994;  accepted  28  July  1995. 
(  nrrespondence:  Dr.  N.  C.  Inestrosa,  Molecular  Neurobiology  Unit, 
Catholic  University  of  Chile,  Casilla  1  14-D,  Santiago.  Chile. 


multispecific  films  and  to  the  microalga  Prasinocladus 
marinus  showed  an  increased  ciliar  movement.  The  find- 
ing that  mucus  and  hemolymph  of  conspecific  adults  and 
ECM  molecules  (mainly  sulfated  polysaccharides)  induce 
the  cessation  of  swimming  of  C.  concholepas  larvae  sug- 
gests a  possible  role  for  cell-surface  receptors  in  mediating 
the  larval  response  of  marine  organisms.  Likewise,  the 
positive  chemotaxis  responses  of  C.  concholepas  larvae  to 
different  microbial  films  suggest  that  microorganisms  may 
have  a  role  in  bringing  larvae  close  to  settlement  inducers 
on  the  marine  benthos. 

Introduction 

Settlement  and  metamorphosis  are  key  steps  during 
the  life  cycle  of  benthic  marine  invertebrates.  A  number 
of  artificial  and  natural  settlement-inducing  substances 
have  been  described  (Pawlik,  1992;  Rodriguez  et  al.. 
1993).  Most  of  the  artificial  inducers  are  neuroactive  mol- 
ecules such  as  neurotransmitters,  neurotransmitter  pre- 
cursors, and  ions  (Morse  et  al.,  1979;  Hirata  and  Hadfield, 
1986;  Yool  et  al..  1986;  Bonar  et  al..  1990).  Natural  in- 
ducers are  associated  with  three  main  sources:  conspecific 
individuals  (e.g..  Pawlik,  1986),  microbial  films  (Maki  et 
al..  1989),  and  prey  species  (Hadfield  and  Pennington, 
1990).  Concerning  the  first  source,  conspecific  mucus  is 
known  to  induce  larval  settlement  in  the  gastropod  Hal- 
iotis  nifescens  (Slattery,  1992).  Moreover,  it  has  been  pro- 
posed that  growth  factors  associated  with  the  mucus  could 
trigger  the  settlement  response  in  at  least  some  molluscan 
species  (Cantillana  and  Inestrosa,  1993;  Rodriguez  et  al., 
1993).  Bacterial  films  have  been  reported  to  induce  larval 
settlement  in  a  number  of  marine  invertebrates  (e.g., 


LARVAL  RESPONSES  TO  SETTLEMENT  CUES  AND  MICROBIAL  FILMS 


273 


Kirchman  el  a/.,  1982).  In  some  cases,  exopolymers  are 
the  stimulus.  These  are  produced  by  bacteria,  possibly  as 
adhesive  factors  during  attachment  to  the  substratum 
(Maki  ct  ul,.  1989).  In  other  cases,  the  active  inductive 
factor  from  bacterial  supernatants  could  be  ammonium 
ion  (NH/)  (Bonar  ct  ai,  1990).  For  example,  oyster  larvae 
exposed  to  solutions  of  NH4C1  exhibit  stereotypical  set- 
tlement behavior  similar  to  that  which  normally  precedes 
metamorphosis  (Coon  ct  ul..  1990). 

Morse  and  Morse  (1991)  reported  that  the  morpho- 
genetic  molecule  for  a  scleractinian  coral  larvae  is  a  sul- 
fated  glycosaminoglycan.  In  spite  of  this  finding,  the  pos- 
sibility that  extracellular  matrix  (ECM)  macromolecules 
play  a  role  in  the  settlement  of  marine  invertebrate  larvae 
has  not  been  widely  explored.  Several  studies  have  ex- 
amined the  behavioral  response  of  gastropod  pre-com- 
petent  larvae  to  different  settlement-inducing  cues.  Results 
indicate  that  pre-competent  larvae  are  able  to  show  some 
of  the  typical  settlement  behaviors  observed  during  the 
metamorphosis  of  competent  ones,  such  as  ciliary  arrests 
and  contractions  of  the  velar  lobes,  when  exposed  to  set- 
tlement-inducing substances  (Arkett  ct  ai.  1987;  Barlow, 
1990).  The  pre-competent  larvae  sometimes  retract  the 
velum  into  the  shell,  probably  due  to  overstimulation 
(Barlow,  1990).  As  a  consequence  of  the  settlement  be- 
haviors described  above,  larvae  sink  and  remain  tran- 
siently inactive  on  the  substratum. 

The  prosobranch  mollusc  Concholepas  concholepas 
("loco"),  an  economically  important  benthic  marine  re- 
source along  the  Chilean  coast,  is  in  danger  of  extinction 
resulting  from  overexploitation  (Castilla.  1988).  We  have 
been  studying  this  species  to  generate  basic  information 
that  will  eventually  allow  us  to  culture  it  (Urrea  ct  ul.. 
1992;  Cantillana  and  Inestrosa.  1993:  Inestrosa  ct  ai. 
1993a,b;  Campos  ct  ai.  1994).  We  previously  showed  that 
an  excess  of  K+  induces  metamorphosis  in  planktonic  as 
well  as  in  laboratory -reared  larvae  of  C.  concholepas 
(Inestrosa  ct  ai.  1993a;  Campos  ct  ai.  1994). 

Here  we  report  the  effect  of  conspecific  mucus  and  he- 
molymph.  ECM  macromolecules.  sulfated  polysaccha- 
rides,  and  ammonium  ion  on  the  behavior  of  early  pre- 
competent  C.  concholepas  larvae.  Likewise,  we  report  on 
the  behavioral  response  of  pre-metamorphic  larvae  to  dif- 
ferent microbial  films  isolated  from  a  native  area  of  re- 
cruitment of  C.  concholepas. 

Materials  and  Methods 

Experimental  animals 

Adult  specimens  and  egg  capsules  of  Concholepas 
concholepas  were  collected  from  the  subtidal  zone  off  the 
central  Chilean  coast  (Las  Cruces:  33°  30'  S.  71°  30'  W) 
and  immediately  transported  to  our  laboratory  in  fresh 
seawater.  Capsules  were  maintained  in  aerated,  mem- 


brane-filtered (0.45  urn)  seawater  at  20-22°C  until  hatch- 
ing. For  the  experiments  with  natural  and  artificial  cues, 
early  pre-competent  veliger  larvae  just  hatched  from  cap- 
sules were  acclimated  1-2  days  before  being  used.  For 
experiments  with  microbial  films,  pre-metamorphic  larvae 
were  obtained  from  a  culture  of  75  days  as  described  by 
Riquelme  and  Chavez  (1995).  In  brief,  veliger  larvae  were 
obtained  from  mature  capsules  and  maintained  in  1-1 
bottles  containing  membrane-filtered  (0.22  urn)  seawater 
at  20°C  and  with  a  14: 10  LD  photoperiod  (60  larvae  per 
liter).  The  seawater  was  changed  every  2  days.  The  mi- 
croalga  Isoc/irvsi.s  galbana  was  used  as  food  at  a  density 
of  around  10?  cells  per  liter  of  larval  culture.  Larvae  of 
about  1650  /urn  were  maintained  at  a  density  of  10  larvae 
per  liter  and  acclimated  for  2  days  before  using  in  exper- 
iments. These  larvae  showed  all  the  characteristics  of  the 
pre-metamorphic  stage  of  C.  concholepas  described  by 
DiSalvo(1988). 

Obtaining  mucus  and  hemolymph 

Conspecific  mucus  was  obtained  by  smoothly  scraping 
the  muscular  foot  of  living  adult  C.  concholepas  with  a 
spatula.  After  that,  the  individuals  were  broken  into  pieces 
and  placed  inside  a  funnel:  the  drained  hemolymph  was 
collected.  Both  procedures  were  carried  out  in  a  cold  room 
(4°C).  The  mucus  and  the  hemolymph  were  used  in  ex- 
periments immediately  after  collection. 

Obtaining  microorganisms 

The  microorganisms  used  to  create  microbial  films  were 
isolated  from  the  surface  of  rocks  obtained  in  the  natural 
area  of  recruitment  of  C  concholepas  on  the  north  coast 
of  Chile  (Antofagasta  Bay:  23°  39'  S,  71°  30'  W).  Four 
types  of  microbial  films  were  used  for  larval  behavior  ex- 
periments: ( 1 )  multispecific  bacteria-microalgal  films 
(MBM),  scraped  directly  from  rocks:  (2)  monospecific 
bacterial  films,  constituted  by  a  periphytic  bacterium  able 
to  develop  a  strong  film  on  glass  and  polystyrene  plates: 
(3)  Prasinoclaihis  marinus  films,  produced  by  a  periphytic 
dominant  microalga  present  on  the  rocks;  and  (4)  mul- 
tispecific diatom  films.  To  isolate  bacteria,  different  rocks 
were  scraped.  The  resulting  material  was  inoculated  in 
agar  St  10  for  marine  bacteria  (Ishida  el  ai.  1986)  and 
incubated  at  20°C  for  a  week.  Different  bacterial  strains 
growing  in  St  10  medium  were  recognized  on  the  basis 
of  some  morphological  characters  (size,  shape,  color,  and 
height)  of  their  colonies.  These  bacterial  strains  were  iso- 
lated and  tested  for  their  ability  to  develop  a  strong  film 
on  polystyrene  plates.  Monospecific  bacterial  suspensions 
were  placed  on  petri  dishes  and  rinsed  with  sterile  seawater 
after  24  h.  The  strain  that  was  able  to  remain  attached  to 
plates  after  rinsing  was  considered  a  strong  periphytic 
bacterium. 


274 


S    R.  RODRIGUEZ  ET  AL. 


In  the  case  of  the  microalgal  isolation,  the  scraped  ma- 
terial was  diluted,  inoculated  in  agar  (Provasoli  et  a/., 
1957),  and  incubated  at  20°C  for  2  weeks  with  a  14:10 
LD  photoperiod.  The  dominant  microalgal  species,  Pra- 
sinocladus  marinits,  was  isolated  by  hand  under  a  micro- 
scope and  also  inoculated  in  Provasoli  medium.  This  spe- 
cies and  the  diatoms  were  identified  by  Professor  Gerald 
Boalch  (Citadelhill  Plymouth  Laboratories,  U.K.). 

Preparing  microbial  films 

Pieces  of  C.  concholepas  shell  and  glass  coverslips  were 
offered  as  substrata  to  microorganisms.  Before  being  used, 
substrata  were  washed  with  acid  and  rinsed  with  abundant 
seawater  to  remove  all  tissue  residue.  After  that,  they  were 
deposited  in  bottles  containing  1 50  ml  of  seawater.  This 
material  was  autoclaved  before  being  inoculated  with  the 
different  strains.  The  substrata  were  incubated  with  the 
microorganisms  in  suspension  until  they  developed  film. 
The  substrata  were  washed  with  sterile  seawater  and  im- 
mediately used  in  experiments.  Preliminary  experiences 
showed  that  48  h  of  incubation  was  sufficient  to  create  a 
film  able  to  adhere  after  washing. 

Behavioral  response  bioassays 

Larval  response  to  different  natural  and  artificial  cues. 
Conspecific  mucus  was  spread  over  24-well  culture  plates 
in  a  homogeneous  film.  Twenty  to  thirty  early  pre-com- 
petent  veliger  larvae  were  assayed  per  well  in  a  final  vol- 
ume of  1  ml  of  filtered  seawater.  The  number  of  C.  con- 
cholepas larvae  that  sank  as  a  result  of  a  cessation  of 
swimming  (due  to  ciliary  arrest,  retraction  of  the  velum 
into  the  shell,  or  both)  and  remained  inactive  on  the  bot- 
tom of  the  wells  during  a  2-h  incubation  was  recorded 
using  a  Wild  dissecting  microscope.  The  behavior  of  20 
to  30  control  larvae  maintained  in  wells  containing  noth- 
ing but  1  ml  of  normal  filtered  seawater  was  followed  si- 
multaneously with  each  treatment.  Each  treatment  and 
each  control  were  performed  in  triplicate.  Hemolymph 
was  loaded  into  24-well  culture  plates  (100  ^I/plate)  and 
dried  overnight.  The  same  procedure  was  followed  with 
100  jul  of  solutions  containing  2  ^g  of  fibronectin,  carra- 
geenan,  chondroitin  sulfate,  or  heparin;  or  12  /jg  of  col- 
lagen; or  200  ng  of  gelatin.  Higher  concentrations  of  col- 
lagen and  gelatin  were  used  because  no  larval  response 
was  observed  at  lower  concentrations.  The  wells  were  filled 
with  filtered  seawater  ( 1  ml)  before  20  to  30  larvae  were 
placed  in  each  well.  The  experiments  were  followed  for 
30  min  in  the  case  of  the  hemolymph  and  24  h  for  the 
ECM  components  and  sulfated  polysaccharides.  The 
number  of  larvae  that  sank  and  remained  attached  to  the 
substratum  was  recorded  as  described  above.  A  similar 
experiment  was  carried  out  with  hemolymph  boiled  for 
3  min.  Together  with  each  treatment,  the  behavior  of  20 


to  30  control  larvae  maintained  in  wells  containing  noth- 
ing but  1  ml  of  normal  filtered  seawater  was  followed. 
Each  treatment  and  each  control  were  performed  in  trip- 
licate. 

Larvae  were  exposed  to  a  range  of  concentrations  of 
NH4C1  (i.e.,  2,  5,  8,  and  10  mAf).  A  stock  solution  of 
100  mA/  NH4C1  was  made  in  seawater  and  adjusted  to 
pH  8.0  with  1  N  NaOH.  At  the  beginning  of  each  bioassay, 
enough  stock  solution  was  added  to  a  volume  of  seawater 
(pH  8.0)  to  generate  1  ml  of  the  desired  NH4C1  final  con- 
centration in  24-well  culture  plates  containing  20  to  30 
larvae  per  well.  The  experiments  were  followed  for  30 
min  and  the  number  of  larvae  that  sank  and  remained 
inactive  on  the  bottom  of  wells  was  recorded  as  previously 
described.  Treatments  and  controls  were  performed  in 
triplicate,  as  described  for  the  other  bioassays.  The  ECM 
molecules  and  sulfated  polysaccharides  were  obtained 
from  Sigma  Chemical  Co.  (St.  Louis,  MO). 

The  concentration  of  larvae  used  in  all  the  above  ex- 
periments (i.e.,  20  to  30  larvae/ml)  was  similar  to  the 
concentration  at  which  larvae  were  acclimated  after 
hatching  and  before  the  assays.  In  many  species,  repeated 
encounters  with  others  causes  larvae  stop  swimming  and 
settle  to  the  bottom  of  the  culture  vessel;  thus  the  high 
density  used  in  our  experiments  could  have  affected  the 
results.  However,  the  control  larvae  in  our  assays  were 
never  observed  to  stop  swimming  or  settle  as  a  result  of 
encounters  among  them.  The  time  courses  followed  in 
the  above  experiments  were  different  because  the  assays 
were  carried  out  until  a  clear  response  was  observed. 

Chemotaxis  to  micmhial  films.  Individual  assays  of 
chemotactic  response  to  microbial  films  were  carried  out 
in  sterile  petri  dishes  containing  15  ml  of  sterile  seawater. 
Pre-metamorphic  larvae  and  substrata  containing  micro- 
organisms were  placed  on  opposite  sides  of  the  dishes.  We 
considered  a  response  to  be  positive  when  larvae  moved 
directly  to  the  substrata  and  remained  close  to  them,  and 
negative  when  larvae  moved  to  the  edge  of  the  dishes  or 
remained  close  to  the  starting  point  (random  movement). 
Ten  pre-metamorphic  larvae  were  simultaneously  placed 
for  each  type  of  substratum  and  continuously  observed 
for  1  h  with  a  Wild  dissecting  microscope.  The  controls 
were  carried  out  using  sterile  substrata.  Each  treatment 
was  performed  in  triplicate.  A  G  test  (Sokal  and  Rohlf. 
198 1 )  was  used  for  statistical  analysis. 

Lan'al  activity  in  response  to  microbial  films.  The  ac- 
tivity of  C.  concholepas  larvae  exposed  to  microbial  films 
was  observed.  The  films  were  prepared  on  glass  coverslips 
and  assayed  in  petri  dishes  as  described  above.  An  in- 
creased ciliar  movement  and  the  presence  of  contractions 
of  the  velum  were  used  as  criteria  for  larval  activity.  Larvae 
swimming  with  the  velum  extended  and  retracting  it 
briefly  but  repeatedly  were  categorized  as  presenting  velar 
contractions.  Likewise,  larvae  moving  their  cilia  faster 


LARVAL  RESPONSES  TO  SETTLEMENT  CUES  AND  MICROBIAL  FILMS 


275 


than  the  rate  observed  during  a  normal  swim  were  con- 
sidered to  be  showing  an  increased  ciliary  beating.  Larvae 
were  directly  placed  on  the  films  and  observed  with  a 
Wild  dissecting  microscope;  the  number  showing  an  ac- 
tivity response  was  recorded  at  intervals  of  0-2,  2-4,  4- 
6,  6-8,  and  8-10  min  (hereafter  2,  4.  6.  8.  and  10  min, 
respectively).  A  total  of  30  individual  bioassays  were  car- 
ried out  per  film.  A  G  test  was  used  for  statistical  analysis. 

Results 

Larval  response  to  different  natural  and  artificial  cues 

Mucus  and  hemolymph.  To  learn  about  the  effect  of 
conspecific  natural  substances  on  the  behavioral  response 
of  C.  concholepas.  early  pre-competent  larvae  of  this  gas- 
tropod were  exposed  to  mucus  and  hemolymph.  In  the 
presence  of  conspecific  mucus,  the  veliger  larvae  stopped 
swimming  and  sank.  The  effect  of  mucus  was  time-de- 
pendent and  reached  a  plateau  after  5  min  of  continuous 
exposure  to  the  film  (Fig.  1).  At  this  time,  about  50%  of 
the  larvae  were  inactive  on  the  bottom  of  the  wells  and 
remained  in  this  state  for  30  min  of  incubation  (Fig.  1). 
Thereafter,  the  number  of  larvae  swimming  normally  in- 
creased, leaving  only  10%  of  the  total  larvae  sunk  after  2 
h  (Fig.  1 ).  Larvae  exposed  to  hemolymph  showed  a  pro- 
nounced and  quick  response.  After  just  2  min  of  exposure, 
the  sinking  rate  was  94%  (Fig.  2).  Thereafter,  the  number 
of  larvae  showing  a  cessation  of  swimming  slowly  de- 
creased, resulting  in  a  75%  sinking  rate  after  30  min  (Fig. 
2).  Only  a  few  larvae  remained  inactive  on  the  bottom 
(16%)  after  2  h  of  incubation  (data  not  shown).  Larvae 
exposed  to  boiled  hemolymph  showed  a  response  similar 
to  that  observed  with  normal  hemolymph  during  the  first 


100 


120 


TIME  (min) 


Figure  I.  Mean  percentage  of  early  pre-competent  larvae  of  Con- 
cholepas concholepas  induced  to  sink  by  conspecific  mucus  after  2  h  of 
incubation.  •  =  Mucus,  and  O  =  control. 


12  18  24  30 


TIME    (min) 

Figure  2.  Mean  percentage  of  early  pre-competent  larvae  of 
Concholepas  concholepas  induced  to  sink  by  conspecific  hemolymph. 
•  =  Hemolymph,  O  =  boiled  hemolymph.  and  •  =  control. 


2  min.  However,  the  effect  was  transient  because  only 
50%  of  the  larvae  remained  sunk  after  5  min  of  incubation 
(Fig.  2).  The  number  of  larvae  induced  to  sink  with  boiled 
hemolymph  was  less  than  that  observed  with  normal  he- 
molymph throughout  the  experiment  (Fig.  2). 

ECM  components,  sulfated  polysaccharides,  and 
NH4Cl.  To  study  the  effect  of  some  artificial  cues  on  the 
behavioral  response  of  C.  concholepas,  early  pre-compe- 
tent larvae  of  this  mollusc  were  exposed  to  different  ECM 
molecules  and  sulfated  polysaccharides.  A  positive  and 
time-dependent  response  was  observed  for  all  the  assayed 
molecules.  After  24  h  of  incubation,  collagen,  gelatin,  and 
fibronectin  induced  sinking  rates  of  44%,  67%,  and  89%, 
respectively  (Fig.  3a).  At  the  same  time,  rates  of  70%, 
78%,  and  87%  were  observed  when  larvae  were  exposed 
to  carrageenan,  heparin,  and  chondroitin  sulfate,  respec- 
tively (Fig.  3b).  The  larvae  responded  to  the  sulfated  poly- 
saccharides more  quickly  than  to  the  ECM  molecules, 
reaching  more  than  the  50%  of  the  final  response  after 
just  2  h  of  incubation  (i.e..  sinking  rates  of  39%,  73%,  and 
68%>  with  carrageenan.  heparin,  and  chondroitin  sulfate, 
respectively)  (see  Fig.  3a,  b).  To  test  the  effect  of  NH4C1 
on  the  response  of  C.  concholepas,  early  pre-competent 
larvae  of  this  gastropod  were  exposed  to  different  concen- 
trations of  ammonium  ion.  The  behavioral  response  ob- 
served was  time-  and  dose-dependent  (Fig.  4):  it  increased 
rapidly  during  the  first  2  min,  reaching  sinking  rates  of 
4%,  45%,  48%,  and  84%  at  NH4C1  final  concentrations  of 
2,  5,  8,  and  10  mM,  respectively  (Fig.  4).  Thereafter,  the 
number  of  larvae  that  stopped  swimming  and  remained 
inactive  stayed  relatively  constant,  resulting  in  respective 
rates  of  1%,  39%,  74%,  and  98%  after  30  min  of  incubation 
(Fig.  4).  At  the  end  of  the  experiment,  larvae  exposed  to 


276 


S.  R.  RODRIGUEZ  ET  AL. 


100 


18 


24 


TIME   (h) 


Figure  3.  Mean  percentage  of  early  pre-competent  larvae  of 
Concholepas  concholepas  induced  to  sink  by  extracellular  matrix 
(ECM)  components  and  sulfated  polysaccharides.  (a)  Mean  percentage 
of  C  concholepas  larvae  induced  to  sink  by  ECM  constituents.  • 
=  Fibronectin,  O  =  gelatin.  •  =  collagen,  and  D  =  control,  (b)  Mean 
percentage  of  O  concholepas  larvae  induced  to  sink  by  sulfated  poly- 
saccharides. •  =  Carrageenan,  •  =  chondroitin  sulfate,  O  =  heparin. 
and  D  =  control. 


10  mM  NH4C1  were  washed  and  placed  in  normal  fresh 
seawater  to  see  if  they  would  recover.  Three  hours  later, 
all  larvae  were  observed  swimming  normally  (data  not 
shown). 

Chemotaxis  to  microhial  films.  Microbial  films  are  well 
known  as  settlement  inducers  for  a  number  of  benthic 
marine  invertebrates.  We  studied  the  attraction  responses 
of  pre-metamorphic  C.  concholepas  larvae  exposed  to 
several  such  films.  In  the  chemotaxis  experiment  in  which 
glass  plates  were  coated  with  different  films,  the  larvae 
responded  positively  to  P.  marimis.  multispecific.  diatom, 
and  bacterial  films  after  25  min  of  incubation:  rates  of 
attraction  were 20%.  30%,  20%,  and  20%  respectively  (Fig. 
5a).  At  that  time,  no  significant  difference  was  observed 
among  the  different  films  (G  test  with  3  df).  However, 
after  60  min  of  incubation,  the  respective  rates  of  larval 


attraction  increased  to  40%.,  60%,  60%.  and  30%,  and  a 
significant  preference  was  observed  for  the  multispecific 
and  the  diatom  films  compared  to  the  P.  marimis  and 
bacterial  films  (P  <  0.001,  G  test  with  1  df)  (Fig.  5a). 
When  C.  concholepas  shells  were  used  as  substrata,  a  pos- 
itive response  of  larvae  to  P.  marimis,  multispecific,  and 
diatoms  films  was  observed  after  25  min  of  incubation; 
rates  of  attraction  were  30%,  40%,  and  10%,  respectively 
(Fig.  5b).  At  that  time,  the  attraction  to  the  multispecific 
and  P.  marimis  films  was  significatively  higher  than  to 
the  diatom  films  (P  <  0.001,  G  test  with  1  df).  At  the  end 
of  the  experiment,  the  larvae  showed  a  clear  and  significant 
preference  for  the  multispecific  films  over  the  other  films, 
with  an  80%  rate  of  attraction  (P  <  0.001,  G  test  with  1 
df )  (Fig.  5b).  In  the  experiments  with  both  glass  and  shell 
substrata,  the  larvae  were  not  attracted  to  the  sterile  con- 
trol at  any  time. 

Larval  activity  in  response  to  microbial films.  The  ac- 
tivity of  C.  concholepas  larvae  exposed  to  different  mi- 
crobial films  was  recorded.  Larvae  showing  velar  con- 
traction were  observed  in  the  presence  of  all  the  films 
after  6  min  of  incubation  (Fig.  6a).  The  percentage  of 
larvae  presenting  this  behavior  was,  however,  significa- 
tively higher  for  the  bacterial  films  at  4,  6,  and  8  min  of 
incubation  with  20%,  30%.,  and  30%-,  respectively  (P 
<  0.001,  G  test  with  1  df).  At  the  end  of  the  experiment, 
the  response  to  the  bacterial  film  decreased,  and  no  sig- 
nificant difference  was  observed  among  the  bacterial, 
multispecific,  and  diatom  treatments  (Fig.  6a).  On  the 
other  hand,  the  ciliar  movement  of  larvae  increased  only 
in  the  presence  of  the  multispecific  and  the  P.  marimis 
films  (Fig.  6b).  However,  the  effect  of  the  former  was 
higher  after  8  min  of  incubation.  At  the  end  of  the  ex- 


100 


l==          75- 


50- 


25- 


c/j 


O 


30 


TIME    (min) 


Figure  -4.  Mean  percentage  of  early  pre-competent  larvae  of 
Concholepas  ciincholcpas  induced  to  sink  by  different  concentrations 
of  NH4C1.  •  =  10  mM.  D  =  8  mM.  •  =  5  mA/,  O  =  2  mA/.  and  D 
=  control. 


LARVAL  RESPONSES  TO  SETTLEMENT  CUES  AND  MICROBIAL  FILMS 


277 


GLASS    SUBSTRATE 


o 

G 
I 


< 

> 
cc 
< 


G  P-  marinus 

•  MBM  film 

•  Diatom  film 
M  Bacterial  film 
D  Control 


25 


60 


SHELL    SUBSTRATE 


TIME    (min) 

Figure  5.  Mean  percentage  of  attraction  of  pre-metamorphic  Con- 
fhiilcpas  concholepas  larvae  to  different  microbial  films.  Microbial  films 
prepared  on  glass  (a)  and  C.  concholepas  shells  (b). 


periment.  larvae  exposed  to  the  bacterial  films  exhibited 
an  increased  ciliar  movement,  but  this  response  was  sig- 
nificantly less  than  that  observed  for  the  other  two  treat- 
ments (P  <  0.001,  G  test  with  1  df)  (Fig.  6b). 

Discussion 

Mucus  and  hcmolymph 

Early  pre-competent  larvae  of  Concholepas  concholepas 
stopped  swimming,  sank,  and  remained  inactive  on  the 
bottom  of  the  wells  when  exposed  to  both  mucus  and 
hemolymph  of  adult  individuals.  Traces  of  mobile  animals 
(e.g.,  mucus)  can  influence  the  settlement  of  sessile  ani- 
mals (e.g.,  barnacles)  (Johnson  and  Strathmann.  1989). 
and  conspecific  mucus  induces  larval  settlement  in  the 
abalone  Haliotis  rufescens  (Slattery.  1992).  Structural 
factors  such  as  glycoproteins  and  growth  factors  as  well 
as  bacteria  associated  with  mucus  have  been  suggested  as 
possible  morphogens  involved  in  triggering  the  larval  set- 
tlement response  in  gastropods  (Slattery,  1992;  Cantillana 
and  Inestrosa,  1993).  Recently,  a  heparin-binding  growth 
factor,  which  shows  properties  similar  to  those  of  fibroblast 
growth  factors  (FGF),  has  been  identified  in  the  foot  of 


C.  concholepas  (Cantillana  and  Inestrosa,  1993).  The 
binding  of  basic  FGF  to  high-affinity  receptors  requires 
the  presence  of  an  ECM  component  (i.e.,  heparan  sulfate 
proteoglycans)  (Yayon  el  ai,  1991 ).  Therefore,  it  is  pos- 
sible that  growth  factors  in  the  mucus  of  mollusc  species 
could  be  interacting  with  ECM  molecules  (mainly  sulfated 
polysaccharides)  and  then  with  high-affinity  growth  factor 
receptors.  On  the  other  hand,  it  has  been  hypothesized 
that  lectins  (i.e..  sugar-binding  proteins  or  glycoproteins 
of  non-immune  origin  that  agglutinate  cells  or  precipitate 
glycoconjugates)  may  be  involved  in  the  settlement  and 
metamorphosis  of  marine  invertebrate  larvae  (Maki  and 
Mitchell,  1985).  Lectins  have  been  reported  in  the  mucus 
of  different  fish  species  (Kamiya  and  Shimizu,  1980;  Ka- 
miya tY  a/.,  1988)  as  well  as  in  the  hemolymph  of  a  number 
of  marine  invertebrates  such  as  starfish  (Kamiya  et  al., 
1992),  barnacles  (Kamiya  et  al..  1987).  and  isopods 
(Kaim-Malka,  1993).  Therefore,  lectins  present  in  the 
mucus  and  hemolymph  may  be  another  factor  mediating 
the  larval  settlement  of  C  concholepas  and  other  species. 
The  effect  of  heated  hemolymph  was  clearly  lower  than 
that  observed  with  unheated  hemolymph  during  most  of 
the  incubation  period.  Kamiya  et  al.  (1992)  found  that 
the  hemagglutinating  activity  of  lectins  was  heat  labile  in 


30- 


24- 


|§       '8 


I 


rj  P.  marinus 

•  MBM  film 

|  Diatom  film 

^  Bacterial  film 

D  Control 


10 


30- 

b 

~      24~ 

I       18' 

£       12- 

D         6  " 

1 

o 

n  - 

'  ^m   I  I 

•FT  1 

BTl 

•n 

•  r  i 

2  4  6  8  10 

TIME  (min) 

Figure  6.  Mean  percentage  of  pre-metamorphic  Concholepas  con- 
cholepas larvae  showing  velar  contractions  and  an  increased  ciliary  beating 
in  response  to  microbial  films,  (a)  Mean  percentage  of  C.  concholepas 
larvae  showing  velar  contractions,  (b)  Mean  percentage  of  C  concholepas 
larvae  showing  an  increased  ciliary  beating. 


278 


S.  R.  RODRIGUEZ  ET  AL 


the  hemolymph  of  a  starfish,  decreasing  or  disappearing 
when  exposed  to  high  temperatures.  A  similar  effect  could 
have  occurred  in  our  case.  Why  the  boiled  hemolymph 
initially  affects  larvae  in  the  same  way  that  the  unheated 
hemolymph  does  is  not  clear — maybe  there  are  two  types 
of  cues  or  responses;  in  any  case  further  studies  are  nec- 
essary to  clarify  this  matter.  The  transient  effect  produced 
by  both  natural  substances  in  larvae  of  C.  concholepas 
may  be  related  to  some  kind  of  habituation  to  the  cue; 
alternatively,  it  may  be  due  to  a  deficient  larval  response, 
given  the  presence  of  an  immature  signal-transduction 
mechanism. 

ECAt  macromoleailes,  sulfated  polysaccharides,  and 
NH4Cl 

A  positive  and  time-dependent  behavioral  response  was 
observed  in  early  pre-competent  larvae  of  C.  concholepas 
after  exposure  to  ECM  molecules  and  sulfated  polysac- 
charides. In  both  cases  larvae  sank  and  remained  attached 
to  the  substratum.  The  effect  of  sulfated  molecules  was 
faster  than  that  of  the  other  ECM  components.  Morse 
and  Morse  ( 1991 )  identified  the  molecule  that  is  biolog- 
ically active  as  a  morphogen  for  a  scleractinian  coral  larvae 
(i.e.,  Agaricia  humilis)  as  a  sulfated  glycosaminoglycan. 
Moreover,  they  showed  that  some  sulfated  polysaccharides 
such  as  K-  carrageenan,  fucoidan,  and  keratan  sulfates  in- 
duce the  metamorphosis  of  A.  humilis,  but  chondroitin 
sulfates  and  heparin  do  not  (Morse  and  Morse,  1991).  It 
was  previously  demonstrated  that  larvae  of  C.  concholepas 
incubated  in  artificial  seawater  in  the  absence  of  sulfate 
or  in  presence  of  a  specific  sulfation  inhibitor  show  a  de- 
crease in  their  mobility  (Urrea  et  a/..  1992;  Brandan  et 
al.  1995).  The  results  presented  in  this  work  strongly  sug- 
gest that  sulfated  polysaccharides  play  a  role  in  aspects  of 
the  settlement  of  C  concholepas  larvae.  Heparin,  which 
produced  one  of  the  most  rapid  response  in  larvae  of  C. 
concholepas,  can  interact  with  growth  factors  (Yayon  et 
al..  1991 ).  In  this  context,  factors  associated  with  the  mu- 
cus of  larvae  may  be  important  in  this  interaction.  The 
results  described  in  this  paper  suggest  that  larvae  of  C. 
concholepas  are  able  to  interact  with  macromolecules 
found  in  the  ECM,  especially  those  that  are  sulfated. 

A  dose-  and  time-dependent  response  was  observed 
when  early  pre-competent  larvae  of  C  concholepas  were 
exposed  to  NH4+.  Larvae  were  able  to  recover  their  mo- 
bility after  an  extensive  washing  with  fresh  seawater.  NH4+ 
has  been  described  as  an  important  inducer  of  settlement 
behavior  in  oyster  larvae  (Coon  et  al.,  1990).  Likewise,  it 
has  been  found  that  NH4+  is  the  active  inductive  factor 
associated  with  bacterial  supernatants  (Bonar  et  al..  1990). 
Following  in  this  vein,  marine  zones  rich  in  dissolved  or- 
ganic matter  would  represent  important  areas  where  high 
settlement  of  benthic  marine  invertebrates  may  occur 
(Morse,  1990). 


Since  in  many  species  repeated  encounters  with  others 
cause  the  larvae  to  stop  swimming  and  settle  to  the  bottom 
of  the  culture  vessel,  it  is  possible  that  the  high  larval 
density  used  in  our  experiments  affected  the  results  ob- 
tained here.  However,  repeated  observations  of  the  be- 
havior of  control  C.  concholepas  larvae  during  the  assays 
never  showed  that  larvae  stopped  swimming  or  settled  as 
the  result  of  encounters  among  them.  This  observation 
represents  a  behavioral  pattern  of  remarkable  interest, 
making  our  results  all  the  more  notable. 

Chemotaxis  and  larval  activity  in  response  to  microbial 
films 

Our  results  clearly  show  that  microbial  films,  especially 
multispecific  ones,  are  able  to  attract  pre-competent  larvae 
of  C.  concholepas.  This  response  is  preferentially  observed 
in  the  presence  of  multispecific  films  attached  to  shell 
substrata.  Moreover,  larvae  exposed  to  these  films  show 
increased  ciliary  beating.  The  role  of  bacterial  films  in  the 
settlement  of  marine  invertebrate  larvae  has  been  widely 
studied  (e.g..  Kirchman  et  al.,  1982;  Maki  and  Mitchell, 
1985;  Maki  et  al.,  1989;  Bonar  et  al..  1990).  However, 
the  effect  of  microbial  films  on  the  attraction  of  larvae 
and  the  eventual  role  of  these  films  in  bringing  larvae  near 
to  the  marine  bottom  has  not  received  much  attention 
(Pawlik,  1992).  Our  results  suggest  a  quite  important  role 
for  microbial  films  in  attracting  larvae  of  Concholepas 
concholepas.  This  attraction  could  favor  the  approach  of 
larvae  to  the  chemical  metamorphic  inducers  on  the  ma- 
rine bottom.  The  higher  response  of  larvae  to  multispecific 
films  on  shell  than  on  glass  was  perhaps  due  to  a  better 
attachment  of  the  bacteria  to  an  irregular  surface.  Also  it 
is  possible  that  the  films  produced  on  the  shell  were  either 
more  numerous  or  had  a  different  composition  than  those 
on  the  glass. 

In  this  paper  we  have  provided  evidence  that  pre-com- 
petent larvae  of  Concholepas  concholepas  exposed  to  dif- 
ferent natural  and  artificial  cues  exhibit  behaviors  (i.e., 
ciliary  arrests,  contraction  of  the  velar  lobes,  and  retraction 
of  the  velum  into  the  shell)  similar  to  those  described  for 
competent  veliger  larvae  of  other  gastropod  species  during 
metamorphosis.  Likewise,  they  showed  different  degrees 
of  attraction  to  different  microbial  films  isolated  from  a 
native  recruitment  zone  of  this  species.  This  information 
on  natural  and  artificial  metamorphic  inducers  of  C.  con- 
cholepas larvae  may  be  of  paramount  importance  in  de- 
veloping successful  methods  for  culturing  this  overex- 
ploited  species. 

Acknowledgments 

This  work  was  supported  by  FONDECYT  Grants  3502/ 
89,  0651/91,  and  19406/94  to  Dr.  N.  C.  Inestrosa  and 
0997/92  to  Dr.  C.  Riquelme,  and  by  IPS  Grant  1407-3F 


LARVAL  RESPONSES  TO  SETTLEMENT  CUES  AND  MICROBIAL  FILMS 


279 


to  Dr.  E.  Brandan.  We  thank  Prof.  Gerald  Boalch  from 
Citadelhill  Plymouth  Labs.  U.K..  for  identifying  microal- 
gae  and  diatoms.  During  this  study  S.  R.  Rodriguez  was 
a  Research  Fellow  from  DIUC.  He  is  now  a  Fellow  from 
Fundacion  Andes  (Dept.  of  Ecology). 

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Ultrastructural  Localization  of  Antho-RWamides  I  and 

II  at  Neuromuscular  Synapses  in  the  Gastrodermis 

and  Oral  Sphincter  Muscle  of  the  Sea  Anemone 

Calliactis  parasitica 

JANE  A.  WESTFALL1,  KELLEY  L.  SAYYAR1,  CAROL  F.  ELLIOTT1 
AND  CORNELIS  J.  P.  GRIMMELIKHUIJZEN2 

1 Department  of  Anatomy  and  Physiology.  Kansas  State  University.  Manhattan.  Kansas  66506  and 

2Depanment  of  Cell  Biology  and  Anatomy.  University  of  Copenhagen. 

DK-2100  Copenhagen  0.  Denmark 


Abstract.  Light  microscopic  studies  have  shown  that  the 
sea  anemone  neuropeptides  Antho-RWamides  I  (<Glu- 
Ser-Leu-Arg-Trp-NH2)  and  II  «Glu-Gly-Leu-Arg-Trp- 
NH:)  are  located  in  neurons  associated  with  the  oral 
sphincter  muscle  of  the  sea  anemone  Calliactis  parasitica. 
In  the  present  ultrastructural  study,  using  the  immunogold 
technique,  we  found  Antho-RWamide-like  material  in  the 
granular  vesicles  of  neurons  that  make  synaptic  contacts 
with  the  myonemes  of  both  gastrodermal  and  oral 
sphincter  muscle  cells  of  Calliactis.  Gastrodermal  nerve 
cells  contained  immunoreactive  granular  vesicles  aver- 
aging 149.3  ±4.1  nm  in  diameter;  smaller  granular  ves- 
icles (47.5  ±  2.5  nm)  were  present  at  a  labelled  synapse. 
Neurites  associated  with  the  sphincter  muscle  had  im- 
munoreactive granular  vesicles  averaging  78.8  ±  3.3  nm 
in  diameter  with  smaller  granular  vesicles  (63  ±  4.4  nm) 
at  three  labelled  neuromuscular  synapses.  All  Antho- 
RWamide-immunoreactive  vesicles  were  irregularly 
granular,  unlike  the  typical  dense-cored  vesicles  observed 
at  some  other  synapses  in  sea  anemones.  No  evidence  was 
found  of  storage  or  release  at  nonsynaptic  sites  (paracrine 
secretion). 

The  Antho-RWamide  immunoreactive  neurites  inner- 
vate the  sphincter  muscle  fibers  directly  rather  than 
through  intermediate  neuronal  pathways.  This  is  the  first 
ultrastructural  evidence  of  a  neuropeptide  at  a  coelenterate 
neuromuscular  synapse. 

teceived  15  June  1995;  accepted  21  September  1995. 

-unions:  BSA,  bovine  serum  albumin;  PBS,  phosphate-buffered 
saline. 


Introduction 

Nervous  systems  first  appeared  in  cnidarians  or  in  a 
closely  related  ancestor  group.  The  basic  plan  of  the  cni- 
darian  nervous  system  is  a  diffuse  network  of  nerve  cells, 
but  in  some  members  of  this  group,  such  as  medusae, 
nerve  cells  also  can  aggregate  in  nerve  plexuses,  nerve 
rings,  or  sense  organs.  Sea  anemones  have  complex  neu- 
ronal nets  and  nerve  plexuses  in  both  the  inner  and  outer 
epithelial  layers  (Grimmelikhuijzen  and  Westfall,  1995). 
From  sea  anemones,  a  variety  of  neuropeptides,  including 
the  closely  related  Antho-RWamide  I  (<Glu-Ser-Leu-Arg- 
Trp-NH:)  and  Antho-RWamide  II  (<Glu-Gly-Leu-Arg- 
Trp-NH2)  have  been  isolated  (Graff  and  Grimmelik- 
huijzen, 1988a.  b:  Grimmelikhuijzen  et  a/..  1992).  The 
Antho-RWamides  are  present  in  neurons  of  many  body 
regions  of  sea  anemones,  but  Antho-RWamide-immu- 
noreactive  neurons  are  especially  dense  in  the  upper  body 
column,  where  they  innervate  the  oral  sphincter  muscle 
(Graff  and  Grimmelikhuijzen.  1988a;  Grimmelikhuijzen 
el  a/..  1989,  1992).  The  oral  sphincter  muscle  is  a  ring  of 
circular  muscle  fibers  embedded  in  the  gelatinous  middle 
layer,  the  mesoglea.  of  the  upper  body  wall.  During  periods 
of  danger  and  environmental  stress,  it  contracts  to  close 
the  animal  and  protect  the  retracted  apical  tentacles.  The 
cell  bodies  of  the  Antho-RWamide-positive  neurons  in- 
nervating the  sphincter  appear  to  be  located  in  the  gas- 
trodermis  (endoderm)  of  the  upper  body  wall,  whereas 
their  processes  project  across  the  mesoglea  and  ramify 
into  long,  fine  projections  paralleling  the  circular  bundles 
of  sphincter  muscle  fibers  (Graff  and  Grimmelikhuijzen, 


280 


ANTHO-RWAMIDE  AT  SEA  ANEMONE  SYNAPSES 


281 


1988a:  Gnmmelikhuijzen  et  at..  1989.  1992).  No  synaptic 
contacts  between  neurons  and  muscle  fibers  can  be  seen 
at  the  light  microscope  level. 

In  physiological  experiments,  the  Antho-RWamides 
(10~8.U)  induced  tonic  contractions  in  isolated  oral 
sphincter  muscle  rings  and  cells  isolated  from  the  sphincter 
(McFarlane  et  at..  1991).  Taken  together,  these  data  in- 
dicate that  the  Antho-RWamides  are  transmitters  at  neu- 
romuscular  synapses. 

Electron  microscopic  "immunogold"  techniques,  using 
neuropeptide  antisera  and  colloidal  gold-conjugated  sec- 
ondary antibodies,  have  permitted  the  ultrastructural  lo- 
calization of  neuropeptides  in  dense-cored  or  granular 
vesicles  of  a  variety  of  cnidarian  neurons  (Koizumi  et  at.. 
1 989;  Singla  and  Mackie,  1 99 1 :  Westfall  and  Grimmelik- 
huijzen.  1993).  Antho-RFamide  (<Glu-Gly-Arg-Phe- 
NH;),  the  first  sea  anemone  neuropeptide  to  be  isolated, 
was  demonstrated  in  dense-cored  vesicles  of  bidirectional, 
interneuronal  synapses  of  sea  anemones  (Westfall  and 
Grimmelikhuijzen,  1993).  In  the  present  study,  using  the 
immunogold  technique  with  an  antiserum  against  the 
common  C  terminus  of  the  Antho-RWamides,  we  were 
able  to  label  granular  synaptic  vesicles  at  neuromuscular 
junctions  of  sea  anemones.  This  strongly  supports  our 
hypothesis  that  the  Antho-RWamides  are  transmitters  at 
some  cnidarian  neuromuscular  synapses. 

Materials  and  Methods 

Three  specimens  of  the  sea  anemone  Calliaetis  para- 
silica  (sent  from  Roscoff  Station  Biologique,  France) 
were  anesthetized  using  0.3  M  MgCl:.  Once  relaxation 
was  sufficient,  the  animals  were  cut  using  Personna  Gem 
super  stainless  steel  blades. 

For  light  microscopy,  one  animal  was  placed  in  2.5% 
glutaraldehyde  in  0.05  M sodium  cacodylate,  pH  7.4,  and 
cut  longitudinally  in  half.  Photographs  were  taken  using 
an  OM-2S  Olympus  camera  attached  to  a  Wild  model 
M75  zoom  stereomicroscope  to  locate  the  sphincter  mus- 
cle. Longitudinal  slices  of  the  oral  sphincter  from  the  other 
half  of  the  animal  were  processed,  embedded  in  paraffin, 
sectioned,  mounted  on  glass  slides,  and  stained  with  hae- 
matoxylin  and  eosin.  Photographs  were  taken  of  the 
sphincter  muscle  using  an  Aristoplan  image  analysis  light 
microscope. 

For  electron  microscopy,  two  animals  were  cut  longi- 
tudinally, and  the  lower  body  columns  removed.  Several 
longitudinal  slices  were  cut,  starting  at  one  edge  and  pro- 
ceeding serially.  Each  slice  contained  a  few  tentacles.  The 
slices  were  placed  in  one  of  two  fixatives:  4%  paraform- 
aldehyde — 0.1%  glutaraldehyde  in  O.I.I/  phosphate 
buffer,  pH  7.4;  4%  paraformaldehyde— 0. 1%  glutaralde- 
hyde in  0.1  A/  phosphate  buffered  saline,  pH  6.5  for 
30  min,  and  then  pH  1 1.0  for  3  h  (Berod  et  ill..  1981 ). 


All  tissues  were  rinsed  in  0.1  M  phosphate  buffer,  pH 
7.4,  dehydrated  in  ethanol,  and  then  in  acetone;  infiltrated 
overnight  in  a  mixture  of  Epon  and  Araldite;  and  cut  into 
small  segments  for  final  embedding  by  taking  horizontal 
slices  down  the  length  of  the  oral  sphincter  starting  at  the 
region  near  the  tentacles. 

Thin  longitudinal  sections  of  sphincter  muscle  were  cut 
with  a  diamond  knife  and  mounted  on  Formvar-coated, 
100-mesh,  nickel  grids.  The  sections  were  rinsed  in  doubly 
distilled  water  (ddH:O),  then  exposed  to  saturated  sodium 
metaperiodate  for  30  min  to  open  antigenic  sites.  After  a 
ddH:O  rinse,  the  sections  were  exposed  to  normal  goat 
serum  diluted  1 :20  with  PBS-Tween-BSA  buffer  to  block 
nonspecific  antigenic  sites.  They  were  incubated  for  1  h 
with  rabbit  antiserum  #2061  against  Antho-RWamide, 
diluted  1:50-1:200  with  buffer. 

After  rinsing  in  buffer,  the  sections  were  immunogold 
stained  for  1  h  in  goat  anti-rabbit  IgG  conjugated  to  either 
5  or  15  nm-gold  particles,  diluted  in  buffer  1:10-1:40.  Af- 
ter rinsing  in  buffer  with  BSA,  then  in  PBS,  they  were 
postfixed  for  15  min  in  2%  glutaraldehyde  in  PBS  and 
rinsed  in  ddH:O.  The  sections  were  further  stained  in  7% 
uranyl  acetate  in  70%.  ethanol,  then  in  Reynolds  lead  ci- 
trate and  examined  in  a  Philips  400  transmission  electron 
microscope.  Because  only  2-3  sections  covered  a  grid, 
and  usually  10  grids  were  used  per  experiment,  the  search 
for  synapses  was  slow  and  laborious. 

Control  sections  were  exposed  to  Antho-RWamide 
antiserum  (1:200).  which  had  been  incubated  overnight 
in  100  Mg/ml  of  Antho-RWamide. 

Antiserum  #2061  directed  against  the  C  terminus  (Arg- 
Trp-NH;)  of  both  Antho-RWamides  I  and  II  was  prepared 
as  described  by  Grimmelikhuijzen  (1985).  Arg-Trp-NH2 
was  a  customer  synthesis  by  Bachem  (Bubendorf,  Swit- 
zerland). Only  antisera  against  Arg-Trp-NH:  and  no  other 
antisera  against  the  other  sea  anemone  Arg-X-NH2  pep- 
tides  stained  neurons  in  the  sphincter  muscle  (see  e.g., 
Fig.  2  of  Grimmelikhuijzen  et  at..  1992). 

To  categorize  a  granule  type,  measurements  were  made 
of  10  randomly  selected  granules  or  vesicles  per  gastro- 
dermal  neuron  or  sphincter  muscle  neurite,  and  four  to 
five  granules  or  vesicles  per  synapse.  The  reason  why  only 
granules  were  measured  in  some  cases  is  owing  to  the  fact 
that  the  two-pH  paraformaldehyde  fixation,  which  worked 
best  for  immunogold  labeling  with  antisera  to  the  Antho- 
RWamides.  caused  some  loss  of  membrane  preservation 
around  many  granules. 

Results 

The  oral  sphincter  muscle  of  Calliactis  parasitica  was 
located  in  a  widened  region  of  the  upper  body  column 
mesoglea  (Fig.  1 ).  It  was  composed  of  multiple  layers  of 
myonemes  forming  the  circular,  smooth,  muscle  fibers 


282 


J.  A.  WESTFALL  ET  AL 


Figure  1.  Longitudinal  section  through  whole  glutaraldehyde-fixed  specimen  of  Calliactis  parasitica 
indicating  thickened  mesoglea  containing  oral  sphincter  muscle  (SM )  at  base  of  tentacles  (T).  Note  epidermis 
(E),  gastrodermis  (G).  septa  (S),  pharynx  (P),  and  basal  disk  (BD).  Bar  =  1000  ^m. 

Figure  2.  Light  micrograph  of  a  cross  section  through  the  oral  sphincter  muscle  (SM).  Note  longitudinal 
section  of  tentacle  (T),  epidermis  (E),  diffuse  bundles  of  myonemes  (between  arrowheads)  within  the  mesoglea, 
gastrodermis  (G).  and  septa  (S).  Bar  =  100  ^m. 


encompassing  the  oral  region.  In  a  longitudinal  section 
of  the  animal,  the  bundles  of  oral  sphincter  myonemes 
extended  one  tenth  of  the  length  of  a  3-cm-long  sea  ane- 
mone and  appeared  to  increase  in  number  near  the  oral 
region  (Fig.  2).  The  myonemes  extended  irregularly  to- 
wards the  epidermis,  but  stopped  abruptly  near  a  band  of 
mesoglea  separating  them  from  the  gastrodermis. 

Using  electron  microscopy,  we  observed  various-sized 
granular  vesicles  in  bipolar-like  nerve  cells  of  the  gas- 
troderrnal  nerve  net.  The  granules,  which  averaged 
149.3  ±  4.1  nm  in  diameter,  were  immunoreactive  to 


Antho-RWamide  (Fig.  3).  The  granules  labeled  with 
both  5  nm  gold  (upper  inset  Fig.  3)  and  15  nm  gold 
(middle  inset)  and  were  present  at  a  neuromuscular 
synapse  (lower  inset).  The  synaptic  vesicles  averaged 
47.5  ±  2.5  nm  in  diameter. 

Nerve  processes  from  the  gastrodermis  crossed  the 
muscle-free  border  of  the  mesoglea  and  entered  into  the 
individual  oblong  bundles  of  oral  sphincter  myonemes 
(Fig.  4).  These  myonemes  were  composed  of  closely 
packed  bundles  of  myofilaments,  aggregated  at  one  side, 
and  of  a  myofilament-free  area  at  the  other  side.  An 


ANTHO-RWAMIDE  AT  SEA  ANEMONE  SYNAPSES 


283 


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i  i .  -    ' 


Figure  3.  Electron  micrograph  of  neuronal  perikaryon  and  neurite  containing  granular  vesicles  im- 
munogold-labeled  with  antisera  to  Antho-RWamide  in  gastrodermal  nerve  plexus.  Note  large  neurite  (N) 
with  various-sized  granular  vesicles  (box)  and  nucleus  (NU)  of  neuron.  Bar  =  1  /*m.  Insets:  High  magnification 
of  boxed  area  of  neurite  with  5  nm  gold  marker  in  large  granules  (arrowhead,  upper  inset),  serial  section  of 
neurite  with  smaller  granules  labeled  with  15  nm  gold  particles  (arrowhead,  middle  inset),  and  5  nm  gold 
particles  in  granular  vesicles  (arrowhead,  lower  inset)  at  a  neuromuscular  synapse  with  transverse  filaments 
in  the  synaptic  cleft  (arrow).  Bar  =  0.25  jim  (upper;  middle)  and  0.1  ^m  (lower). 


284 


J.  A.  WESTFALL  ET  AL 


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Figure  4.  Ultrathin  section  of"  Antho-RWamide  immunoreactive  neurite  (N)  passing  into  a  bundle  of 
oral  sphincter  myonemes  (MY)  in  the  mesoglea  (ME).  Note  small  neurites  with  either  clear  or  granular 
vesicles  (arrowheads).  Bar  =  1  ^m.  Inset:  High  magnification  of  neurite  with  immunogold-laheled  granular 
vesicles.  Bar  =  0.2}  /jm. 


ANTHO-RWAMIDE  AT  SEA  ANEMONE  SYNAPSES 


285 


occasional  nucleus  was  observed  in  these  myonemes,  sug- 
gesting that  they  are  complete  muscle  cells.  The  neurites 
that  invaded  the  sphincter  usually  were  associated  with 
the  myofilament-free  areas  of  the  myonemes. 

Dense,  granular  vesicles,  varying  in  size  and  having  an 
average  diameter  of  78.8  ±  3.3  nm  were  distributed  un- 
equally within  the  slender,  0.2-0.3  /urn-diameter  neurites. 
Groups  of  these  granules  were  immunoreactive  to  Antho- 
RWamide  antisera  using  both  5  and  15  nm  gold  markers 
(inset  Fig.  4).  Most  neurites  observed  within  individual 
bundles  of  myonemes  had  densely  granular  vesicles,  al- 
though occasional  groups  of  nongranular  vesicles  were 
present. 

Typical  neuromuscular  synaptic  foci  were  few  in  num- 
ber and  difficult  to  locate  in  the  oral  sphincter  muscle, 
but  several  putative  immunoreactive  neuromuscular  syn- 
apses were  observed  (Fig.  5).  The  presynaptic  vesicles  were 
aligned  at  the  presynaptic  membrane  opposite  a  series  of 
cross  filaments  in  the  synaptic  cleft  and  a  postsynaptic 
density  (Fig.  5b).  The  synaptic  cleft  ranged  from  9  to 
18  nm  in  width.  Sometimes,  it  took  two-to-three  serial 
sections  through  a  synapse  to  verify  the  presence  of  cross 


filaments  in  the  synaptic  cleft  at  loci  where  granular  ves- 
icles were  gold-labeled  with  antisera  to  Antho-RWamide. 
Occasionally,  vesicles  attached  to  the  presynaptic  mem- 
brane appeared  empty  (Fig.  5b),  although  gold  label  was 
present.  The  synaptic  vesicles  averaged  63  ±  4.4  nm  in 
diameter. 

Experimental  serial  sections,  incubated  in  Antho- 
RWamide  antisera,  had  immunoreactive  granular  vesicles 
in  some  neurites  (Fig.  6a,  b).  Control  sections,  incubated 
in  antisera  which  had  been  incubated  overnight  in  100  ^g/ 
ml  of  Antho-RWamide,  did  not  stain  with  immunogold 
(Fig.  6c).  A  neurite  adjacent  to  those  with  immunoreactive 
granular  vesicles  contained  electron-lucent  vesicles,  which 
were  not  immunoreactive  to  Antho-RWamide  antisera 
(Fig.  6a.  b). 

Discussion 

Both  Antho-RWamides  I  and  II  stimulate  contractions 
in  rings  of  sphincter  muscle  and  in  isolated  sphincter 
muscle  cells  from  Calliactis  parasitica  (McFarlane  el  al., 
1991).  In  this  study,  we  have  found  that  neurons  make 


Figure  5.  Three  examples  (a.  b,  c)  of  neuromuscular  synapses  with  Antho-RWamide-labelled  presynaptic 
granules  and  vesicles  in  oral  sphincter  muscle.  Note  parallel  pre-  and  postsynaptic  densities  (between  arrows, 
a),  cross  filaments  in  synaptic  cleft  (arrow,  b),  and  1 5  nm-gold  label  specific  to  presynaptic  granular  vesicles 
(arrowheads,  c).  Muscle  cell  (M).  Bar  =  0. 1  /jm. 

Figure  6.  Three  serial  thin  sections  through  a  cross-sectioned  bundle  of  oral  sphincter  myonemes  revealing 
immunoreactive  granular  vesicles  in  experimental  (a.bj  and  nonimmunoreactive  granular  vesicles  in  control 
section  (c).  Note  clear  vesicles  without  gold  label  in  adjacent  upper  neunte  (N).  Bar  =  0.25  ^m. 


286 


J.  A.  WESTFALL  ET  AL. 


morphologically  identifiable  synapses  with  the  sphincter 
muscle  cells  and  that  these  synapses  contain  Antho- 
RWamide-immunoreactive  granular  vesicles.  This  sup- 
ports our  hypothesis  that  the  Antho-RWamides  are  trans- 
mitters at  the  neuromuscular  junctions  of  the  sphincter. 

In  a  previous  study,  we  located  Antho-RFamide  im- 
munoreactivity  in  granular  vesicles  at  two-way  interneu- 
ronal  synapses  in  the  sea  anemone  Anthopleura  (Westfall 
and  Grimmelikhuijzen,  1993).  Therefore,  peptidergic 
synapses  clearly  play  an  important  role  in  primitive  ner- 
vous systems. 

The  long  slender  neurites,  which  cross  the  mesoglea  to 
innervate  the  sphincter  muscle,  contain  Antho-RWamide 
immunoreactive  granules  of  varying  sizes,  whereas  the 
granular  vesicles  at  synapses  appear  to  be  somewhat 
smaller.  The  synthesis  of  vertebrate  neuropeptides  follows 
a  stepwise  pattern:  a  prepropeptide  is  formed  in  the  rough 
endoplasmic  reticulum.  then  reduced  in  the  Golgi  com- 
plex to  a  smaller  propeptide  and  is  eventually  cleaved 
into  the  active  peptides  in  vesicles  (Brownstein,  1982). 
This  pattern  of  synthesis  and  reduction  of  the  Antho- 
RWamide  precursor  may  also  occur  in  sea  anemone  neu- 
rons, and  it  may  explain  the  difference  in  size  between 
granules  in  the  neurites  and  in  the  synapses.  Similarly, 
an  immunoreactive  nucleated  nerve  cell  in  the  gastro- 
dermal  nerve  plexus  has  both  large  and  small  granular 
vesicles,  the  latter  being  denser.  Slightly  smaller  granular 
vesicles  have  been  observed  at  an  immunoreactive  gastro- 
dermal  neuromuscular  synapse. 

Sea  anemone  muscles  undergo  spontaneous  autono- 
mous movements  similar  to  those  of  the  smooth  muscle 
of  the  vertebrate  intestine,  which  result  in  constriction 
and  extension  of  this  tube-like  structure  (Parker,  1919). 
Autonomic  nerve  fibers,  which  innervate  smooth  muscle 
of  the  vertebrate  intestine,  do  not  always  terminate  with 
morphologically  distinct  neuromuscular  synapses  (Ja'nig. 
1978).  A  similar  situation  might  exist  in  the  cnidarian 
smooth  muscles.  However,  some  neuromuscular  junc- 
tions do  occur  in  cnidarians  and  can  be  recognized  by  the 
presence  of  dense-cored  or  clear  vesicles  (80-100  nm  in 
diameter)  aligned  on  the  presynaptic  side  of  a  pair  of  elec- 
tron-dense synaptic  membranes  separated  by  a  1 5-25  nm- 
wide  cleft  (Westfall,  1973). 

In  this  study  of  gastrodermal  and  oral  sphincter  neu- 
romuscular synapses  of  the  sea  anemone  Calliactis  par- 
asitica,  there  are  two  to  four  or  more  granular  vesicles 
aligned  at  paired,  electron-dense,  synaptic  membranes 
separated  by  a  1 5-nm-wide  cleft  traversed  by  a  series  of 
cross  filaments.  The  neuromuscular  synapses  are  similar 
ultrastructurally  to  the  interneuronal  synapses  in  the  sea 
anemone  nerve  plexus  (Westfall,  1970,  1987).  Electron- 
lucent  vesicles,  which  do  not  label  with  antisera  to  Antho- 
RWamide,  are  present  in  a  few  other  neurites.  Although 
nothing  is  known  about  the  putative  neurotransmitter 


substances  at  sea  anemone  neuromuscular  synapses  with 
electron-lucent  vesicles,  this  study  demonstrates  Antho- 
RWamide  immunoreactivity  in  granular  vesicles  at  neu- 
romuscular synapses. 

Ross  (1960a,b)  has  stated  that  adrenaline  causes  con- 
traction in  sphincter  and  circular  muscle  preparations  of 
Calliactis  parasitica  and  Metridium  senile:  Wood  and 
Lentz  (1964)  have  claimed  that  adrenaline  is  present  in 
the  mesenteries  of  Metridium.  Other  studies  have  sug- 
gested that  catecholamines  (Dahl  el  ai.  1963;  Anctil  et 
al.,  1984;  DeWaele  et  ai.  1987;  Umbriaco  et  ai.  1990) 
and  DOPA  (Carlberg.  1983)  are  present  in  nerve  cells  of 
various  anthozoa.  Taurine-like  immunoreactivity  has 
been  reported  in  the  motor  nerve  net  of  the  scyphozoan 
jellyfish  Cyanea  capillata  (Carlberg  et  ai.  1995).  Thus, 
besides  Antho-RWamides.  other  neuromuscular  trans- 
mitters also  may  be  present  in  the  anthozoans. 

Acknowledgments 

Contribution  No.  95-570-J  of  the  Kansas  Agricultural 
Experiment  Station.  We  thank  Shelly  Christenson  in  the 
Diagnostic  Medicine/Pathobiology  Histopathology  Lab- 
oratory for  instruction  on  the  tissue  processor  and  staining 
apparatus,  and  Dr.  J.  W.  Sharp  for  instruction  on  the 
Image  Analysis  System.  This  study  was  funded  by  NSF 
grant  IBN-9 120161  to  J.  A.  Westfall  and  by  a  grant  from 
the  Danish  Natural  Science  Research  Council  to  C.  J.  P. 
Grimmelikhuijzen. 

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Isolation  and  Partial  Characterization  of  the  Pink  and 
Blue  Pigments  of  Pocilloporid  and  Acroporid  Corals 

SOPHIE  G.  DOVE,  MISAKI  TAKABAYASHI,  AND  OVE  HOEGH-GULDBERG 

School  ol  Biological  Sciences,  Building  A08,  University  of  Sydney,  2006  NSW  Australia 


Abstract.  The  compounds  responsible  for  the  pink  and 
blue  colors  of  two  families  of  hermatypic  corals  (Pocil- 
loporidae,  Acroporidae)  from  the  southern  Great  Barrier 
Reef  were  isolated  and  biochemically  characterized.  Iso- 
lation of  the  pink  pigment  from  Pocillopora  damicomis 
(named  pocilloporin,  Xmax  =  560  nm,  390  nm)  revealed 
that  it  was  a  hydrophilic  protein  dimer  with  a  native  mo- 
lecular weight  of  approximately  54  kD  and  subunits  of 
28  kD.  The  subunits  are  not  linked  by  disulfide  bonds. 
Attempts  to  dissociate  the  chromophore  from  the  protein 
proved  unsuccessful.  Denaturing  the  protein  with  heat 
(60°C)  or  5%  sodium  dodecyl  sulfate  (SDS)  removed  the 
560-nm  absorbance  peak  without  introducing  a  detectable 
bathochromic  shift.  In  acetone,  ethanol,  ether,  and  chlo- 
roform, the  pigment  precipitates  out  of  solution,  leaving 
a  colorless  supernatant.  These  properties  suggest  that  the 
protein  and  chromophore  are  covalently  linked.  Ion  anal- 
ysis revealed  that  the  pigment  does  not  have  metal  ions 
chelated  to  it.  Coral  pigments  were  also  isolated 
from  pink  morphs  of  other  pocilloporids.  Seriato/wra 
hy\th\  (Xmax  =  560  nm)  and  Stylophora  pistillata  (Xmax 
=  560  nm);  and  from  bluish  regions  of  the  acroporids, 
Acropom  formosa  (blue:  Xmax  =  590  nm)  and  Acropora 
digitijem  (purple;  Xmax  =  580  nm).  With  the  exception  of 
A.  formosa,  all  the  corals  examined  had  pigments  with 
the  same  native  (54  kD)  and  subunit  (28  kD)  molecular 
weights  as  those  of  P.  damicomis.  A.  formosa  pigment 
has  a  native  molecular  weight  of  about  82.6  kD  and  three 
subunits  of  28  kD.  The  pigments  isolated  from  each  of 
these  coral  species  have  properties  similar  to  those  de- 
scribed for  P  damicomis.  Isolation  and  biochemical  pu- 
rification of  the  pigment  enabled  the  exploration  of  the 
function  of  the  pink  pigment.  Three  possibilities  were 
eliminated.  The  compound  does  not  act  as  (i)  a  photo- 
protectant  for  shielding  the  photosynthetic  pigments  of 


Received  15  November  1994;  accepted  5  September  1995. 


symbiotic  zooxanthellae  against  excessive  irradiances,  (ii) 
a  fluorescent  coupling  agent  for  amplifying  the  levels  of 
photosynthetically  active  radiation  available  for  resident 
zooxanthellae.  or  (iii)  a  UV-screen  against  the  high  UV 
levels  of  shallow  tropical  marine  environments. 

Introduction 

The  vivid  colors  of  reef-building  corals  and  other  in- 
vertebrates are  among  the  most  conspicuous  elements  of 
a  living  coral  reef.  With  this  in  mind,  it  is  perhaps  sur- 
prising that  so  little  is  known  about  the  identity  and  role 
of  color  in  reef-associated  organisms  (Czeczuga,  1983). 
The  pigmentation  of  reef-building  corals  occurs  in  the 
skeleton  of  some  species  and  in  the  ectodermal  and  en- 
dodermal  tissues  of  others  (Kawaguti,  1944;  Takabayashi 
and  Hoegh-Guldberg.  1995).  The  chemical  identities  of 
compounds  responsible  for  coral  color  are  known  only 
in  a  few  cases.  Pigments  associated  with  some  hydrocoral 
and  scleractinian  coral  skeletons  have  been  identified  as 
carotenoprotein  complexes  (Fox  and  Wilkie,  1970;  Fox, 
1972;  Ronneberg  et  a/.,  1979).  Red  and  green  caroteno- 
protein complexes  are  also  present  in  askeletal  cnidarians 
such  as  Actinia  euitina  and  Epiactis  prolifera  (Czeczuga, 
1983).  The  blue  pigment  from  the  skeleton  of  the  hydro- 
coral  Heliopora  caerulea  has  been  identified  as  a  calcium- 
bonded  biliverdin,  which  belongs  to  the  tetrapyrrole  group 
of  pigments  (Tixier,  1945,  cited  in  Fox  and  Wilkie,  1970). 
Among  the  most  prominent  pigments  associated  with  the 
tissues  of  corals  are  the  pinky-mauve  pigments  that  are 
typical  of  the  Pocilloporidae,  Acroporidae,  Poritidae, 
Fungiidae,  and  Meruliniidae  (5  out  of  the  16  families  of 
reef-building  corals.  Veron.  1986).  Although  several  skel- 
etal pigments  have  been  purified  and  identified,  the  nature 
of  these  tissue-associated  pigments  in  corals  remains 
unexplored. 

Tissue-based  pigments  have  been  extracted  from  corals 
in  early  studies  using  distilled  water  or  buffer  solutions 


THE  PIGMENTS  OF  HERMATYPIC  CORALS 


289 


(Kawaguti.  1944;  Shibata.  1969).  hut  further  purification 
and  characterization  have  not  been  attempted.  The  Indo- 
Pacific  coral  Pocillopora  damicomis  from  One  Tree  Island 
(southern  Great  Barrier  Reef)  shows  a  range  of  colony 
colors  from  pink  to  brown.  In  this  case,  the  color  has  been 
identified  as  due  to  a  hydrophilic  compound  found  in  the 
cells  of  the  coral;  the  compound  is  inducible  by  visible 
light  in  the  pink  morph  of  P.  damicomis  (Takabayashi 
and  Hoegh-Guldberg,  1995).  Interestingly,  the  presence 
of  this  compound  in  pink  morphs  is  associated  with  several 
physiological  characteristics  such  as  reduced  growth  rates 
(Takabayashi  and  Hoegh-Guldberg.  1995)  and  superior 
competitive  abilities  relative  to  the  brown  morph  (Taka- 
bayashi. 1994).  Despite  these  correlations,  the  exact  iden- 
tity and  function  of  this  pigment  remain  elusive. 

In  this  study,  the  pink  pigment  from  the  tissues  of  P. 
damicomis  was  isolated  and  characterized  and  its  function 
was  explored.  Pigment  complexes  were  also  isolated  from 
the  pink  morphs  of  the  related  pocilloporids  Stylophora 
pistillata  and  Seriatopora  hystrix,  and  the  blue  regions  of 
colonies  of  the  acroporids  Acropora  formosa  and  A.  dig- 
itifera.  The  color  in  all  five  cases  is  associated  with  a  similar 
protein  complex,  which  in  the  case  of  P.  damicomis  does 
not  appear  to  function  as  a  photoprotectant,  U  V-screening 
agent,  or  fluorescent  coupling  pigment. 

Materials  and  Methods 

This  is  the  first  study  to  purify  the  pigment  complex 
associated  with  the  pink  color  of  Pocillopora  damicomis. 
To  simplify  the  description  of  this  compound  in  the  fol- 
lowing text,  the  compound  is  referred  to  hereafter  as  "po- 
cilloporin."  Similar  compounds  from  other  species  (i.e., 
with  the  same  molecular  weight,  subunit  size,  or  both) 
will  be  referred  to  as  "pocilloporin-like"  compounds. 

Purification  of  pocilloporin  and  pocilloporin-like 
compounds 

Corals  (Pocillopora  damicomis,  Seriatopora  hystrix, 
Stylophora  pistillata,  Acropora  digit ifera.  and  A.  formosa) 
were  collected  at  a  depth  of  2  m  from  One  Tree  Island 
lagoon  near  the  One  Tree  Island  Research  Station  (Uni- 
versity of  Sydney)  at  the  southern  end  of  the  Great  Barrier 
Reef,  Australia,  in  May  1994.  Pigments  were  extracted 
by  immersing  coral  branches  in  0.06  M  KH2PO4, 0.06  M 
K:HPO4  pH  6.65  (phosphate  buffer  ==  "raw  extract"; 
Takabayashi  and  Hoegh-Guldberg,  1995)  for  24  h  at  4°C. 
Raw  extracts  were  concentrated  and  partially  purified  by 
centrifugation  (Centrifuge  17RS.  Heraeus  Sepatech) 
through  a  Centricon  30  (Amicon,  molecular  weight  cutoff 
=  30  kD.  time  and  speed  determined  by  volume  and 
Centricon  specifications).  Wavelengths  of  maximum  ab- 
sorbance  (Xmax)  and  protein  concentrations  were  deter- 
mined spectrophotometrically  (Pharmacia  Ultrospec  III 
and  Autofill  III;  Whitaker  and  Granum,  1980). 


Gel  filtration.  Pocilloporin  (from  P.  damicomis,  Xmax 
=  560  nm)  and  pocilloporin-like  proteins  (from  Seriato- 
pora hystrix,  Stylophora  pistillata.  and  the  Acropora  spe- 
cies, Xmax  =  560-590  nm)  were  further  purified  by  gel 
filtration  on  a  Superose  FPLC  column  (Pharmacia,  12 
HR  10/30).  The  sample  was  eluted  from  the  column  with 
phosphate  buffer  pH  6.65  at  a  flow  rate  of  0.5  ml  min~' 
and  the  absorbance  of  the  protein  was  monitored  using  a 
multi-wavelength  detector  (Model  490E;  Millipore- Wa- 
ters, Australia).  The  major  peaks  were  collected  and  an- 
alyzed by  polyacrylamide  gel  electrophoresis  in  the  pres- 
ence of  sodium  dodecyl  sulfate  (SDS-PAGE)  to  determine 
subunit  molecular  weights.  Collected  fractions  were  rerun 
through  the  HPLC  to  determine  purity  (symmetry  and 
overlay  of  280-nm  peak  and  560-nm  peak)  and  the  ex- 
tinction coefficient  for  pocilloporin  at  560  nm  (see 
below). 

SDS-PAGE  gel  electrophoresis.  Polyacrylamide  gel 
electrophoresis  (15%  running  gel)  in  the  presence  of  so- 
dium dodecyl  sulfate  (SDS-PAGE)  was  performed  using 
a  modification  of  the  method  described  by  Laemmli 
( 1970).  In  general,  /5-mercaptoethanol  (5%)  was  added  to 
all  samples  and  the  samples  were  boiled  for  5  min  prior 
to  loading  on  the  gel.  However,  to  study  the  effects  of 
boiling  and  reducing  conditions  on  the  mobility  of  sample 
subunits  during  electrophoresis.  boiled  and  unboiled 
samples  were  run  in  the  presence  and  absence  of  |8-mer- 
captoethanol,  sodium  dodecyl  sulfate  (SDS),  or  both.  In 
these  experiments,  and  others  involving  SDS-PAGE,  pro- 
tein subunits  were  revealed  by  Coomassie  blue  staining 
(Righetti  et  ai,  1990).  All  gels  used  Biorad  low  molecular 
weight  standards. 

Properties  oj  isolated  compounds 

Relationship  between  pocilloporin  and  co-eluting  pro- 
tein. To  investigate  whether  pocilloporin  was  a  protein, 
gel  filtration  was  done  on  extracts  of  regions  of  two  col- 
onies of  P.  damicomis  that  varied  in  the  intensity  of  pink 
color.  These  extracts  were  used  to  investigate  the  rela- 
tionship between  absorbance  at  560  nm  and  co-eluting 
protein.  The  relationship  between  protein  abundance 
(280-nm  absorbance.  Dawson  et  ai,  1986)  and  pigment 
(560-nm  absorbance)  was  measured  by  relating  the  area 
of  a  defined  280  slice  (Fig.  4A)  to  that  of  the  corresponding 
560  slice  (Maxima  820  software;  Millipore-Waters.  Aus- 
tralia). All  chromatograms  were  collected  on  the  same 
day  to  minimize  the  effects  of  changes  in  column  perfor- 
mance. The  same  start  and  end  time  points  were  used  for 
delimiting  chromatogram  slices. 

Measurement  of  extinction  coefficient  at  560  nm  for  po- 
cilloporin. The  extinction  coefficient  of  pocilloporin,  t_S60, 
was  measured  using  Beer's  law  (Nobel,  1983).  where  the 
path  length  of  the  detector  (Model  490E;  Millipore-Wa- 
ters, Australia)  was  1  cm.  and  where  the  values  for  A560 


290 


S.  G.  DOVE  ET  AL. 


and  the  molar  concentration  (A/)  of  pocilloporin  were 
derived  from  280-nm  and  560-nm  chromatograms  of  pu- 
rified pocilloporin  (Fig.  1  A).  The  molar  concentration  of 
pocilloporin  was  calculated  in  the  following  manner.  The 
amount  of  pocilloporin  (micrograms)  was  calculated  by 
converting  the  area  of  a  very  slim  "slice"  of  the  280-nm 
chromatogram  (Area  A.  Fig.  1  A)  to  protein  concentration, 
using  a  relationship  previously  determined  between  the 
total  area  under  a  280-nm  chromatogram  and  known 
amounts  of  protein  from  several  different  colonies  ofPi>- 
ctllopora  damicomis  injected  through  the  column  (protein 
in  micrograms  =  8.89  X  area  +  0.10,  r2  =  0.95).  The 
volume  of  each  slice  was  calculated  by  multiplying  the  .v- 
axis  (time  elapsed.  Fig.  1A)  of  the  slice  by  the  flow  rate 
(0.5  ml-  min"1).  The  resulting  concentration  of  pocillo- 
porin (grams  per  liter)  was  then  converted  into  the  molar 
concentration  (A/)  of  pocilloporin  by  using  the  native 
molecular  weight  of  pocilloporin  (=  54  kD,  see  Results). 
This  method  was  used  to  determine  the  extinction  coef- 
ficient because  it  required  only  relatively  small  amounts 
of  protein  and  thus  could  be  applied  to  only  the  purest 
of  fractions  (determined  by  observation  of  the  symmetrical 
overlay  of  the  560-nm  and  280-nm  chromatograms). 

To  verify  the  validity  of  the  above  method,  the  extinc- 
tion coefficient  for  pocilloporin  at  560  nm  was  also  de- 
termined using  a  more  conventional  technique  employing 
two  methods  of  measuring  protein  concentration  (Brad- 
ford, 1976;  Whitaker  and  Granum.  1980).  Five  aliquots 
of  raw  extract  that  had  been  molecular  weight  filtered 
(using  Centricons)  were  injected  into  the  gel  filtration  col- 
umn, and  the  pocilloporin  fractions  collected.  The  col- 
lected fractions  were  pooled  and  concentrated,  and  the 
absorbances  were  measured  at  235,  280,  and  560-nm  with 
a  spectrophotometer  (Pharmacia  Ultraspec  III). 

Ion  content  of  pocilloporin.  Many  chromophores  in- 
clude a  chelated  metal  ion  (Fox,  1979).  To  determine 
whether  pocilloporin  has  a  constituent  metal  ion.  the  ion 
content  of  pure  pocilloporin  was  investigated.  Pocilloporin 
was  purified  by  gel  filtration  as  described  above.  About 
20 /jg  of  protein  (10^1)  was  placed  in  70%  nitric  acid 
(AristaR,  BHD  Chemicals)  for  4  h  at  95°C.  and  diluted 
to  3  ml  of  0.7%,  HNO_,  with  Milli-Q  distilled  water.  Ion 
content  was  then  determined  by  inductively  coupled 
plasma  mass  spectroscopy  (ICP-MS;  Elan  5000,  Perkin 
Elmer)  using  the  total  quant  peak-hopping  option.  The 
blank  contained  10^1  of  phosphate  buffer  eluant  from 
HPLC  heated  in  70%  nitric  acid  (AristaR,  BHD  Chemi- 
cals) and  diluted  to  3  ml  of  0.7%  HNO,  with  Milli-Q  dis- 
tilled water. 

Thermal  li'^iliiy  of  pigment  compound.  The  pocillo- 
porin fraction  w  is  collected  as  it  eluted  from  the  column. 
Fractions  were  concentrated  by  centrifugation  through  a 
Centricon  30.  The  concentrated  sample  was  aliquoted  into 
25-^1  proportions  in  0.6-ml  Eppendorf  microcentrifuge 
tubes.  Samples  were  then  held  in  a  water  bath  at  temper- 


Time  elapsed  (mm) 


\  Area  A 


0018 


22 


24  26  28 

Elution  time  (min) 


560  nm 
280  nm 

30 


B 

'  005 
0045 

C/320  =  (A320/  A560  '  '  ^560 

?  absorbance 

004 
0.035 

003 

A560                              /          \ 

HI 

0.025 

A320          /'                 \"~-. 
.-  T_/                    \    ""•  320  nm 

cc 

002 

2 

24                         26                         28                         30 

Elution  time  (mm) 

Figure  1.  (A)  Example  of  purified  pocilloporin  showing  symmetrical 
overlay  of  560  nm  and  280  nm  chromatograms  and  labeled  parameters 
used  in  the  calculation  of  the  extinction  coefficient  at  560  nm.  (B)  Cal- 
culation of  the  extinction  coefficient  for  pocilloporin  at  320  nm  from 
the  extinction  coefficient  at  560  nm. 


atures  of  40°,  60°,  and  100°C  for  10  min  before  being 
injected  onto  the  column.  A  control  (RT,  Fig.  5)  sample 
was  injected  without  incubation  in  the  water  bath.  Peak 
area  was  determined  at  both  560  nm  and  280  nm,  and 
the  ratio  of  560  nm  to  280  nm  was  determined  (Maxima 
820  software;  Millipore-Waters.  Australia).  In  a  separate 
experiment,  pocilloporin  was  introduced  into  the  (60°C) 
cell  of  a  spectrophotometer,  heated  to  60°C.  and  main- 
tained at  this  temperature  for  10  min,  during  which  it  was 
scanned  from  200  nm  to  900  nm  once  every  80  s  to  de- 
termine changes  in  absorbance  profile  with  heating  (Phar- 
macia Ultraspec  III). 

Solubility  of  pocilloporin  and  pocilloporin-like  com- 
pounds. Ethanol.  acetone,  ether,  and  chloroform  were 
added  to  separate  phosphate  buffer  extracts  (50%-  v/v  each) 
of  the  coral  tissues.  Solubility  was  checked  by  looking  for 
a  precipitate  in  samples  (1.5  ml)  after  vortexing  and  al- 
lowing samples  to  settle. 

Effect  of  denaturing  agents  on  pigment.  Five  percent 
SDS  was  added  to  phosphate  buffer  extract  of  pigment 
from  P.  damicomis.  The  raw  extract,  and  the  extract  after 
the  addition  of  the  denaturing  agent,  were  scanned  from 
200  nm  to  700  nm  (Pharmacia  Ultraspec  III)  to  measure 
any  bathochromic  shifts  (Xmax  shifts).  Phosphate  buffer 
extracts  from  all  five  coral  species  were  acidified  with 


THI    PIGMENTS  OF  HFRMATYPIC  CORALS 


291 


H,PO4  and  neutralized  with  NaOH.  Samples  were  vor- 
texed  and  spun  for  1  min  in  an  Eppendorf  microcentrifuge 
E,  prior  to  spectrophotometric  and  visual  examination. 
The  extinct  ion  coefficient  ami  the  contribution  ot  pocil- 
loporin  to  the  total  absorbance  of  Pocillopora  damicornis 
at  320  nm.  The  extinction  coefficient  of  pocilloporin  at 
320  nm  (t^o)  was  calculated  by  multiplying  the  e560  for 
pocilloporin  by  the  ratio  of  the  absorbance  at  320  nm  to 
that  at  560  nm  for  four  purified  samples  (Fig.  IB).  The 
greatest  possible  contribution  of  pocilloporin  to  the  total 
UV  absorbance  (320  nm)  was  measured  in  the  following 
manner  for  five  pink  colonies  of  Pocillopora  damicornis. 
The  total  absorbance  at  320  nm  was  measured  for  raw 
extracts  after  they  were  filtered  through  glass  fiber  filters 
(Millipore)  to  remove  suspended  material  (no  color  re- 
mained on  the  filter).  The  filtered  raw  extracts  contain 
both  mycosporine  amino  acids  (Matthews,  1993)  and  po- 
cilloporin. The  portion  of  the  total  320-nm  absorbance 
due  to  pocilloporin  was  calculated  from  the  absorbance 
at  560  nm  of  the  raw  filtered  extract  multiplied  by  the 
ratio  of  e,:o  to  e5t>0  (only  the  pocilloporin  fraction  of  raw 
extracts  absorbs  at  560  nm.  Fig.  2A).  This  value  was  then 
expressed  as  a  percentage  of  the  total  absorbance  at 
320  nm. 

Fluorescent  emission  measurement.  The  fluorescent 
emission  (between  390  and  750  nm)  of  pocilloporin  was 
measured  for  excitation  at  390  and  560  nm  (both  absorp- 
tion maxima)  with  a  luminescence  spectrometer  (LS50B. 
Perkin  Elmer).  Phosphate  buffer  extracts  of  pink  (0.3  mg/ 
ml  pocilloporin  by  Beer's  law  from  e560  with  A390  =  0.505) 
and  brown  (0.1  mg/ml  pocilloporin  with  A390  =  0.519) 
P.  damicornis  branches,  and  a  partially  purified  extract 
of  pink  branches  (retentate  after  centrifugation  through 
a  Centricon  30:  3.7  mg/ml  pocilloporin)  were  used  for 
these  measurements.  If  pocilloporin  is  to  act  as  an  acces- 
sory photosynthetic  pigment  via  fluorescence,  then  the 
concentrations  of  pocilloporin  used  in  these  measure- 
ments should  yield  measurable  fluorescence  (cf.  0.15  jug/ 
ml  chlorophyll  with  A436  =  0.006  provided  a  detectable 
fluorescent  response  for  excitement  at  436  nm). 

Results 

Purification  of  pocilloporin  from  Pocillopora  damicornis 

Phosphate  buffer  extracts  of  pink  and  brown  morphs 
of  P.  damicornis  had  similar  complex  280-nm  chromato- 
grams  (Fig.  2A,  D).  The  key  features  of  these  chromato- 
grams  were  as  follows:  (i)  A  peak  that  also  absorbs  at 
320  nm  (data  not  shown)  eluting  at  37  min  (Fig.  2A,  D; 
MAA),  which  corresponds  to  a  molecular  mass  of  about 
1.3kD.  The  1.3-kD  peak  contained  mycosporine-like 
amino  acids  (MA As)  as  shown  by  CIS  reverse  phase 
HPLC  (data  not  shown).  Previously,  MAAs  have  been 
shown  to  elute  though  gel  nitration  columns  at  times  that 


o> 
o 

a 

o 
</> 

.O 
(0 
0) 


0) 

or. 


Pink  morph                         Brown  morph 

A 

0.1 

MAA  '.                           „  . 

u-             MAA 

>Vii 

\, 

008 

008 

I 

i! 

ji 

0.06 

.'  i                  0.06 

•  : 

0.04 
002 

YP                 /   \ 
T                  :     i       ,         0.04 
.•-•.    /      \     ''"I 

I      J\ 

10      20      30      40      50            10     20      30      40      50 

0.16 

B. 

E. 

0.12 

I                   0.12 

1          I        ^ 

0.08 

S            i       i\                  0.08 

i;       ,  \  /  s 

!\     f   \  }  \ 

0.04 

I  '•..-•••''    L'"'       \.     .,n        0.04 

1  I        j          \  /       ', 

10      20      30      40      50            10      20    1  1  30      40      50 

C.  20-fold  concentration              F.  1  000-fold  concentration 

0.04 

0.06 

t 

0.04 

i 

n 
i\ 

0.02 

r\  —  ~                 002 

JV..-A..  _... 

10      20      30      40      50  10      20      30      40      50 

Retention  time  (min) 

Figure  2.  Comparison  of  the  pink  and  brown  morphs  of  Pocillopora 
(liimicornis,  showing  the  purification  steps  for  the  560-nm  chromatogram 
peak  (pocilloporin).  (A  and  D)  Phosphate  buffer  pigment  extracts:  ('„ 
indicates  void  volume:  MAA  indicates  UV-absorbing  peak.  (B  and  F.) 
Retentate  after  centrifugation  through  a  Centricon  30:  ||  indicates  560- 
nm  fraction  collected.  (C  and  F)  Chromatograms  of  fractions  collected 
from  B  and  E.  respectively.  Note  concentration  difference  between  C 
and  F. 


correspond  to  compounds  of  about  1 .3  kD  despite  having 
an  actual  molecular  weight  of  0.4  kD  (Matthews,  1993). 
(ii)  A  void  volume  peak  (v0)  that  contains  proteins  of  mo- 
lecular mass  greater  than  2000  kD  and  absorbs  slightly  at 
560  nm  (Fig.  2A-F).  (iii)  A  group  of  overlapping  peaks 
occurring  between  20  and  30  min  that  absorb  at  280  nm 
(Fig.  2A,  B,  D,  E).  In  this  region,  the  pink  morph  of  P. 
damicornis  has  a  major  560-nm  absorbing  peak  at 
26.8  min  with  a  small  peak  on  the  front  shoulder 
(Fig.  2C). 

Centrifugation  of  the  phosphate  extract  through  a  Cen- 
tricon 30  reduced  the  relative  quantity  of  UV-absorbing 
compound  (cf.  Fig.  2B,  E  with  Fig.  2A,  D).  Further  pu- 
rification of  the  pink  fraction  revealed  that  it  co-eluted 
with  a  280-nm  absorbing  fraction,  presumably  protein 
with  a  native  molecular  weight  of  about  54  kD  (Fig.  2C). 
Fractions  collected  at  the  same  elution  time  (i.e.,  corre- 
sponding to  54  kD  proteins)  from  the  brown  morphs  of 
/-"  damicornis  did  not  show  significant  absorbance  at 
560  nm  (Fig.  2F),  even  when  concentrated  1000-fold  (as 
opposed  to  20-fold  for  the  pink  morph)  from  the  previous 
step  (Fig.  2E,  B).  Concentrating  the  purified  fractions  from 
the  brown  morph  by  1000-fold  resulted  in  an  amplifica- 
tion of  contaminating  proteins  (see  end  of  next  paragraph). 


292 


S.  G.  DOVE 


SDS-PAGE  of  pocilloporin  fraction  (Fig.  2C)  from  a 
pink  morph  of  P.  damicornis  showed  one  major  band 
with  a  molecular  weight  of  28  kD  (Fig.  3;  lanes  4,  5).  Other 
bands  that  are  present  were  due  to  contamination  inas- 
much as  their  appearance  is  dependant  on  the  start  and 
end  time  of  fraction  collection  (cf.  narrow  collection  pe- 
riods shown  in  Fig.  3;  lanes  4,  5,  with  broader  collection 
period  in  Fig.  8  P.  damicornis:  lane  5).  The  presence  or 
absence  of  /3-mercaptoethanol  or  boiling  or  SDS  did  not 
affect  the  mobility  of  this  28-kD  band  (data  not  shown), 
suggesting  that  disulfide  bonds  are  not  involved  in  the 
linking  of  the  subunits  in  the  native  protein.  SDS-PAGE 
of  the  same  gel  filtration  fraction  taken  from  a  brown  P. 
damicornis  morph  (Fig.  2F)  showed  a  very  faint  band  at 
28  kD  amongst  a  smear  of  other  bands  with  a  wide  mo- 
lecular weight  range  (Fig.  3;  lanes  1,  2). 

Properties  of  pocilloporin  and  related  compounds 

Relationship  between  pocilloporin  and  co-eluting  pro- 
tein. The  association  between  the  absorbance  at  560  nm 
and  co-eluted  protein  was  investigated  to  strengthen  the 
conclusion  that  pocilloporin  was  a  protein  present  in  pig- 
mented  coral  morphs  but  absent  in  unpigmented  morphs. 
The  280-nm  chromatograms  of  brown  fragments  of  P. 
damicornis  had  a  concave  shape  in  the  region  of  the  max- 
imal absorbance  at  560  nm  (26.8  min.  Fig.  4A).  The  280- 
nm  chromatograms  of  pink  fragments  of  P.  damicornis 
had  a  convex  shape  in  this  region  (Fig.  4A).  The  shape  of 
these  curves  suggests  the  absence  of  a  protein  in  the  brown 
morph  that  is  present  in  the  pink  morph  of  P.  damicornis. 
That  this  protein  is  bound  to  or  part  of  the  pigmented 
compound  is  supported  by  the  strong  positive  correlation 


Brown  morph 
4  M9        8  MQ 


Pink  morph 
4  M9        8  fj 


97- 
66- 

45- 


30- 
21  - 

14- 


Figure  3.  Fifteen  percent  SDS-PAGE  of  560-nm  fraction  from  pink 
and  brown  morphs  of  Pocillopora  damicornis:  Lane  I,  2.  brown  morph 
(4  and  8  >ig.  respectively);  lane  3.  Biorad  low  MW  standards;  lane  4.  5, 
pink  morph  (4  and  8  Mg.  respectively).  The  band  corresponding  to  a  28- 
kD  subunit  is  indicated  by  an  arrow. 


Pink  280  nm 


B 

160 

0) 

I  120 
|  .-•'  Brown  280  nm      o 

Pink  560  nm  !£      80 

O 

P     40 


Brown  560  nm 


26         27          28 
Elution  Time  (min) 


-60    -30      0      30     60 
Area  of  280  slice 


Figure  4.  Relationship  between  absorbance  at  560  nm  and  280  nm. 
(A)  Chromatograms  of  phosphate  buffer  extracts  from  pink  and  brown 
branches  of  Pocilloporu  damicurnis:  shaded  slices  show  area  used  in  280 
determination.  (B)  Linear  relationship  between  area  of  560  slice  and  area 
of  280  slice:  r  =  0.97;  (•)  colony  1:  (O)  colony  2. 


between  the  560-nm  absorption  and  280-nm  absorption 
(Fig.  4B:  linear  relation  with  r2  =  0.97). 

Extinction  coefficient.  The  extinction  coefficient  (t560) 
of  pocilloporin  was  determined  directly  from  three  in- 
dependent chromatograms  of  purified  pocilloporin  (e.g.. 
Fig.  1  A)  and  was  34059  ±  1635cm"1  A/"' (mean  ±  SEM). 
The  extinction  coefficient  measured  using  a  spectropho- 
tometer  applied  to  purified  protein  from  five  HPLC  runs 
was  3 1950  cm  '  A/"1  (using  the  method  of  Whitaker  and 
Granum,  1980,  to  measure  protein)  and  32900  cirT1  M~[ 
(using  the  method  of  Bradford,  1976.  to  measure  protein). 
The  three  methods  resulted  in  extinction  coefficients  for 
pocilloporin  that  were  not  statistically  different  (P>  0.05), 
hence  verifying  the  validity  of  the  first  method. 

Metal  ion  analysis.  The  association  of  metal  ions  with 
pocilloporin  was  investigated  using  ICP-MS.  Total-quant 
analysis  of  the  ion  content  of  pocilloporin  samples  re- 
vealed no  ions  occurring  at  significantly  greater  levels  than 
background;  therefore,  pocilloporin  does  not  have  an  ac- 
companying metal  ion  in  its  structure. 

Thermal  lability  of  pocilloporin.  Chromatograms  at 
280  nm  and  560  nm  of  pocilloporin  from  a  broad  collec- 
tion around  560  nm  are  asymmetrical  (Fig.  5A),  dem- 
onstrating that  the  fraction  (in  this  case)  was  contaminated 
with  proteins  other  than  pocilloporin.  No  changes  occured 
to  pocilloporin  when  it  was  heated  to  40°C  for  10  min. 
When  heated  to  60°C,  280-nm  and  560-nm  absorbance 
decreased,  as  did  the  relative  amount  of  560-nm  absor- 
bance to  280-nm  absorbance.  The  decrease  in  the  ratio 
of  absorbance  at  560  nm  to  280  nm  may  be  due  to  the 
fact  that  the  contaminants  are  more  thermally  stable  than 
pocilloporin.  At  100°C,  there  was  a  further  decrease  in 
both  280-  and  560-nm  absorbances  (Fig.  5A).  Spectro- 
photometric  scans  of  pocilloporin  reveal  that  no  batho- 
chromic  shifts  accompanied  the  loss  of  280-nm  and  560- 
nm  absorbance  (Fig.  5B;  note,  baseline  shift  for  sample 
maintained  at  60°C  for  10  min).  Heating  raw  extract  to 
100°C  changes  the  solution  to  pale  yellow.  This  correlated 


THi:  PKiMHNTS  OF  HERMATYPIC  CORALS 


293 


A 

0.045 

g       RT         40  C 

loo'c 
I        1 

o 

x                     60<c 

$                                1  j 
5                                1          I 

RT         A                        0.045 

40°C          l| 

absorbance 

o 
b  P 
-••  o 
_L  en  w 

_JL_  °°3 

0.015 

A 

0     20     30     40     50             10     20     30     40     50 

0) 

1  0.045 

<D 

rr 
0.03 

0.015 
1 

6tfC                                               °'045 

/I                            0.03 
^^.^JsL-™™^^ 

100°C 

0.015 

0     20     30     40     50             10     20     30     40     50 

Retention  time  (mins) 

B 

0.4 

CD 
O 

c 

280 
V                     390 

J3 

o  0.2 

c/> 

.a 

txx   <"• 

0 
2C 

~ 

0                   400                   600                   800 

Wavelength  (nm) 

Figure  5.  Thermal  lability  of  560-nm  fraction  from 
Jamicornis:  RT  =  control  chromatogram;  40°C  =  chromatogram  after 
heating  sample  to  40°C  for  10  min:  60°C  =  chromatogram  after  heating 
sample  to  60°C  for  10  min:  !00°C  =  chromatogram  after  heating  sample 
to  IOO°C  for  10  min.  (— )  280-nm.  (xxxx)  560-nm  chromatograms.  Bar 
chart,  showing  ratio  of  560-nm  peak  area  to  280-nm  peak  area  at  each 
temperature.  (B)  Wavelength  scan  of  pocilloporin  at  different  tempera- 
tures: (  — )  cell  at  25°C.  (xxxx)  cell  at  60°C.  (- - -)  cell  at  60°C  after 
10  min.  Arrows  indicate  approximate  position  of  Xmlx  in  each  case. 


well  with  the  observation  that  the  390-nm  peak  was  rel- 
atively unaffected  by  temperature. 

So/iihilily  in  .so/vents  and  the  effects  of  denaturing  agents 
on  pigment  compounds.  For  pink  P.  damicomis,  5^  SDS 
gave  a  white  pellet  and  a  colorless  supernatant  with  no 
absorbance  between  360  and  700  nm.  Ethanol  and  ace- 
tone gave  a  pinky-purple  precipitate  and  a  colorless  su- 
pernatant. The  supernatant  had  no  peak  absorbance  be- 
tween 360  and  700  nm,  the  precipitate  redissolved  in 
phosphate  buffer,  and  the  solution  had  peak  absorbances 
(Ama\)  at  560  and  approximately  385  nm.  Colored  extracts 
from  all  coral  species  examined  were  insoluble  in  ethanol, 
acetone,  ether,  and  chloroform.  In  all  coral  species  ex- 
amined, acidification  (pH  4.8)  or  alkalization  (pH  1 1.2) 
of  phosphate  buffer  extract  altered  the  color  of  solutions 
of  pocilloporin  to  pale  orange,  with  further  acidification 
or  alkalization  turning  solutions  yellow  and  giving  rise  to 
a  yellow  precipitate. 


The  importance  of  UV-absorbance  by  pocilloporin. 
The  (320  for  pocilloporin  was  calculated  to  be  14889 
±64  cm"1  A/~'  (mean  ±  SEM;  /;  =  4  chromatograms). 
The  proportion  (percent)  of  the  total  absorbance  at 
320  nm  in  raw  extracts  that  was  due  to  pocilloporin  was 
1.60  ±  0.49%  (mean  ±  SEM;  n  =  5). 

Fluorescent  emission  measurement.  Fluorescence  (390- 
750  nm)  was  not  emitted  for  excitation  at  560  nm,  from 
either  unpurified  extracts  of  brown  and  pink  P.  damicomis 
colonies  or  partially  purified  extracts  of  pocilloporin.  Some 
fluorescence  was  observed  for  samples  excited  at  390  nm. 
Brown  fragments  of  P.  damicomis  fluoresced  at  450  nm 
and  480  nm.  Pink  fragments  fluoresced  only  at  450  mm. 
The  intensity  of  the  fluorescence  was,  however,  unrelated 
to  the  concentration  of  pocilloporin  in  the  sample  (pink 
and  brown  extracts  with  the  same  390-nm  absorbance 
fluoresced  with  the  same  intensity  at  450  nm).  Fluores- 
cence, as  a  result  of  390-nm  excitation,  was  therefore  due 
to  compounds  other  than  pocilloporin  in  raw  and  partially 
purified  extracts  of  P.  damicomis. 

Comparison  of  pocilloporin-like  compounds:  native  and 
subunit  molecular  weights  within  and  between  families 

Stylophora  pistillata,  Seriatopora  hystrix.  Acropora 
digitifera,  and  A.  formosa  have  280-nm  chromatograms 


280  nm  chromatogram 
of  raw  extract 


Seriatopora  riysfr/x 


Absorbance  profile  of 
pocilloporin-like  fraction 


Seriatopora  hystrix 


10         20         30         40 

015    styl/ophora  pistillata 


50 


10         20         30         40 
Acropora  digitifera 


50 


002 


006 
004 
0.02 


10        20         30        40 
Acropora  formosa 


50 


10        20         30        40 
Retention  time  (mm) 

Figure  6. 


50 


2.5 
2 

1.5 
1 

05 
0 

25 
2 

15 
1 

05 
0 

0.4 
0.3 
02 
0.1 
0 


400          600          800 
Styllophora  pistillata 


400 


600 


Acropora  digitifera 

^578 


400          600 
Acropora  formosa 

588^ 


800 


400          600          800 
Wavelength(nm) 

Chromatograms  of  raw  phosphate  buffer  extracts  from  four 
different  coral  species  at  280  nm;  I  '„.  void  volume;  MAA.  mycosporine- 
like  amino  acids.  Ama>  determinations  of  isolated  pocilloponn-like  com- 
pounds. Arrows  indicate  approximate  Xmax.  Vertical  dotted  line  indicates 
position  of  280-nm  absorbance. 


294 


S.  G.  DOVE  ET  AL 


similar  to  those  of  Pocillopora  damicornis.  All  five  species 
have  a  peak  related  to  the  presence  of  mycosporine-like 
amino  acids  that  elutes  at  about  37  min  (MAA,  Fig.  6), 
a  group  of  peaks  that  elute  between  20  and  30  min.  and 
a  void  volume  peak  that  elutes  at  approximately  14  min 
(I ,,.  Fig.  6).  S.  hystrix  and  S.  pistillata  had  Xmax  about 
equal  to  560  nm  (Fig.  6).  A.formosa  had  Xmax  about  equal 
to  578  nm  and  a  shoulder  at  about  550  nm  (Fig.  6).  In 
the  blue  Acropora  species.  A.  formosa,  there  is  a  shift  in 
the  native  molecular  weight  (MW)  of  the  pigment  from 
54  kD  (native  MW  of  P.  damicornis,  S.  hystrix.  S.  pistil- 
lata. and  Acropora  digitifera)  to  82.6  kD  (cf.  peak  position 
relative  to  dotted  line.  Fig.  7).  However,  in  all  coral  species 
examined,  SDS-PAGE  of  corresponding  gel  filtration 
fractions  showed  a  single  band  with  a  subunit  molecular 
weight  of  about  28  kD  (Fig.  8,  data  not  shown  for  A.  dig- 
itifera). The  front  shoulder  fractions,  which  absorb  min- 
imally at  X  =  max  (560-590)  nm  and  variably  at  400  nm 
(Fig.  7),  show  a  more  complex  banding  pattern  that  con- 
tains both  the  28-kD  subunit  and  a  40-kD  subunit 
amongst  a  smear  of  other  bands  (Fig.  8:  S  pistillata.  lane 
1;  P  damicornis,  lane  1;  S.  hystrix,  lane  5).  Gel  filtration 
chromatograms  suggest  that  compounds  absorbing  at 
about  400  nm  are  more  closely  associated  with  a  peak 
whose  approximate  molecular  weight  is  1 1 2  kD  than  with 
the  pocilloporin  or  pocilloporin-like  compound  peak 
(Fig.  7). 

Discussion 

The  colors  that  typify  many  members  of  the  animal 
kingdom  have  a  variety  of  roles  that  range  from  crypsis 
to  courtship  (McFall-Ngai,  1990;  Dawkins  and  Guilford, 
1993).  Although  the  role  of  color  has  been  explored  ex- 
tensively in  some  groups  (e.g..  insects:  Endler.  1981;  birds: 
Owen,  1980;  fish:  Neal,  1993),  an  understanding  of  the 
function  of  color  in  others  is  lacking.  Reef-building  corals 
fall  into  the  latter  category.  This  study  is  the  first  attempt 
to  isolate  and  biochemically  characterize  the  compounds 
responsible  for  the  pink  and  blue  colors  of  two  prominent 
families  of  reef-building  corals  (Pocilloporidae.  Acropor- 
idae).  Once  the  protein  dimer  from  Pocillopora  damicornis 
(pocilloporin)  had  been  isolated  and  characterized,  its 
function  was  explored,  and  several  functions  suggested  by 
early  studies  were  solidly  rejected. 

Biochemical  structure  of  pocilloporin 

SDS-polyacrylamide  gel  electrophoresis  and  gel  filtra- 
tion of  pocilloporin  and  pocilloporin-like  compounds 
suggest  that  the  pigments  from  Pocillopora  damicornis. 
Seriatopora  hystrix.  Stylophora  pistillata,  and  Acropora 
digitifera  are  protein  dimers  with  native  molecular  weights 
of  about  54  kD  and  subunits  of  28  kD  (Figs.  1-3,  7.  8). 
The  pigment  from  the  blue  regions  of  A.  formosa  is  a 
trimer  with  a  native  molecular  weight  of  about  82.6  kD 


Pocillopora  damicornis 
purple 

112  kD       54  kD 

f 


Pocillopora  damicornis 
pink 


112  kD 


54  kD 


10     20     30     40     50   10     20     30     40     50 


(0 

-e 

o 

(/) 

.o 
nj 


o> 
cr 


Stylophora  pistillata 
pink 


Seriatopora  hystrix 
pink 


Acropora  digitifera 
purple 


50   10     20     30     40     50 

Acropora  formosa 

82  kD 


10  20  30  40  50       10  20  30  40  50 

Retention  time  (min) 

Figure  7.  400-nm  ( — )  and  560-nm  ( — )  chromatograms  from  Cen- 
tncon  10  (Amicon)  centrifuged  phosphate  buffer  extracts  from  Pocillopora 
damicornis,  Stylophora  pistillata,  Seriatopora  hystrix,  Acropora  digitifera, 
and  .4.  formosa.  Dotted  line  shows  approximate  position  of  54-kD  eluting 
protein. 


and  subunits  of  28  kD  (Figs.  7,  8).  The  subunits  in  the 
case  of  P.  damicornis  are  not  linked  by  disulfide  bonds 
in  the  native  protein. 

All  evidence  supports  the  existence  of  a  strong  stoi- 
chiometric  link  between  the  protein  and  chromophore  in 
pocilloporin.  The  nature  of  that  link  can  be  explained  in 
several  ways.  That  is,  the  absorbance  at  560  nm  could  be 
due  to  (i)  a  non-protein  compound  that  is  coincidentally 
eluting  with  a  same-sized  protein  without  any  chemical 
bonding  between  them;  (ii)  a  chromophore  that  is  non- 
covalently  bonded  with  a  protein  to  form  a  chromophore- 
protein  complex,  and  (iii)  a  chromophore  covalently 
bonded  to  the  protein. 

The  first  option  can  be  eliminated  by  the  strong  cor- 
relation between  the  absorption  at  560  nm  and  the  con- 
centration of  co-eluting  54-kD  protein  from  colonies 
differing  in  the  expression  of  the  pink  pigment  (Fig.  4). 
In  other  words,  the  more  of  the  particular  protein  there 
is,  the  greater  the  absorbance  at  560  nm.  Furthermore, 
a  28-kD  subunit  existed  in  significant  amounts  only  in 
the  fractions  with  a  high  absorbance  at  560  nm  and  not 
in  fractions  that  had  no  absorbance  at  560  nm  (e.g., 
extracts  of  brown  colonies.  Fig.  3).  The  second  option, 
that  the  chromophore  is  non-covalently  linked  to  the 
protein,  is  questionable  on  the  grounds  that  the  chro- 
mophore is  not  easily  separated  from  the  protein. 


THE  PIGMENTS  OF  HERMATYPIC  CORALS 


295 


Stylophora  pistil lata        Acropora  formosa 


28  kD 


-97 
-66 

-45 
-30 
-21 


-14 
12345  12345 

Pocillopora  damicornis     Seriatopora  hystrix 

-97 
-66 


28  kD 


-    —30 
-21 


1 


3      4 


1 


-14 


Figure  8.  Fifteen  percent  SDS-PAGE  of  isolated  pigment  (pink  or  blue)  tractions  from  different  coral 
species.  Slvlophura  pistillala:  lane  I,  front  shoulder  of  560-nm  peak  (8  ^g):  lane  2.  Standards;  lanes  3-5, 
main  560-nm  fraction  with  2,  4,  8  Mg,  respectively.  Acropora  formosa:  lanes  1-4  main  580-nm  fraction  with 
8.  6,  4,  2  ^g,  respectively.  Pocillopora  damicornis:  lane  1 ,  front  shoulder  of  560  peak  (4  ^g);  lane  2,  Standards; 
lanes  3-5.  main  560-nm  fraction  with  2,  4,  6  ^g.  respectively.  Serialopora  hvstrix  lanes  1-3,  main  560-nm 
fraction  with  6.  4,  2  n%.  respectively:  lane  3,  Standards;  lane  5,  front  shoulder  of  560-nm  peak  (8  ^g>. 


Treatments  such  as  5%  SDS,  heat,  and  relatively  non- 
polar  solvents  such  as  acetone  liberate  carotenoids  and 
other  chromophores  from  their  conjugate  proteins,  re- 
sulting in  bathochromic  shifts  in  the  absorbance  of  the 
pigment  whilst  the  protein  either  denatures  (5%  SDS. 
heat)  or  precipitates  out  of  nonpolar  solvents  (Cheese- 
man  et  al,  1967;  Fox,  1979;  Milicua  el  ai.  1985;  Garate 
etal..  1986;Zagalskye/a/.,  1989;  Zagalsky  et  al..  1991). 
No  such  reaction  was  found  with  pocilloporin  or  the 
pocilloporin-like  compounds  of  this  study.  Treatments 
with  these  agents  simply  resulted  in  precipitation  of  the 
pigment  complex  from  solution. 

Pocilloporin-like  compounds  from  the  Pocilloporidae 
and  Acroporidae 

Several  other  biochemical  properties  of  pocilloporin 
and  pocilloporin-like  compounds  were  identified  in  this 


study.  The  compounds  were  hydrophilic,  in  agreement 
with  the  work  of  Kawaguti  (1944)  and  Shibata  ( 1969), 
who  both  extracted  similar  coral  pigments  in  water.  It 
is  curious,  however,  that  Shibata  ( 1969)  did  not  find  an 
absorption  peak  at  560  nm  in  the  water  extracts  of 
Pocillopora  sp.;  the  main  absorbance  peak  found  in  that 
study  was  at  480  nm.  However,  Shibata  did  not  classify 
the  species  of  Pocillopora  he  used,  and  his  description 
of  the  morph  as  red  rather  than  pink  suggests  that  it 
may  have  been  a  differently  pigmented  form  than  the 
ones  used  in  the  present  study.  Shibata  (1969)  also  de- 
scribed pigments  from  Acropora  sp.  as  having  absor- 
bance peaks  at  560  nm  and  590  nm.  Our  results  for  two 
species  of  Acropora  are  similar:  A.  digitifera  had  a  Xmax 
at  578  nm  and  a  shoulder  at  550  nm.  Similarly,  A.  for- 
mosa had  a  Amax  at  588  nm  and  a  shoulder  at  550  nm 
(Fig.  6). 


296 


S.  G.  DOVE  ET  AL. 


The  present  study  did  not  determine  the  biochemical 
pigment  group  to  which  the  proteins  pocilloporin  and 
pocilloporin-like  compounds  belong;  however,  some  op- 
tions seem  unlikely.  No  metal  ions  were  found  associated 
with  pocilloporin,  thus  eliminating  it  from  the  group  of 
metal-bearing  biochromes  such  as  the  colored  copper 
proteins  and  metal-chelated  tetrapyrroles  (Fox,  1 979).  The 
absence  of  metal  ions  cannot  be  attributed  to  inadequate 
sample  size.  Assuming  1  metal  ion  per  protein  molecule, 
we  would  expect  readings  in  the  range  of  parts  per  million 
for  the  amount  of  protein  introduced  into  the  ICP-MS. 
This  amount  is  well  above  the  detection  capabilities  of 
the  ICP-MS,  which  can  measure  ion  concentrations  down 
to  the  range  of  parts  per  billion  (Henshaw  el  ai,  1989). 
The  absorbance  of  pocilloporin  at  560  nm  is  significantly 
reduced  by  short  ( 10-min)  exposures  to  60°C  without  any 
accompanying  bathochromic  shift  (Fig.  5).  This  extreme 
thermal  sensitivity  suggests  that  this  absorbance  is  not 
due  to  more  thermally  stable  compounds  such  as  carot- 
enoids  (e.g..  those  that  retain  absorbance  even  when 
heated  at  temperatures  up  to  120°C  for  1  h.  Lee  el  al. 
1987,  1990).  Pocilloporin.  therefore,  does  not  appear  to 
involve  a  carotenoid  component  and  is  probably  not  a 
carotenoprotein. 

Why  be  pigmented?  Possible  roles  of  pocilloporin 

Investigation  of  the  biochemical  characteristics  of  po- 
cilloporin did  not  reveal  its  function.  However,  several 
possible  functions  are  firmly  rejected  and  a  narrow  range 
of  possibilities  remains.  The  following  hypotheses  about 
the  function  of  pocilloporin  are  rejected. 

Pocilloporin  as  a photoprotectant.  In  his  seminal  study 
of  coral  color,  Kawaguti  (1944)  proposed  that  pigments 
may  shade  zooxanthellae  from  excessive  sunlight  that 
might  otherwise  lead  to  photobleaching  of  constituent 
photosynthetic  pigments.  The  results  of  the  present  study 
are  counter  to  this  idea.  The  absorption  spectrum  of  po- 
cilloporin suggested  that  it  did  not  protect  against  pho- 
tobleaching of  the  major  photosynthetic  pigments  because 
its  absorbance  spectrum  does  not  coincide  with  those  of 
chlorophyll  a  and  e  (Nobel.  1983;  Kirk,  1994)  and  is,  if 
anything,  curiously  complementary  to  these  compounds. 
Thus,  a  role  for  pocilloporin  as  a  photoprotectant  for  the 
photosynthetic  pigments  of  the  zooxanthellae  is  unlikely. 

Pocilloporin  as  an  accessory  photosynthetic  pigment.  If 
pocilloporin  were  an  accessory  photosynthetic  pigment, 
the  ene ru\  absorbed  by  it  would  have  to  be  transferred  to 
the  plun  'ems  in  the  zooxanthellae  to  be  used  for  pho- 
tosynthesis This  transfer  could  occur  in  two  ways.  The 
first  way  is  by  the  direct  energy  transfer  (resonance  energy 
transfer).  In  this  case,  light  energy  absorbed  by  one  mol- 
ecule is  passed  to  the  reaction  centers  in  the  photosystems 
through  a  chain  of  energy  transfers  between  closely  ad- 
jacent molecules  (Nobel,  1983).  This  could  happen  only 


if  pocilloporin  were  in  proximity  to  the  photosystems. 
However,  most  (if  not  all)  of  the  pocilloporin  is  associated 
with  the  coral  tissues  (Takabayashi  and  Hoegh-Guldberg, 
1995)  and  not  with  the  zooxanthellae.  Consequently,  the 
direct  transfer  of  excitation  energy  by  resonance  transfer 
from  pocilloporin  to  photosystems  is  impossible.  A  second 
method  of  energy  transfer  is  through  fluorescent  coupling. 
In  this  case,  pocilloporin  might  absorb  light  at  non-pho- 
tosynthetic  wavelengths  and  re-fluoresce  them  at  wave- 
lengths suitable  for  absorption  by  the  primary  photosyn- 
thetic pigments  (Kawaguti,  1944;  Schlichter  et  al..  1988). 
This  type  of  transfer  has  been  proposed  for  pigments  as- 
sociated with  the  animal  tissues  of  the  deep-water  coral 
Leptoseris  fragilis,  which  transforms  short-wavelength, 
non-photosynthetically  active  radiation  into  longer  wave- 
length, photosynthetically  active  radiation,  which  is  reab- 
sorbed  by  the  photosynthetic  pigments  of  the  symbiotic 
dinoflagellates  (Schlichter  and  Fricke,  1991).  This  indirect 
means  of  energy  transfer  is,  however,  unlikely  for  pocil- 
loporin, which  is  not  fluorescent  for  excitation  at  either 
of  its  Xmax. 

Pocilloporin  as  a  U\  '-screening  pigment.  The  last  pos- 
sibility is  that  pocilloporin  might  have  UV-absorbing  ca- 
pabilities in  addition  to  its  ability  to  absorb  visible  light. 
This  is  also  unlikely  because  the  absorbance  of  pocillo- 
porin within  the  UV  region  of  light  was  minimal  (1.6% 
of  the  total  absorbance  of  raw  extracts  at  320  nm).  Com- 
pounds such  as  the  mycosporine-like  amino  acids  that  are 
abundant  in  corals  are  far  more  potent  UV-B-screening 
agents  (Dunlap  and  Chalker,  1986),  a  fact  that  is  clear 
from  the  comparison  of  the  6320  of  pocilloporin 
( 14,889  cm  '  M~])  with  the  f3:u  of  a  typical  mycosporine- 
like  amino  acid  (e.g..  e32o  of  palythine  =  36,200  cm"1  M~\ 
Dunlap  and  Chalker,  1986).  These  MAAs,  especially  pal- 
ythine (Xmax  =  320  nm),  are  probably  responsible  for  more 
than  95%  of  the  absorption  by  the  coral  at  320  nm  (Dun- 
lap  and  Chalker,  1986;  Dunlap  et  ai.  1988). 

The  role  of  pocilloporin  remains  elusive.  Pocilloporin 
may  act  as  an  agent  that  enhances  the  abilities  of  the  pig- 
mented morph  to  resist  fouling  or  predation  or  to  compete 
successfully  (Lang,  1971,  1973;  Sheppard,  1979,  1982).  It 
is  interesting  to  note  that  Rinkevich  and  Loya  (1983)  re- 
ported the  purple  morph  of  Stylophora pistillata  from  the 
Red  Sea  to  be  competitively  superior  to  the  yellow  morph 
of  the  same  species,  even  when  they  were  not  touching. 
Takabayashi  (1994)  found  a  similar  trend  in  the  compet- 
itive abilities  of  the  pink  morph  of  Podllopora  damicornis. 
In  this  case,  the  pink  morph  won  significantly  more  of 
the  contests  between  pink  and  brown  colonies  in  grafting 
experiments,  and  a  distinct  pink  band  was  often  observed 
in  the  tissue  of  pink  colonies  at  the  contact  sites  between 
nonsimilar  colonies.  Pocilloporin,  therefore,  might  func- 
tion in  the  coral's  immunological  and  chemical  defense 
systems. 


THE  PIGMENTS  OF  HERMATYPIC  CORALS 


297 


Acknowledgments 

The  authors  thank  Dr.  Tariq  Khan  for  help  with  the 
fluorospectroscopy,  and  P.  Snitch  at  Royal  Prince  Alfred 
Hospital  (Sydney)  for  access  to  the  ICP-MS.  This  study 
was  supported  by  an  Australian  Postgraduate  Research 
Scholarship  to  SD  and  a  grant  from  the  Great  Barrier 
Reef  Marine  Park  Authority  (ENCORE  program)  and 
Australian  Research  Council  to  OHG.  This  is  publication 
#6  in  the  ENCORE  series. 

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Veron,  J.  E.  N.  1986.  Corals  nl  Australia  and  The  Indo-Pacific.  Angus 
and  Robertson  Publishers,  Sydney. 

Zagalsky,  P.  F.,  F.  Haxo,  S.  Hertzberg,  and  S.  Liaaen-Jensen.  1989. 
Studies  on  a  blue  carotenoprotein,  linckiacyamn.  isolated  from 
the  starfish  Linckia  laevigata  (Echinodermata:  Asteroidea).  Comp. 
Biochem  Physiol  93B:  339-353. 

Zagalsky,  P.  F.,  E.  E.  Eliopoulos.  and  J.  B.  C.  Findlay.  1991.  The 
lobster  carapace  carotenoprotein.  alpha-crustacyanin.  A  possible  role 
for  try  ptophan  in  the  bathochromic  spectral  shift  of  protein-bound 
astaxantin.  Biochem  J  274:  79-83. 


Reference:  Bin/.  Bull  189:  298-307.  (December,  1995) 


Bleaching  Patterns  of  Four  Species  of 
Caribbean  Reef  Corals 

W.  K.  FITT  AND  M.  E.  WARNER 
Institute  of  Ecology.  University  of  Georgia,  Athens,  Georgia  30602 


Abstract.  Bleaching  of  reef  corals,  involving  loss  of 
symbiotic  algae  (=  zooxanthellae),  loss  of  algal  pigments, 
or  both,  has  been  linked  to  temperature  stress.  In  this 
study  the  effects  of  high  temperature  and  light  on  zoox- 
anthellae living  in  the  Caribbean  reef  corals  Montastrea 
annularis,  M.  cavernosa,  Agaricia  agaricites,  and  A.  la- 
marcki  were  studied.  Pieces  of  coral  colonies  were  incu- 
bated at  ambient  seawater  temperature  (26°  ±  1  °C),  and 
at  30°,  32°,  and  34°C.  Symbiotic  algae  from  M.  annularis. 
a  species  of  coral  from  the  forereef  that  commonly 
bleaches,  showed  the  following  sequence  of  events  when 
exposed  to  natural  light  at  32°C:  loss  of  photosynthetic 
potential  measured  as  fluorescence  yield,  corresponding 
reduction  of  both  oxygen  production  per  zooxanthella 
and  P:R  (photosynthesis:respiration)  ratio,  and  subse- 
quent reduction  in  density  of  algae  in  relation  to  surface 
area  of  the  coral.  These  parameters  were  not  significantly 
reduced  and  no  deaths  occurred  for  M.  annularis  or  any 
other  coral  species  maintained  at  26°  or  30°C.  However, 
the  sequence  of  events  was  condensed  to  less  than  24  h 
when  M.  annularis  was  subjected  to  34°C  seawater,  except 
that  there  was  little  if  any  reduction  in  algal  density  before 
tissue-sloughing  and  death  occurred  between  10  and  24  h. 
Loss  of  significant  amounts  of  chlorophyll  a  per  alga  was 
not  evident  for  any  corals  except  those  maintained  at  34°C 
longer  than  10  h.  In  contrast,  symbiotic  algae  in  A/,  cav- 
ernosa, a  species  that  rarely  bleaches  in  nature,  showed 
only  slight  reductions  in  photosynthesis  and  fluorescence 
yield,  and  no  significant  loss  of  algal  cells  or  chlorophyll 
a,  when  maintained  in  seawater  at  32°C  for  2  days.  Thus 
zooxanthellae  in  M.  cavernosa  appeared  to  be  less  affected 
by  sublethal  high-temperature  stress.  Similar  contrasting 
patterns  of  bleaching  were  seen  in  zooxanthellae  from  the 
plating  coral  Agaricia  lamarcki.  which  often  bleaches 


Received  6  December  1994;  accepted  14  September  1995. 


during  the  late  summer  and  fall,  compared  with  zooxan- 
thellae from  A.  agaricites.  a  coral  which  bleaches  less  fre- 
quently. In  addition.  A/,  annularis  exposed  to  sublethal 
high  temperatures  and  ambient  light  bleached  faster  than 
those  kept  in  dimmer  light,  supporting  past  field  observa- 
tions suggesting  that  light  energy  is  an  important  compo- 
nent of  bleaching  in  nature.  When  A/,  annularis  was  ex- 
posed to  different  wavelengths  of  natural  light  at  32°C,  the 
fluorescence  yield  declined  more  quickly  in  the  presence 
of  higher  energy  UV-A  and  blue  light  than  with  other  pho- 
tosynthetically  active  radiation.  Natural  levels  of  UV-B  had 
little  effect  in  this  study.  These  data  suggest  that  the  patterns 
of  bleaching  seen  in  nature  may  be  at  least  partially  ex- 
plained by  different  tolerances  of  the  symbiotic  algae  in 
the  corals,  and  that  light  plays  a  significant  role  in  bleaching. 

Introduction 

Two  major  ecological  events  during  the  last  decade  fo- 
cused the  attention  of  coral  reef  researchers  on  the  sus- 
ceptibility of  corals  and  associated  reef  organisms  to  the 
potentially  devastating  effects  of  elevated  seawater  tem- 
peratures. The  first  was  the  1982-1983  El  Nino  Southern 
Oscillation  (ENSO),  during  which  many  hard  and  soft 
corals  from  the  Great  Barrier  Reef,  the  Central  Pacific, 
and  eventually  the  Eastern  Pacific  bleached  as  seawater 
temperatures  rose  2°-6°C  above  normal  (Glynn,  1983, 
1984;  Oliver,  1985;  Harriot,  1985;  Fisk  and  Done.  1985; 
Coffroth  ct  al..  1990:  Glynn  and  D'Croz,  1990).  Subse- 
quent coral  death  was  common:  up  to  97%  of  the  species 
harboring  symbiotic  algae  were  reported  dead  on  some 
reefs  (Glynn  and  D'Croz,  1990). 

The  Caribbean-wide  "bleaching  event"  of  1987  again 
drew  attention  to  warm-water  stress  in  the  marine  envi- 
ronment, this  time  coupled  with  concerns  that  global 
warming  might  be  one  of  the  causes  (Williams  and  Wil- 
liams. 1988).  Though  bleaching  was  extensive,  total  loss 


298 


BLEACHING  OF  REEF  CORALS 


299 


of  zooxanthellae  from  coral  tissues  was  rare,  as  was  death 
of  entire  coral  colonies  (see  references  in  Fitt  el  al.,  1993; 
Porter  and  Meier,  1992).  Most  bleached  corals  recovered 
their  normal  coloration  within  a  year  (Szmant  and  Gass- 
man,  1990;  Fitt  ct  al..  1993).  The  results  of  both  of  these 
events  are  consistent  with  the  notion  that  corals  and  other 
associated  invertebrates  are  living  close  to  their  physio- 
logical upper  thermal  limits  during  summer  months,  so 
that  even  the  smallest  increase  in  seawater  temperature 
may  have  an  effect  if  the  exposure  time  is  long  enough 
(Coles  end..  1976). 

Virtually  all  studies  of  bleaching  support  the  supposi- 
tion that  summertime  bleaching  is  at  least  partially  linked 
to  the  high  temperatures  (e.g..  Yongeand  Nichols,  193  la; 
Jokiel  and  Coles,  1977;  Jaap,  1979;Glynn,  1984;  Lasker 
ei  al..  1984;  Hoegh-Guldberg  and  Smith.  1989;  Glynn 
and  D'Croz.  1991;  Gates  el  al,  1992;  Jokiel  and  Coles, 
1990;  Fitt  et  al..  1993).  Two  of  the  best  examples  of  the 
role  of  temperature  involve  laboratory  experiments,  one 
simulating  El  Nino  conditions  in  the  Eastern  Pacific 
(Glynn  and  D'Croz,  1990)  and  the  other  mimicking  the 
effects  of  the  thermal  discharge  system  of  a  power  gen- 
erator in  Hawaii  (Jokiel  and  Coles,  1977).  Both  studies 
clearly  showed  the  immediate,  adverse  effects  on  corals 
of  abnormally  high  temperatures  (>32°C),  as  well  as  more 
subtle  bleaching  during  long-term  exposure  to  tempera- 
tures only  1°-2°C  above  normal  ambient  (e.g.,  30°C). 

The  relative  importance  of  other  environmental  factors 
on  bleaching  is  more  contentious.  Low  salinity  and  high 
levels  of  natural  light  sometimes  show  synergistic  effects 
in  connection  with  high  temperatures  near  the  limits  of 
tolerance  for  corals  (Coles  and  Jokiel,  1978;  cf.  Hoegh- 
Guldberg  and  Smith,  1989).  In  addition,  high  doses  of 
ultraviolet  light  induce  bleaching  without  increased  tem- 
perature (Jokiel.  1980;  Gleason  and  Wellington,  1993). 
Although  the  role  of  light  in  bleaching  is  interesting,  little 
is  currently  known  about  the  role  of  light  quantity  and 
quality,  especially  in  relation  to  photosynthetic  action 
spectra  of  the  symbiotic  algae.  For  instance,  UV-B  block- 
ing compounds  have  been  described  and  characterized 
(Dunlap  and  Chalker,  1986).  and  show  the  expected  de- 
crease in  concentrations  with  depth  (Dunlap  et  al..  1988). 
However,  protection  by  these  compounds  from  UV-A 
light  (ca.  320-400  nm)  is  generally  limited  at  wavelengths 
greater  than  350  nm,  where  photosynthetic  pigments  in 
zooxanthellae  begin  absorbing  light  (Jeffrey  and  Haxo, 
1968;  Dunlap  et  al..  1988).  This  leaves  the  coral  and  sym- 
biotic algae  exposed  to  longer  UV-A  wavelengths  and  blue 
light  (ca.  400-450)  (Dunlap  et  al..  1988),  as  well  as  to 
other  photosynthetically  active  radiation  (PAR). 

One  of  the  most  perplexing  aspects  of  coral  bleaching 
is  that  some  species  seem  to  lose  color  frequently  and 
quickly  during  bleaching  events,  whereas  others  never 
seem  to  bleach.  For  instance,  the  Caribbean  reef-building 


coral  Montastrea  ami/arris  is  one  of  the  first  species  to 
appear  discolored  during  bleaching  events,  whereas  A/. 
cavernosa  rarely  bleaches  (Jaap,  1979,  1985).  Although 
differential  tolerance  of  host  tissue  to  environmental  stress 
may  explain  these  patterns,  it  is  also  possible  that  different 
species  or  types  of  zooxanthellae  (see  Trench,  1993)  ex- 
hibit different  tolerances  to  temperature  and  light  (cf.  Fitt, 
1985). 

One  explanation  proposed  for  high-temperature 
bleaching  is  that  the  host  digestive  cells  detach  from  the 
mesoglea,  carrying  zooxanthellae  out  of  the  coelenteron, 
in  a  fashion  seen  in  cnidarians  exposed  to  cold  water  stress 
(Gates  et  a!..  1992).  Other  investigators  have  found  that 
cultured  zooxanthellae  placed  in  temperatures  equal  to 
or  higher  than  32°C  show  decreased  photosynthetic  effi- 
ciency (Iglesias-Prieto  et  al..  1992);  this  observation  sug- 
gests that  the  algae,  and  not  just  the  host,  are  responsible 
for  the  breakdown  of  the  symbiosis  during  bleaching. 
There  is  still  no  consensus  as  to  which  of  the  symbiotic 
partners  is  more  affected  by  high  temperature.  In  this  study 
we  address  some  of  these  issues  by  documenting  the  se- 
quence of  events  occurring  in  zooxanthellae  living  sym- 
biotically  with  four  species  of  Caribbean  reef  corals,  and 
show  that  both  light  quantity  and  quality  can  be  important 
environmental  factors  in  bleaching. 

Materials  and  Methods 

Collection  and  maintenance  of  animals 

Intact  colonies  of  the  reef  corals  Agaricia  agaricites,  A. 
lamarcki.  Montastrea  anmilaris,  and  M.  cavernosa  were 
collected  from  a  depth  of  14-16  m  on  the  forereef  off  the 
Discovery  Bay  Marine  Laboratory  in  Jamaica  in  the  early 
morning  (0700-0800)  in  February  and  March  of  1993 
and  1994.  Within  1  h  of  collection  each  colony  was  broken 
into  eight  pieces,  each  with  a  surface  area  of  5-10  cm2, 
and  placed  into  one  of  four  water-jacketed  acrylic  incu- 
bation chambers  containing  about  3.5  1  of  seawater.  The 
clear  plastic  chambers  were  exposed  to  ambient  light;  their 
open  tops  were  covered  with  three  layers  of  screen  to  re- 
duce the  maximum  light  intensities  to  slightly  less  those 
found  at  14-16  m  on  the  reef.  Light  intensities  at  noon 
on  a  cloudless  day  on  the  reef  at  15  m  were  measured  on 
three  occasions  and  ranged  between  500-600  juE  m~2s~', 
maximum  intensities  measured  in  the  chambers  under 
the  screen  were  400-475  ^E  m  2s  '.  During  the  experi- 
ments, fresh  unfiltered  seawater  flowed  into  the  chambers 
at  ca.  1 50  m  min~ '  and  vigorous  aeration  from  aquarium 
pumps  and  air  stones  kept  the  water  well  mixed.  Ambient 
seawater  temperatures  were  26.0°  ±  1.0°C.  Coral  pieces 
were  allowed  to  equilibrate  in  the  chambers  for  5-15  min 
at  ambient  temperature  before  the  start  of  each  experi- 
ment. Under  ambient  temperature  and  light,  control 


300 


W    K.  FITT  AND  M.  E.  WARNER 


pieces  maintained  in  chambers  showed  no  adverse  or  vis- 
ible effects  for  at  least  4  days. 

Experimental  protocol 

Coral  pieces  were  placed  in  one  of  four  chambers,  each 
starting  out  at  ambient  seawater  temperature  (26.0°C 
±  1 .0°C).  In  three  of  the  chambers,  aquarium  heaters  were 
used  to  raise  the  temperature  over  a  period  of  about  1  h. 
Temperatures  were  kept  at  30°,  32°,  and  34°C  ±  0.5°C. 
A  minimum  of  four  replicate  colonies  were  tested  from 
each  species.  At  least  two  pieces  of  each  replicate  colony 
were  placed  into  each  of  the  four  chambers  so  that  samples 
could  be  taken  at  different  times.  Coral  pieces  maintained 
at  26°,  30°,  and  32°C  were  processed  at  about  24  and 
48  h.  Coral  pieces  exposed  to  34°C  were  sampled  3- 
5  times  during  the  first  24-h  period.  Pieces  of  coral  were 
processed  for  physiological  testing  and  biomass  deter- 
minations as  detailed  below. 

Light  quality  and  quantity  experiments 

Pieces  of  six  replicate  heads  of  A/,  annularis  used  in 
experiments  testing  the  effects  of  light  quality  and  quantity 
were  collected  from  a  patch  reef  (1-2  m  deep)  oft"  Key 
Largo,  Florida.  Coral  pieces  were  placed  in  glass  petri 
dishes  in  a  32°C  temperature  bath  with  aeration,  where 
the  seawater  was  changed  at  least  every  4  h  throughout 
the  experiment.  The  quality  and  quantity  of  natural  am- 
bient light  were  adjusted  with  screens  and  glass  cut-oft" 
filters  (Melles  Girot).  Corals  were  exposed  to  one  of  the 
following  conditions:  natural  light  with  no  filters,  natural 
light  without  UV-B  (>320  nm),  natural  light  without  UV- 
A  and  B  (>395  nm),  or  natural  light  without  UV  or  blue 
light  (>495  nm).  Two  layers  of  window  screen  covered 
the  entire  waterbath  to  reduce  the  maximum  exposure 
level  to  slightly  lower  than  that  found  ///  situ 
(<700  nE  m:s~').  Some  coral  pieces  (control)  were  main- 
tained under  two  layers  of  window  screen  without  filters, 
but  at  26°C. 

Physiological  testing  and  biomass  determinations 

Coral  tissue  and  zooxanthellae  were  removed  from  the 
coral  skeleton  with  a  Water-Pik  and  subsamples  of  the 
homogenate  taken  for  zooxanthellae  counts  and  chloro- 
phyll a  determinations.  The  remaining  homogenate  was 
filtered  through  three  layers  of  cheesecloth  and  centrifuged 
at  1500  X  g  for  3  min.  The  pellet  was  resuspended  and 
washed  (recentrifuged)  with  fresh  filtered  (0.45  nm)  sea- 
water  (FSW)  at  least  three  times,  or  until  few  animal  frag- 
ments were  seen  amongst  the  zooxanthellae  in  micro- 
scopic observations. 

Cleaned  zooxanthellae  were  resuspended  in  FSW  at 
densities  between '0.5  and  1.0  X  106  zooxanthellae  per 


milliliter.  Respiration  rates  in  the  dark  and  photosynthetic 
rates  at  450  ^E  m  :s  '  (above  saturation)  were  determined 
with  a  YSI  oxygen  meter  equipped  with  low-volume  (2- 
10  ml)  chambers  and  magnetic  stirrers.  Respiration  and 
net  photosynthesis  rates  were  added  together  to  give  gross 
photosynthesis  rates  and  standardized  to  number  of 
zooxanthellae.  Gross  photosynthesis:respiration  (P:R)  ra- 
tios were  calculated  from  these  rates. 

Chlorophyll  fluorescence  of  zooxanthellae  suspensions 
was  measured  with  a  Turner  fluorometer,  after  a  10-min 
incubation  in  darkness.  The  ratio  of  fluorescence  obtained 
with  additions  of  DCMU  (10~5  A/)  in  relation  to  that 
without  DCMU  was  calculated.  Long-term  kinetics  of 
chlorophyll  fluorescence  of  zooxanthellae  living  within 
the  host  were  recorded  with  a  pulse  amplitude  modulation 
fluorometer  (model  PAM  101,  103;  Waltz).  The  corals 
were  dark-adapted  for  10  min  under  the  fiber  optic  bundle 
of  the  tluorometer  prior  to  measurement.  The  initial  fluo- 
rescence (Fo)  was  measured  by  exposing  the  coral  to  a 
weak  pulse  of  red  light  (<1  juE  m2s~').  Maximum  fluo- 
rescence (Fm)  was  then  determined  by  applying  a  1-s  pulse 
of  intense  white  light  (>500  juE  ITT  s~').  The  maximum 
variable  fluorescence  was  calculated  as  Fv  =  Fm  —  Fo. 
The  value  Fv/Fm  is  used  to  indicate  the  photosynthetic 
efficiency  and  is  proportional  to  the  quantum  yield. 

Chlorophyll  a  was  extracted  with  acetone  by  the  method 
of  Jeffrey  and  Humphrey  ( 1975).  Total  chlorophyll  a  was 
calculated  from  absorbance  at  663  and  630  nm  and  stan- 
dardized per  algal  cell  extracted. 

Zooxanthellae  densities  were  calculated  from  the  total 
number  of  zooxanthellae  and  the  surface  area  of  the  coral. 
Number  of  zooxanthellae  was  determined  from  replicate 
(8-10)  hemacytometer  counts.  Surface  area  was  deter- 
mined by  covering  the  surface  of  the  coral  with  aluminum 
foil,  weighing  the  toil,  and  applying  a  standard  curve  re- 
lating aluminum  weight  to  area. 

Results 

High-temperature  stress  in  ambient  light 

The  response  of  corals  and  their  symbiotic  algae  to  high- 
temperature  stress  varied  with  species  of  coral,  but  fol- 
lowed a  similar  pattern  (Figs.  1-4).  The  pattern  is  best 
illustrated  with  Montastrea  annularis  maintained  at  32°C: 
photosynthetic  rates  and  potential  (fluorescence  ratio 
p+DCMiyp-DCMU)  as  we,|  as  p.R  (photosynthesis:  respi- 
ration) ratio  all  decreased  before  any  significant  change 
in  density  of  zooxanthellae  was  evident.  At  34°C  it  took 
less  than  24  h  for  photosynthesis,  fluorescence  ratios,  and 
the  P:R  to  decrease  to  0.  The  zooxanthellae  density  and 
chlorophyll  a  content  per  zooxanthella  changed  little  at 
34°C  before  coral  death  was  first  observed  at  19  h.  Zoo- 
xanthellae from  A/,  annularis  maintained  at  30°C  differed 
little  from  zooxanthellae  isolated  from  freshlv  collected 


BLEACHING  OF  REEF  CORALS 
Montastrea  annularis  Montastrea  cavernosa 


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Figure  1.  Patterns  of  bleaching  of  zooxanthellae  in  the  corals  Momasirea  anniilanx  and  At  cavernosa 
exposed  to  seawater  temperatures  of  26°C  (control).  30°,  32°.  and  34°C:  gross  photosynthesis:  respiration 
ratio  (P:R),  gross  photosynthesis  (Pg),  and  fluorescence  ratio  (F+DCMU/F-DCMU)  in  relation  to  exposure  time. 
All  data  points  are  means  ±  SD.  n  =  4.  unless  otherwise  noted.  *  =  significantly  (P  <  0.05.  ANOVA) 
different  from  controls. 


302 


W    K.  FITT  AND  M.  E.  WARNER 


corals  or  those  maintained  at  ambient  seawater  temper- 
ature (26°  ±  1°C)  for  2  days.  Chlorophyll  a  per  zooxan- 
thella  increased  slightly  over  the  2-d  experiment,  probably 
due  to  photoadaptation  to  the  experimental  light  inten- 
sities, which  were  slightly  lower  than  the  light  intensities 
in  situ  (Fig.  2). 

M.  cavernosa  responded  somewhat  differently  to  in- 
creases of  temperature  in  the  light  than  did  M.  annularis. 
No  significant  reductions  in  the  density  of  zooxanthellae 
were  observed  over  53  h  at  any  temperature  (Fig.  2).  Pho- 
tosynthesis, fluorescence,  and  the  P:R  ratio  at  26°,  30°, 
and  32°C  remained  relatively  stable,  except  that  there 
was  a  25%-50%  decrease  in  these  parameters  at  53  h  at 
32°C  (Fig.  1).  In  contrast,  photosynthesis,  fluorescence, 
and  P:R  of  the  zooxanthellae  from  M.  cavernosa  declined 
rapidly  at  34°C  in  a  fashion  similar  to  that  seen  in  zoo- 
xanthellae from  A/,  annularis.  Chlorophyll  a  per  zoo- 
xanthella  increased  slightly  throughout  the  experiment  at 
all  temperatures  except  34°C,  at  which  values  remained 
the  same  or  decreased  slightly  (Fig.  2).  Zooxanthellae  from 
Agaracia  lamarcki  appeared  to  be  less  tolerant  to  seawater 
temperatures  of  32°  and  34°C  than  were  zooxanthellae 
from  A.  agaricites  (Figs.  3-4).  Photosynthesis  and  the  flu- 
orescence ratio  of  zooxanthellae  from  A.  lamarcki  de- 
clined faster  at  34°C  than  zooxanthellae  from  A.  agaricites 
(Fig.  3).  At  32°C,  photosynthesis  and  fluorescence  ratios 
decreased  significantly  for  both  species  (Fig.  3),  and  zoo- 
xanthellae density  was  about  half  of  that  from  corals  main- 
tained at  30°  or  26°C  (Fig.  4).  Chlorophyll  a  per  zoo- 
xanthella  did  not  change  significantly  (P  >  0.05,  ANOVA) 
at  any  temperature  for  either  coral,  except  for  a  marked 
decrease  for  A.  lamarcki  at  32 °C  for  48  h  (Fig.  4). 

High-temperature  stress  and  light 

When  pieces  of  A/,  annularis  were  exposed  to  different 
wavelengths  of  light  at  32°C,  those  experiencing  the  largest 
decrease  in  fluorescence  ratio  (Fv/Fo)  received  wave- 
lengths in  the  UV-A  range  (320-400  nm)  or  blue  to  blue- 
green  light  (395-495  nm)  (Fig.  5A).  Rates  of  decrease  in 
fluorescence  ratio  were  no  different  with  or  without  nat- 
ural levels  of  UV-B  light  (<320  nm)  in  these  experiments 
(ANOVA,  P  >  0.05).  Control  corals  (those  maintained  at 
26°C  in  natural  light  with  no  filters)  showed  no  change 
in  fluorescence  ratio  throughout  the  experiment. 

Fluorescence  ratios  (Fv/Fo)  of  intact  M.  annularis  ex- 
posed to  32°C  declined  faster  when  exposed  to  higher 
intensity  than  lower  intensity  of  natural  light  (Fig.  5B). 
Interestingly,  a  "recovery"  trend  was  observed  in  the  same 
experiment  and  in  three  similar  experiments  (not  in- 
cluded) during  periods  of  cloudy  weather. 

Discussion 

This  study  shows  that  symbiotic  dinoflagellates  living 
inside  of  reef  corals  exhibit  a  marked  decline  in  their  pho- 


tosynthetic  capacity  and  oxygen  evolution  when  exposed 
to  higher  than  normal  temperatures  (32°,  34°C)  in  natural 
light  for  relatively  short  periods  of  time.  Reductions  in 
photosynthesis  and  corresponding  flow  of  electrons  be- 
tween photosystems  II  and  I,  as  indicated  by  fluorescence 
ratios,  preceded  any  significant  reductions  in  density  of 
zooxanthellae  in  the  reef-building  corals  Monlastrea  an- 
nularis, Agaricia  lamarcki,  and  A.  agaricites.  In  addition, 
zooxanthellae  from  A/,  cavernosa  and  A.  agaricites  ap- 
peared to  be  more  tolerant  to  the  experimental  temper- 
ature regimes,  showing  reduced  photosynthetic  compe- 
tence after  longer  exposure  times  (>24  h).  There  was  no 
significant  reduction  in  symbiont  density  in  M.  cavernosa 
over  the  course  of  the  experiment  (48-55  h),  though 
probably  they  too  would  eventually  lose  symbiotic  algae 
that  were  not  photosynthetically  functional.  The  data 
correspond  to  the  bleaching  patterns  seen  in  the  field;  M. 
annularis,  and  A.  lamarcki  commonly  lose  color  during 
bleaching  events,  whereas  A/,  cavernosa  rarely  bleaches 
and  A.  agaricites  sometimes  bleaches.  The  results  of  this 
study  suggest  that  the  differences  seen  in  nature  in  bleach- 
ing of  coral  species  may  be  due  to  the  different  physio- 
logical tolerances  of  their  specific  symbiotic  algae. 

Though  it  has  been  clear  for  some  time  that  the  fluo- 
rescence patterns  and  photosynthetic  rates  of  cultured 
zooxanthellae  are  altered  at  moderate  increases  above 
control  temperatures  (e.g.,  32°  vs.  26°C)  (Iglesias-Prieto 
et  u/..  1992).  there  has  been  debate  as  to  the  mechanism 
of  bleaching  in  relation  to  mode  of  release  of  the  zoo- 
xanthellae from  the  coral  and  the  relative  health  of  the 
symbiont  and  host  (Gates  et  ai,  1992).  Hoegh-Guldberg 
and  Smith  ( 1989)  clearly  showed  that  bleaching  of  corals 
can  occur  without  loss  of  zooxanthellae,  especially  when 
high  light  intensities  "photo-bleach"  the  algal  pigments. 
However,  most  bleaching  events  in  nature  involve  heat 
stress  with  full  solar  radiation,  and  the  loss  of  both  sym- 
biotic dinoflagellates  and  their  photosynthetic  pigments 
has  been  documented  (Kleppel  et  a/.,  1989;  Porter  et  ai, 
1989).  In  our  study,  chlorophyll  a  content  per  zooxan- 
thella  varied  little,  in  spite  of  up  to  55  h  of  exposure  to 
temperatures  as  high  as  34°C.  These  results  are  similar 
to  those  of  Hoegh-Guldberg  and  Smith  (1989),  also  in- 
volving short-term  laboratory  experiments  on  corals  ex- 
posed to  elevated  temperatures,  but  in  contrast  to  field 
observations  made  during  natural  bleaching  events  in  the 
Virgin  Islands  and  southern  Florida  which  showed  re- 
ductions of  chlorophyll  a  ranging  from  50%  to  80%  (Porter 
et  ai,  1989;  Kleppel  et  ai.  1989).  The  only  reductions 
seen  in  chlorophyll  content  in  this  study  occurred  at  34°C 
at  longer  exposure  times,  suggesting  that  pigment  loss 
during  bleaching  occurs  after  physiological  damage  to 
photosynthesis.  During  short-term  (days)  laboratory  ex- 
periments, zooxanthellae  from  the  more  sensitive  sym- 
bioses  appear  to  leave  the  host  before  or  during  loss  of 


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BLEACHING  OF  REEF  CORALS 

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Figure  2.  Patterns  of  bleaching  of  zooxanthellae  in  the  corals  Mimtaslrca  anniiluris  and  M.  cavernuMi 
exposed  to  seawater  temperatures  of  26°C  (control),  30°,  32°,  and  34°C:  chlorophyll  a  per  zooxanthella 
and  zooxanthella  density  in  relation  to  exposure  time.  All  data  points  are  means  ±  SD.  n  =  4,  unless 
otherwise  noted.  *  =  significantly  (P  <  0.05,  ANOVA)  different  from  controls. 


photosynthetic  pigmentation.  Longer  exposures  (weeks) 
to  elevated  seawater  temperatures  (>30°C)  typically  in- 
volve loss  of  chlorophyll  a  per  zooxanthellae  co-occurring 
with  relatively  low  rates  of  zooxanthellae  expulsion  and 
a  decrease  in  density  of  zooxanthellae  (Glynn  and  D'Croz, 
1990).  Thus,  loss  of  photosynthetic  pigments  appears  to 
be  a  normal  step  in  warm-water  bleaching  in  nature,  and 
one  that  indicates  algal  stress. 

When  Hoegh-Guldberg  and  Smith  (1989)  used  chlo- 
rophyll data  taken  from  water  surrounding  the  corals  to 
calculate  release  rates  of  zooxanthellae  from  the  heat- 
stressed  (30°,  32°C)  Pacific  corals  Stylophora  pistillata 
and  Serialopora  hystrix.  expulsion  rates  increased  by  a 
factor  of  2  to  10,  but  only  the  corals  maintained  at  32°C 
showed  significant  decreases  in  density  of  zooxanthellae. 
In  the  present  study,  zooxanthellae  density  in  Montastrea 


annularis,  Agaricia  lamarcki.  and  A.  agaricites  decreased 
significantly  only  after  photosynthesis  and  enhanced 
zooxanthellar  fluorescence  decreased.  Zooxanthellae  from 
;\/.  cavernosa  were  apparently  more  resistant  to  the  higher 
temperatures  than  zooxanthellae  in  M.  annularis,  in  that 
zooxanthellae  density  did  not  change  over  the  2  days  of 
exposure  to  32°C.  However,  at  least  some  of  the  zooxan- 
thellae in  M.  cavernosa  held  2  days  at  32°C  showed  re- 
duced photosynthetic  capacity,  and — on  the  basis  of  the 
experiments  with  M.  annularis — densities  might  be  ex- 
pected to  decrease  after  longer  exposure  times.  Similarly. 
Glynn  and  D'Croz  (1990),  who  documented  steady  de- 
creases in  density  of  zooxanthellae  from  Pocillopora 
damicornis  at  30°  and  32°C.  found  that  the  effects  were 
evident  (significantly  different  from  controls)  only  after 
2  weeks  or  more. 


304 


W.  R.  FITT  AND  M  E.  WARNER 


Agaricia  lamarcki 


Agaricia  agaricites 


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40 


Time  (h) 


Time   (h) 


Figure  3.  Patterns  of  bleaching  of  zooxanthellae  in  the  corals  Agaric/a  af>ariciles  and  A  lamarckii  exposed 
to  seawater  temperatures  of  26°C  (control),  30°,  32°,  and  34°C:  gross  photosynthesis:  respiration  ratio  (P: 
R).  gross  photosynthesis  (Pg),  and  fluorescence  ratio  (F+DCMu/F-DCMu)  in  relation  to  exposure  time.  All  data 
points  are  means  ±  SD..  /;  =  4,  unless  otherwise  noted.  *  =  significantly  (P  <  0.05.  ANOVA)  different  from 
controls.  Symbols  as  in  Figures  1  and  2. 


X 

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BLEACHING  OF  REEF  CORALS 
Agaricia  lamarcki  Agaricia  agaricites 


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Figure  4.  Patterns  of  bleaching  of  zooxanthellae  in  the  corals  Agaricia  agariatf*  and  A  lamarckii  exposed 
to  seawater  temperatures  of  26°C  (control),  30°.  32°.  and  34°C:  chlorophyll  a  per  zooxanthella  and  zoo- 
xanthella  density  in  relation  to  exposure  time.  All  data  points  are  means  ±  SD.  /;  =  4,  unless  otherwise 
noted.  *  =  significantly  (P  <  0.05,  ANOVA)  different  from  controls.  Symbols  as  in  Figures  I  and  2. 


Light  and  dark  rates  of  zooxanthellar  expulsion  are 
identical  in  Stylophora  pistillata  and  Scriatopora  hystnx 
maintained  at  ambient  temperature  (Hoegh-Guldherg  and 
Smith.  1989).  In  contrast,  corals  maintained  at  high  tem- 
peratures in  the  light  exhibit  higher  rates  of  expulsion 
(Hoegh-Guldberg  and  Smith.  1989),  resulting  in  reduced 
densities  of  zooxanthellae  at  32°C  (Hoegh-Guldberg  and 
Smith.  1989;  this  study).  The  quantity  of  light  makes  a 
major  difference  in  the  kinetics  of  warm-water-induced 
bleaching;  zooxanthellae  kept  in  dim  light  take  longer, 
and  often  require  higher  temperatures,  to  achieve  the  same 
level  of  bleaching  as  seen  in  brighter  light  (Fig.  2  this  study. 
Warner  and  Fitt.  unpub.).  The  quality  of  light  is  also  a 
factor  in  bleaching.  Although  the  effects  of  large  and  sud- 
den increases  in  UV-B  can  be  devastating  to  zooxanthellae 
in  corals  (Lesser  ct  ai,  1990;  Gleason  and  Wellington. 


1993).  most  shallow-water  corals  have  UV-protective 
mycosporine-like  amino  acids  (MA As)  that  screen  out 
such  dangerous  wavelengths.  Much  more  likely  sources 
of  synergistic  light  energy  for  bleaching  are  longer  wave- 
length UV-A  (wavelengths  not  screened  out  by  MAAs) 
and  blue  light,  both  important  in  photosynthesis  and 
therefore  not  screened  out  by  the  coral  host  (Dunlap  el 
ai.  1988).  Preliminary  experiments  show  that  blue  light 
also  promotes  bleaching  of  some  types  of  cultured  zoo- 
xanthellae much  more  effectively  than  the  same  amount 
of  light  at  any  other  part  of  the  visible  spectrum  (Fitt  and 
Warner,  unpub.). 

It  is  not  clear  at  present  whether  coral  death  is  solely  a 
function  of  animal  tissue  death,  or  if  lack  or  dysfunction 
of  zooxanthellae  may  trigger  or  exacerbate  events  preced- 
ing host  tissue  sloughing  and  coral  death.  That  the  latter 


306 


W.  K.  FITT  AND  M.  E.  WARNER 


0.75 


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>320  nm 
>395  nm 
>495  nm 
Control 
Reef 


-O 


High  light 
Low  lightt 


0.2 


75 


100 


Time  (h) 


Figure  5.  Fluorescence  ratios  (Fm/F0)  of  zooxanthellae  in  Moniaslrca  annularix  collected  from  the  reef 
(28°C)  or  exposed  to  seawater  temperatures  of  26°C  (control)  and  32°C  (all  other  datal  under  different 
wavelengths  (A)  and  intensities  (B)  of  natural  light  in  relation  to  exposure  time.  Neutral-density  screens 
were  used  to  adjust  maximum  intensities  to  54%  of  air  ambient  (high  light)  in  all  experiments.  Cut-off  filters 
were  used  to  adjust  wavelength  (A),  and  additional  neutral-density  screens  reduced  ambient  light  to  19%  of 
airambient  (low  light)  in  (B).  All  data  points  are  means  ±  SD,  n  =  6.  *  =  significantly  different  from  control 
(A.  ANOVA)  or  low  light  intensities  (B,  Student's  /  test). 


can  occur  in  nature  was  illustrated,  on  a  somewhat  longer 
time  scale,  in  the  Eastern  Pacific  after  extensive  coral 
bleaching  during  the  El  Nino  Southern  Oscillation 
(ENSO)  event  of  1982- 1983  (Glynn,  1983,  1984).  Before 
the  widespread  local  and  regional  deaths  of  the  corals,  no 
zooxanthellae  remained  in  the  tissues  of  Poci/lopora 
damicomis  and  Millepora  spp.  Temperatures  only  a  few 
degrees  above  normal  ambient  will  kill  reef  corals.  Mayer 
(1914)  found  that  all  the  reef  corals  tested  in  the  Dry 
Tortugas.  at  the  end  of  the  Florida  reef  tract,  died  when 
exposed  for  an  hour  to  temperatures  between  36°  and 
38°C  dui  ing  the  summer.  In  this  early  study,  the  organ- 
isms that  died  at  the  lowest  temperatures  tested  (M.  an- 
nularis,  A.  lamarcki.  and  the  hydrocoral  Millepora  sp.) 
are  the  same  sp  \  ies  that  are  the  first  to  react  during  nat- 
ural bleaching  events  (e.g.,  Williams  and  Bunkley-Wil- 


liams,  1988),  lending  support  to  the  notion  that  the  dif- 
ferential bleaching  of  zooxanthellate  cnidarians  exposed 
to  moderately  high  temperatures  in  nature  reflects  the 
tolerances  of  their  particular  zooxanthellae. 


Acknowledgments 

We  thank  Drs.  R.  Smith  and  G.  Schmidt  for  technical 
assistance  during  portions  of  this  study,  and  W.  Wiebe 
for  comments  on  the  manuscript.  This  research  was  par- 
tially supported  by  grants  from  NSF  (OCE  9203327, 
OCE  9349773),  ONR  (N00014-92-J-1734),  and  NOAA 
(CMRC  95-3041).  Contribution  #2  from  the  Key  Largo 
Marine  Research  Laboratory1  and  #575  from  the  Discov- 
ery Bay  Marine  Laboratory. 


BLEACHING  OF  REEF  CORALS 


307 


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Reference:  Bid  Bull  189:  308-319.  (December.  1995) 


Filtration  and  Utilization  of  Laboratory-Cultured 

Bacteria  by  Dreissena  polymorpha,  Corbicula 

fluminea,  and  Camnculina  texasensis 

H.  SILVERMAN.  E.  C.  ACHBERGER1,  J.  W.   LYNN,  AND  T.  H.  DIETZ 

Department  of  Zoology  and  Physiology:  *  Department  of  Microbiology. 
Louisiana  Slate  University.  Baton  Rouge.  Louisiana  70803 


Abstract.  Dreissena  polymorpha  consumed  about  6 
x  108  Escherichiu  co/i  from  20  ml  of  artificial  pondwater 
( APW)  in  30  min  under  laboratory  conditions.  The  clear- 
ance rate  per  mussel  was  1 43  ±  25  ml  g" '  dry  tissue  min  ' 
The  E.  coli  used  in  these  studies  ranged  from  about  1.7 
to  2.9  (im  in  length.  15S-labeled  E.  coli  were  used  to  dem- 
onstrate that  bacteria-derived  nutrients  were  incorporated 
into  mussel  tissue.  Electrophoretic  analysis  of  mussel  and 
bacterial  proteins  on  12%  polyacrylamide  gels  allowed  the 
visual  determination  of  incorporation  of  labeled  amino 
acids  into  bivalve  proteins  and  demonstrated  that  intact 
bacteria  were  not  simply  trapped  in  mussel  tissues.  The 
conversion  of  bacterial-labeled  amino  acids  into  mussel 
protein  was  about  26%.  Similarly,  we  demonstrated  that 
D.  polymorpha  can  use  other  bacterial  species  ranging  in 
size  from  about  1.3  to  4.1  ^m.  including  Citwbacter 
freundii.  Enterobacter  aerogenes,  Serratia  marcescens. 
Bacillus  megaterium,  and  B  suhlilus.  The  ability  of  D. 
polymorpha  to  take  up  E.  coli  was  compared  with  that  of 
two  other  freshwater  mussels,  Corbicula  fluminea  and 
Carunculina  texasensis.  On  a  mussel-dry-weight  basis,  D. 
polymorpha  cleared  bacteria  30  to  100  times  faster  than 
Corbicula  fluminea  and  Carunculina  texasensis.  respec- 
tively. The  ability  to  filter  E.  coli  appears  to  be  related  to 
the  architecture  of  the  cirri  on  the  latero-frontal  cells  of 
the  gill.  Cirri  from  Corbicula  and  Dreissena  are  similar 
in  size,  but  Dreissena  has  a  larger  gill  compared  to  the 
tissue  dry-weight,  and  has  10:  times  more  cirri  than  found 
in  Corbicula.  Carunculina.  the  unionid  representative,  has 


Received  5  January  1995;  accepted  14  September  1995. 

Abbreviations:  APW — artificial  pondwater;  TCA — trichloroacetic  acid: 
PAGE — polyacrylamide  gel  electrophoresis;  SDS— sodium  dodecyl  sul- 
fate;  SEM — scanning  electron  microscopy 


smaller  and  fewer  cirri,  and  has  relatively  limited  ability 
to  capture  E.  coli. 

Introduction 

Dreissena  polymorpha  (Pallas)  has  successfully  colo- 
nized much  of  the  Great  Lakes  region  of  North  America. 
It  is  currently  achieving  the  same  success  in  the  Ohio, 
Tennessee,  and  Mississippi  river  drainages  (unpubl.  obs.). 
This  organism  can  filter  large  amounts  of  water  in  a  rel- 
atively short  period,  eliminating  or  greatly  reducing  the 
abundance  of  zooplankton  and  phytoplankton  (Stanczy- 
kowska  el  a/..  1976:  Maclsaac  el  al.  1992;  Leach,  1993; 
Bunt  et  al..  1993).  Previous  reports  indicate  that  D. 
polvmorpha  selects  food  particles  in  the  size  range  1 5-40 
^m  (Ten  Winkel  and  Davids,  1982),  and  filters  particles 
>2  urn  with  almost  100%  relative  efficiency  (J0rgensen 
et  al..  1984).  Capture  of  particles  ranging  in  size  from  0.7 
^m  (Sprung  and  Rose,  1988)  to  750  urn  (Ten  Winkel  and 
Davids,  1982)  has  been  reported. 

Initial  capture  of  particles  occurs  on  the  gills  of  eula- 
mellibranch  bivalves  and  is  mediated  by  the  ciliary  me- 
chanical systems  associated  with  the  gill  filaments.  While 
all  eulamellibranch  gills  are  organized  into  filaments,  the 
structure  and  organization  of  specialized  ciliary  append- 
ages associated  with  the  filaments  vary  from  species  to 
species  (Atkins,  1938;  Morton,  1983).  All  have  lateral  cil- 
iated cells  that  are  generally  believed  to  be  responsible  for 
moving  water  through  the  gill.  The  latero-frontal  cells  are 
positioned  between  the  frontal  surface  of  the  filament  and 
the  lateral  ciliated  cells.  The  ciliary  appendages  on  these 
cells  range  from  simple  cilia  in  some  species,  to  longer, 
stiffer  cilia  in  other  species,  to  fused  plates  of  cilia  in  still 
others.  In  bivalves  with  cirri,  the  number  of  cilia  per  plate 
varies  with  species:  11-12  fused  cilia  in  Carunculina  te.\- 


308 


UPTAKE  OF  BACTERIA  BY  FRESHWATER  BIVALVES 


309 


asensis.  22-26  in  Myii/ns  ediilis.  and  38-42  fused  cilia  in 
D.  polymorpha  ( Atkins.  1938:  Moore.  1971:  Owen.  1974). 
These  structures,  together  with  even  more  distally  located 
abfrontal  and  frontal  cilia,  act  in  concert  to  capture  and 
move  particles.  The  ciliated  structures,  the  water  currents 
they  produce,  and  the  mucus  produced  by  the  gills  and 
palps  move  food  particles  toward  the  mouth  (Beninger  et 
al..  1992:  1993;  Ward  ct  ai.  1993).  On  the  basis  of  struc- 
ture alone,  some  investigators  have  indicated  that  cirri  or 
latero-frontal  cilia  act  as  a  mechanical  filtering  device  that 
can  explain  particle  trapping  in  various  bivalve  species 
(Owen,  1974:  Owen  and  McCrae.  1976:  Silvester  and 
Sleigh.  1984).  However,  the  actual  mechanism  of  particle 
capture  remains  a  topic  of  disagreement.  Jorgensen  (1976, 
1982,  1989)  has  argued  that  such  descriptions  fail  to  ac- 
count for  fluid  movements  and  the  complex  currents  as- 
sociated with  the  gill.  Recent  endoscopic  studies  directly 
demonstrated  that  mucus  is  involved  in  transporting  par- 
ticles after  their  capture  by  the  gill  (Beninger  et  al..  1992: 
Ward  et  al..  1993). 

In  this  study,  we  describe  controlled  laboratory  exper- 
iments aimed  at  assessing  how  well  D  polymorpha  niters 
bacteria,  and  we  compare  the  results  to  those  for  other 
freshwater  mussels.  The  experiments  tested  the  ability  of 
D.  polymorpha.  Cantnculina  texasensis  (a  unionid  rep- 
resentative), and  Corbicidafluminea  to  utilize  laboratory- 
cultured  Escherichia  coli  as  a  sole  nutrient  source.  Under 
laboratory  conditions  D.  polymorpha  showed  rapid  filtra- 
tion and  incorporation  of  "S-labeled  E.  coli.  The  clearance 
of  bacteria  from  artificial  pondwater  (APW)  was  faster  in 
Dreissena  than  in  either  Corbicitla  Jhiminea  or  Camn- 
culina  texasensis.  These  differences  in  filtration  in  labo- 
ratory studies  suggest  that  the  ability  to  use  natural-sized 
bacteria  in  the  environment  may  differ  substantially 
among  these  freshwater  species. 

Materials  and  Methods 

Animals 

Dreissena  polymorpha  [range  17-25  mm  length;  1.225 
±  0.027  g  total  live  weight  (mean  ±  SE):  0.016  ±  0.000  g 
dry  tissue:  n  =  160];  was  collected  from  the  Mississippi 
River  from  screens  at  the  Dow  Chemical  Plant  in  Pla- 
quemine,  Louisiana.  The  unionid  Carunculina  texasensis 
(23-26  mm  length;  2.453  ±  0.073  g  total  live  weight:  0.090 
±  0.006  g  dry  tissue:  n=  10)  was  collected,  under  permit, 
from  a  pond  in  Baton  Rouge.  Louisiana:  and  Corhici/la 
fluminea  (23-25  mm  length;  7.871  ±  0.233  g  total  live 
weight;  0.368  ±  0.012  g  dry  tissue;  n  =  36)  was  collected 
from  the  Tangipahoa  River  in  southern  Mississippi.  All 
species  were  kept  under  laboratory  conditions  in  aerated 
artificial  pondwater  (APW;  0.5  NaCl.  0.4  CaCl2,  0.2 
NaHCO3, 0.05  KC1  in  mA/)  with  Dreissena  in  APW  con- 
taining 0.2  mM  Mg2SO4  (Dietz  et  al..  1994).  Animals  were 


maintained  unfed  in  the  laboratory  for  five  days  be- 
fore use. 

Labeling  ol  bacteria 

Escherichia  coli  JM83  (Messing.  1979)  was  used  for 
most  of  the  experiments.  For  15S-labeling,  E.  coli  were 
grown  in  a  chemically  defined  medium  containing  5  g 
glucose,  8 10  mg  NH4C1,  and  82  mg  MgCl2  •  7H:O  per  liter 
of  0.05  ;\/  potassium  phosphate  buffer.  pH  7.2.  Added  to 
this  was  5  ml  of  a  trace  salts  solution  containing 
CaCl2  •  2H2O  (2  g),  MnSO4  •  H2O  ( 1  g),  and  FeSO4  •  7H,O 
(0.5  g)  dissolved  in  one  liter  of  0. 1  M  HC1.  For  growth  of 
E.  coli  JM83,  it  was  necessary  to  add  20  Mg/ml  L-prolme 
and  5  Mg/ml  thiamine.  final  concentrations,  to  the  me- 
dium. The  carbon  source,  trace  salts  solution.  L-proline, 
and  thiamine  were  sterilized  separately  from  the  rest  of 
the  medium.  To  label  the  bacteria.  E.  coli  were  grown  at 
37°C  with  shaking  aeration  for  at  least  four  generations 
in  the  above  medium  containing  5  ^Ci/ml  carrier-free 
Na235SO4  (Dupont  NEN).  The  final  cell  density  of  cultures 
was  approximately  1.3-2  X  109  bacteria/ml.  Labeled  bac- 
teria were  collected  by  centrifugation.  washed  once  in  the 
growth  medium  without  carbon  source,  and  stored  in 
APW  at  a  concentration  of  3  X  109  bacteria/ml.  The  cells 
were  stored  on  ice  in  pondwater  until  use.  Following  this 
initial  transfer,  the  bacteria  did  not  experience  any  ad- 
ditional osmotic  shock  and  survived  for  weeks.  E.  coli 
grown  in  this  medium  were  2.3  ±  0.6  yum  long  and  0.9 
±  0. 1  Mm  wide  (n  =  50)  and  did  not  clump. 

Incorporation  of  35S  during  growth  ofE.  coli  was  mea- 
sured with  a  liquid  scintillation  counter.  The  bacteria  were 
precipitated  in  10%  trichloroacetic  acid  (TCA)  and  col- 
lected on  a  glass  fiber  filter.  Greater  than  70%  incorpo- 
ration of  the  label  was  routine. 

To  determine  if  filtration  of  E.  coli  by  D.  polymorpha 
was  novel  for  freshwater  bivalves  or  whether  other  bac- 
terial species  would  be  similarly  filtered,  several  other 
bacteria  differing  in  size  ( 1 .3-4. 1  urn  in  length)  and  shape 
were  tested.  A  similar  35S-labeling  protocol  was  used  to 
label  Citrobacter  freimdii.  Enterobacter  aerogenes,  Ser- 
ratia  marcescens,  Bacillus  subtilus.  and  B.  megateriwn. 
The  concentration  of  bacteria  in  all  suspensions  was  de- 
termined by  direct  microscopic  count  using  a  hemocy- 
tometer.  Cell  dimensions  for  labeled  bacteria  were  mea- 
sured from  photomicrographs. 

Escherichia  coli  feeding  experiments 

All  feeding  experiments  were  carried  out  in  individual 
containers  (test  tubes)  aerated  for  the  duration  of  the  ex- 
periment. Individuals  of  similar  size  were  selected  by 
weighing,  then  placed  in  separate  test  tubes  containing 
20  ml  of  APW.  The  experiment  was  started  by  the  addi- 
tion of  bacteria  as  soon  as  the  bivalves  began  siphoning. 


310 


H    SILVERMAN  ET  AL 


Siphoning  typically  began  within  10  min  of  placing  ani- 
mals in  the  test  tube  containing  pondwater. 

Each  test  tube  had  3  X  107  bacteria/ml  representing 
about  1.7  X  105dpm  35S/ml  (200^1  stock  bacterial  cell 
suspension).  Each  day  the  E.  co/i  stock  solution  (in  APW 
and  held  on  ice)  was  centrifuged  and  resuspended  in  APW. 
The  discarded  supernatant  was  assayed  for  35S.  Using  this 
assay  procedure,  we  found  that  E.  coli  did  not  deteriorate, 
and  that  all  35S  (>99%)  added  to  an  experimental  tube 
was  associated  with  intact  bacteria  in  the  APW  and  not 
with  breakdown  products  in  the  supernatant.  Control 
tubes  without  bivalves  received  labeled  bacteria,  were  aer- 
ated for  20  min,  and  analyzed.  The  bath  solutions  from 
these  tubes  were  passed  through  a  Millipore  filter 
(0.22  ^m)  to  trap  the  bacteria.  Virtually  all  of  the  label  in 
each  tube  (99.5%)  was  on  the  filter,  while  the  supernatant 
contained  1319  ±  85  dpm/ml  (n  =  15).  Thus,  <0.5%  of 
the  radioactivity  was  in  the  non-particulate  material  of 
the  assay  medium  (similar  results  were  obtained  by  cen- 
trifuging  the  assay  medium;  less  than  1%  of  the  radioac- 
tivity remained  in  the  supernatant). 

The  bivalve  filtration  studies  were  initiated  by  collecting 
a  sample  (to)  of  the  bath  exactly  45  s  after  inoculation  of 
the  pondwater  with  bacteria.  This  time  interval  was  re- 
quired for  mixing  and  was  previously  determined  both 
visually  (methylene  blue)  and  by  tracking  the  distribution 
of  labeled  bacteria  in  test  tubes  without  an  animal  present. 
Individual  test  tubes  were  usually  sampled  initially  and 
at  the  end  to  avoid  disturbing  the  animals,  with  final  sam- 
ples collected  after  5  to  90  min,  depending  on  the  bivalve 
species.  The  final  sample  was  taken  by  mixing  the  tube 
and  taking  a  100-^1  sample.  For  all  samples,  35S  radio- 
activity was  determined  with  a  liquid  scintillation  counter 
(Wiegman  el  ai,  1975).  Additional  controls  for  these  ex- 
periments consisted  of  dried  shells  or  rinsed,  formalin- 
fixed  whole  animals  placed  in  individual  containers  and 
handled  as  described  above.  In  none  of  the  controls  was 
radioactivity  significantly  reduced  in  the  bath.  At  the  con- 
clusion of  the  experiments,  animals  were  removed  from 
their  shells  and  dried  overnight  to  constant  weight  at  90°C. 
Radioactive  35S  adsorbed  to  control  (fixed)  animal  tissue 
or  shell  was  less  than  100  dpm.  Incorporation  of  label 
was  not  detectable  in  the  control  tissue  analyses  (see 
below). 

At  the  end  of  some  experiments,  each  animal  was  re- 
moved from  the  tube  and  rinsed  2-3  times  in  >500  ml 
pondwater  containing  no  label,  then  placed  in  a  separate 
beaker  containing  at  least  1 50  ml  of  APW.  The  label  was 
allowed  to  K-  incorporated  into  the  animal  for  48  h,  with 
two  additional  changes  (rinsed  3  X  500  ml)  of  APW  each 
day.  Usually  se  era!  hundred  dpm/ml  were  released  into 
the  APW  by  an  animal  during  a  12-h  period.  After  48-h, 
each  animal  was  removed  from  the  bath  and  rinsed  several 
times  in  APW  and  a  blood  sample  taken  by  pericardia! 


puncture  (Fyhn  and  Costlow,  1975).  Blood  sampling  lo- 
cation was  previously  determined  by  dissection  of  D. 
polymorpha  to  identify  appropriate  landmarks.  A  syringe 
needle  (15.9  mm,  26  ga)  was  inserted  between  the  valves 
between  the  inhalant  and  exhalant  siphons  and  into  the 
pericardial  cavity  located  in  the  vicinity  of  the  posterior 
margin  of  the  hinge  (Dietz  el  ai,  1994).  To  collect  >  100  n\ 
of  blood,  equal  to  10-20%  of  the  animal's  wet  weight,  the 
syringe  needle  had  to  be  rotated  to  prevent  tissue  from 
occluding  the  needle  orifice.  The  osmolality  of  the  APW 
was  4  mOsm  and  the  water  contained  about  300  dpm/ 
ml  radioactivity  at  the  time  of  collection  of  blood  from 
the  bivalves.  Measured  osmolarity  of  the  blood  ranged 
between  40  and  42  mOsm,  within  the  normal  range  pre- 
viously reported  (Dietz  et  a/.,  1994),  and  radioactivity  in 
the  blood  was  1  to  4  orders  of  magnitude  higher  than  that 
found  in  APW.  These  data  suggest  little  if  any  contami- 
nation of  blood  samples  by  mantle  cavity  fluid. 

Animals  were  dissected  free  of  the  shell  and  the  tissue 
was  dried  overnight  at  90°C  and  weighed.  The  tissue  was 
homogenized  in  3%  TCA,  and  the  TCA-precipitable  pellet 
was  washed  twice  in  TCA  and  re-pelleted.  The  final  su- 
pernatant was  discarded  and  the  pellet  dissolved  in  1  M 
NaOH.  A  sample  of  digested  tissue  was  assayed  for  ra- 
dioactivity and  an  aliquot  analyzed  for  protein  content 
using  a  BioRad  protein  determination  procedure.  This 
allowed  us  to  determine  the  amount  of  J5S  incorporated 
following  the  feeding  experiments.  The  48-h  rinse  in  large 
volumes  of  APW  was  used  to  allow  passage  of  any  bacteria 
that  might  have  remained  in  the  gut,  and  also  allowed 
time  for  incorporation  of  radioactive  label  into  clam  pro- 
teins. 

Variations  in  the  basic  experimental  protocol  included 
experiments  designed  to  compare  clearance  rates  in  dif- 
ferent volumes  of  bath  (with  constant  bacterial  concen- 
tration) and  at  different  bacterial  concentrations  (while 
keeping  bath  volume  constant).  Volumes  tested  were  20, 
60,  and  120  ml.  Even  with  the  small  20-ml  bath  volume, 
the  reduction  of  bacterial  radiolabel  followed  first-order 
exponential  kinetics.  The  use  of  a  small  volume  reduced 
the  amount  of  radiolabeled  waste  fluid  produced.  In  ad- 
dition, we  maintained  the  bath  at  a  constant  20  ml  while 
increasing  the  number  of  bacteria  present.  The  latter  ex- 
periments were  designed  to  assess  maximal  bacterial 
clearance  under  laboratory  conditions. 

Finally,  in  an  attempt  to  assess  whether  repeated  ex- 
posure to  bacteria  led  to  satiation,  we  exposed  some 
animals  to  6  X  108  E.  coli  in  20  ml  of  bath,  assayed 
bacterial  disappearance  from  the  medium  over  30  min, 
waited  30  min  and  added  another  6  X  108  cells;  this  pro- 
cess was  repeated  until  we  determined  that  the  animals 
were  no  longer  removing  bacteria  from  the  bath  at  the 
initial  rate. 


UPTAKE  OF  BACTERIA  BY  FRESHWATER  BIVALVES 


311 


Electrophoretic  analysis  of  35S-labeled  proteins 

To  demonstrate  the  assimilation  of  bacterial  sources  of 
sulfur-containing  amino  acids  by  D.  polymorpha.  a  few 
animals  were  dissected  directly  into  liquid  nitrogen  after 
48-h  pulse  labeling.  Each  animal,  as  well  as  a  separate 
sample  of  the  ^S-labeled  E.  coli  (109  cells),  was  placed  in 
400  M!  of  ice-cold  buffer  ( 10  mMTris-HCl,  1  mA/EDTA, 
10  mAl  NaCl,  pH  7.8)  with  protease  inhibitor,  0.7  mg/ 
ml  phenylmethylsufonyl  fluoride.  The  samples  were  par- 
tially homogenized  by  sonic  disruption  using  the  microtip 
probe  of  an  ultrasonic  processor  (Model  W220,  Heat  Sys- 
tems-Ultrasonic, Inc.).  Samples  received  four  10-s  bursts 
at  a  power  setting  of  2  and  were  cooled  on  ice  for  3  min 
between  treatments.  Following  sonication,  the  samples 
were  centrifuged  at  16,000  X  g  for  3  min,  and  the  super- 
natant fluid  was  collected  on  ice.  For  each  cell-free  extract, 
the  TCA  precipitable  radioactivity  was  determined,  and 
the  protein  content  was  assayed.  Proteins  in  the  cell-free 
extracts  were  separated  by  electrophoresis  on  a  1 2%  poly- 
acrylamide  gel  (PAGE)  according  to  the  method  of 
Laemmli  (1970).  D.  polymorpha  samples  contained  ap- 
proximately 100,000  dpm  in  60  ^g  of  protein.  Following 
electrophoresis,  samples  were  visualized  by  staining  with 
Coomassie  brilliant  blue  and  by  autoradiography  with 
Kodak  X-OMAT  AR  film.  Molecular  weight  standards 
(Broad  range  SDS-PAGE  Standards,  BioRad  Laborato- 
ries) stained  with  Coomassie  brilliant  blue  were  used  to 
define  the  banding  patterns  of  the  samples. 

Cirral  structure 

Differences  in  cirral  structure  and  distribution  were  ex- 
amined and  described  using  scanning  electron  microscopy 
(SEM).  Tissue  fixation  followed  modification  of  previously 
described  procedures  (Richard  el  al.  199 1 ).  Animals  were 
fixed  for  1  h  in  2%  glutaraldehyde  in  phosphate  buffer 
(35-60  mOsm  as  appropriate  to  match  blood  osmolality 
of  the  bivalve  species).  Gills  were  excised  and  exposed  to 
glutaraldehyde  for  an  additional  hour,  rinsed  in  buffer, 
and  postfixed  in  1%  osmium  tetroxide.  Gills  were  dehy- 
drated in  an  ethanol  series,  critical-point  dried,  mounted 
on  aluminum  stubs  using  carbon  tape,  and  sputter-coated 
with  gold/palladium  (20  nm).  Gills  were  examined  using 
a  Cambridge  200  scanning  electron  microscope.  For  cal- 
culations of  cirral  size  and  gill  surface  areas  it  was  nec- 
essary to  measure  the  shrinkage  associated  with  tissue 
processing  for  SEM  examination.  For  these  purposes, 
several  gills  from  each  species  were  carefully  excised  and 
photographed  using  a  dissecting  microscope.  The  tissue 
was  fixed  and  critical-point  dried  as  described  above,  then 
re-photographed  to  allow  determination  of  tissue  shrink- 
age under  our  tissue  preparation  procedures. 


13.0-1 


0.8H 


^   0.6 

Q_ 

?   0.4 
o 

o    0.24 


0.0 


0    20  40  60  80 
time,  min 


8.0 


25  50 

time  (min) 


75 


100 


Figure  1.  Inset  is  the  time-dependent  removal  of  Escherichia  coli 
from  pondwater  by  Drei.isena  polymorpha  (filled  squares).  Corhicula 
fluminea  (open  squares),  or  Carunculina  texasensis  (open  circles).  The 
vertical  lines  represent  ±1  standard  error.  The  slope  of  the  line  of  the 
time-dependent  natural  logarithmic  transformation  of  radioactive  bacteria 
concentration  in  the  APW  is  the  rate  constant  (min"').  Each  mussel  was 
placed  in  20  ml  of  pondwater  containing  6  x  10s  bacteria  labeled  with 
15S.  Each  point  represents  the  mean  ±  standard  error  for  at  least  10 
separate  animals.  The  t,/2  was  7.1,  16.5,  and  77.0  min  forD.  polymorpha. 
Corbicula  fluminea,  and  Carunculina  texasensis.  respectively. 


Calculations  and  statistical  analysis 

Disappearance  of  radioactive  label  from  the  bath  was 
exponential  and  followed  first-order  rate  kinetics  (rate 
constant  =  ln(D0/D,)  •  t  ',  D  was  the  dpm/ml  at  times  0 
and  t).  The  half  time  was  calculated  from  the  expression 
t1/2  =  ln(2)/(rate  constant).  Bacterial  clearance  rate  (C, 
ml  min"')  for  each  mussel  was  calculated  using  the  equa- 
tion C  =  V/t-ln(D,,/Dt),  where  V  is  volume  in  ml,  D0 
and  D,  are  bacteria  radiolabel  concentrations  defined 
above  ( Riisgard,  1988).  Weight-specific  clearance  (dry  soft- 


312 


H.  SILVERMAN  ET  AL. 
Table  I 


Pale  of35S-labeled  E.  coli  uptake  by  three  species  of  freshwater  bivalves 


Bivalve  species 


Dry  tissue,  mg 


Rate  constant,  min~ 


Clearance,  ml  g  '  dry  tissue  min 


Dreissena  polymorpha 

20 

16  ±  lc 

-0.098  ±  0.011" 

143.1  ±24.6a 

Corbicula  fluminea 

32 

368  ±  12" 

-0.043  ±  0.002" 

4.4  ±  0.6" 

Carunculina  texasensis* 

10 

90  ±  6b 

-0.009  ±  0.002C 

1.3  +0.2C 

Data  expressed  as  mean  ±  standard  error.  Values  within  a  column  having  different  letters  were  significantly  different  (P  <  0.01 )  using  an  unpaired 
Student's  (-test. 

*  The  same  animals  were  sampled  repeatedly  at  30-min  intervals  for  90  min. 


tissue  mass)  data  were  expressed  as  ml  g  '  dry  tissue  min  ' . 
In  some  cases  we  determined  the  rate  constants  and  cal- 
culated clearance  from  a  linear  regression  of  ln(dpm/ml) 
as  a  function  of  time  to  compare  rate  constants  or  to 
calculate  clearance  between  species  or  experimental  treat- 
ments. Differences  between  species  or  treatments  were 
determined  by  ANOVA  and  were  considered  significant 
if  P  <  0.05.  Fisher's  protected  least  significant  difference 
(PLSD)  was  used  to  determine  differences  between  average 
values  within  a  group  having  a  significant  ANOVA.  Dif- 
ferences between  regression  slopes  were  determined  using 
Student's  /-test. 

Results 

Filtration  of  E.  coli  by  freshwater  bivalves 

Freshwater  bivalves  removed  E.  coli  from  pondwater 
with  first-order  exponential  kinetics  (Fig.  1).  The  size 
of  the  cultured  E.  coli  was  2.3  ±  0.6  /urn  long  and  0.9 
±0.1  ^m  wide.  The  bacterial  concentration  used  in 
these  experiments  was  approximately  3  X  107  per  ml 
in  20  ml  APW,  and  10-36  animals  of  each  species  were 
used  to  obtain  the  average  values.  The  slope  of  the  lines 
represents  the  rate  constants  for  removal  of  bacteria  by 
the  different  bivalve  species  (Table  I).  The  rate  constants 
differed  statistically  (P  <  0.01 )  for  each  species,  and  D 
polymorpha  had  the  highest  rate  and  the  shortest  \U2 
(Fig.  1 ).  On  a  dry  weight  basis,  clearance  rates  also  were 
highest  for  D.  polymorpha  and  lowest  for  Carunculina 
texasensis.  The  rate  of  clearance  exhibited  by  D,  poly- 
morpha was  30- 1 00  times  greater  than  that  of  the  other 
two  species. 

Incorporation  of  radioactive  bacterial  nutrients  in 
bivalve  proteins 

Disappearance  of  radiolabel  from  the  bath  indicated 
that  the  mussels  were  capable  of  removing  the  particles 
from  the  water  column,  but  did  not  necessarily  indicate 
that  the  particles  and  their  associated  nutrients  were  as- 
similated. Mussels  hold  food  particles  in  their  digestive 
tube  and  perhaps  in  the  mantle  cavity  for  days  without 


digesting  them.  In  addition,  we  have  observed  living  algae 
escaping  into  the  water  column  from  pseudofeces  released 
by  unionids  two  days  after  feeding  (S.  J.  Nichols,  pers. 
comm.;  unpub.  obs.).  However,  35S  label  was  accumulated 
into  the  body  fluids  of  the  animals  48  h  after  the  pulse 
feeding  (Table  II).  Given  the  amount  of  radiolabel  ob- 
served in  the  blood  of  the  animals,  it  was  unlikely  that 
the  blood  samples  were  contaminated  by  the  APW.  The 
APW  bath  containing  each  D.  polymorpha  had  only  302 
±  35  dpm/ml  (n  =  5)  at  the  end  of  the  48-h  period.  Each 
of  these  bivalve  species  had  label  in  the  blood,  and  this 
fluid  is  presumably  maintained  in  a  sterile  condition  by 
the  animal.  Thus,  label  was  likely  to  be  attributable  to 
dissolved  35S-containing  matter  (amino  acids,  polypep- 
tides,  etc.).  Further,  48  h  after  Dreissena  were  exposed  to 
bacteria,  a  few  animals  were  fed  Ch/orel/a.  After  algal 
feeding  (30  min),  the  gut  tubule  was  visibly  green,  and 
was  dissected  from  the  animal  for  measurement  of  radio- 
activity. Only  bacteria  in  the  gut  contents  would  be  ra- 
diolabeled  and  detected  by  scintillation  counting,  and  in 
no  case  were  counts  above  background  recorded. 

To  determine  whether  the  radioactivity  in  the  mussels 
was  in  the  form  of  bacterial  protein  or  had  been  converted 
into  mussel  protein,  we  used  PAGE  analysis  to  compare 
mussel  tissue  proteins  with  E.  coli  proteins.  The  presence 
of  E.  coli  proteins  in  the  PAGE  gels  of  mussel  tissue  would 
indicate  that  intact  bacteria  were  associated  with  the  mus- 
sel body.  Figure  2,  an  autoradiograph  of  a  corresponding 


Table  II 

•"S  accumulated  in  the  blood  of  freshwater  mussels  48  h  after  they 
were  fed  35S-labek-d  E.  coli  (3  X  107  bacteria/ml)  for  60  nun 


Bivalve  species 


Blood,  dpm/ml 


Dreissena  polymorpha 
Corbicula  tlummea 
Carunculina  texasensis 


835070  ±  224170s 

145694  ±  51875b 

1840  ±  387C 


Mean  ±  standard  error,  n  =  5  for  each  species.  The  means  with  different 
letters  are  significantly  different  using  the  unpaired  Student's  /-test  (P 
<  0.05). 


UPTAKE  OF  BACTERIA  BY  FRESHWATER  BIVALVES 


313 


A    B 


Figure  2.  Autoradiograph  of  a  12%  polyacrylamide  gel  used  to  elec- 
trophoretically  separate  proteins  isolated  from  the  homogenized  whole 
body  of  Dreissena  polymorpha.  Mussels  were  allowed  48  h  to  assimilate 
"S-laheled  Escherichia  coli  after  a  20-min  feeding  (individual  mussels 
represented  in  lanes  A-E).  Lane  F  represents  the  soluhilized  protein  frac- 
tion of  the  35S-laheled  E  cull  used  in  the  feeding  experiments.  Molecular 
weight  markers  were  located  from  the  stained  gel.  Note  the  similarity 
between  individual  D  polymorpha  (lanes  A-E)  and  the  distinctive  dif- 
ference between  these  lanes  and  the  E  coli  lane.  There  was  no  evidence 
for  the  presence  of  any  of  the  recognized  E.  coli  proteins  in  the  D 
polymorpha  lanes  (the  heavy  E  coli  bands  at  about  50  kDa  and  95-100 
kDa  were  not  present  in  lanes  A-E).  Conversely,  many  of  the  major 
labeled  proteins  in  the  mussel  tissue  do  not  appear  in  the  E  coli  band. 


polyacrylamide  gel,  does  not  show  any  overlap  between 
E.  coli  proteins  (lane  F)  and  label  incorporated  into  mussel 
proteins  (A-E,  each  lane  representing  an  individual  D. 
polymorpha).  The  incorporation  of  35S  into  mussel  pro- 
teins indicated  the  assimilation  of  bacterial  components; 
E.  coli  proteins  were  not  present  in  the  bivalve  samples. 
The  lack  of  bacterial  contamination  allowed  us  to  de- 
termine the  incorporation  characteristics  of  nutrients  into 
mussel  tissue.  Several  individuals  of  each  species  were  al- 
lowed to  feed  on  labeled  bacteria,  removed  to  pondwater 
(500  ml.  changed  twice  daily)  containing  no  bacteria  or 
label,  and  allowed  to  assimilate  label  for  48  h  following 
the  pulse  exposure  to  radiolabeled  bacteria.  Whole  animals 
were  homogenized,  then  precipitated  in  TCA,  and  the 
precipitate  was  dissolved  in  1  M  NaOH.  The  35S  incor- 
porated into  macromolecules/total  35S  removed  from  the 
bath  was  0.29  ±  0.02  (n  =  4),  0.23  ±  0.07  (n  =  4),  and 
0.28  ±  0.02  (n  =  5)  for  D.  polymorpha.  Corbiada  Jlit- 
minea.  and  Carunculina  texasensis,  respectively.  Thus, 
regardless  of  mussel  species,  about  26%  of  the  label  that 


disappeared  from  the  bath  was  incorporated  into  mussel 
protein  48  h  after  the  pulse  feeding  experiment.  There 
were  no  significant  differences  between  animal  species  in 
the  proportion  of  35S  assimilated  into  mussel  protein  from 
E.  coli.  Formalin-fixed  control  tissue  had  no  detectable 
radioactivity. 

I  'olume  of  water  cleared  of  bacteria 

Increasing  the  volume  of  fluid  available  for  clearance 
by  D.  polymorpha  while  maintaining  a  constant  concen- 
tration of  bacteria  per  milliliter  allowed  us  to  determine 
clearance  rates  over  different  volumes,  but  with  differing 
quantities  of  total  bacteria  present  in  the  APW  (Fig.  3). 
The  rate  constants  (slope)  for  the  different  volumes  were 
20ml,  -0.098  ±  0.01  miiT1,  60ml,  -0.029  ±  0.001 
min~';  120  ml,  -0.009  ±0.001  min'1.  These  slopes  were 
significantly  different  from  each  other  (P  <  0.01).  When 
expressed  as  clearance  (ml  g~'  dry  tissue  min~'),  the  values 
were  20  ml,  143  ±  25;  60  ml,  189  ±  26;  and  120  ml,  113 
±11.  These  weight-specific  clearance  rates  were  not  sig- 
nificantly different  from  each  other  (P  >  0.05).  Because 
D.  polymorpha  in  this  data  set  weighed  about  15  ±  1  mg 
(n  =  55),  the  animals  cleared  1.7-2.8  ml  animal"1  min~' 
under  all  experimental  conditions,  or  roughly  2.4-4  liters 
of  water  per  day  for  an  average  animal  (about  8.9  1  g~' 
dry  tissue  day~'). 

By  varying  the  concentration  of  E.  coli  in  a  constant 
bath  volume  and  determining  the  time-dependent  (5-60 
min)  removal  of  particles  from  the  suspension,  we  cal- 
culated the  average  clearance  rates  (Fig.  4).  These  data 


13.0-1 


20  40  60 

time  (min) 


80 


Figure  3.  Clearance  of  35S-labeled  Escherichia  coli  by  Dreissena 
polymorpha  from  different  volumes  of  pondwater  containing  3 
X  10'  bacteria/ml.  Open  squares  indicate  experiments  in  20  ml  volume, 
filled  squares  in  60  ml,  and  open  circles  in  120  ml  of  pondwater.  The 
vertical  line  indicates  ±  1  standard  error.  The  slopes  of  the  lines  were 
significantly  different  (P  <  0.0 1 )  but  when  multiplied  by  the  bath  volume 
and  normalized  to  dry  tissue  weight  the  clearance  values  ranged  from 
1 13  to  189  ml  g~'  min*1  and  were  not  significantly  different  (P  >  0.05). 


314 


H.  SILVERMAN  ET  AL. 


100. On 


80.0- 


°>       60.0- 


40.0- 


<u 
O 


20.0- 


o.c 


10 


15 


20 
-7 


25 


E.  coli  concentration,  x  10    /ml 


Figure  4.  Clearance  of  Escherichia  coli  from  20  ml  of  artificial 
pondwater  by  Dreixxena  polymorpha  as  a  function  of  bacterial  concen- 
tration. Note  that  bacterial  clearance  appeared  to  be  approximately  con- 
stant between  3.7  and  1 1. 1  x  I07  bacteria/ml  but  was  reduced  at  higher 
bacteria  concentrations.  The  vertical  lines  represent  ±  1  standard  error. 


indicate  that  D.  polymorpha  had  a  relatively  constant  rate 
of  bacterial  clearance  when  exposed  to  bacterial  concen- 
trations ranging  from  3.7  to  1 1  X  107  bacteria/ml,  but  at 
concentrations  greater  than  18  X  107  bacteria/ml  the  ap- 
parent rate  of  bacterial  nitration  was  significantly  reduced 
(P<0.0\). 

When  bacteria  concentration  was  kept  at  or  below  3 
X  107  bacteria/ml,  no  pseudofeces  were  observed  over  60 
min.  When  higher  concentrations  of  bacteria  were  used, 
pseudofeces  production  was  visually  observed  after 
roughly  60  min  of  exposure.  Thus  at  the  higher  bacteria 
concentrations,  pondwater  was  cleared  of  radioactivity 
but  some  of  the  label  was  deposited  in  pseudofeces  rather 
than  accumulated  by  the  animal. 

Maximal  uptake  of  bacteria  by  D.  polymorpha 

In  a  few  experiments,  we  followed  the  disappearance 
of  E.  coli  from  the  bath  under  the  standard  conditions 
described  in  Figure  1,  but  allowed  the  mussel  to  remain 
in  the  container  and  added  another  aliquot  of  bacteria  to 
the  APW  after  30  min.  The  bath  was  sampled  at  t0  and 
t30to  measure  isotope  uptake.  This  procedure  was  repeated 
until  the  animal  was  no  longer  removing  bacteria  at  the 
initial  rate.  Under  these  conditions  five  separate  feedings 
were  required  before  the  removal  of  bacteria  by  the  mussel 
was  reduced  (data  not  shown). 

Clearance  of  other  bacterial  species 

To  determine  whether  bacterial  species  other  than  E. 
coli  could  be  filtered,  specimens  of  D.  polymorpha  were 
exposed  to  a  variety  of  laboratory  strains  of  several  bac- 
terial species  of  different  sizes  (0.8-1.1  ^m  width.  1.3- 
4.1  i/rn  length),  following  the  protocol  used  to  produce 


Figure  1.  Table  III  indicates  the  rates  of  clearance  of  the 
bacterial  species  by  D.  polymorpha,  and  the  dimensions 
of  each  species  including  E.  coli.  In  all  cases  except  one, 
55-91%  of  the  bacterial  cells  were  removed  from  the  bath 
in  30  min  and  the  calculated  clearance  rates  were  50- 
80  ml  g~'  dry  tissue  min"1.  The  exception  was  Bacillus 
megaterium,  which  formed  large  chains  of  bacteria.  Some 
of  these  bacterial  chains  settled  to  the  bottom  of  our  ex- 
perimental containers  and  were  not  available  in  the  APW 
for  the  mussel  to  filter.  In  this  case,  the  corresponding 
clearance  rate  was  significantly  lower  than  for  the  other 
bacterial  species. 

Effects  of  laboratory  storage  of  mussels  on  bacterial 
uptake 

Separate  experiments  were  performed  on  animals  ac- 
climated to  the  laboratory  for  various  periods.  Clearance 
rates  of  E.  coli  by  D.  polymorpha  were  not  significantly 
different  whether  the  mussels  were  tested  18  h  after  they 
were  collected  from  the  Mississippi  River  or  after  they 
had  been  stored  in  the  laboratory,  unfed,  for  a  week:  105 
±  17  (n  =  10)  versus  108  ±  24  (n  =  10)  ml  g~'  dry  tissue 
min  '  18  h  after  collection  and  one  week  later,  respec- 
tively. Furthermore,  these  clearance  rates  were  not  sig- 
nificantly different  from  that  of  D.  polymorpha  maintained 
in  the  laboratory,  unfed,  for  several  weeks. 

Cirral  structure 

Briefly,  the  cirrus  organelle  is  composed  of  two  plates 
of  fused  cilia  and  beats  from  a  flexed  to  an  extended  po- 
sition (Moore,  1971;  Owen,  1974).  In  the  flexed  position 
the  cirri  bend  up  over  the  frontal  surface  of  the  filament. 
In  the  extended  position,  the  cirri  lie  in  the  internlament 


Table  III 

Mean  cell  dimensions  (n  >  50  for  each  bacterium)  for  bacteria  used 
in  Dreissena  polymorpha  studies  measuring  the  clearance 
Inim  20  ml  APW 


Cell  dimensions 

Dry 

tissue. 

Clearance, 
ml  g~'  dry 

Bacterium  species 

(L  • 

W)Mm 

mg 

tissue  min"' 

Citrobacter  freundii 

1.3 

+ 

0.3 

X 

0.8 

±0 

1 

12 

±  1 

64 

±  12(9)' 

Enterobacter  aerogenex 

1.5 

f 

0.3 

X 

0.9 

±0, 

1 

17 

±  ? 

50 

±  10  (9)a 

Serratiu  marcescens 

1.8 

+ 

0.5 

X 

0.8 

±0 

1 

13 

±  1 

52 

±12(8)" 

Escherichia  coli 

2.3 

+ 

0.6 

• 

0.9 

±0 

1 

18 

±  4 

80 

±  1  1  (  1  1  )a 

Bacillus  megaterium* 

3.6 

+ 

1.0 

X 

1.1 

±0. 

•) 

15 

±  1 

14 

±  1  (9)" 

Bacillus  suhtilus 

4.1 

± 

0.8 

X 

0.9 

±0.2 

14 

±  1 

59 

±  6  (9)a 

Mean  ±  standard  error  (n).  The  chains  did  not  remain  in  suspension 
during  the  course  of  the  experiment.  Clearance  values  with  different 
letters  are  significantly  different  using  Fisher's  PLSD  test. 

*  Bacteria  in  this  culture  were  present  in  chains  with  an  average  length 
of  14.5  ±  3.7  jim. 


UPTAKE  OF  BACTERIA  BY  FRESHWATER  BIVALVES 


315 


space  opposing  the  cirri  from  the  adjacent  filament.  When 
extended,  each  cirrus,  along  with  its  adjacent  cirri,  forms 
a  filtration  trap  that  does  not  allow  movement  of  particles 
down  into  the  ostia  of  the  gill.  Trapped  particles  are  passed 
to  the  frontal  cilia  when  the  cirri  bend  into  their  flexed 
position.  Differences  in  the  structure  of  the  cirri  of  D. 
polymorpha.  Corbicula  fluminea,  and  Carunculina  tex- 
asensis  are  shown  in  Figure  5.  The  free  tips  of  the  cirral 
cilia  form  the  filtration  traps,  and  the  spaces  between  cil- 
iary tips  are  in  the  0.5-Mm  range  for  Dreissena.  Note  the 
smaller  cirral  structure  of  C.  te.\asensis  compared  with 
the  other  two  species. 

Even  with  an  osmotically  balanced  fixative,  gill  shrink- 
age in  Carunculina  texase nsis  was  16.6%  (dorsal-ventral, 
long  axis  of  the  filament)  and  38.9%  (anterior-posterior); 
14.4%  and  33.4%  in  Corbicula  fluminea:  and  23.0%  and 
25.2%  in  D.  polymorpha.  In  Carunculina  and  Corbicula 
the  differential  shrinkage  between  length  and  width  is  due 
to  the  relatively  rigid  connective  tissue  support.  Carun- 
culina manifests  this  support  as  calcified  chitinous  rods 
supporting  the  filaments.  D.  polymorpha  shows  less  sup- 
port, and  shrinkage  was  more  uniform. 

Discussion 

Dreissena  polymorpha  was  capable  of  filtering  and  in- 
gesting large  numbers  of  laboratory-cultured  bacteria  from 
the  water  column.  On  an  individual  animal  basis  with  no 
standardization  for  size  differences  among  animals,  Cor- 
bicula fluminea  cleared  E.  colial  a  significantly  lower  rate 
(P  <  0.01)  than  did  D.  polymorpha.  but  at  a  higher  rate 
than  did  Carunculina  texasensis.  When  standardized  on 
the  basis  of  dry  tissue  weight,  the  bacterial  clearance  rate 
was  30-100  times  higher  in  D.  polymorpha  than  in  the 
other  two  bivalve  species.  Standardization  on  the  basis  of 
dry  tissue  weight  provides  a  convenient  normalization  for 
clearance  study  comparisons.  However,  surface  area  of 
the  gill  is  likely  to  be  physiologically  important  with  regard 
to  the  actual  mechanism  of  particle  capture.  Both  Cor- 
bicula fluminea  and  Carunculina  texasensis  had  gill  sur- 
face areas  that  were  about  1 .5  mrrr/mg  dry  tissue,  whereas 
D.  polymorpha  had  about  14  mrrr/mg  dry  tissue  (Table 
IV).  When  gill  surface  area  was  used  as  a  normalizing 
factor,  it  was  apparent  that  the  dreissenid  species  had  a 
greater  ability  to  capture  E.  coli  than  did  the  other  two 
species.  A  regression  analysis  comparing  E.  coli  clearance 
from  the  APW  with  cirri  g~'  dry  tissue  among  the  bivalve 
species  was  highly  significant  (r  =  0.998;  P  <  0.05),  with 
100-200  times  more  cirri  in  D  polymorpha  than  in  the 
other  bivalves. 

In  addition  to  gill  surface  area,  another  major  difference 
among  these  three  species  was  the  complexity  of  the  latero- 
frontal  cirri  associated  with  the  gill  filaments.  Gill  cirri  lie 
between  the  lateral  ciliated  cells  and  the  surface  of  the 


filaments.  The  lateral  ciliated  cells  and  perhaps  the  mus- 
culature in  eulamellibranch  gills  provide  the  force  for  wa- 
ter movement.  Although  there  is  considerable  debate  on 
the  exact  mechanism  of  particle  capture  (hydrodynamic 
vs.  direct  mechanical  contact;  see  Nielsen  et  ai,  1993), 
the  movement  of  cirri,  frontal  and  abfrontal  cilia  are  ap- 
parently coordinated  to  intercept,  capture,  and  move  par- 
ticles. Recent  endoscopic  work  indicates  that  ciliary  ac- 
tivity, mucus,  and  water  current  are  important  in  trans- 
porting particles  toward  the  mouth  once  they  are  captured 
(Beninger  et  ai.  1993;  Ward  et  ai.  1993).  When  the  cirri 
are  extended,  they  are  directly  in  the  path  of  water  flow 
between  the  filaments.  The  cirri  are  positioned  to  trap 
particles  and  direct  them  toward  the  filament  apex  and 
the  frontal  cilia  when  the  cirri  are  flexed  (unpub.  obs.). 
In  the  extended  position,  cirri  form  an  effective  filtration 
"trap"  or  "net."  Unfortunately,  the  words  trap  and  net 
may  seem  to  exclude  the  possibility  of  considering  water 
current  generation  by  the  cirri  as  part  of  the  filtration 
mechanism  or  "trap."  However,  if  the  term  is  defined  to 
mean  "barrier"  or  "intercepting  unit"  then  the  comments 
here  are  compatible  with  previous  endoscopic  and  mi- 
croscopic observations  of  particle  transport  and  particle 
"bouncing"  (J0rgensen,  1976;  Ward  et  ai,  1993). 

The  scanning  electron  micrographs  demonstrate  clearly 
that  the  cirri  of  D.  polymorpha  and  Corbicula  fluminea 
are  more  complex  than  those  of  Carunculina  texasensis. 
The  cirri  in  the  first  two  species  are  composed  of  two 
ciliary  plates  containing  as  many  as  42  cilia  per  plate  (un- 
pub. obs.).  The  cirri  of  Carunculina  are  less  complex, 
consisting  of  1 1-13  cirri  per  plate,  and  not  as  long  or  as 
rigid  as  those  in  the  other  species  (Fig.  5;  Table  IV).  The 
organization,  or  number  of  cirri  along  the  filament  (cirri/ 
mm),  also  was  much  reduced  in  Carunculina  compared 
to  the  other  species.  Although  the  shorter,  less  organized 
cirri  were  apparently  able  to  intercept  some  E.  coli,  C. 
texasensis  captured  E.  coli  less  effectively  than  did  the 
other  two  species.  The  enhanced  ability  of  the  species  with 
more  complex  cirri  to  filter  E.  coli  is  consistent  with  the 
hypothesis  that  the  cirri  are  acting  as  particle-capturing 
structures  (Owen.  1974;  Owen  and  McCrae,  1976).  This 
is  likely  to  be  particularly  true  for  small  (<2  ^m)  particles. 
Indeed,  using  laser  confocal  microscopy  to  study  in  vitro 
gill  strips,  we  have  observed  the  interaction  between 
0.75  ^m  fluorescent  particles  and  individual  cirri  at  a  res- 
olution approaching  0.2  ^m  (unpub.  obs.).  Morphometric 
analysis  also  indicates  that  when  adjusted  for  animal  dry 
weight,  the  number  of  cirri  in  a  standard-sized  Corbicula 
fluminea  is  similar  to  that  in  Carunculina  texasensis,  and 
is  about  two  orders  of  magnitude  less  than  that  of  D.  po- 
lymorpha (Table  IV).  On  a  dry-weight  basis,  D.  polymor- 
pha had  a  clearance  rate  two  orders  of  magnitude  higher 
than  Carunculina  texasensis  and  30  times  greater  than 
Corbicula  fluminea. 


316 


H.  SILVERMAN  ET  AL 


Ax.tJ>:.      , 

muiut 


Figure  5.     Scanning  electron  micrographs  of  gill  filaments  from  Drcixxcna  polynwrpha.  Corbiculajluminea, 

and  (.aninciilina  lexaiensis.  (a)  Lower  magnification  of  a  D  pitlynuirpha  gill  filament  and  portions  of  the 


UPTAKE  OF  BACTERIA  BY  FRESHWATER  BIVALVES 


317 


Table  IV 

\ltnplionicliic  characteristics  etf  gills  from  representative  bivalves  used  in  bacterial  clearance 


Animal  dry 

Live  gill  area. 

Cilia 

Cirri/mm2 

Cirri/mm2 

Cirri/mg 

Bivalve  species 

mass,  mg 

mm2  per  mg  dry  tissue 

per  cirri 

dry  gill 

live  gill 

dry  tissue 

l>reissena  /'o/rmix/'/k; 

13.3 

13.72 

38-42 

44.161 

16.863 

45,556 

(.'nrhicnlii  Ihiminen 

392 

1.44 

32-33 

35.802 

20,430 

330 

I  'arunculina  ti'.\asen\i.\ 

100 

1.68 

11-13 

11.263 

5.526 

189 

Many  authors  have  shown  that  clearance  rates  of  bi- 
valves are  dependent  on  the  number  of  particles  in  the 
water  column  (Morton.  1971:  Walz,  1978).  Foster- 
Smith  ( 1975)  demonstrated  that  assimilation  efficiency 
was  related  to  the  number  of  particles  taken  up  by  ma- 
rine bivalves.  The  same  author  noted  that  pseudofeces 
are  produced  at  particle  concentrations  associated  with 
high  assimilation  efficiency.  Walz  (1978)  documented 
a  threshold  of  clearance  below  which  Dreissena  does 
not  produce  pseudofeces.  We  observed  that  no  pseu- 
dofeces were  produced  in  30  min  at  bacterial  concen- 
trations that  stimulated  maximal  particle  clearance 
rates.  This  observation,  coupled  with  our  experimental 
pulse-washout  procedures  designed  to  minimize  recy- 
cling of  label,  allowed  us  to  estimate  the  incorporation 
of  35S  directly  from  bacteria.  Despite  the  differences  in 
filtering  ability  among  the  three  species,  the  incorpo- 
ration of  1:~S  derived  from  E.  coli  was  similar.  Even 
though  the  absolute  quantity  of  E.  coli  ingested  differed 
among  the  species,  the  amount  of  label  incorporated 
into  mussel  protein  per  total  label  ingested  was  about 
26%  and  did  not  differ  among  the  three  species. 

The  present  study  indicates  that,  when  the  results  are 
normalized  on  a  whole-animal  basis,  D.  polymorpha 
and,  to  a  lesser  extent,  Corbicula  fluminea  were  better 
able  to  use  E.  coli  than  was  the  unionid  species  studied. 
However,  and  perhaps  more  importantly,  on  a  dry- 
weight  basis  D.  polymorpha  was  substantially  better  than 


either  of  the  other  species  in  clearing  E.  coli  from  the 
APW.  The  differences  in  ability  to  use  the  bacterial  food 
source  appear  to  correlate  rather  well  to  the  different 
structures  associated  with  particle  capture  in  these  spe- 
cies. The  gill  of  Dreissena  is  large  compared  to  the  size 
of  its  body,  and  it  has  138  times  as  many  cirri  as  found 
in  Corbicula  fluminea.  However,  D.  polymorpha  cleared 
bacteria  only  32  times  faster  than  did  Corbicula  flu- 
minea. Corbicula  may  move  more  water  per  gill  surface 
area  to  achieve  the  observed  rate  of  bacterial  particle 
capture  (see  Way  et  al.,  1990).  The  differences  in  clear- 
ance rate  described  here  are  for  a  single  bacterial  species 
under  laboratory  conditions,  and  provide  no  informa- 
tion on  particle  selection  by  these  bivalves.  The  current 
study  also  does  not  address  the  transport  of  particles 
once  they  are  captured. 

We  recalculated  a  clearance  rate  from  the  data  obtained 
by  Reeders  et  al.  (1989)  for  D.  polymorpha  fed  a  mixed 
population  of  algae.  Clearance  values  ranged  from  80  to 
180  ml  g~'  dry  tissue  min'1;  the  clearance  rate  we  found 
for  laboratory-cultured  E.  coli  was  within  this  range. 
Moreover,  in  a  study  in  which  several  marine  bivalves 
were  selected  on  the  basis  of  cirri  size,  retention  efficiencies 
for  particles  >5  /urn  were  similar  in  all  species  (Riisgard, 
1988).  However,  small  (2  /urn)  particles  were  poorly  re- 
tained by  bivalves  with  no  cirri,  but  species  with  large  cirri 
retained  similar-sized  particles  with  30-70%  efficiency 
(Riisgard.  1988). 


adjacent  filaments.  Note  the  presence  of  cirri  in  both  the  extended  (arrowheads)  and  flexed  (arrows)  positions. 
In  the  flexed  position  the  cirri  tips  are  located  over  the  frontal  cilia  (F).  The  cirri  in  D  polvmorpha  can 
extend  across  the  interfilament  space.  Cirri  from  opposite  sides  of  the  filament  in  the  fully  flexed  position 
can  cover  most  of  the  frontal  surface  of  the  filament,  (h)  Higher  magnification  of  the  interfilament  space 
covered  by  two  sets  of  cirri  from  adjacent  filaments.  Note  the  "net"  that  is  formed  by  the  38-42  cirri  tips, 
(c)  A  gill  filament  from  Corbicula  fluminea  showing  extended  cirri  (arrowheads),  flexed  cirri  (arrows),  and 
frontal  cilia  (F).  The  filtration  net  formed  by  extended  cirri  is  apparent.  The  length  of  the  cirri  indicate  that 
in  the  extended  position  the  cirri  span  the  interfilament  space.  There  are  a  large  number  of  cilia  tips  (32- 
33)  associated  with  a  single  cirri.  Corbicula  fluminea  also  has  frontal  cirri  (C)  located  among  the  frontal 
cilia,  (d)  A  gill  filament  from  Caruneulina  lexasensis  showing  cirri  in  the  extended  position  (arrowheads) 
and  in  the  flexed  position  (arrows)  on  opposite  sides  of  the  frontal  cilia  (F).  Cirri  from  an  adjacent  filament 
at  the  bottom  of  the  micrograph  are  in  a  partially  flexed  position.  Note  that  individual  cirri  have  only  12- 
13  free  cilia  tips  per  cirri  plate  (Bars:  a  =  20  ^m;  b  =  5  ^m;  c  =  10  nm:  d  =  10  urn). 


318 


H.  SILVERMAN  ET  AL. 


Determination  of  the  environmental  relevance  of  our 
observations  will  require  further  study.  All  the  bacteria 
used  in  these  experiments  were  from  laboratory  strains. 
Bacteria  in  nature  tend  to  be  smaller  (<1  /jm)  than  those 
grown  in  the  laboratory  (see  Table  III),  and  the  numbers 
of  bacteria  present  in  the  water  column  will  differ  from 
environment  to  environment  as  well  as  from  the  con- 
centrations studied  here  (Prieur  el  at.,  1990).  However, 
all  of  the  following  have  been  documented:  (1)  Food 
particle  density  is  an  important  determinant  of  clear- 
ance rate  of  bacterial-sized  particles  in  Dreissena.  This 
is  not  unusual  and  has  been  reported  for  a  number  of 
bivalves  feeding  on  many  different  particles  sizes,  in- 
cluding algae,  diatoms,  bacteria,  and  detritus  (Walz, 
1978).  (2)  Dreissena  effectively  filtered  all  strains  of 
laboratory-sized  bacteria  (1-4 /urn  in  length).  (3)  The 
26%  efficiency  of  conversion  of  E.  coli  proteins  into 
bivalve  proteins  is  well  within  the  limits  associated  with 
an  energetically  feasible  food  source.  (4)  The  rate  at 
which  freshwater  bivalves  cleared  laboratory  bacteria 
was  directly  related  to  the  size  and  number  of  cirri  per 
gill  surface  area. 

The  design  of  this  study  was  purposely  simplified  to 
consider  only  a  single  component  of  the  bivalve  feeding 
mechanism — that  of  particle  capture  or  clearance.  How- 
ever, these  experiments  raise  several  questions  about  the 
biology  of  freshwater  bivalves  that  require  further  study. 
For  example,  are  these  species  able  to  derive  significant 
nutrients  from  natural-sized  bacteria  in  the  water  column 
during  or  between  algal  blooms?  Many  studies  have  dem- 
onstrated that  bivalves  use  bacteria  present  in  the  water 
column  (e.g.,  Mikheev  and  Sorokin,  1966;  Lucas  et  a/., 
1987;  Prieur  el  til.,  1990).  In  particular,  Mytilm  echilis,  a 
marine  species  with  complex  cirri,  rapidly  clears  E.  coli 
from  the  water  column  in  laboratory  experiments  in  which 
the  bacterium  was  presented  as  the  sole  particle  source  or 
in  the  presence  of  algae  (Birbeck  and  McHenery,  1982; 
McHenery  and  Birbeck,  1985).  Do  pseudofeces  deposited 
on  the  substrate  serve  as  a  nutrient  resource  to  enhance 
bacterial  populations  in  a  microhabitat  around  freshwater 
bivalves  as  has  been  observed  in  marine  bivalves  (Crosby 
et  ill..  1990;  reviewed  by  Prieur  et  ai.  1990)?  Thus,  the 
potential  of  freshwater  bivalves  to  use  bacteria  could  be 
an  important  factor  influencing  the  distribution  of  these 
bivalves  and  merits  continued  study. 

Acknowledgments 

The  authors  thank  Julie  Cherry,  Ron  Bouchard,  and 
Justin  Marque/  for  their  outstanding  technical  assistance. 
We  thank  T.  R.  LeBlanc  at  the  Dow  Chemical  Company 
(Plaquemine,  LA)  for  helping  us  collect  zebra  mussels. 
Cory  Thompson  participated  in  early  experiments  on 
support  from  a  grant  to  improve  undergraduate  education 


from  the  Howard  Hughes  Medical  Institute  to  LSU.  K. 
Carman  provided  useful  comment  and  discussion.  We 
give  special  tribute  to  S.  J.  Nichols  for  the  many  hours 
she  has  spent  sharing  her  insight  on  mussel  feeding  with 
us.  The  work  was  supported  by  Louisiana  Sea  Grants  R/ 
ZM-l-PD  and  NA46RG0096  project  R/ZMM-1. 

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Reference:  Bio/  Bull  189:  320-329.  (December.  1995) 


The  Effect  of  Salinity  and  Temperature  on  Spawning 

and  Fertilization  in  the  Zebra  Mussel  Dreissena 

polymorpha  (Pallas)  from  North  America 


PETER  P.  FONG1,  KEIICHIRO  KYOZUKA2,  JILL  DUNCAN,  STACY  RYNKOWSKI, 
DANIEL  MEKASHA,  AND  JEFFREY  L.  RAM* 

Department  of  Physiology.  U'ayne  State  University.  Detroit.  Michigan  48201 


Abstract.  Zebra  mussels  have  dispersed  from  their  orig- 
inal site  of  introduction  in  the  Great  Lakes  into  the  Mis- 
sissippi River,  Hudson  River,  and  other  watersheds  in 
which  they  will  encroach  upon  brackish  water  estuaries. 
To  investigate  their  likelihood  of  reproductive  success  in 
such  estuaries,  we  investigated  the  conditions  of  temper- 
ature, salinity,  and  acclimation  under  which  spawning 
and  fertilization  could  occur.  Reproductive  function  of 
mussels  that  were  acclimated  to  salinities  up  to  7.0  parts 
per  thousand  (ppt)  at  12°,  20°,  and  27°C  for  1  to  21  days 
was  tested.  Reproductive  function  of  non-acclimated 
mussels  that  had  been  maintained  in  fresh-water  aquaria 
was  also  tested  in  various  salinities.  Spawning  was  induced 
by  serotonin,  previously  demonstrated  to  elicit  spawning 
of  fertile  gametes  in  fresh  water.  Successful  fertilization 
was  indicated  by  oocyte  cleavage  after  adding  sperm.  Non- 
acclimated  mussels  spawned  in  salinities  of  1.75  and 
3.5  ppt  at  12°,  20°.  and  27°C.  but  not  at  7.0  ppt.  Fertil- 
ization using  gametes  from  non-acclimated  mussels  oc- 
curred only  in  fresh  water  and  at  1.75  ppt.  Acclimation 
for  as  little  as  2  days  enhanced  spawning.  Fertilization 
rate  in  a  salinity  of  3.5  ppt  improved  within  4  days  of 
acclimation  and  continued  at  a  high  level  for  as  long  as 
21  days  of  acclimation.  Although  animals  acclimated  for 
4  days  in  3.5  ppt  spawned  readily  when  tested  in  salinities 
as  high  as  7.0  ppt,  almost  no  fertilization  occurred  in 
7.0  ppt.  The  reduction  in  fertilization  at  increasing  salin- 
ities may  be  due  in  part  to  reduced  sperm  motility.  Un- 


Received  22.  August  1994:  accepted  14  September  1995. 

*To  whom  all  correspondence  should  be  sent. 

Present  address:  'Department  of  Biology.  Gettysburg  College.  Get- 
tysburg, PA  17325. 2Asamushi  Marine  Biological  Station,  Tohoku  Uni- 
versity, Asmushi,  Aomori,  039-34,  Japan. 


fertilized  oocytes  remain  intact  for  hours  in  fresh  water; 
however,  in  salinities  as  low  as  0.7  ppt,  unfertilized  oocytes 
tended  to  rupture  within  2  hours.  These  data  show  that 
although  sudden  increases  in  salinity  produce  an  imme- 
diate decrease  in  the  reproductive  capacity  of  zebra  mus- 
sels, acclimation  to  brackish  water  can  occur,  and  zebra 
mussels  may  be  able  to  reproduce  in  brackish  water  below 
7.0  ppt. 

Introduction 

Zebra  mussels  (Dreissena  polymorpha)  have  spread 
rapidly  throughout  North  America  since  their  accidental 
introduction  in  the  mid-1980s  (Hebert  etal..  1989).  Their 
geographical  distribution  and  the  factors  controlling  it 
have  been  the  focus  of  a  number  of  studies  (Mackie  el 
al..  1989;  Griffiths  etal..  1 99 1 ;  Strayer,  1991;  Ramcharan 
el  al..  1992).  Strayer  and  Smith  (1993)  predicted  that  the 
downstream  movement  of  zebra  mussels  will  eventually 
carry  them  to  estuarine  sections  of  North  America.  At 
present,  zebra  mussels  occupy  brackish  waters  in  the 
Hudson  River  estuary  at  West  Haverstraw,  New  York 
(Walton,  1992;  D.  L.  Strayer,  pers.  comm.),  and  with 
populations  already  well  established  in  the  Susquehanna 
and  Mississippi  Rivers,  areas  such  as  Northern  Chesa- 
peake Bay  and  the  Mississippi  River  delta  are  at  particular 
risk  of  invasion. 

Unlike  most  freshwater  bivalves,  zebra  mussels  are  free- 
spawners,  releasing  large  numbers  of  gametes  directly  into 
the  water,  where  fertilization  occurs  (Sprung,  1987). 
However,  environmental  tolerance  to  salinity  may  not  be 
the  same  for  gametes  as  for  adults.  Thus,  even  though 
adult  zebra  mussels  may  be  capable  of  withstanding  a 
range  of  salinities,  these  conditions  may  be  suboptimal 
or  lethal  for  freshly  released  gametes  and  may  interfere 


320 


SALINITY  EFFECTS  ON  ZEBRA  MUSSEL  REPRODUCTION 


321 


with  fertilization.  Similar  situations  are  common  in 
brackish-water  organisms.  In  the  viviparous  polychaete 
Neanthes  limnicola.  higher  salinities  reduce  the  number 
of  juveniles  born  and  probably  interfere  with  oogenesis 
(Fong  and  Pearse,  1992).  In  the  brackish-water  macro- 
phyte  Myriophylhim  crispatitm.  higher  salinities  block 
both  sexual  and  asexual  reproduction  (James  and  Hart, 
1993).  Higher  salinities  reduce  reproductive  capacity  in 
brackish-water  populations  ofDaphnia  magna  (Arner  and 
Koivisto,  1993).  Eggs  of  the  yellow  perch  (Percaflavescens) 
suffer  higher  mortality  with  increasing  salinity  (Victoria 
c/  al.,  1992).  In  the  striped  mullet  (Mugil cephalus),  lower 
salinity  reduces  fertilization  and  sperm  motility  (Lee  et 
al..  1992).  Thus  salinity  may  have  salient  effects  on  re- 
productive processes,  including  spawning  and  fertilization, 
in  zebra  mussels. 

Estuarine  and  brackish-water  populations  of  adult  zebra 
mussels  occur  throughout  Europe  in  both  tidal  and  non- 
tidal  bodies  of  water  (Ludyanskiy  et  al.,  1993);  however, 
no  experiments  to  date  have  examined  the  reproductive 
potential  of  zebra  mussels  in  different  salinities.  If  repro- 
duction is  reduced  or  inhibited  in  estuarine  areas,  then 
maintenance  of  adult  populations  in  these  areas  will  de- 
pend largely  upon  larvae  derived  from  upstream  sources, 
and  local  recruitment  will  be  negligible.  Such  information 
is  directly  relevant  for  modeling  zebra  mussel  spread  (e.g., 
Neary  and  Leach,  1992;  Ramcharan  et  al..  1992)  in  all 
North  American  estuaries  at  risk  of  invasion,  as  well  as 
some  brackish  inland  lakes.  In  the  present  paper,  we  report 
on  the  effects  of  salinity  and  temperature  on  spawning 
and  fertilization  in  salinity-acclimated  and  non-acclimated 
zebra  mussels. 

Materials  and  Methods 

Acclimation  and  spawning  experiments 

Animals  ( 1 3-25  mm  in  length)  were  collected  on  several 
occasions  in  1994,  from  late  May  to  the  end  of  June,  from 
western  Lake  Erie  at  Monroe,  Michigan,  (41°  54TM,  83° 
23^).  Mussels  were  immediately  transported  to  the  lab- 
oratory and  maintained  in  a  thermostatically  controlled 
70-gallon  aquarium  at  12°C  until  use.  This  main  holding 
tank  was  originally  filled  with  Detroit  tap  water,  and  at 
intervals  the  water  was  partially  replaced  (up  to  20%  per 
week)  from  the  same  source.  As  a  result  of  feeding,  sedi- 
ments associated  with  animals,  the  animals  themselves, 
etc.,  average  ion  concentrations  in  the  aquarium  water  are 
somewhat  higher  than  those  in  the  tap  water.  This  water 
has  been  measured  to  contain  1.0  mAf  sodium,  0.25  mM 
potassium,  and  1.4  mAf  calcium  (Walker  and  Ram,  1994). 

For  acclimation  to  different  conditions  of  temperature 
and  salinity,  clusters  of  animals  were  transferred  to  sep- 
arate aquaria  with  the  requisite  conditions.  Before  being 
used  in  an  experiment,  animals  were  individually  trans- 


ferred to  vials  having  the  temperature/salinity  combina- 
tion being  tested,  and  except  for  acute  tests,  were  main- 
tained in  individual  vials  under  these  conditions  for  1  day 
before  testing.  A  schematic  diagram  of  a  typical  sequence 
of  holding,  acclimating,  and  testing  conditions  is  illus- 
trated in  Figure  1 . 

Specifically,  about  five  clusters  (8  cm  diameter)  of 
mussels  were  placed  into  2.5-gallon  aquaria  equipped  with 
aquarium  heaters  and  cascading  water  pumps  for  circu- 
lation. Groups  of  animals  were  acclimated  to  12  different 
temperature-salinity  combinations:  aquaria  were  main- 
tained at  12°,  20°,  or  27°C,  and  at  each  temperature  sep- 
arate aquaria  contained  either  aquarium  water  (AW,  water 
from  our  main  holding  tank),  or  water  of  a  higher  salinity 
( 1 .75,  3.5,  or  7.0  ppt).  Desired  salinities  were  achieved  by 
diluting  Instant  Ocean  with  AW. 

Animals  were  acclimated  for  up  to  21  days  without 
added  food,  and  the  water  was  changed  every  3  days.  Food 
was  withheld  so  as  not  to  introduce  additional  variables 
in  water  quality.  In  histological  studies  of  Dreissena 
starved  for  30  days,  Bielefeld  (1991)  reported  that  gonads 
were  relatively  resistant  to  degeneration  compared  to 
digestive  gland  and  that  the  gonad/body  quotient  de- 
creased only  slightly.  Furthermore,  as  reported  in  this  pa- 
per for  controls  (e.g.,  AW,  20°C),  spawning  and  fertiliza- 
tion continued  to  occur  at  high  levels  even  after  animals 
were  maintained  without  food  for  up  to  2 1  days. 

In  our  first  experimental  series,  spawning  tests  (de- 
scribed below)  were  conducted  on  days  0  (no  acclimation), 
1,  2.  6,  and  13.  Zebra  mussels  did  not  survive  at  7.0  ppt 
for  more  than  2  days.  Thus,  after  2  days  of  acclimation, 
mussels  to  be  tested  for  spawning  in  7.0  ppt  were  taken 
from  the  3.5-ppt  aquaria  and  transferred  to  individual 
vials  containing  water  at  7.0  ppt  for  24  h  before  spawning 
was  tested. 

Spawning  experiments  on  acclimated  animals  were  re- 
peated with  a  second  group  of  animals,  but  in  this  case, 
long-term  acclimation  to  salinities  greater  than  3.5  ppt 
was  preceded  by  exposure  to  intermediate  salinities,  as 
described  below.  Animals  tested  for  spawning  on  days  0 
(no  acclimation)  and  1  were  acclimated  in  AW,  1.75,3.5, 
and  7.0  ppt  at  12°,  20°,  and  27°C.  On  day  4,  mussels 
from  AW,  1 .75,  and  3.5  ppt  were  tested  along  with  mussels 
tested  in  6.0  ppt  that  had  been  acclimated  for  3  days  in 
3.5  ppt  and  then  transferred  to  vials  containing  6.0  ppt 
for  the  final  24  h  preceding  testing.  On  day  7,  animals 
previously  acclimated  in  1.75  ppt  were  transferred  to 
3.5  ppt  and  those  previously  in  3.5  ppt  were  transferred 
to  5.0  ppt  for  an  additional  7  days.  On  the  14th  day  of 
acclimation,  some  animals  in  5.0  ppt  were  transferred  to 
vials  containing  6.0  and  7.0  ppt  and  were  tested  for 
spawning  on  the  following  day. 

Spawning  was  tested  by  exposing  animals  to  10~3  M 
serotonin  (5-hydroxytryptamine;  5-HT),  which,  as  shown 


322 


P.  P.  FONG  ET  AL. 


Lake  Erie 


70  gallon  holding  tank,  AW 
(animals  held  1  -  4  weeks, 
with  feeding) 


T  PC)     1 2 
salinity 


2.5  gal 
aquaria 
(0-21  days, 

12     12     12    20    20    20    20    27    27    27    27    no  feeding) 
.75   3.5    7.0    AW   1.75   3.5    7.0    AW   1.75   3.5    7.0 


t 


a 

a 

0 

a 

0 

0 

a 

0 

0 

a 

vial  for  individual  animal 


water  bath  (e.g.,   20  C) 


After  24  h,     test  for  spawning 
in  individual  vials. 

Figure  1.  Diagram  of  the  experimental  design  for  testing  spawning  responsiveness  of  zebra  mussels 
acclimated  to  various  conditions  of  salinity  and  temperature.  Testing  of  spawning  responses  of  individual 
animals,  illustrated  here  for  10  animals  from  just  one  temperature/salinity  condition,  was  similarly  performed 
for  all  other  conditions.  Non-acclimated  animals  (0  days  in  the  2.?-gal  aquaria)  went  directly  from  the  70- 
gal  holding  tank  of  aquarium  water  (AW)  into  individual  vials  for  testing  with  serotonin  after  I  h  in  their 
individual  temperature/salinity  conditions. 


by  Ram  et  al.  (1993),  reliably  elicits  spawning  in  fresh 
water.  About  24  h  prior  to  the  spawning  test,  acclimated 
animals  were  transferred  to  vials  (1  mussel/vial,  10-15 
animals  per  salinity/temperature  condition)  containing 
9  ml  of  water  at  the  same  salinity  and  temperature  in 
which  they  had  previously  been  acclimated.  Temperature 
was  maintained  by  immersing  the  vials  in  thermostatically 
controlled  water  baths.  The  test  was  begun  by  the  addition 
of  1  ml  of  1(T2  M  5-HT  to  each  vial  (for  a  final  concen- 
tration of  10~3A/),  and  spawning  was  assessed  visually 
and  microscopically  for  up  to  4  h  after  5-HT  addition. 
After  4  h,  the  gonads  of  all  nonspawners  were  dissected 
and  their  sex  and  reproductive  maturity  determined  under 
compound  microscopy  to  calculate  percent  spawning  in 
each  group.  Non-acclimated  animals  were  tested  with 
identical  procedures  except  that  they  were  transferred 
from  the  main  holding  aquarium  (AW,  1 2°C)  to  vials  at 
the  appropriate  salinity  and  temperature  and  tested  for 
spawning  by  adding  5-HT  1  h  after  transfer. 

To  investigate  whether  salinity  had  an  effect  on  the 
motility  of  freshly  released  sperm,  we  exposed  sperm  from 


non-acclimated  mussels  spawned  in  AW  to  salinities  of 
1.75,  3.5,  and  7.0  ppt  at  room  temperature  (23°C).  Sperm 
from  salinity-acclimated  mussels  were  similarly  observed 
in  their  respective  spawning  salinities.  Sperm  motility  was 
assessed  by  visual  observation  with  a  compound  micro- 
scope (400X).  Sperm  were  classified  as  either  highly  motile 
(the  usual  state  of  sperm  in  AW),  reduced  in  motility,  or 
immotile. 

Fertilization  experiments 

We  tested  the  effect  of  salinity  on  fertilization  of  oocytes 
obtained  from  salinity-acclimated  and  non-acclimated 
zebra  mussels.  Acclimated  (for  4  days  and  2 1  days  at  20°C) 
and  non-acclimated  mussels  (n  =  30  mussels  in  each  sa- 
linity) were  placed  in  individual  20-ml  vials  in  9  ml  of 
water  having  salinities  of  AW,  1.75,  3.5,  and  7.0  ppt.  All 
animals  tested  in  7.0  ppt  were  initially  acclimated  in 
3.5  ppt  and  then  transferred  to  7.0  ppt  overnight  at  20°C 
before  spawning  was  induced.  To  each  vial,  1  ml  of  10~2  M 
5-HT  was  added  until  spawning  occurred. 


SALINITY  EFFECTS  ON  ZEBRA  MUSSEL  REPRODUCTION 


323 


Sperm  suspensions  were  made  by  mixing  equal  volumes 
of  sperm  from  3  to  5  males  at  each  salinity  into  a  separate 
vial.  These  sperm  suspensions  were  then  diluted  to  a  den- 
sity of  10'°  sperm/ml  measured  with  a  hemacytometer. 
A  volume  of  10  ^1  of  diluted  sperm  suspension  was  added 
to  wells  of  a  24-well  culture  plate  containing  1.0  ml  of 
oocyte  suspension  (see  below)  for  a  final  concentration  of 
108  sperm/ml.  At  this  sperm  concentration,  high  fertil- 
ization rates  (>75%)  can  be  obtained  (data  in  this  paper 
and  unpublished  data  by  K.  Kyozuka  in  this  laboratory), 
although  polyspermy  may  also  be  occurring  (Misamore 
etal.  1994). 

Egg  suspensions  from  single  females  were  prepared  by 
counting  the  number  of  spawned  oocytes  in  a  20-jul  pipette 
and  adjusting  the  volume  to  get  a  concentration  of 
1000  oocytes/ml.  Oocytes  were  then  diluted  by  50%  for 
a  final  concentration  of  500  oocytes  per  well  in  1.01  ml 
of  sperm-egg  water,  or  1.0  ml  of  egg  suspension  alone 
(no-sperm  controls  in  the  2 1-day  acclimation  experiment). 
All  fertilization  tests  were  done  at  room  temperature 
(23°C).  Oocytes  were  observed  at  200X  on  an  inverted 
microscope  up  to  3.5  h  after  the  addition  of  sperm.  Be- 
tween 67  and  124  oocytes  from  each  well  were  assessed 
for  fertilization,  as  indicated  by  cleavage  to  at  least  the 
two-cell  stage  within  3.5  h.  For  mussels  acclimated  for  21 
days,  we  also  enumerated  the  number  of  oocytes  that  had 
ruptured. 

Statistical  analysis 

Effects  of  acclimation  to  various  salinity  and  temper- 
ature combinations  were  tested  in  two  similar,  albeit  not 
identical,  spawning  experiments.  Similarly,  fertilization 
replicates  used  oocytes  from  individual  animals  indepen- 
dently tested,  but  the  animals  had  been  acclimated  to- 
gether in  group  tanks  in  two  nonidentical  experiments. 
Since  the  lack  of  identical  replicates  of  acclimation  con- 
ditions prevents  us  from  applying  inferential  statistics 
validly,  conclusions  are  drawn  on  the  basis  of  the  consis- 
tency of  the  findings  over  similar  conditions,  refraining 
from  using  inferential  statistics  when  these  cannot  be  val- 
idly applied,  as  recommended  by  Hurlbert  (1984).  Ac- 
cordingly, some  statements  regarding  whether  a  response 
is  "higher"  or  "lower"  are  made  based  on  consistency  or 
direction  of  the  data,  unaccompanied  by  a  statistical  analy- 
sis. Tests  on  non-acclimated  animals,  however,  are  true 
replicates  of  experimental  conditions  because  all  animals 
came  from  a  common  holding  tank,  and  each  animal  was 
independently  exposed  in  its  own  vial  to  a  given  temper- 
ature/salinity condition.  For  fertilization,  the  percentage 
of  fertilized  oocytes  from  several  females  was  analyzed  by 
one-way  ANOVA.  For  spawning,  analyses  for  dichoto- 
mous  data  (spawning  vs.  no  spawning  of  individually 
tested  animals;  Fisher's  exact  test)  were  used  to  determine 


if  the  occurrence  of  spawning  was  randomly  distributed 
(null  hypothesis).  Extensive  experience  with  testing 
spawning  in  zebra  mussels  in  this  laboratory  in  a  large 
number  of  independent  experiments  over  several  years 
(Ram  et  a!..  1993;  Fong  el  ai.  1993,  1994)  has  indicated 
that  under  standard  conditions  (AW,  20-25°C,  during 
June  to  mid-August)  the  percentage  of  animals  spawning 
in  response  to  10 ~3  M  5-HT  ranged  between  60%  and 
100%.  Furthermore,  when  10-15  animals  were  tested  in 
independent  replicates  of  each  experimental  condition, 
percentage  differences  in  spawning  of  >30%  between 
treatments  were  usually  statistically  significant. 

Results 

Acclimation  and  spawning  experiments 

Results  of  the  first  acclimation  experiment,  including 
data  from  non-acclimated  animals  tested  in  the  same  se- 
ries, are  illustrated  in  Figure  2.  The  percentage  of  animals 
spawning  was  as  high  as  90%  in  AW  at  20°C.  Without 
acclimation,  spawning  in  7.0  ppt  occurred  in  <10%  of  the 
animals  tested,  significantly  lower  than  in  AW  at  all  tem- 
peratures (Fisher's  exact  test,  for  12°C,  P  <  0.006;  for 
20°C,  P  <  0.00007;  for  27°C,  P  <  0.04.  Fig.  2A).  Inter- 
mediate salinity  conditions  ( 1 .75  ppt,  3.5  ppt)  did  not  dif- 
fer markedly  from  spawning  tested  in  AW  at  all  temper- 
atures, although  there  is  a  clear  tendency,  occurring  both 
with  and  without  acclimation,  for  spawning  to  occur  in 
a  higher  percentage  of  animals  at  20°C  than  at  either  12° 
or  27°C. 

With  acclimation,  spawning  rates  in  the  various  salin- 
ities and  temperatures  tended  to  maintain  the  same  rel- 
ative positions,  with  the  most  notable  change  being  a  rise 
to  more  than  80%  in  spawning  rates  tested  in  7.0  ppt  after 
13  days  of  acclimation.  With  1-day  acclimation,  7.0  ppt 
was  still  inhibitory  to  spawning  (Fig.  2B).  After  acclima- 
tion for  2  days,  spawning  rates  were  higher  in  all  condi- 
tions except  for  7.0  ppt  at  27°C.  These  increases  in  percent 
spawning  from  1  day  to  2  days  of  acclimation  were  par- 
ticularly notable  in  7.0  ppt  at  both  12°C  and  20°C  (com- 
pare 7.0  ppt  in  Fig.  2B  to  Fig.  2C).  By  the  6th  day  of 
acclimation,  mussels  in  all  conditions  spawned  at  a  high 
percentage,  and  this  continued  to  the  13th  day  (Fig. 
2D,  E). 

A  repeat  of  the  acclimation  experiment  yielded  similar 
results.  Without  acclimation  and  with  a  1-day  acclimation. 
7.0  ppt  was  inhibitory  to  spawning  (Figs.  3A,  B).  As  in 
the  first  experiment,  where  differences  in  spawning  as  a 
function  of  temperature  were  present,  higher  spawning 
rates  were  observed  at  20°C  than  at  either  12°C  or  27°C. 
By  the  4th  day  of  acclimation,  mussels  in  all  groups 
spawned  at  a  high  percentage  (>70%,  Fig.  3C)  at  all  tem- 
peratures. Mussels  tested  on  the  15th  day  had  been  ac- 


324 


P.  P.  FONG  ET  AL 


No  acclimation 


B 


1   day  acclimation 


100 

0^ 

80 

01 

c 

60 

-^                 ^01.75 

a 

""-    \ 

40- 

"  -  ^\      AW 

20 

~'§3.5 

A                                  7.0 

1  ^t  

IE  "i 

12                         20                         27 

Temperature  (C°) 

U                                2  day  acclimation 

100 

-^'D^-^__^ 

D-  '  ^'^^fr'<  x^~^r^^o  AW 

80 

^^"^  ^^                        "  "*    ~"~"~O  1.75 

O) 

c 

x''*"                     ^ 

'c 

60 

0^                                                         VD  3.5 

D 

a. 

00 

40 

^ 

A 

20 

n 

7.0 

1  1  1  A  

12  20  27 

Temperature  (C°) 


100- 

_---°^ 

o.     S0 

C 

"'^XD^X^O  ,,5 

|     60- 

D 
Q. 

if>     40- 

O    AW 
X 

20- 

%D    3.5 

A                                    7.0 

12                           20                           27 

Temperature  (C°) 

1  00  - 

U                              6  day  acclimation 

On 

a.     80 
c 

~~C^O    1.75 
°    AW 

§      60- 

o 
D. 
(n     40 

fr? 

20- 

o 

12                       20                       27 

Temperature  (C°) 

1  00 

t,                              13  day  acclimation 
AW  k  1  75                    rj                              n 

o.     80 
c 

A         -'-'                 A 

-      3.5  D'                                                           a   7.0 

|      60 

o 

Q. 

i/l      40 

20 
n 

12  20  27 

Temperature  (C°) 

Figure  2.  Effect  of  salinity,  temperature,  and  duration  of  acclimation  on  serotonin  (10~3  A/)-induced 
spawning.  Zebra  mussels  were  acclimated  in  aquarium  water  (AW),  1.75,  3.5,  and  7.0  ppt  at  12°.  20°,  and 
27°C  and  tested  for  spawning  after  (A)  no  acclimation  to  any  salinity/temperature  condition  (previously 
maintained  in  AW,  12°C);  (B)  1-day  acclimation;  (C)  2-day  acclimation:  (D)  6-day  acclimation;  and  (E)  13- 
day  acclimation.  In  the  1 3-day  acclimation  experiment,  mussels  tested  for  spawning  in  7.0  ppt  were  acclimated 
for  12  days  in  3.5  ppt  and  then  transferred  to  7.0  ppt  for  1  day  before  testing.  Each  point  represents  the 
percentage  of  animals  spawning  out  of  12-15  animals  tested  for  each  salinity /temperature/duration  condition. 


climated  to  a  gradual  increase  in  salinity,  and  all  groups 
spawned  at  frequencies  of  80%  or  higher  (Fig.  3D). 

The  motility  of  sperm  from  non-acclimated  mussels 
was  affected  by  salinity.  Sperm  taken  from  aquarium  water 
and  transferred  directly  to  either  3.5  or  7.0  ppt  exhibited 
either  greatly  reduced  motility  or  no  movement  at  all. 
Sperm  transferred  to  1.75  ppt  had  noticeably  reduced 
motility.  Sperm  spawned  from  mussels  that  had  been  ac- 
climated to  higher  salinities  for  1  to  2  days  also  showed 


reduced  motility  or  were  completely  immotile  in  all  series. 
By  the  6th  day  of  acclimation,  however,  no  obvious  re- 
duction in  sperm  motility  was  observed  at  any  salinity. 

Fertilisation  experiments 

Although  1.75  ppt  had  no  acute  or  long-term  effect  on 
fertilization,  higher  salinities  had  inhibitory  effects  that 
could  be  at  least  partially  reversed  by  acclimation  (Fig. 


SALINITY  EFFECTS  ON  ZEBRA  MUSSEL  REPRODUCTION 


325 


No  acclimation 


B 


12  20  27 

Temperature  (C°) 


1   day  acclimation 


1  UU 

XU-  ^                                                                                           1  UU  ' 

AW  O—  "                  ,'Q-~ 

0,      80 

^  a-^^--'     ""  1-75   ~                                                  en     80 

_,-'' 

c 

C>"^jr                                                                                c. 

3.5  D'' 

|      60 

D3.5                                                                                     1       60- 

o 

D 

CL 

Q. 

in     40 

in      40- 

20 

7.0                                           2Q  . 

A  '"                      A 

n 

1  1  *  n- 

7.0  A 

12  20  27 

Temperature  (C°) 


100- 


80 


60 


40 


20 


4  day  acclimation 

-D: _^-O      AW 

-a--^--"~^-o  1.75 

-.O-^"^  • D  3.5 

A  6.0 


12  20  27 

Temperature  (C°) 


D 


1  5  day  acclimation 


^.z^^*        ~~  -~-^^ 

u 

O-"^''^'^ 

0 

a> 

80- 

es^' 

_c 

c 

60- 

o 

D-- 

D 

3.5 

o. 
t/) 

40- 

o  — 
o  — 

O 
O 

5.0 
6.0 

K 

A 

A 

7.0 

20- 

n- 

12  20  27 

Temperature  (C°) 


Figure  3.  Results  of  a  second  experiment  on  effects  of  salinity,  temperature,  and  duration  of  acclimation 
on  serotonin  ( 10~3  A/ (-induced  spawning.  Zebra  mussels  were  acclimated  in  aquarium  water  (AW)  and 
various  salinities  at  12°,  20°,  and  27°C  and  tested  for  spawning  after  (A)  no  acclimation  to  any  salinity/ 
temperature  condition  (previously  maintained  in  AW,  I2°C;  (B)  1-day  acclimation;  (O  4-day  acclimation: 
and(D)  15-day  acclimation.  Mussels  tested  for  spawning  in  6.0  ppt  after  4  days  acclimation  were  acclimated 
for  3  days  in  3.5  ppt  and  then  transferred  to  6.0  ppt  for  1  day  before  testing.  In  the  15-day  acclimation  tests, 
all  mussels  were  acclimated  gradually  to  increasing  salinities  from  1.75  ppt  up  to  their  final  test  salinity  as 
described  in  the  text.  All  other  animals  in  the  experiments  were  acclimated  in  their  indicated  salinities  (AW, 
1.75.  3.5.  and  7.0  ppt)  and  temperatures  (12°,  20°,  and  27°C)  throughout  the  acclimation  period.  Each 
point  represents  the  percentage  of  animals  spawning  out  of  10-15  animals  tested  for  each  salinity/temperature/ 
duration  condition. 


4).  For  non-acclimated  mussels,  one-way  ANOVA  of  the 
effect  of  salinity  on  log-transformed  data  of  percent  fer- 
tilization gave  F)A2  =  74.2.  P  <  0.0001  in  experiment  1 
and  F38  =  361.8,  P  <  0.0001  in  experiment  2.  The  per- 
centage of  oocytes  fertilized  in  both  3.5  ppt  and  7.0  ppt 
was  significantly  lower  than  in  both  AW  and  1.75  ppt 
(Fisher's  LSD.  P  <  0.05  for  all  four  comparisons  in  both 
experiments).  For  acclimated  mussels,  7.0  ppt  was  still 
inhibitory  to  fertilization  after  a  4-day  acclimation;  how- 
ever, the  point  to  be  noted  is  that  the  fertilization  rate  in 
3.5  ppt  now  overlapped  in  range  with  the  fertilization  rate 
in  AW.  The  fertilization  rate  at  3.5  ppt  rose  from  1.4% 
±  0.5%  (mean  ±  SE)  in  non-acclimated  mussels  to  37.6% 
±  13.6%  in  4-day-acclimated  mussels.  Similarly,  after  21 
days  acclimation,  fertilization  rates  in  3.5  ppt  increased 
to  57%  ±  35%.,  compared  to  0%-  in  non-acclimated  con- 
trols. 

During  the  course  of  the  above  fertilization  experi- 
ments, we  noticed  that  oocytes  often  ruptured  at  higher 


salinities.  These  observations  were  quantified  in  the  latter 
of  the  two  experiments  (21 -day  acclimation  and  its  non- 
acclimated  control.  Fig.  5).  There  was  a  significant  increase 
in  rupturing  with  elevated  salinity  in  both  acclimated  and 
non-acclimated  mussels,  with  and  without  sperm  (one- 
way ANOVAs:  for  acclimated  mussels  without  sperm, 
/•:.,_,  =  61.1,  P  =  0.0001:  with  sperm.  F2,,3  =  5.46,  P 
=  0.001;  for  non-acclimated  mussels  without  sperm,  F3>8 
=  6.2,  P  =  0.01;  with  sperm,  F3.7  =  8.9,  P  =  0.008).  The 
decrease  in  oocyte  rupturing  that  occurred  in  the  presence 
of  sperm  was  apparently  due  to  a  protective  effect  of  fer- 
tilization. Thus,  in  1.75  ppt,  in  which  fertilization  occurs 
at  a  high  rate  (Fig.  4),  addition  of  sperm  significantly  re- 
duced the  percentage  of  oocytes  rupturing  from  >80%  to 
<20%  (P  <  0.05).  The  effect  of  sperm  addition  on  the 
percentage  of  ruptured  oocytes  in  non-acclimated  mussels 
was  not  apparent  in  3.5  and  7.0  ppt  because  almost  no 
fertilization  occurred  at  these  two  salinities.  After  2 1  days 
of  acclimation,  in  which  fertilization  occurs  at  a  higher 


326 


P.  P.  FONG  ET  AL 


A 


No  acclimation 


100 

^    80- 

41               |3 

<D 

</) 

I    \^         j\    sperm,  expt.  2 

N 

+i     60- 

>v        j     \ 

t 

a> 

c 

0 

expt.   1       l\  \ 

LL. 

®     40- 

4      \x 

*B 

c 

\     \ 

s^X 

\    \ 

20- 

V 

n. 

V           T 

no  sperm            \  o              4    ^ 

A  A  4   •          .         A  

AW          1.75  3.5  7.0 

Salinity  (parts  per  thousand) 


rate  in  3.5  ppt,  a  significant  protective  effect  of  sperm  was 
apparent  in  both  1.75  ppt  and  3.5  ppt  (P  <  0.05). 

Because  unfertilized  oocytes  in  AW  rarely  ruptured, 
and  rupturing  in  1.75  ppt  occurred  in  >60%  of  oocytes 
whether  acclimated  or  not,  we  wondered  what  the  lower 
limit  of  oocyte  sensitivity  to  increased  salinity  was.  To 
investigate  this  question,  oocytes  freshly  spawned  in  AW 
were  exposed  to  salinities  of  AW,  0.175,  0.35,  0.7,  1.0, 
and  1.75  ppt,  without  sperm,  in  a  24-well  culture  plate 
and  observed  at  intervals  of  1  to  2  h.  No  rupturing  oc- 
curred after  1  h;  however,  some  oocytes  in  1.75  ppt  ap- 
peared shrivelled.  Within  2  h,  oocytes  began  to  rupture 
and,  as  illustrated  in  Figure  6,  the  percentage  of  ruptured 
oocytes  increased  with  increasing  salinity  (linear  regres- 
sion, /•  =  0.66,  P  <  0.001). 


100- 


B 


4— day  acclimation 
(expt.   1) 


o  Fertilizec 
mean  ±  SE 

60- 
40- 

6 

°\  5    i 

x.       i    sperm    1 

20- 

r    '\  . 

n 

6 

i                          I                          ,         ....     .1.        - 

L/ 


AW  1.75  3.5  7.0 

Salinity  (parts  per  thousand) 


21—  day  acclimation 


100- 

I 

(expt.  2' 

6   •. 

^    80- 

1     -^ 

LiJ 

5'< 

>^^ 

sperm 

+i     60- 

"  -« 

» 

c 

5 

o 

*     40- 

J^ 

20- 

n- 

no  sperm 

A  -A  A.  —  )  

AW  3.5 

Salinity  (parts  per  thousand) 

Figure  4.  Effect  of  salinity  on  fertilization.  Percentage  of  oocytes 
fertilized  when  exposed  to  sperm  in  aquarium  water  (AW)  and  salinities 
of  1.75.  3.5,  or  7.0  ppt  after  (A)  no  prior  acclimation  of  source  animals 
(data  from  two  separate  experiments):  (B)  4-day  acclimation  of  source 
animals  to  various  salinities  at  20°C  (experiment  I);  and  (C)  21-day 
acclimation  of  source  animals  to  various  salinities  (experiment  2).  Ex- 
periment 2  included  "no  sperm"  controls  with  each  group,  none  of  which 


Discussion 

This  is  the  first  study  to  examine  the  effects  of  salinity 
directly  on  the  reproductive  mechanisms  of  zebra  mus- 
sels. Previous  studies  focused  on  the  viability  of  larvae 
(Setzler-Hamilton  and  Wright,  1994)  and  adults  (e.g.. 
Mackie  and  Kilgour.  1992),  and  on  biogeographic  data 
relating  distribution  to  salt  concentrations  (Walton, 
1992;  Strayer  and  Smith,  1993).  The  present  study  has 
demonstrated  that  although  acute  exposure  of  zebra 
mussels  or  their  gametes  to  higher  saline  conditions  is 
detrimental  to  spawning,  fertilization,  sperm  motility, 
and  oocyte  integrity,  acclimation  of  mussels  to  elevated 
saline  levels  over  a  period  of  days  results  in  significant 
improvement  in  these  indicators  of  reproductive  func- 
tion. 

The  improvement  in  reproductive  function  with 
acclimation  was  particularly  striking  at  3.5  ppt,  the  in- 
termediate level  of  salinity  tested.  In  response  to  5-HT, 
non-acclimated  zebra  mussels  can  spawn  gametes  in 
salinities  from  1 .75  to  3.5  ppt,  but  only  rarely  in  7.0  ppt 
(Figs.  2  and  3).  Although  gametes  obtained  under  these 
acute  conditions  can  be  consistently  fertilized  at 
1.75  ppt,  fertilization  in  3.5  ppt,  without  prior  accli- 
mation, was  practically  nil  (Fig.  4).  Acclimation  of  an- 
imals in  3.5  ppt  resulted  in  increased  fertilization  rates 
when  tested  in  3.5  ppt.  Moreover,  after  acclimation  of 
animals  at  3.5  ppt  for  4  or  more  days,  spawning  could 
be  elicited  by  5-HT  in  salinities  as  high  as  7.0  ppt  (Fig. 
2  and  subsequent  experiments).  Nevertheless,  fertiliza- 
tion rates  of  oocytes  obtained  in  7.0  ppt  after  accli- 


resulted  in  apparent  cleavage  and  development  (the  end-point  for  de- 
termining that  fertilization  had  occurred).  Points  and  error  bars  represent 
the  mean  ±  SE  percent  of  cleaved  oocytes  observed  within  3.5  hours  of 
the  addition  of  sperm.  Sample  sizes  (numbers  of  females  from  which 
oocytes  were  obtained  and  tested  in  separate  wells)  are  given  adjacent 
to  error  bars. 


SALINITY  EFFECTS  ON  ZEBRA  MUSSEL  REPRODUCTION 


327 


No  acclimation 

n  =  3  for  all  points 


fr? 


100- 

^    80-- 
«  £ 

.2+1     60  •• 
Q-  c 

^    ®     40-- 
K  ^ 

20- 
0-- 


AW  1.75  3.5  7.0 

Salinity  (parts  per  thousand) 

D  21 -day  acclimation 


no  sperm 


sperm 


AW  1.75  3.5 

Salinity  (parts  per  thousand) 

Figure  5.  Effect  of  salinity  on  oocyte  integrity.  Percentage  of  oocytes 
ruptured  was  determined  within  3.5  h  of  spawning,  for  oocytes  tested 
with  and  without  sperm  present  in  aquarium  water  (AW),  and  various 
salinities.  (A)  Oocvtes  were  from  animals  not  previously  acclimated  and 
were  tested  in  AW  and  salinities  of  1 .75,  3.5,  and  7.0  ppt,  n  =  3  females 
per  group.  (B)  Oocytes  were  from  animals  previously  acclimated  21  days 
at  20°C  to  AW  and  salinities  of  1.75  and  3.5  ppt  and  were  tested  in  the 
same  salinities  to  which  they  had  been  acclimated.  Sample  sizes  (number 
of  females  from  which  oocytes  were  obtained  and  tested  in  separate 
wells)  are  given  adjacent  to  error  bars.  Points  and  error  bars  represent 
the  mean  ±  SE  percent  of  oocytes  ruptured. 


mation  remained  depressed  (Fig.  4).  Thus  the  repro- 
ductive function  of  zebra  mussels  acclimated  for  several 
days  to  salinities  as  high  as  3.5  ppt  recovers  to  normal 
levels  for  several  reproductive  parameters  but,  at  least 
within  the  time-temperature  ranges  tested,  reproductive 
function  at  the  highest  salinity  (7.0  ppt)  is  still  signifi- 
cantly below  normal. 

The  acute  reduction  in  fertilization  with  increasing  sa- 
linity may  be  due  in  part  to  reduced  sperm  motility.  Sperm 
from  non-acclimated  mussels  were  either  immotile  or 
moving  slowly  in  3.5  and  7.0  ppt.  with  some  reduction 


in  motility  at  1.75  ppt.  Moreover,  mussels  acclimated  for 
up  to  2  days  showed  similar  immotility  in  all  salinities. 
Sperm  from  males  acclimated  for  6  days  showed  no  re- 
duction in  motility.  and  this  may  explain  the  significant 
increase  in  fertilization  in  acclimated  animals  (albeit  tested 
at  4  and  2 1  days  acclimation)  compared  to  non-acclimated 
animals  tested  in  the  same  salinity.  It  may  also  explain 
the  trend  towards  higher  fertilization  rates  between  4-day 
and  21 -day  acclimation.  In  the  present  study,  fertilizations 
were  carried  out  in  about  I  ml  of  water  in  24-well  culture 
plates.  This  small  volume  would  increase  the  likelihood 
of  even  motility-deficient  sperm  encountering  eggs.  In  the 
field,  where  volumes  in  which  sperm  may  encounter  oo- 
cytes are  much  larger,  reduced  motility  of  sperm  may 
reduce  fertilization  success  further  than  observed  here. 

Reductions  in  fertilization  success  may  also  be  due  to 
osmotic  effects  on  oocytes.  Rupture  of  unfertilized  oocytes 
was  evident  in  salinities  as  low  as  0. 1 75  ppt  and  increased 
significantly  with  salinity  even  at  moderate  saline  levels. 
Although  oocytes  generally  took  1  to  2  h  to  rupture, 
shrinkage  in  saline  solutions  was  present  (the  exact  timing 
and  dimensions  of  such  physical  changes  were  not  re- 
corded) before  rupture.  Thus,  increased  saline  levels  may 
reduce  the  likelihood  of  fertilization  by  inducing  patho- 
logical changes  in  oocytes  between  the  time  that  they  are 
exposed  to  the  saline  solution  and  the  time  that  they  en- 
counter sperm.  Osmotic  effects  may  also  explain  the  re- 
duced motility  of  non-acclimated  sperm.  We  need  to 
know  more  about  the  normal  longevity  of  sperm  and  egg 
viability  and  the  normal  latency  between  spawning  and 
fertilization  in  the  field  before  we  can  evaluate  the  im- 
portance of  salinity-induced  oocyte  rupture  on  reproduc- 
tive success. 


100- 


80- 


LJ 
(/I 


Q-  c 

3     o 

o:   <o 


AW  0.5  1.0  1.5 

Salinity  (parts  per  thousand) 


2.0 


Figure  6.  Effect  of  salinity  on  integrity  of  unfertilized  oocytes.  Female 
mussels  were  spawned  in  aquarium  water  (AW),  and  then  oocytes  were 
exposed  to  AW  and  various  salinities  up  to  1.75  ppt.  Oocytes  (1  12  to 
1  44  oocytes  from  each  female  under  each  condition)  were  observed  after 
1  h.  Points  represent  the  mean  ±  SE  percent  of  oocytes  ruptured  for 
oocytes  from  4  females.  Linear  regression  gives  r  =  0.66,  with  P<  0.001. 


328 


P.  P.  FONG  ET  AL. 


Fertilization  protected  oocytes  from  salinity-induced 
rupture.  Two  possible  mechanisms  for  this  protection  are 
(1)  that  secretion  of  a  fertilization  envelope  provides  some 
ionic/osmotic  protection,  and  (2)  that  after  fertilization, 
solutes  (sugars,  proteins,  ions)  accumulate  and  provide 
protection. 

Temperature  had  a  modulating  effect  on  spawning  in 
different  salinities.  Without  acclimation,  animals  were 
more  likely  to  spawn  at  20°C  than  at  either  12°C  or  27°C. 
This  was  true  for  three  of  four  salinities  in  experiment  1 
(Fig.  2A)  and  all  four  salinities  in  experiment  2  (Fig.  3A). 
The  higher  response  at  20°C  is  in  agreement  with  the 
results  of  a  previous  experiment  (Ram  el  a/..  1993),  in 
which  the  rate  of  spawning  in  AW  was  higher  at  20°C 
that  at  12°C  or  27°C.  These  data  indicate  that  sudden 
rises  in  temperature  into  the  upper  twenties  (°C)  could 
inhibit  reproductive  function  in  zebra  mussels,  a  sugges- 
tion that  may  explain  some  of  the  spawning  variations 
observed  in  field  studies  (unpub.  data).  However,  with 
longer  acclimation  to  higher  temperatures,  spawning  rates 
under  all  conditions  rose  to  high  levels  at  all  salinities. 
Thus,  despite  the  somewhat  greater  inhibitory  effects  of 
salinity  at  high  temperatures  in  non-acclimated  mussels, 
if  high  temperatures  are  sustained  over  long  periods, 
spawning  responses  can  recover  to  maximal  levels  within 
the  range  of  salinity  and  temperature  studied. 

Salinity  is  a  limiting  factor  in  the  distribution  of  adult 
zebra  mussels  in  European  brackish  waters  (Strayer  and 
Smith.  1993).  In  North  America,  zebra  mussels  have 
spread  downstream  in  the  Hudson  River  estuary  as  far  as 
West  Haverstraw,  New  York,  where  salinities  reach  4- 
6  ppt  (Walton,  1 992).  In  studies  of  adult  mortality,  Mackie 
and  Kilgour  ( 1992)  found  that  the  96-h  LC50  for  salinity 
(Instant  Ocean)  at  19°C  was  7.6  ppt  for  non-acclimated 
mussels.  Acclimated  zebra  mussels  survived  longer,  but 
at  lower  test  temperatures.  Setzler-Hamilton  and  Wright 
(1994)  reported  that  D-hinge  larvae  of  cultured  zebra 
mussels  survived  at  least  48  h  at  22°C,  but  that  survival 
ranged  from  only  20%  at  18°C  and  4  ppt  to  0.2%  at  18°C 
and  8.0  ppt.  The  results  of  our  experiments  together  with 
these  previous  observations  suggest  that  the  salinity/tem- 
perature regime  for  zebra  mussel  survival  is  somewhat 
less  than  7.0  ppt  and  less  than  27°C,  and  that  mussels 
can  reproduce  successfully  at  a  salinity  of  3.5  ppt.  Thus, 
brackish-water  populations  of  adult  zebra  mussels  could 
result  from  both  downstream  transport  of  larvae  and  local 
recruitment. 

This  is  one  of  the  few  investigations  into  the  salinity 
tolerance  of  reproductive  mechanisms  in  an  invertebrate 
that  abounds  in  fresh  water.  Animals  living  in  fresh  water 
must  have  special  adaptations  to  withstand  the  osmotic 
stress  of  low  salinity.  Furthermore,  if  exposure  to  brackish 
waters  is  a  possibility,  then  mechanisms  may  have  evolved 
for  surviving  and  reproducing  in  conditions  of  increasing 


or  varying  salinity.  In  most  fresh-water  bivalves,  fertiliza- 
tion normally  occurs  within  the  suprabranchial  chamber 
of  the  female,  from  which  brooded,  parasitic  glochidia 
larvae  are  released.  Zebra  mussels  represent  a  rare  non- 
crustacean,  freshwater  invertebrate  with  external  fertil- 
ization and  planktotrophic  larval  development.  However, 
the  osmoregulatory  mechanisms  that  allow  zebra  mussel 
gametes  and  larvae  to  tolerate  extremely  dilute  conditions 
and  to  acclimate  (as  shown  here)  to  brackish-water  con- 
ditions are  unknown.  It  would  be  of  interest  to  determine 
how  zebra  mussel  gametes  are  protected  from  osmotic 
stress  in  fresh  water  and  what  changes  take  place  during 
acclimation. 

Acknowledgments 

This  research  was  sponsored  in  part  by  grants  from 
the  Michigan  Sea  Grant  College  Program  (NOAA 
NA16RC04 17-01)  and  from  NIH  (RR-08167).  JD  and 
SR  received  partial  support  from  the  Wayne  State  Uni- 
versity Department  of  Physiology  SURF  Program. 

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Reference:  Biol.  Bull  189:  330-339.  (December,  1995) 


Existence  of  Three  Mechanisms  for  Blocking  Polyspermy 
in  Oocytes  of  the  Mussel  Mytilus  edulis 

TATSURU  TOGO1-2*,  KENZI  OSANAI1,  AND  MASAAKI  MORISAWA2 

lAsamushi  Marine  Biological  Station,  Tohokit  University,  Asamushi,  Aomori  039-34,  Japan,  and 
2Misaki  Marine  Biological  Station,  University  of  Tokyo,  Misaki,  Miura,  Kanagawa  238-02,  Japan 


Abstract.  We  found  the  existence  of  a  three-step  mech- 
anism to  block  polyspermy  in  the  oocyte  of  the  mussel 
Mytilus  edulis.  When  the  oocytes  were  inseminated  within 
30  min  after  spawning,  they  underwent  monospermic 
fertilization  over  a  wide  range  of  sperm-oocyte  ratios  up 
to  5  X  103.  A  transient  depolarization  of  the  oocyte  plasma 
membrane  (fertilization  potential)  was  observed  imme- 
diately after  insemination.  Low-sodium  seawater  induced 
polyspermy  and  decreased  the  amplitude  of  the  fertiliza- 
tion potential,  suggesting  the  existence  of  a  fast  block  to 
polyspermy  that  is  dependent  on  depolarization  of  the 
plasma  membrane.  When  the  fertilized  oocytes  were  in- 
seminated again  at  a  sperm-oocyte  ratio  that  is  great 
enough  to  give  a  high  rate  of  polyspermy  in  initial  insem- 
ination, many  sperm  could  not  undergo  the  acrosomal 
reaction  and  thus  could  not  penetrate  fertilized  oocytes. 
The  remaining  sperm  underwent  an  acrosomal  reaction 
and  the  acrosomal  process  protruded  through  the  vitelline 
coat,  but  it  did  not  fuse  with  the  oocyte  plasma  membrane. 
These  findings  suggest  the  existence  of  two  strategies  con- 
stituting a  late  polyspermy  block:  suppression  of  acroso- 
mal reaction  and  block  of  contact  or  fusion  between  the 
plasma  membranes  of  sperm  and  oocyte. 

Introduction 

The  success  of  fertilization  and  the  subsequent  devel- 
opment of  the  zygote  require  the  fusion  of  a  single  male 
pronucleus  with  a  female  pronucleus.  Penetration  of  the 


Received  10  April  1995;  accepted  31  July  1995. 

*  To  whom  correspondence  should  he  addressed  at  Misaki  Marine 
Biological  Station,  University  of  Tokyo. 

Abbreviations:  ASW,  artificial  seawater,  DAPI,  4',6-diamidino-2- 
phenylindole;  GalNAc,  /V-acetylgalactosamine;  NaFSW,  sodium-free 
artificial  seawater;  NSW.  natural  seawater,  Rs/n,  sperm-oocyte  ratio. 


spermatozoon  into  an  oocyte  is  controlled  through  several 
mechanisms  (Jaffe  and  Gould,  1985).  Rothschild  and 
Swann  ( 1952)  first  suggested  the  existence  of  a  fast  poly- 
spermy block  at  the  plasma  membrane  of  the  oocyte  in 
the  sea  urchin.  Jaffe  (1976)  demonstrated  that  the  block 
was  mediated  electrically.  The  fast  electrical  polyspermy 
block  has  been  further  observed  in  many  animal  species, 
such  as  starfish  (Miyazaki  and  Hirai,  1979),  the  echiuroid 
Urechis  (Gould-Somero  el  ai,  1979),  amphibians  (Cross 
and  Elinson,  1980;  Grey  et  al.,  1982;  Iwao,  1989),  the 
nemertean  Cerebratulus  (Kline  et  ai.  1985),  crab  (Gou- 
deau  and  Goudeau,  1989a),  lamprey  (Kobayashi  et  al., 
1994),  and  ascidians  (Goudeau  et  al.,  1994),  as  well  as  in 
algal  protists,  among  the  fucoid  seaweeds  (Brawley,  1991). 

After  fusion  between  the  plasma  membranes  of  the  ga- 
metes, the  extracellular  coat  of  the  oocyte  is  altered  and 
forms  a  fertilization  envelope  to  prevent  sperm  penetra- 
tion (zona  reaction  in  mammals).  A  late  polyspermy  block 
mechanism  may  also  operate  at  the  level  of  the  oocyte 
plasma  membrane  in  Urechis.  Paul  and  Gould-Somero 
( 1976)  found  that  the  acrosomal  process  of  supernumerary 
sperm  penetrated  through  the  vitelline  coat  into  the  peri- 
vitelline  space  of  the  fertilized  oocyte,  but  sperm  could 
not  fuse  with  the  oocyte  plasma  membrane.  The  block  at 
the  plasma  membrane  is  also  found  in  the  nemertean 
Cerebratulus  (Kline  et  al.,  1985),  in  mammals  (e.g.,  Hor- 
vath  et  al.,  1993),  and  in  the  surf  clam  Spisula  (Ziornek 
and  Epel,  1975). 

The  existence  of  the  electrical  block  in  bivalves  was 
suggested  in  Spisula  by  Finkel  and  Wolf  (1980),  who 
found  that  the  depolarization  of  the  oocyte  plasma  mem- 
brane occurs  soon  after  insemination  and  that  low-sodium 
seawater  induces  polyspermy.  A  vitelline  coat  and  cortical 
granules  are  present  in  the  oocyte  cortex  in  bivalves,  but 
there  is  no  evidence  demonstrating  the  formation  of  a 


330 


POLYSPFRMY  BLOCK  IN  MYTILUS  OOCYTE 


331 


fertilization  envelope  by  their  structural  changes  (Longo, 
1 983;  Alliegro  and  Wright,  1983:  Longo  etui.  1993):  thus 
the  involvement  of  both  structures  in  the  late  polyspermy 
block  is  uncertain.  However,  a  complete  polyspermy  block 
has  been  demonstrated  in  oocytes  ofSpisula  from  which 
the  vitelline  coat  was  removed,  suggesting  that  a  complete 
block  to  polyspermy  occurs  at  the  oocyte  plasma  mem- 
brane in  this  species  (Ziomek  and  Epel,  1975). 

In  contrast  to  Spisula,  the  mussel  Mytilus  gallopro- 
vincialis  was  described  as  lacking  a  complete  mechanism 
to  block  polyspermy  (Dufresne-Dube  ct  a/..  1983).  Du- 
fresne-Dube el  al.  (1983)  also  found  induction  of  poly- 
spermy in  M.  galloprovincialis  by  lowering  the  concen- 
tration of  sodium  ions  in  seawater,  but  no  fertilization 
potential  was  observed.  We  demonstrate  here  a  complete 
polyspermy  block  in  Mytilus  edulis  and  describe  its  three 
steps:  ( 1 )  a  fast  electrical  block,  (2)  a  suppression  of  the 
acrosomal  reaction,  and  (3)  a  block  of  contact  or  fusion 
of  the  plasma  membrane  in  the  gametes. 

Materials  and  Methods 
Artificial  seawater 

Artificial  seawater  (ASW)  consisted  of  450  mA/  NaCl, 
9.4  mA/  KC1,  10.2  mA/CaCl,,  48.2  mA/  MgSO4,  5.4  mM 
NaHCOi.  When  sodium-free  artificial  seawater  (NaFSW) 
was  prepared,  NaCl,  KG,  and  NaHCO,  were  replaced 
with  455.4  mM  choline  chloride  (Nacalai  Tesque  Inc.), 
4mA/  KC1.  and  5.4mA/  KHCO,,  respectively.  Low- 
sodium  ASWs  were  prepared  by  mixing  ASW  and 
NaFSW.  The  pH  was  adjusted  to  8.3  with  1  N  NaOH  for 
ASW  or  1  N  KOH  for  NaFSW  and  low-sodium  ASWs 
just  prior  to  use. 

Gametes 

Specimens  of  the  mature  mussel  Mytilus  edulis  were 
collected  from  November  to  April  in  the  vicinity  of 
Asamushi  Marine  Biological  Station  (Aomori  Prefecture), 
Tohoku  University,  and  Misaki  Marine  Biological  Station 
(Kanagavva  Prefecture),  University  of  Tokyo.  They  were 
kept  in  aquaria  at  10°C.  Spawning  of  oocytes  and  sperm 
was  induced  by  transferring  the  mussels  to  warm  seawater 
at  25°C.  When  the  mussels  started  spawning,  they  were 
returned  to  natural  seawater  (NSW)  at  10°C.  The  oocytes 
were  washed  several  times  with  NSW,  ASW,  or  low-so- 
dium ASWs  according  to  the  experiments.  Concentrations 
of  oocytes  were  determined  by  counting  the  number  of 
oocytes  in  5-/ul  glass  capillary  tubes.  Sperm  were  collected 
"dry"  and  stored  at  4°C.  Sperm  suspensions  were  prepared 
by  diluting  the  dry  sperm  with  NSW,  ASW,  or  low-sodium 
ASWs.  Concentrations  of  sperm  in  the  suspensions  were 
determined  by  counting  the  number  of  sperm,  fixed  with 


1%  glutaraldehyde,  in  a  hemacytometer.  Sperm-oocyte 
ratio  (Rs/0)  in  the  medium  at  insemination  was  an  absolute 
ratio.  All  experiments  were  carried  out  at  room  temper- 
ature (18°-20°C). 

Assay  of  polyspermy 

To  remove  supernumerary  sperm  bound  to  the  surface 
of  the  oocyte,  the  inseminated  or  re-inseminated  oocytes 
were  washed  with  NSW  containing  0.001%  sodium  do- 
decyl  sulfate  at  20  min  after  the  first  insemination.  Oocytes 
were  subsequently  fixed  with  a  3: 1  mixture  of  methanol 
and  acetic  acid  for  1  h  at  room  temperature.  After  the 
oocytes  were  washed  with  distilled  water,  they  were  stained 
with  1  Mg/ml  DAPI  (4'.6-diamidino-2-phenylindole)  (100 
Mg/ml  in  dimethyl  sulfoxide  as  a  stock)  for  30  min  to 
observe  incorporated  sperm  nuclei.  The  rate  of  poly- 
spermy was  indicated  by  the  percentage  of  oocytes  that 
included  multiple  sperm  nuclei.  The  mean  number  of 
sperm  nuclei  included  in  an  oocyte  was  determined  by 
counting  the  number  of  decondensed  sperm  nuclei  in  an 
oocyte.  At  least  100  oocytes  were  observed  under  a  flu- 
orescence microscope  (Olympus,  BH-2). 

Re-insemination  experiments 

When  oocytes  were  inseminated  with  a  low  Rs/0  (light 
insemination;  Rs/0  =  5  X  102  —  1  X  103),  fertilized  oocytes 
were  monospermic.  At  various  periods  up  to  5  min  after 
initial  insemination,  the  monospermic  oocytes  were 
inseminated  again  with  a  high  Rs/0  (heavy  insemination: 
R5/0  =  8  X  103  -  1  X  10")  sufficient  to  give  a  high  rate  of 
polyspermy  in  initial  insemination.  The  length  of  time 
during  which  the  sperm  remained  monospermic  upon 
heavy  re-insemination  was  taken  as  the  completion  time 
for  the  polyspermy  block. 

Assay  of  sperm  binding 

Fertilized  and  unfertilized  oocytes  were  fixed  with  1% 
glutaraldehyde  in  NSW  at  30  s  after  the  heavy  insemi- 
nation (Rs/0  =  8  X  103  -  1  x  104),  and  then  the  number 
of  bound  sperm  was  counted  by  scanning  the  entire  oocyte 
surface  under  n  Nnmarski  microscope  (Nikon,  OPTI- 
PHOT).  Obse:  \  :i ':••  ms  were  made  on  at  least  100  oocytes 
per  experiment. 

Assay  ofucrosome  reaction 

Half  of  the  oocytes  from  a  female  were  lightly  insem- 
inated (Rs/0  =  5  X  102  -  1  X  103).  The  fertilized  oocytes 
and  remaining  unfertilized  oocytes  were  washed  several 
times  with  NSW  at  10  min  after  the  initial  light  insemi- 
nation. Both  unfertilized  and  fertilized  oocytes  were  in- 
seminated at  the  Rs/0  of  3.5  -  4.0  X  103,  and  then  they 


332 


T.  TOGO  ET  AL 


were  fixed  with  1%  glutaraldehyde  at  5  min  after  the  in- 
semination. Because  the  acrosome  of  Mytilus  sperm  is 
large  (see  Fig.  8),  acrosome-intact  were  easily  differentiated 
under  a  Nomarski  microscope  from  acrosome-reacted 
sperm  and  were  easily  removed  after  fixation.  Thus,  an 
appropriate  volume  of  the  suspension  was  mounted  on 
the  glass  slide,  and  the  number  of  acrosome-reacted  and 
acrosome-intact  sperm  in  randomly  selected  samples  of 
sperm  (both  bound  and  unbound  on  the  oocyte  surface) 
was  counted  under  the  microscope.  As  a  control,  glutar- 
aldehyde-fixed  oocytes  and  sperm  were  mixed,  and  the 
number  of  acrosome-reacted  sperm  was  counted. 

Measurement  of  membrane  potential 

To  make  microelectrodes,  glass  tubing  containing  a 
glass  fiber  was  pulled  with  a  microelectrode  puller  (Nar- 
ishige.  PN-3).  and  back-filled  with  3  M  KG.  Resistance 
of  the  electrode  was  40-60  M12.  As  shown  in  Figure  1. 
the  chamber  was  filled  with  medium  such  as  NSW,  ASW, 
or  low-sodium  ASWs,  which  was  connected  to  ground 
via  an  Ag-AgCl  electrode.  An  oocyte  was  held  by  sucking 
on  the  tip  of  a  capillary  that  was  placed  in  the  chamber. 
Electrode  penetration  was  achieved  by  lowering  the  elec- 
trode to  the  oocyte  surface  perpendicularly  and  applying 
an  "oscillating"  current.  Recordings  were  made  with  a 
microelectrode  amplifier  (Nihon  Kohden,  MEZ-7200),  an 
oscilloscope  (Hitachi,  V-212),  and  a  chart  recorder  (Hi- 
tachi, 200).  After  measurements  of  the  membrane  poten- 
tial, each  oocyte  was  transferred  into  a  hole  of  a  96-well 
culture  plate,  and  the  first  cleavage  (about  80  min  after 
insemination  at  18°C)  was  observed.  Oocytes  were  con- 
sidered to  be  polyspermic  when  the  first  cleavage  was  ab- 
normal. When  cleavage  did  not  occur,  or  if  the  micro- 
electrode  resistance  changed  at  the  end  of  a  measurement 
compared  to  the  initial  value,  the  data  were  not  used. 

Electron  microscopy 

According  to  the  method  of  Einsenman  and  Alfert 
( 1 982),  gametes  were  prefixed  for  10  min  in  seawater  con- 
taining 1%  glutaraldehyde  (Nacalai  Tesque  Inc.)  and 
0.05%  osmium  tetroxide  (TAAB)  or  in  0.2  M  sodium 
cacodylate  buffer  (pH  7.2)  containing  1%  glutaraldehyde 
and  0.05%  osmium  tetroxide,  0.1  M  NaCl,  and  0.35  M 
sucrose.  Then,  the  samples  were  fixed  for  1  h  in  0.2  M 
sodium  cacodylate  buffer  (pH  7.2)  containing  4%.  glutar- 
aldehyde, 0.1  M  NaCl,  and  0.35  M  sucrose.  Postfixation 
was  performed  in  0.2  A/  sodium  cacodylate  buffer  (pH 
7. 2)  containing  1%  osmium  tetroxide  and  0.3  M  NaCl  for 
1  h.  After  the  fixations,  the  samples  were  dehydrated  in 
a  graded  series  of  ethanol  and  embedded  in  Spurr's  resin 
(Polysciences  Inc.).  Thin  sections  were  cut  with  a  Porter- 
Blum  MT-1  ultramicrotome,  stained  with  2%  aqueous 


Figure  1.  The  chamber  for  the  electrophysiological  measurements. 
The  oocyte  (Oo)  was  held  with  a  suction  capillary  tube  (C),  and  the 
microelectrode  (E)  was  advanced  into  the  oocyte  vertically. 


uranyl  acetate  and  Reynolds'  lead  citrate  (Reynolds, 
1963),  and  observed  with  a  Hitachi  H-500  transmission 
electron  microscope. 

Results 

Polyspermy  block  mechanism  in  the  oocyte  of 
Mytilus  edulis 

When  oocytes  were  collected  within  30  min  after 
spawning  (fresh  oocytes)  and  inseminated  with  sperm, 
90%  of  the  oocytes  were  monospermic  until  Rs/0  reached 
5  X  103;  insemination  with  higher  concentration  of  sperm 
(Rs/0  is  above  5  X  103)  resulted  in  polyspermy  (Fig.  2). 
The  incidence  of  polyspermy  increased  when  oocytes  were 
aged  in  seawater  before  insemination,  though  the  time  at 
which  oocytes  become  polyspermic  varied  from  batch  to 
batch  (data  not  shown).  Typical  data  are  shown  in  Figure 
3.  When  oocytes  collected  more  than  30  min  after  spawn- 
ing (old  oocytes)  were  inseminated  at  a  Rs/0  of  2  X  103, 
the  number  of  sperm  penetrating  the  oocyte  increased 
with  time,  reaching  a  mean  of  3.47  when  60-min-old  oo- 
cytes were  inseminated;  insemination  with  a  low  Rs/0  (9 


I 


o    1  0 
<§ 


fc 

a 
(fl 

'o 

ci 


n 
v 


10 


POLYSPERMY  BLOCK  IN  MYTILL'S  OOCYTE 

100 


I    50 


o 

Q. 


333 


10  10  10 

Sperm  -  Oocyle  Ratio 


1  0 


Figure  2.  The  number  of  penetrating  sperm  per  fresh  oocyte  insem- 
inated with  various  sperm-oocyte  ratios.  Note  that  heavy  insemination 
induced  polyspermy. 


(3.47) 


1 1 .09) 


(1.01) 


30  60  90 

Time  after  Spawning  (min) 


120 


Figure  3.  Increase  in  the  rate  of  polyspermy  with  passage  of  time 
after  spawning.  The  mean  number  of  sperm  nuclei  per  oocyte  is  in  pa- 
rentheses. Rs/0  was  9  x  102  (»)  or  2  x  103  (0). 


X  10:)  resulted  in  monospermic  fertilization  even  in  old 
oocytes  (Fig.  3).  The  number  of  penetrated  sperm  in  an 
oocyte  increased  when  Rs/0  was  increased  and  the  time  of 
insemination  was  delayed;  the  number  of  penetrated 
sperm  was  2.68,  3.6 1 ,  and  4.90  when  oocytes  of  the  other 
batch  were  inseminated  at  50,  90,  and  120  min  after 
spawning,  whereas  the  number  of  sperm  was  2.46  when 
fresh  oocytes  were  inseminated  with  Rs/0  of  1  X  104  (data 
not  shown).  These  data  suggest  that  a  mechanism  to  block 
polyspermy  exists  in  the  oocyte  of  Afytilus  edidis.  and 
that  it  weakens  with  the  passage  of  time  after  spawning. 
The  number  of  penetrated  sperm  in  a  fresh  oocyte  that 
was  inseminated  under  heavy  insemination  conditions 
was  2.79  (Fig.  4).  However,  when  monospermic  oocytes 
made  by  fertilization  with  light  insemination  of  fresh  oo- 
cytes were  re-inseminated  at  a  higher  Rs/0  (8  X  103  --  1 
X  104;  heavy  insemination)  at  1 5  and  30  s  after  the  initial 
light  insemination,  the  number  of  penetrated  sperm  per 
oocyte  was  1 .38  and  1.18,  respectively  (Fig.  4).  From  these 
results  and  data  from  two  other  batches  of  oocytes  (data 
not  shown),  the  completion  time  for  polyspermy  block 
was  concluded  to  be  30  s.  Thus  fresh  oocytes  acquire  a 
block  to  polyspermy  very  rapidly  after  fertilization. 

The  fas!  polyspermy  hlock  by  fertilization  potential 

Both  the  depolarization  of  the  oocyte  plasma  mem- 
brane and  the  polyspermy  block  in  the  marine  inverte- 
brates and  fucoid  seaweeds  are  known  to  be  suppressed 
in  low-sodium  ASW  (Gould-Somero  el  al.  1979;  Jaffe, 
1980;  Kline  el  al.  1985:  Brawley,  1991).  When  the  oocytes 
of  M.  eduliswere  lightly  inseminated  in  low-sodium  ASW, 
they  became  polyspermic  (Fig.  5).  In  ASW.  1.04  sperm 
penetrated  the  oocyte,  but  this  number  was  increased  by 


lowering  the  sodium  ion  concentration  in  ASW,  suggest- 
ing that  a  sodium-dependent  depolarization  causes  the 
fast  polyspermy  block  in  M.  edidis. 

It  is  difficult  to  insert  the  microelectrode  in  Afytilus 
oocytes  because  the  plasma  membrane  of  the  oocyte  is 
easily  broken  by  mechanical  treatments.  In  the  present 
study,  only  eight  measurements  of  the  fertilization  poten- 
tial were  obtained  without  damaging  the  oocytes.  The 
membrane  potential  of  unfertilized  Afytilus  oocytes  was 
-66.0  ±  2.2  mV  (n  =  5)  in  NSW  (Fig.  6A,  Table  I).  Upon 
insemination,  the  plasma  membrane  of  the  oocyte  rapidly 


u 


E 

I 

w 

I  ' 

I 

3 
Z 

§        ° 


1 


Time  of  Re-insemination  (min) 

Figure  4.  The  number  of  penetrating  sperm  per  oocyte  re-insemi- 
nated at  various  times  after  initial  insemination.  Values  are  the  mean 
of  100  oocyles  from  a  single  batch.  Time  zero  represents  the  number  of 
sperm  nuclei  per  oocyte  when  unfertilized  oocytes  were  heavily  (•)  or 
lightly  (D)  inseminated. 


334 


T.  TOGO  ET  AL. 


6  r 


100  200  300  400 

Na*  Concentration  (niM) 


500 


Figure  5.  Induction  of  polyspermy  in  low-sodium  ASW.  Oocytes 
were  incubated  in  low-sodium  ASWs  for  10  min,  and  inseminated  at 
R,/0  of  5  x  102  -  1  x  103.  Values  are  the  mean  ±  SEM  of  five  experiments. 


depolarized  to  +59.4  ±  2.2  mV  (n  =  5)  and  then  repo- 
larized  to  a  steady  resting  potential  of  -68.0  ±  3.4  mV 
(n  =  5)  at  28.0  ±  5.2  s  (n  =  5)  after  the  onset  of  depolar- 
ization (Fig.  6A,  Table  I).  After  measurement,  all  the  oo- 
cytes  became  normal  2-cell-stage  embryos  (Table  I).  A 
series  of  oscillating  spikes  of  depolarization  (arrows  in 
Fig.  6A)  was  observed  after  the  membrane  potential  re- 
turned to  a  resting  potential  in  all  oocytes  measured  in 
NSW.  These  spikes  may  be  due  to  sperm  contacts  with 
the  oocyte  surface,  since  the  frequency  of  the  spikes  in- 
creased as  the  concentration  of  sperm  was  increased  (data 
not  shown). 

When  the  concentration  of  sodium  in  ASW  was  re- 
duced to  50  mAf,  the  resting  potential  of  unfertilized  oo- 
cytes was  —79.3  ±  5.3  mV  («  =  3).  The  membrane  of  the 
oocytes  depolarized  upon  insemination,  but  the  peak 
value  in  low-sodium  ASW  was  remarkably  lower  (-56.0 
±  5.0  mV,  n  =  3)  than  that  in  NSW  (Fig.  6B,  Table  I). 
All  the  oocytes  transferred  into  normal  ASW  after  the 
measurement  in  low-sodium  ASW  exhibited  abnormal 
cleavage  (Table  I).  Although  we  attempted  to  examine 
fertilization  under  current-clamp  or  voltage-clamp  con- 
ditions, the  experiments  were  unsuccessful  because  of  the 
difficulty  of  electrode  penetration. 

The  late  polyspermy  block 

When  the  fertilized  oocytes  were  heavily  re-inseminated 
(Rs/0  =  8  X  103  -  1  X  104)  at  1  min  after  an  initial  light 
insemination  (Rs/0  =  5  X  102  -  1  X  103),  the  number  of 
bound  sperm  was  smaller  (0.46)  than  that  in  the  oocytes 
initially  inseminated  at  the  same  Rs/0  (Rs/0  =  8  X  103  -  1 
X  104)  (1.0  at  time  zero  in  Fig.  7).  The  number  of  bound 


sperm  was  reduced  to  0.32,  0.3 1 ,  or  0.32  when  the  oocytes 
fertilized  with  a  light  insemination  were  heavily  re-insem- 
inated at  2,  5,  or  10  min  after  initial  insemination  in  NSW, 
respectively.  When  fixative  (e.g.,  glutaraldehyde)  was 
added,  almost  all  the  acrosome-intact  sperm  on  the  oocyte 
surface  were  removed  (data  not  shown).  These  results 
suggest  that  a  mechanism  to  prevent  sperm  binding 
through  suppression  of  the  acrosomal  reaction  developed 
shortly  after  fertilization. 

For  investigating  the  relationship  between  sperm  bind- 
ing and  the  acrosomal  reaction,  the  rate  of  the  acrosomal 
reaction  of  the  re-inseminated  sperm  was  investigated  (Fig. 
8).  When  unfertilized  oocytes  were  inseminated  at  a  R5/0 
of  3.5  —  4  X  103  and  fixed  at  5  min  after  the  insemination, 
the  rate  of  the  acrosomal  reaction  was  77%.  However,  this 
rate  was  reduced  to  about  27%  when  fertilized  oocytes 
(10  min  after  initial  insemination)  were  re-inseminated 
at  Rs/0  of  3.5  -  4  X  103  and  fixed  at  5  min  after  the  re- 
insemination.  Light  microscopic  observations  of  living 
samples  also  showed  that  the  rate  of  the  acrosome  reaction 
was  higher  for  sperm  on  the  surface  of  an  unfertilized 
oocyte  than  for  those  on  a  fertilized  oocyte  (Fig.  8B,  C). 
These  results  suggest  that  an  acrosomal-reaction-inducing 
activity  is  lower  on  the  surface  of  the  fertilized  oocytes 
than  on  unfertilized  oocytes.  As  a  result,  sperm  hardly 
undergo  the  acrosome  reaction  (Fig.  8)  and  bind  on  the 
surface  of  the  fertilized  oocyte  (Fig.  7). 

When  the  surfaces  of  the  oocytes  that  were  re-insemi- 
nated (Rs/0  =  3.5  -  4  X  103)  at  5  min  after  initial  light 
insemination  were  observed  with  a  transmission  electron 
microscope,  bound  sperm  had  undergone  the  acrosomal 
reaction,  and  the  acrosomal  process  reached  the  oocyte 
plasma  membrane  through  the  vitelline  coat.  Typical  su- 
pernumerary sperm  are  shown  in  Figure  9.  We  examined 
several  serial  sections  of  supernumerary  sperm,  but  we 
did  not  find  fusion  between  the  supernumerary  sperm 
and  the  fertilized  oocyte.  Furthermore,  the  fertilization 
cone  associated  with  fertilizing  sperm  was  not  observed 
in  these  sections  or  in  other  single  sections.  Therefore,  we 
are  certain  that  fertilization  of  the  supernumerary  sperm 
that  underwent  the  acrosomal  reaction  was  prevented  at 
the  level  of  the  oocyte  plasma  membrane. 

Discussion 

Conflicting  results  have  been  reported  on  the  poly- 
spermy block  mechanism  in  bivalves.  The  oocyte  of  the 
surf  clam  Spisula  has  a  complete  mechanism  to  block 
polyspermy  (Ziomek  and  Epel,  1975;  Longo,  1976a). 
Dufresne-Dube  el  ai  (1983),  however,  obtained  mono- 
spermic  fertilizations  in  the  mussel  Mytilus  galloprovin- 
cia/is  only  when  the  oocytes  were  inseminated  with  a  very 
low  concentration  of  sperm  at  a  range  of  Rs/0  between 


POLYSPERMY  BLOCK  IN  MYTll.L'S  OOCYTE 


+60 


+40 


+20 


I 


£     -20 
01 


-40 


-60 


-80 


i 
Sp 


335 
B 


1  min 


Figure  6.  Typical  patterns  of  membrane  depolarization  of  Mylilus  oocytes  at  fertilization  in  NSW  (A) 
or  low-sodium  ASW  (B).  Sperm  suspension  was  added  to  the  chamber  containing  an  oocyte  at  Sp.  Sodium 
concentration  in  low-sodium  ASW  was  50  m.M.  Arrows  show  depolarization  spikes. 


Fertilization  potential  characteristics  in  Mytilus  edulis 


Table  I 


Seawater 

Resting  potential 
before  fertilization 
(mV) 

Resting  potential 
after  fertilization 
(mV) 

Peak  value 

(mV) 

Duration  of 
fertilization  potential3 
(s) 

n" 

% 
polyspermy 

NSW 
50  mA/-Na+ 

-66.0  ±  2.2 
-79.3  ±  5.3 

-68.0  +  2.2 
-90.0  ±  2.3 

+  59.4  ±  2.2 
-56.0  ±  5.0 

28.0  ±  5.2 
48.7  ±  4.8 

5 
3 

0(0/5) 
1  00  (3/3) 

Values  are  mean  ±  SE. 

1  Time  for  which  the  membrane  potential  was  more  positive  than  resting  potential  before  fertilization. 

b  Number  of  measurements. 


336 


T.  TOGO  ET  AL 


1.0  r 


a 
w 

•o 

§ 
m 


0.5 


2345678 
Time  of  Re-insemination  (min) 


10 


Figure  7.  Sperm  binding  to  fertilized  oocytes.  Fertilized  oocytes  were 
heavily  re-inseminated  at  appropriate  times  and  fixed  at  30  s  after  re- 
insemmation.  Time  zero  represents  heavily  inseminated  sperm  binding 
to  unfertilized  oocytes  (control).  Values  are  represented  as  a  ratio  of  the 
control  value  (mean  ±  SEM  of  six  experiments). 


10'  and  2  X  10:,  suggesting  that  no  complete  block  to 
polyspermy  is  established  in  this  species.  In  Mytihts  ednlis, 
we  found  here  that  the  oocyte  exhibits  a  complete  block 
to  polyspermy  after  fertilization  (Fig.  4),  but  that  the  block 
lasts  only  30  min  after  spawning.  Because  the  incidence 
of  polyspermy  increases  with  time  after  oocytes  are 
spawned  (Fig.  3),  use  of  old  oocytes  for  insemination 
would  lead  to  the  same  conclusion  for  At.  edulis  as 
Dufresne-Dube  et  al.  (1983)  reached  for  M.  gallo- 
provincialis—i.e.,  that  the  species  lacks  a  mechanism  for 
complete  polyspermy  block. 

In  the  brown  alga  Fucus  (Brawley,  199 1 )  and  in  marine 
invertebrates  such  as  the  sea  urchin,  the  starfish,  the 
echiuroid  Urechis.  and  the  nemertean  Cerebratnlus  (Jaffe, 
1976,  1980;  Miyazaki  and  Hirai,  1979;  Gould-Somero  et 
al.,  1979;  Kline?/  al..  1985),  the  membrane  of  the  oocyte 
depolarizes  at  fertilization.  In  crustaceans,  hyperpolariza- 
tion  was  observed  at  fertilization  (Goudeau  and  Goudeau, 
1989a,  b).  All  the  above  reports,  except  those  for  crusta- 
ceans, also  showed  that  polyspermic  fertilization  occurs 
in  low-sodium  ASW  by  suppression  of  the  depolarization, 
and  that  fertilization  is  inhibited  when  the  membrane  po- 
tential is  clamped  at  a  positive  value.  Depolarization  of 
the  oocyte  plasma  membrane  also  occurred  at  fertilization 
in  M.  edulis.  and  less  depolarization  and  higher  poly- 
spermic fertilization  were  found  in  low-sodium  ASW 
(Figs.  5  and  6,  Table  I),  suggesting  that  an  electrical  event 
at  the  plasma  membrane  acts  as  the  fast  polyspermy  block 
in  the  oocyte  of  Mytihts. 

The  fast  electrical  block  is  not  absolute,  and  thus  high 
sperm  concentrations  can  sometimes  overcome  it  (see 


Jaffe  and  Gould,  1985).  An  effective  late  block  appears 
following  the  fast  electrical  block  (see  Fig.  12  in  Brawley, 
1991).  In  many  species  except  bivalves,  this  late  block 
usually  accompanies  morphological  changes  in  the  cortex 
of  the  oocyte  such  as  cortical  granule  breakdown  or  ele- 
vation of  the  fertilization  envelope  (Longo,  1983;  Jaffe 
and  Gould,  1985).  During  the  formation  of  the  fertiliza- 
tion envelope,  sperm-oocyte  binding  is  impaired  by  en- 
zymes released  from  the  fertilized  oocyte,  and  late  poly- 
spermy block  is  established.  For  example,  during  cortical 
reaction  after  fertilization,  the  egg  of  the  sea  urchin  releases 
proteases  that  cause  separation  of  sperm  and  egg  ( Vacquier 
et  al..  1972,  1973).  Sperm  detachment  also  occurs  before 


100  n 


o 


3 

DC 
0) 

O 

in 
o 


50  - 


0   J 


(A) 


T 


UF 


Cont. 


Figure  8.  Acrosomal  reaction  in  sperm  used  to  inseminate  unfertil- 
ized or  fertilized  oocytes.  (A)  Control  represents  the  acrosome  reaction 
of  fixed  sperm  inseminating  fixed  oocytes.  Values  are  the  mean  ±  SEM 
of  four  experiments.  (B)  On  the  surface  of  an  unfertilized  oocyte,  many 
round  acrosome-reacted  sperm  (arrows)  are  seen.  (C)  On  the  surface  of 
a  fertilized  oocyte.  pear-shaped  acrosome-intact  sperm  (arrowheads)  are 
seen.  The  photographs  were  taken  within  1  min  after  re-insemination. 
When  fixative  was  added,  almost  all  acrosome-intact  sperm  on  the  oocyte 
surface  were  removed  (not  shown).  Bar  =  20  tim. 


POLYSPERMY  BLOCK  IN  M\TILl'S  OOCYTE 


337 


MV 


JC 


figure  9.  Transmission  electron  micrographs  of  supernumerary  sperm  on  the  surface  of  the  fertilized 
oocyte.  (A,  B)  Acrosome-reacted  sperm  at  the  oocyte  surface.  Sperm  dissolved  the  vitelline  coat  (VC),  and 
protruded  the  acrosomal  process  (AP)  to  the  oocyte  plasma  membrane  (PM),  but  the  acrosomal  process  did 
not  fuse  with  the  oocyte  plasma  membrane.  (C)  Some  acrosome-intact  sperm  were  observed  on  the  surface 
of  the  fertilized  oocyte  without  being  removed  during  fixation.  A  =  acrosome.  N  =  nucleus.  JC  =  jelly  coat, 
MV  =  microvilli.  Bar  =  1  nm. 


cell-wall  formation,  and  destruction  of  the  sperm  "recep- 
tor" with  the  glycosidase  released  from  the  egg  after  fer- 
tilization is  hypothesized  to  occur  in  Fiicnx  (Brawley, 
1991).  In  ascidians  (Ascidia.  Phallusia.  Halocynthia), 
sperm  recognize  A-acetylglucosamine  residues  coming  out 
onto  the  vitelline  coat  during  fertilization  (Lambert,  1986; 
Honegger,  1982,  1986;  Matsuura  et  a/..  1993).  Although 
the  type  of  cortical  reaction  observed  in  sea  urchins  is 
lacking  in  ascidian  eggs  (Rosati  et  al,  1977),  the  oocytes 
rapidly  release  A'-acetylgulucosaminidase  after  fertiliza- 
tion, blocking  the  binding  of  the  sperm  to  the  vitelline 
coat  (Lambert,  1986,  1989;  Lambert  and  Goode,  1992). 
Neither  ascidians  (Rosati  et  al..  1977)  nor  many  bivalve 
species,  including  M.  edulis  (Longo.  1983:  Alliegro  and 
Wright,  1983;  Longo  el  al.,  1993),  display  the  morpholog- 
ical changes  of  the  oocyte  cortex  seen  during  fertilization 
in  sea  urchins.  Nevertheless,  in  M.  edulis.  the  late  poly- 
spermy  block  that  follows  the  fast  electrical  mechanism  is 
established  by  30  s  after  fertilization  since  the  duration  of 
the  fertilization  potential  is  30  s  (Table  I).  One  stage  of  the 
late  polyspermy  block  in  the  species  seems  to  be  inhibition 
of  sperm-oocyte  binding  through  suppression  of  the  ac- 


rosomal reaction  (Figs.  7  and  8).  The  acrosome  in  sperm 
on  the  surface  of  the  oocyte  can  be  in  one  of  two  states: 
unreacted  or  reacted  (Figs.  8  and  9).  Therefore,  it  is  possible 
that  the  oocyte  loses  its  ability  to  induce  an  acrosome  re- 
action after  penetration  by  a  fertilizing  spermatozoon.  In 
M.  galloprovincialis,  the  acrosomal  reaction  is  reportedly 
triggered  when  sperm  recognize  /V-acetylgalactosamine 
(GalNAc)  residues  on  the  oocyte  (Focarelli  et  al..  1991). 
Perhaps  Mytilus,  like  the  ascidians  (Lambert  and  Goode, 
1992),  release  some  enzyme  from  its  oocyte  so  that  the 
"receptor"  molecule  necessary  to  induce  the  acrosomal  re- 
action is  destroyed  or  covered,  resulting  in  an  inhibition 
of  sperm-oocyte  binding  through  suppression  of  the  ac- 
rosomal reaction.  In  our  preliminary  study  in  M.  edulis. 
contrary  to  the  report  of  Focarelli  et  al.  ( 1 99 1 ),  neither 
fertilization  nor  sperm-binding  were  inhibited  in  the  pres- 
ence of  GalNAc  (Togo  and  Morisawa,  unpub.  data). 
Treatment  of  oocytes  with  GalNAc-binding  lectins  (DBA 
and  SBA)  also  failed  to  inhibit  these  processes  (Togo  and 
Morisawa,  unpub.  data).  Further  studies  on  the  "receptor" 
prerequisite  for  the  induction  of  the  acrosomal  reaction  in 
Mvtilus  will  be  needed. 


338 


T.  TOGO  ET  AL 


Some  sperm  could  undergo  an  acrosomal  reaction  and 
bind  to  the  fertilized  oocyte  (Fig.  7),  although  the  rate  of 
reaction  was  reduced  in  re-inseminated  sperm  (Fig.  8). 
Electron  microscopic  observations  showed  the  acrosomal 
process  of  sperm  penetrating  the  oocyte  by  digesting  the 
vitelline  coat  with  sperm  lysin  (Fig.  9).  However,  we  could 
observe  neither  fusion  between  the  acrosomal  process  and 
the  oocyte  plasma  membrane  nor  formation  of  the  fer- 
tilization cone  associated  with  fertilizing  sperm  (Fig.  9), 
suggesting  that  the  plasma  membrane  of  the  oocyte  may 
change  after  fertilization.  This  kind  of  change  would  block 
the  sperm-oocyte  fusion  and  has  been  described  in  other 
animals  such  as  the  echiuroid  Urechis  (Paul  and  Gould- 
Somero,  1976),  the  nemertean  Cerebratithts  (Kline  el  ai, 
1985),  mammals  (e.g.,  Horvath  el  al.,  1993),  and  the  surf 
clam  Spisula  (Ziomek  and  Epel,  1975;  Longo,  1976a). 
Complete  polyspermy  block  at  the  plasma  membrane  was 
reported  in  Spisula:  oocytes  from  which  the  vitelline  coat 
had  been  removed  were  still  monospermic  (Ziomek  and 
Epel,  1975).  In  M.  ednlis.  however,  polyspermic  fertiliza- 
tion occurred  even  when  the  vitelline  coat  was  removed 
by  actinase  E,  suggesting  that  the  oocyte  of  the  species 
has  an  incomplete  mechanism  to  block  polyspermy  at  the 
level  of  the  oocyte  plasma  membrane  (Kyozuka  and  Os- 
anai,  1994).  Changes  of  the  plasma  membrane  at  fertil- 
ization was  reported  by  Longo  (1976b),  who  observed  a 
twofold  increase  in  the  number  of  particles  on  the  pro- 
toplasmic face  of  the  plasma  membrane  in  freeze-fracture 
replicas  of  Spisula  oocytes.  Nevertheless,  the  relationship 
between  the  increase  in  particles  and  the  block  to  poly- 
spermy at  the  oocyte  plasma  membrane  remains  obscure. 

Bivalves  such  as  Spisula  (Finkel  and  Wolf,  1980)  and 
M.  galloprovincialis  (Dufresne-Dube  el  al.,  1983)  are 
thought  to  produce  a  fast  polyspermy  block  by  depolar- 
izing the  oocyte  plasma  membrane;  the  same  mechanism 
was  found  here  in  AI.  ednlis.  Data  from  re-insemination 
experiments  (Ziomek  and  Epel,  1975;  Longo,  1976a)  in- 
dicate that  a  late  polyspermy  block  mechanism  is  found 
in  Spisula  as  well.  As  reported  here,  M.  ednlis  clearly 
shows  a  complete  late  block  following  the  electrically  me- 
diated fast  block.  Monospermy  is  ensured  after  the  fast 
polyspermy  block  by  the  collaboration  of  the  two  strate- 
gies— suppression  of  the  acrosome  reaction  of  the  super- 
numerary sperm  and  blockage  of  sperm  entry  at  the 
plasma  membrane. 

Acknowledgments 

We  thank  Drs.  H.  Nakamura,  Akkeshi  Marine  Biolog- 
ical Station,  Hokkaido  University,  and  T.  Nawata,  College 
of  Medical  Sciences,  Tohoku  University,  for  their  valuable 
advice  and  support  for  the  electrophysiological  investi- 
gations. We  also  thank  the  staff  of  Asamushi  Marine  Bio- 


logical Station,  Tohoku  University,  and  Misaki  Marine 
Biological  Station,  University  of  Tokyo,  for  supplying  the 
mussels.  This  work  was  supported  in  part  by  Grants-in- 
Aid  from  the  Ministry  of  Education,  Science  and  Culture 
of  Japan  to  Dr.  M.  Morisawa  (No.  03404004). 

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In  Vivo  Effects  of  Dopamine  and  Dopaminergic 

Antagonists  on  Testicular  Maturation  in  the 

Red  Swamp  Crayfish,  Procambams  clarkii 


RACHAKONDA  SAROJINI,  RACHAKONDA  NAGABHUSHANAM, 
AND  MILTON  FINGERMAN* 

Department  of  Ecology.  Evolution,  and  Organismal  Biology. 
Tulane  University.  New  Orleans.  Louisiana  70118 


Abstract.  In  vivo,  dopamine  (DA)  inhibits  testicular 
maturation  in  the  red  swamp  crayfish,  Procambams  clar- 
kii. Crayfish  given  DA  injections  had  a  smaller  testicular 
index,  smaller  testicular  lobes,  fewer  mature  sperm,  and 
less-well-developed  androgenic  glands  than  did  the  control 
crayfish  given  physiological  saline.  Males  administered  5- 
hydroxytryptamine  (5-HT)  or  a  DA  receptor  blocker,  spi- 
perone  or  pimozide,  showed  enhanced  testicular  matu- 
ration and  more  highly  developed  androgenic  glands  than 
did  the  control  crayfish.  When  equimolar  amounts  of  5- 
HT  and  DA  were  co-injected,  the  actions  of  DA  and  5- 
HT  were  found  to  be  antagonistic.  These  results  can  be 
explained  by  assuming  not  only  that  5-HT  triggers  release 
of  the  gonad-stimulating  hormone  (GSH)  but  that  DA 

(a)  triggers  release  of  the  gonad-inhibiting  hormone  (GIH), 

(b)  inhibits  GSH  release,  or  (c)  does  both  (a)  and  (b),  with 
GSH  and  GIH  affecting  the  androgenic  glands  directly, 
thereby  regulating  release  of  the  androgenic  gland  hor- 
mone that  has  the  well-established  role  of  stimulating  tes- 
ticular maturation  and  spermatogenesis. 

Introduction 

Biogenic  amines  function  as  neurotransmitters  in  a 
wide  array  of  animals  (Werman,  1966;  Gerschenfeld. 
1973;  Fingerman,  1985).  Among  the  demonstrated  roles 
of  at  least  some  of  the  biogenic  amines  in  crustaceans  is 
regulation  of  release  of  neurohormones  (Fingerman  and 
Nagabhushanam,  1992;  Fingerman  et  ai.  1994). 

The  presence  of  the  biogenic  amines  5-hydroxytrypt- 
amine  (5-HT)  and  dopamine  (DA)  in  the  nervous  systems 


Received  8  May  1995;  accepted  14  September  1995. 
*To  whom  correspondence  should  be  sent. 


of  crustaceans,  including  crayfishes,  is  well  established. 
5HT-like  immunoreactivity  in  the  central  nervous  system 
of  the  red  swamp  crayfish  Procambams  clarkii.  the  species 
used  in  the  present  study,  was  demonstrated  by  several 
investigators  (Fujii  and  Takeda,  1988;  Arechiga  et  al.. 
1990;  Real  and  Czternasty,  1990).  In  addition,  5-HT  has 
been  identified  and  quantitatively  measured  by  high  per- 
formance liquid  chromatography  (HPLC)  in  Procambams 
clarkii  by  Kulkarni  and  Fingerman  (1992).  Using  the  crab 
Carcimis  macnas.  Kerkut  et  al.  (1966)  provided  the  first 
convincing  evidence  for  the  existence  of  DA  in  the  nervous 
system  of  a  crustacean.  Neurons  with  DA-like  immuno- 
reactivity have  been  visualized  in  the  crayfish  Orconectes 
limosiis  (Elekes  et  al..  1988),  the  lobster  Homarus  gam- 
mams  (Barthe  el  al..  1989).  and  Procambams  clarkii 
(Mercier  et  al..  1991).  By  use  of  HPLC,  Elofsson  et  al. 
(1982)  showed  the  presence  of  DA  in  the  nervous  system 
of  the  crayfish  Pacifastacus  leniusculus. 

In  decapod  crustaceans  the  major  neuroendocrine 
component  of  the  eyestalk,  the  medulla  terminalis  X-or- 
gan-sinus  gland  complex,  is  the  source  of  the  gonad-in- 
hibiting hormone  (GIH)  (Panouse,  1943).  In  contrast,  a 
gonad-stimulating  hormone  (GSH)  is  present  in  the  brain 
and  thoracic  ganglia  (Otsu.  1960,  1963;  Eastman-Reks 
and  Fingerman,  1984).  Data  from  this  laboratory  provide 
the  basis  for  the  hypothesis  that  5-HT  triggers  release  of 
GSH  in  both  sexes  of  the  fiddler  crab  Uc  •apugilator(  Rich- 
ardson et  al..  1991;  Sarojini  et  al..  1993)  and  in  Procam- 
bams clarkii  (Kulkarni  and  Fingerman,  1992;  Sarojini  et 
al.  1994).  On  the  other  hand,  DA  has  so  far  been  found 
to  antagonize  the  gonad-stimulating  action  of  5-HT  in 
females  of  Procambams  clarkii  (Sarojini  et  al..  1995a) 
and  in  males  of  Uca  pitgilator  (Sarojini  el  al..  1995b). 


340 


CRAYFISH  TESTICULAR  MATURATION 


341 


In  male  crustaceans,  in  addition  to  the  two  neurohor- 
mones,  GSH  and  GIH,  the  androgenic  gland  hormone 
(AGH)  has  a  major  role  in  the  control  of  spermatogenesis. 
The  function  of  the  androgenic  gland  in  controlling  de- 
velopment and  maturation  of  the  reproductive  system  and 
secondary  sexual  characteristics  in  male  crustaceans  was 
first  described  by  Charniaux-Cotton  ( 1954).  Initiation  of 
spermatogenesis  is  due  to  circulating  AGH  (Payen,  1973). 
Spermatogenesis  stops  when  the  androgenic  glands  are 
removed  (Charniaux-Cotton,  1964;  Puckett,  1964;  Na- 
gamine  el  til..  1980).  Removal  of  both  eyestalks,  thereby 
removing  the  source  of  GIH,  results  in  hypertrophy  of 
the  androgenic  glands  and  precocious  spermatogenesis 
(Meusy,  1965;  Demeusy,  1967;  Payen  ct  a/..  1971).  Thus. 
GIH  appears  to  exert  its  effect  on  the  testes  indirectly,  by 
inhibiting  the  androgenic  glands.  On  the  other  hand,  a 
GSH  is  required  to  activate  the  androgenic  glands  for 
spermatogenesis  to  occur  (Juchault  and  Legrand.  1965), 
a  process  that  Payen  ( 1 980)  referred  to  as  a  positive  control 
of  the  androgenic  glands  by  a  neurohormone.  Gupta  el 
ul.  (1989)  suggested  from  their  studies  of  the  crab  Para- 
tclp/iusa  hydrodromus  that  the  inactive  phase  of  the  testes 
is  due  to  an  increase  in  the  hemolymph  liter  of  GIH  with 
concomitant  decreases  in  the  liters  of  GSH  and  AGH. 

In  view  of  the  facts  that  5-HT  stimulates  gonadal  mat- 
uration in  both  male  and  female  Procambarus  clarkii  and 
DA  antagonizes  this  action  of  5-HT  in  females  of  this 
species,  this  investigation  was  designed  to  determine  (a) 
whether  DA  inhibits  testicular  maturation  in  Procambarus 
clarkii,  (b)  whether  5-HT  and  DA  act  antagonistically  on 
gonadal  maturation  and  spermatogenesis  in  the  male 
crayfish,  and  (c)  whether  the  androgenic  glands  will  be 
affected  when  DA  or  a  dopaminergic  receptor  blocker  is 
injected.  This  is  the  first  report  that  shows  injection  of 
DA  affects  the  androgenic  glands  of  any  crustacean. 

Materials  and  Methods 

Experimental  animals 

Specimens  of  the  red  swamp  crayfish,  Procambarus 
clarkii,  were  purchased  from  a  local  seafood  dealer.  In 
the  laboratory  they  were  maintained  in  freshwater  tanks 
where  the  water  was  recirculated  constantly  through  sand 
filtration  units.  Male  intermolt  crayfish  with  a  carapace 
length  of  30-35  mm  and  a  body  weight  of  1 1-12  gm  were 
used  for  these  experiments.  The  crayfish  were  maintained 
at  a  room  temperature  of  24  ±  2°C.  with  12  h  of  light 
daily,  from  8:00  A.M.  to  8:00  P.M.,  and  were  fed  com- 
mercial crayfish  food  daily. 

Drugs 

5-HT  creatinine  sulfate,  DA  hydrochloride.  spiperone, 
and  pimozide  were  purchased  from  the  Sigma  Chemical 


Company  (St.  Louis,  MO).  The  drugs  were  dissolved  in 
crayfish  physiological  saline  (Van  Harreveld,  1936).  To 
prepare  the  stock  solution  of  spiperone  a  few  drops  of 
acetic  acid  were  added  to  facilitate  solubilization.  When 
DA  was  used  1  X  10~6  mol,  1  X  10~7  mol  and  I 
X  10~s  mol  per  crayfish  were  injected.  The  amounts  of  5- 
HT,  si  '  and  pimozide  injected  were  1  X  10~6  mol 

per  crayfish.  The  volume  injected  into  each  crayfish  was 
100  pi. 

The  testicular  index  (TI)  was  determined  for  each  cray- 
fish used  in  these  experiments  according  to  the  standard 
formula: 


Weight  of  the  testes 
Weight  of  the  crayfish 


^ 


The  testes  and  androgenic  glands  were  removed  from  each 
of  the  crayfish  used  in  these  experiments  after  the  crayfish 
were  weighed  at  the  time  of  sacrifice.  When  these  organs 
were  removed  the  testes  were  weighed.  The  testes  and 
androgenic  glands  were  then  fixed  for  24  h  in  Bouin's 
fluid,  dehydrated  in  an  alcoholic  series,  and  embedded  in 
paraffin  (m.p.  56°-58°C).  Sections  (7  ^m)  were  cut  and 
stained  with  Delafield's  hematoxylin  followed  by  coun- 
terstaining  with  alcoholic  eosin  (Bancroft  and  Stevens, 
1982).  The  diameters  of  50  testicular  follicles  (^m)  in  the 
testes  of  each  male  were  measured  by  use  of  a  compound 
microscope  fitted  with  an  ocular  micrometer.  The  number 
of  mature  sperm  per  follicle  was  also  determined.  The 
diameters  (urn)  of  50  cells  in  each  androgenic  gland  were 
likewise  measured.  The  experiments  were  performed  twice 
and  the  averaged  results  are  presented  in  the  figures  where 
each  value  represents  the  mean  for  20  crayfish,  except  for 
bars  1C  and  SC  in  Figure  7  which,  as  we'll  explain  below. 
represent  the  means  for  40  crayfish.  The  data  were  ana- 
lyzed by  means  of  Student's  /-test  with  significance  set  at 
the  95%  confidence  interval.  Standard  errors  of  the  means 
were  also  calculated. 

Results 

Effect  of  D  ion  the  testes 

To  determine  the  response  of  the  testes  to  DA.  each 
time  the  experiment  was  done  50  male  crayfish  were  di- 
vided into  five  groups  of  10  each.  The  first  group  served 
as  the  initial  control,  and  this  group  of  crayfish,  which 
received  no  treatment,  was  sacrificed  on  the  first  day  of 
the  experiment.  The  initial  control  crayfish  were  weighed, 
and  then  their  testes  and  androgenic  glands  were  dissected 
out.  Then,  as  stated  above,  the  paired  testes  were  weighed, 
and  the  testes  and  androgenic  glands  were  fixed  in  Bouin's 
fluid.  A  simultaneous  control  group  received  only  phys- 
iological saline  in  100  /ul  doses.  The  last  three  groups  ran 
concurrently  with  the  simultaneous  control  group  and 


342 


R.  SAROJINI  ET  AL. 


0.10 


X 

_ 
- 
z 

ai 
< 
— 


C/5 

- 


100 


0.00 


1C 


sc 


DA 


DA 
TREATMENT 

Figure  1.  Effect  of  different  doses  (1  x  ICT8,  1  x  10"',  and  1 
X  10~6  mol  per  crayfish)  of  dopamine  (DA)  on  the  mean  testicular  index 
of  the  crayfish,  Pnuwnharus  darkii.  1C.  initial  control;  SC.  simultaneous 
control.  Error  bars  are  SEM  Bar  SC  is  significantly  (P  <  0.05)  larger 
than  bars  1C.  1CT6  DA,  and  1(T7  DA.  Bar  10  *  DA  is  significantly  (P 
<  0.05)  larger  than  bar  10""  DA. 


- 


Q 
— 
O 


t/3 
- 


10  - 

DA 
TREATMENT 


10 
DA 


10  'c 
DA 


Figure  2.  Effect  of  different  doses  (1  x  10~8,  1  x  10~7,  and  1 
•>  10~6  mol  per  crayfish)  of  dopamine  (DA)  on  the  mean  testicular  lobe 
diameter  of  the  crayfish.  Proiwnharux  darkii.  1C.  initial  control;  SC. 
simultaneous  control.  Error  bars  are  SEM.  Bar  SC  is  significantly  larger 
(P  <  0.05)  than  bars  1C.  lO'6  DA.  and  10~7  DA. 


received  1  X  10~6  mol,  1  X  1(T7  mol,  and  1  X  1(T8  mol 
DA  per  crayfish  respectively  in  lOO/ul  doses.  Injections 
were  administered  on  the  1st,  5th,  and  10th  days.  The 
simultaneous  control  group  and  those  given  DA  were  sac- 
rificed on  the  1 5th  day  and  processed  in  the  same  manner 
as  the  initial  control  group. 

The  TI  and  mean  testicular  lobe  diameter  of  the  si- 
multaneous control  group  were  significantly  larger  than 
the  corresponding  values  of  the  initial  control  group, 
showing  that  during  the  15  days  of  the  experiments  the 
testes  were  undergoing  maturation  (Figs.  1,2).  Further- 
more, the  simultaneous  control  testes  contained  mature 
sperm  whereas  the  initial  control  testes  had  none  (Fig.  3). 
The  TI  and  mean  testicular  lobe  diameter  of  the  crayfish 
that  received  1  X  1(T6  mol  DA  injections  were  signifi- 
cantly smaller  than  the  corresponding  values  for  the  si- 
multaneous control  crayfish  that  received  only  physio- 
logical saline.  Furthermore,  there  were  no  mature  sperm 
in  the  testicular  lobes  of  the  crayfish  that  received  the 
injections  of  1  X  1CT6  mol  DA  in  contrast  to  the  simul- 
taneous control  crayfish.  The  crayfish  that  received  injec- 
tions of  the  two  lower  doses  of  DA  ( 1  X  1(T7  mol  and  1 
X  10~8  mol)  also  had  a  smaller  TI  and  mean  testicular 
lobe  diameter  than  the  simultaneous  control  crayfish,  but 
only  the  difference  between  the  testicular  lobe  diameter 
of  the  simultaneous  controls  and  the  crayfish  that  received 
injections  of  1  X  10  7  mol  DA  was  statistically  significant. 
The  testes  of  the  crayfish  that  received  injections  of  the 
two  lower  doses  of  DA  contained  mature  sperm,  but  sig- 
nificantly fewer  than  in  the  simultaneous  control  group. 


It  is  evident  from  Figures  1-3  that  DA  inhibited  testicular 
maturation.  The  responses  to  the  three  concentrations  of 
DA  used  strongly  suggest  that  this  inhibition  is  dose-re- 
lated, as  in  Figures  1  and  3  where  the  inhibition  produced 
by  1  X  10~6  mol  DA  per  crayfish  is  significantly  greater 
than  that  produced  by  1  X  10~8  mol  DA  per  crayfish. 


S 
at 
- 

— 

C/3 

a 
at 


— 

O 


a 
— 
S 

3 
Z 


2  - 


1C 


SC 


DA 
TREATMENT 


Figure  3.  Effect  of  different  doses  (1  X  10~8,  1  X  10"',  and  1 
•  10~6mol  per  crayfish)  of  dopamine  (DA)  on  the  mean  number  of 
mature  sperm  per  follicle  in  the  testes  of  the  crayfish,  Prociunhanix  darkii. 
1C.  initial  control;  SC,  simultaneous  control.  Error  bars  are  SEM.  Bar 
SC  is  significantly  larger  (P  <  0.05)  than  bars  1C,  10'"  DA.  IO"7  DA. 
and  10~8  DA.  Bars  10~7  DA  and  10~8  DA  are  significantly  (P  <  0.05) 
larger  than  bar  Ifr6  DA. 


CRAYFISH  TESTICULAR   MATURATION 


343 


0.30 


200  • 


0.00 


TREATMENT 

Figure  4.  Effect  of  different  treatments  on  the  mean  testicular  index 
of  the  crayfish,  Procambants  clarkii-  1C,  initial  control;  SC,  simultaneous 
control;  SP,  1  X  10~6  mol  spiperone  per  crayfish:  PI.  1  x  10~6  mol  pim- 
ozide per  crayfish;  5-HT,  1  X  10"6  mol  5-HT  per  crayfish;  COMB,  com- 
bination of  1  x  10~6mol  DA  per  crayfish  +  1  X  10"6  mol  5-HT  per 
crayfish.  Error  bars  are  SEM.  Bar  SC  is  significantly  (P  <  0.05)  larger 
than  bar  1C.  but  bar  SC  is  significantly  (P  <  0.05)  smaller  than  bars  SP. 
PI.  5-HT.  and  COMB.  Bar  5-HT  is  significantly  (P  <  0.05)  larger  than 
bar  COMB. 


5-HT 


COMB 


TREATMENT 


Figure  5.  Effect  of  different  treatments  on  the  mean  testicular  lobe 
diameter  of  the  crayfish.  Pnicninhariis  clarkii.  1C.  initial  control;  SC. 
simultaneous  control;  SP,  1  X  ICT'mol  spiperone  per  crayfish;  PI,  1 
x  10~6  mol  pimozide;  5-HT.  1  x  10~6  mol  5-HT  per  crayfish;  COMB, 
combination  of  I  x  10~6  mol  DA  per  crayfish  +  1  X  1CT6  mol  5-HT  per 
crayfish.  Error  bars  are  SEM.  Bar  SC  is  significantly  (P  <  0.05)  larger 
than  bar  1C.  but  bar  SC  is  significantly  (P  <  0.05)  smaller  than  bars  SP, 
PI.  5-HT.  and  COMB.  Bar  5-HT  is  significantly  (P  <  0.05)  larger  than 
bar  COMB. 


Effects  of  the  DA  receptor  Mockers  spiperone  ami 
pimozide.  5-HT  alone,  and  5-HT  in  combination  with 
DA  on  the  testes 

For  each  replicate  of  this  set  of  experiments,  6  groups 
of  10  crayfish  were  selected  from  the  stock.  One  group 
served  as  the  initial  control:  the  crayfish  of  this  group 
were  treated  in  the  same  way  as  the  initial  control  crayfish 
of  the  DA  dose-response  experiment.  The  crayfish  in  the 
simultaneous  control  group  received  physiological  saline 
in  100  jul  doses.  Two  groups  received  1  X  10""  mol  of  the 
DA  receptor  blockers  spiperone  and  pimozide  respectively 
in  100  n\  doses.  Another  group  received  1  X  10  6  mol  of 
5-HT  per  crayfish  in  100-^1  doses  and  the  last  group  re- 
ceived 1  X  10~6  mol  DA  in  50-^1  doses  +  1  X  10  6  mol 
5-HT  in  50-jil  doses  per  crayfish,  respectively.  Injections 
were  administered  on  the  1st,  5th,  and  10th  days.  All  the 
crayfish  that  received  injections  were  sacrificed  on  the  1 5th 
day,  and  their  testes  were  processed  in  the  same  manner 
as  those  of  the  initial  control  group. 

As  in  the  previous  experiment,  the  Tl  and  testicular 
lobe  diameter  of  the  simultaneous  control  crayfish  were 
significantly  larger  than  the  corresponding  values  for  the 
initial  control  group  (Figs.  4,  5)  and,  although  the  initial 
control  testes  had  no  mature  sperm,  the  simultaneous  con- 
trol testes  did  have  some  mature  sperm  (Fig.  6).  For  the 
crayfish  that  each  received  1 00-/ul  injections  of  1  X  10~6  mol 
of  either  of  the  DA  receptor  blockers  (spiperone  or 


100 


90  - 


80  - 


P       60  - 


50  - 


O       40  - 

« 

W       30  - 


20  - 


10  - 


1C  SC  SP  PI  5-HT        COMB 

TREATMENT 

Figure  6.  Effect  of  different  treatments  on  the  mean  number  of  ma- 
ture sperm  per  follicle  in  the  testes  of  the  crayfish.  Procambarus  clarkii, 
1C.  initial  control;  SC.  simultaneous  control;  SP.  1  X  10'6  mol  spiperone 
per  crayfish:  PI,  I  x  10~6  mol  pimozide  per  crayfish;  5-HT.  1  X  10~6  mol 
5-HT  per  crayfish:  COMB,  combination  of  1  X  I0~6  mol  DA  per  crayfish 
+  1  X  10~6  mol  5-HT  per  crayfish.  Error  bars  are  SEM.  Bar  SC  is  sig- 
nificantly (P  <  0.05)  larger  than  bar  1C,  but  bar  SC  is  significantly  (P 
<  0.05)  smaller  than  bars  SP.  PI.  5-HT,  and  COMB.  Bar  5-HT  is  sig- 
nificantly (P  <  0.05)  larger  than  bar  COMB. 


344 


R.  SAROJINI  ET  AL 


pimozide).  the  TI  and  mean  testicular  lobe  diameter  were 
significantly  larger  than  the  corresponding  values  for  the 
simultaneous  control  crayfish  that  received  physiological 
saline  alone.  Furthermore,  there  was  a  statistically  signif- 
icant greater  number  of  mature  sperm  in  the  testicular 
follicles  of  the  crayfish  that  received  either  spiperone  or 
pimozide  than  in  the  simultaneous  control  crayfish.  It  is 
clear  from  these  results  that  spiperone  and  pimozide  in- 
duced testicular  maturation. 

The  TI  and  mean  testicular  lobe  diameter  of  the  crayfish 
that  received  100  n\  of  1  X  10  h  mol  5-HT  were  signifi- 
cantly larger  than  the  corresponding  values  for  the  si- 
multaneous control  crayfish  (Figs.  4.  5),  and  the  number 
of  mature  sperm  in  the  testes  of  the  crayfish  given  5-HT 
was  also  significantly  greater  than  for  the  simultaneous 
control  crayfish  (Fig.  6).  The  combination  of  equimolar 
amounts  of  DA  and  5-HT  produced  significant  increases 
in  the  TI,  testicular  lobe  diameter,  and  sperm  count  but 
significantly  less  than  did  5-HT  alone.  These  results  show 
that  DA  and  5-HT  act  antagonistically,  but  DA  was  not 
able  to  inhibit  completely  the  stimulatory  action  of  5-HT. 

Effects  of  DA,  DA  antagonists,  5-HT  alone,  and  5-HT 
in  combination  with  DA  on  the  androgenic  gland 

The  androgenic  glands  of  the  initial  control  crayfish 
consisted  of  only  a  few  cords  of  cells  closely  associated 
with  the  vas  deferens.  These  cells  had  a  thin  rim  of  ho- 
mogeneous cytoplasm  around  the  nucleus.  The  cells  of 
the  simultaneous  control  crayfish  were  significantly  larger 
than  those  in  the  initial  control  glands  (Fig.  7).  The  means 
in  Figure  7  for  the  initial  and  simultaneous  controls  rep- 
resent data  from  40  crayfish  versus  20  crayfish  for  the  rest 
of  the  groups  because  the  means  for  the  initial  and  si- 
multaneous controls  are  based  on  the  averages  of  these 
controls  from  the  crayfish  used  in  the  two  sets  of  experi- 
ments that  provided  the  data  for  Figures  1-3  and  4-6. 
The  cells  of  the  androgenic  glands  of  the  crayfish  that 
received  injections  of  1  X  10~6,  1  X  10~7,  or  1  X  10~8  mol 
DA  per  crayfish  were  not  significantly  different  in  size 
from  those  of  the  initial  control  group.  The  cells  in  the 
androgenic  glands  of  all  the  crayfish  that  received  injec- 
tions of  DA,  regardless  of  the  dose  used,  were  significantly 
smaller  than  the  cells  in  the  concurrent  control  glands. 
The  inhibitory  effects  of  the  three  concentrations  of  DA 
on  the  androgenic  glands  do  not  provide  clear  evidence 
of  a  dose-related  response,  although  the  highest  concen- 
tration produced  somewhat  more  inhibition  than  did  the 
two  lesser  doses.  The  androgenic  glands  of  crayfish  that 
received  a  DA  receptor  blocker,  spiperone  or  pimozide, 
5-HT  alone  or  5-HT  in  combination  with  DA  showed 
significantly  greater  development  of  their  androgenic 
glands  over  the  initial  and  simultaneous  controls.  The 
cytoplasm  in  these  enlarged  glands  was  more  dense  and 


5-HT    COMB 


TREATMENT 


Figure  7.  Effect  of  different  treatments  on  the  mean  cell  size  in  the 
androgenic  glands  of  the  crayfish,  Pnvambams  clarkii.  1C,  initial  control; 
SC,  simultaneous  control;  1(T6  DA,  1  X  1(T6  mol  DA  per  crayfish:  10~7 
DA,  1  x  10'7mol  DA  per  crayfish;  10~8  DA,  1  X  l(T8mol  DA  per 
crayfish;  SP.  1  *  10'6  mol  spiperone  per  crayfish;  PI,  1  x  1(T6  mol  pim- 
ozide per  crayfish:  5-HT,  1  X  10~6  mol  5-HT  per  crayfish;  COMB,  com- 
bination of  1  >  ICT'mol  DA  per  crayfish  +  1  X  10~6  mol  5-HT  per 
crayfish.  Error  bars  are  SEM.  Bar  SC  is  significantly  (P  <  0.05)  larger 
than  bars  1C,  10~6  DA,  10"7DA,and  10~8  DA,  but  bar  SC  is  significantly 
(P  <  0.05)  smaller  than  bars  SP,  PI,  5-HT,  and  COMB.  Bar  5-HT  is 
significantly  (P  <  0.05)  larger  than  bar  COMB. 


granular  than  in  either  control  group.  As  with  the  testes, 
while  the  combination  of  5-HT  and  DA  produced  signif- 
icant growth  of  the  androgenic  glands,  this  growth  was 
significantly  less  than  that  produced  by  5-HT  alone,  ad- 
ditional evidence  of  antagonistic  actions  of  DA  and  5-HT 
on  the  reproductive  system  of  male  red  swamp  crayfish. 

Discussion 

The  present  study  demonstrates  for  the  first  time  in  a 
crayfish  an  inhibitory  action  of  DA  on  the  testes.  Fur- 
thermore, this  is  the  first  report  of  the  effect  of  DA  and 
any  of  its  antagonists  on  the  androgenic  glands  of  any 
crustacean.  DA  alone  inhibited  testicular  and  androgenic 
gland  maturation  (Figs.  1-3,  7).  On  the  other  hand,  5- 
HT  and  the  DA  receptor  blockers  spiperone  and  pimozide 
induced  testicular  and  androgenic  gland  maturation 
(Figs.  4-7). 

In  Pmcambantx  clarkii,  as  stated  above,  gonadal  mat- 
uration is  regulated  by  both  stimulatory  and  inhibitory 
neurohormones.  maturation  being  stimulated  by  GSH 
from  the  brain  and  thoracic  ganglia  and  inhibited  by  GIH 
from  the  eyestalk  neuroendocrine  system.  Our  previous 
studies  with  Procambarus  clarkii  (Sarojini  ct  a/.  1993, 
1994)  showed  that  5-HT  stimulates  gonadal  maturation 
in  males  and  females,  presumably  by  stimulating  GSH 


CRAYFISH  TESTICULAR  MATURATION 


345 


release  and  that  DA  inhibits  ovarian  development.  The 
evidence  for  5-HT  and  DA  presence  in  the  nervous  sys- 
tems of  crayfish  (Fujii  and  Takeda,  1988;  Arechiga  el  al. 
1990;  Real  and  Czternasty.  1990;  Mercier  et  al.,  1991; 
Kulkarni  and  Fingerman,  1992)  was  already  demon- 
strated. 

The  roles  of  DA  and  5-HT  in  regulation  of  gonadal 
maturation  in  vertebrates  is  documented.  Goldfish,  Car- 
assius  auratus,  fed  the  DA  agonist  apomorphine  had  el- 
evated plasma  levels  of  growth  hormone  whereas  the  cir- 
culating levels  of  gonadotropic  hormone  were  reduced 
(Wong  et  al..  1993).  Long-term  feeding  of  goldfish  with 
apomorphine  induced  significant  increases  in  both  the 
body  weight  and  length.  5-HT  stimulates  gonadotropic 
hormone  release  in  the  goldfish  (Somoza  et  al..  1988;  So- 
moza  and  Peter,  1991).  This  effect  of  5-HT  may  be  due 
to  direct  action  on  the  gonadotrophs  or  to  inhibition  of 
DA  release  from  nerve  terminals  in  the  pars  distalis.  DA 
inhibits  release  of  this  gonadotropic  hormone  (Yu  and 
Peter,  1992).  Similarly,  DA  appears  to  inhibit  luteinizing 
hormone  release  in  the  frog,  Rana  temporaria  (Sotowska- 
Brochocka  el  al..  1994). 

The  crayfish  that  received  5-HT  alone  had  a  larger  TI 
and  mean  testicular  lobe  diameter  and  also  had  more  ma- 
ture sperm  in  their  testicular  lobes  than  did  the  simulta- 
neous control  group  (Figs.  4-6)  which  is  consistent  with 
the  earlier  results  of  Sarojini  et  al.  (1994).  The  crayfish 
that  received  5-HT  in  combination  with  DA  had  a  sig- 
nificantly larger  TI  and  mean  testicular  lobe  diameter, 
and  also  a  greater  number  of  mature  sperm,  when  com- 
pared with  the  simultaneous  controls  (Figs.  4-6),  but  all 
three  values  were  significantly  smaller  than  the  corre- 
sponding values  of  the  crayfish  given  5-HT  alone.  The 
DA  in  the  mixture  was  not  able  to  antagonize  fully  the 
stimulatory  action  of  the  5-HT.  This  antagonism  between 
the  effects  produced  by  5-HT  and  DA  on  the  testes  is 
reminiscent  of  that  seen  with  the  erythrophores  of  Uca 
pugilator  where  the  pigment-dispersing  effect  of  5-HT  and 
the  pigment-concentrating  effect  of  DA  were  reduced 
when  mixtures  of  5-HT  and  DA  were  co-injected  (Fin- 
german and  Fingerman,  1977). 

The  data  obtained  with  the  DA  antagonists  used  in  the 
present  study  support  the  conclusion  that  DA  inhibits 
testicular  maturation.  Both  spiperone  and  pimozide  pro- 
duced testicular  maturation  (Figs.  4-6).  Presumably,  these 
blockers  prevent  the  action  of  endogenous  DA.  hence 
leading  to  precocious  testicular  maturation. 

The  inhibitory  action  of  DA  on  the  androgenic  glands 
and  testes  in  Procambarus  clarkii  can  be  explained  as  fol- 
lows: DA  has  an  indirect  action  on  the  testes  and  andro- 
genic glands.  We  hypothesize  that  DA  either  (a)  stimulates 
release  of  G1H  from  the  eyestalk  neuroendocrine  system, 
(b)  inhibits  release  of  GSH,  or  (c)  does  both  (a)  and  (b). 
Any  of  these  hypothesized  actions  of  DA  would  result  in 


reduced  AGH  in  the  blood,  resulting  in  at  least  some  in- 
hibition of  testicular  maturation  and  spermatogenesis. 
Experiments  are  currently  in  progress  to  evaluate  these 
suggested  modes  of  action  of  DA.  That  DA  can  have  a 
stimulatory  role  in  the  release  of  a  neurohormone  was 
shown  for  the  red  pigment-concentrating  hormone,  as  re- 
ported by  Fingerman  and  Fingerman  (1977)  and  Quack- 
enbush  and  Fingerman  ( 1 984)  who  performed  in  vivo  and 
//;  vitro  experiments  on  release  of  this  neurohormone  with 
the  fiddler  crab,  Uca  pugilator.  The  concentrations  of  bio- 
genie  amines  used  in  these  experiments  are  quite  like  those 
injected  by  other  investigators  while  studying  the  same 
species,  Procambarus  clarkii.  Livingstone  et  al.  (1980)  in- 
jected 5.7  X  10"6  mol  5-HT  and  6.5  X  10~6  mol  octo- 
pamine  per  crayfish,  and  Arechiga  et  al.  (1990)  injected 
1  X  10~9  to  1  X  10~3  mol  5-HT  per  crayfish. 

Because  DA  inhibited  testicular  maturation  in  Pro- 
cambarus clarkii.  it  is  worth  mentioning  the  potential  ap- 
plication of  DA  analogues  in  crayfish  farming.  Supple- 
menting the  crayfish  diet  with  long-lasting  DA  agonists 
may  slow  reproductive  activity  of  crayfish  and  simulta- 
neously lead  to  enhanced  somatic  growth. 

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Reference:  Biol.  Bull.  189:  347-355.  (December.  1995) 


A  Transient  Exposure  to  Symbiosis-Competent 
Bacteria  Induces  Light  Organ  Morphogenesis 

in  the  Host  Squid 

JUDITH  A.  DOING  AND  MARGARET  J.  McFALL-NGAI* 

Department  of  Biological  Sciences,  University  of  Southern  California, 
Los  Angeles,  California  90089-0371 


Abstract.  Recent  studies  of  the  symbiotic  association 
between  the  Hawaiian  sepiolid  squid  Euprymna  scolopes 
and  the  luminous  bacterium  Vibrio fischeri  have  shown 
that  colonization  of  juvenile  squid  with  symbiosis-com- 
petent bacteria  induces  morphogenetic  changes  of  the  light 
organ.  These  changes  occur  over  a  4-day  period  and  in- 
clude cell  death  and  tissue  regression  of  the  external  cil- 
iated epithelium.  In  the  absence  of  bacterial  colonization, 
morphogenesis  does  not  occur.  To  determine  whether  the 
bacteria  must  be  present  throughout  the  morphogenetic 
process,  we  used  the  antibiotic  chloramphenicol  to  clear 
the  light  organ  of  bacteria  at  various  times  during  the 
initial  colonization.  We  provide  evidence  in  this  study 
that  a  transient,  1 2-hour  exposure  to  symbiosis-competent 
bacteria  is  necessary  and  sufficient  to  induce  tissue  regres- 
sion in  the  light  organ  over  the  next  several  days.  Further, 
we  show  that  successful  entrance  into  the  light  organ  is 
necessary  to  induce  morphogenesis,  suggesting  that  in- 
duction results  from  bacterial  interaction  with  internal 
crypt  cells  and  not  with  the  external  ciliated  epithelium. 
Finally,  no  difference  in  development  was  observed  when 
the  light  organ  was  colonized  by  a  mutant  strain  of  I', 
fischeri  that  did  not  produce  autoinducer,  a  potential  light 
organ  morphogen. 

Introduction 

Prolonged  associations  with  bacterial  symbionts  are 
now  recognized  as  important  phenomena  in  the  devel- 
opmental program  of  many  plant  and  animal  hosts  (for 


Received  6  February  1995;  accepted  10  October  1995. 
*  Corresponding  author. 

Abbreviations:  CEA,  ciliated  epithelial  appendages:  CSW.  California 
seawater;  Cm,  chloramphenicol. 


reviews  see  Schwemmler.  1989;  Hirsch,  1992;  Saffo, 
1992).  In  some  cases,  bacterial  symbioses  may  even  be 
required  for  normal  host  development  or  survival.  For 
example,  enteric  bacteria  provide  essential  enzymes  and 
vitamins  to  their  mammalian  hosts,  and  associations 
with  bacteria  are  required  for  normal  development  of 
the  mammalian  immune  system  (Gordon  and  Pesti. 
1971).  In  other  cases,  though  essential  only  under 
nutrient-poor  conditions,  the  association  is  highly  ben- 
eficial to  the  host's  fitness  in  its  natural  environment, 
such  as  the  symbioses  between  leguminous  plants  with 
nitrogen-fixing  bacteria  or  between  the  weevil  Silophi/iis 
oryiae  and  its  associated  gram-negative  bacteria.  In 
these  partnerships,  the  bacteria  provide  nutritional 
metabolites  to  their  host  (Nardon  and  Grenier,  1991) 
as  well  as  influence  its  development. 

Of  the  known  prokaryote-eukaryote  associations,  much 
progress  has  been  made  toward  the  understanding  of  the 
development  of  plant-bacterial  symbioses,  both  because 
the  plant  hosts  are  easily  maintained  and  manipulated  in 
the  laboratory  and  the  bacterial  symbionts  are  culturable. 
An  animal-bacterial  association  offering  similar  experi- 
mental benefits  is  the  highly  specific  association  between 
the  Hawaiian  sepiolid  squid  Euprymna  scolopes  and  the 
bioluminescent  bacterium  I  'ibrio  fischeri.  This  symbiosis 
provides  an  experimental  system  to  study  the  effect  of 
bacterial  symbionts  on  host  animal  development  (McFall- 
Ngai  and  Ruby,  1991;  Ruby  and  McFall-Ngai,  1992).  In 
the  host  squid,  the  bacteria  are  always  contained  within 
epithelia-lined  crypts  inside  the  light  organ,  which  is 
housed  within  the  mantle  cavity.  However,  the  mor- 
phology of  the  light  organ  in  juvenile  squid  is  much  dif- 
ferent from  that  of  the  adult  (McFall-Ngai  and  Montgo- 
mery, 1990).  and  the  light  organ  undergoes  complex  de- 


347 


348 


J.  A.  DOING  AND  M.  J.  McFALL-NGAI 


velopmental   changes   following  bacterial  colonization 
(Montgomery  and  McFall-Ngai,  1994). 

Upon  hatching,  juvenile  squid  are  aposymbiotic  (with- 
out bacterial  symbionts)  and  normally  acquire  free-living 
V.  fischeri  from  the  surrounding  seawater  within  hours 
(Wei  and  Young,  1989;  McFall-Ngai  and  Ruby,  1991). 
A  substantial  portion  of  the  juvenile  light  organ  epithelium 
is  microvillous  and  ciliated,  bearing  two  lateral  pairs  of 
appendages  (ciliated  epithelial  appendages;  CEA)  that  ap- 
pear to  facilitate  inoculation  of  bacteria  into  the  light  organ 
(Fig.  la;  McFall-Ngai  and  Ruby,  1991;  Montgomery  and 
McFall-Ngai,  1993).  Microscopy  and  high-speed  cine- 
matography have  revealed  that  the  two  appendages  on 
each  side  of  the  light  organ  form  a  ring,  at  the  base  of 
which  are  three  pores  leading  into  three  independent 
crypts  (Fig.  Ib).  Beating  of  the  cilia  entrains  passing  sea- 
water  within  the  ring,  potentially  increasing  the  probability 
that  symbionts  within  the  water  will  enter  the  pores  (M. 
McFall-Ngai  and  R.  Emlet,  unpub.  results).  When  the 
light  organ  has  been  successfully  colonized  by  }'.  fischeri. 
cell  death  is  observed  in  the  CEA  and  regression  of  these 
appendages  occurs  over  a  period  of  four  days.  Four-day- 
old  squid  that  are  not  infected  with  I',  fischeri  do  not 
show  cell  death  nor  regression  of  the  CEA  (Montgomery 
and  McFall-Ngai,  1994).  Therefore,  the  presence  of  sym- 
biosis-competent bacteria  somehow  induces  host  tissues 
that  are  several  cell  layers  away  to  initiate  light  organ 
morphogenesis.  Cell  death  and  the  resulting  regression  of 
the  CEA  are  the  first  observable  events  of  light  organ  mor- 
phogenesis and  therefore  the  first  developmental  evidence 
that  induction  has  occurred. 

In  this  study  we  have  asked  whether  the  presence  of 
bacteria  within  the  light  organ  is  required  continuously 
for  4  days  to  induce  CEA  regression.  Additionally,  we  used 
noninfective  strains  of  I",  fischeri  to  determine  whether 
colonization  of  the  light  organ  is  necessary  for  induction. 
Finally,  we  tested  whether  I',  fischeri  autoinducer,  a  cell 
density-dependent  factor  secreted  by  the  bacteria  and  in- 
volved in  the  production  of  light,  is  required  to  induce 
light  organ  morphogenesis. 

Materials  and  Methods 

Animal  care  and  maintenance 

Adult  squid  were  collected  at  night  from  Kaneohe  Bay, 
Oahu,  HI,  with  dipnets  and  were  transported  back  to  the 
University  of  Southern  California,  Los  Angeles  within  one 
week  of  collection.  Animals  were  maintained  in  a  265- 
liter  recirculating  aquarium  at  23°C,  and  females  were 
mated  approximately  once  a  week.  Egg  clutches,  attached 
to  coral  rocks  or  other  hard  surfaces  by  the  females,  were 
transferred  for  hatching  to  smaller  temperature-controlled 
23°C  aquaria.  To  ensure  that  juvenile  squid  did  not  be- 
come prematurely  infected  with  any  residual  bacteria  that 


B 


Figure  I .  Scanning  electron  micrograph  (A)  of  a  hatchling  light  organ 
with  complete  CEA  and  a  schematic  drawing  (B)  showing  the  position 
of  the  appendages  in  v/vo  and  the  three  pores  in  the  ciliated  epithelium 
(arrow)  with  their  associated  internal  crypts  (approximated  by  dashed 
line).  A,  anterior;  P,  posterior;  h.  hindgut.  Scale  bar  =  100  ^m. 


might  be  associated  with  the  egg  clutch,  squid  were  trans- 
ferred immediately  upon  hatching  through  three  rinses 
with  California  coastal  seawater  (CSW),  which  does  not 
contain  infective  strains  of  r.  fischeri  (McFall-Ngai  and 
Ruby,  1991).  Juveniles  were  used  for  infection  studies 
within  6  h  of  hatching. 

Inoculation  of  squid  with  V.  fischeri  bacteria 

Bacteria  were  grown  to  log  phase  in  a  seawater-based 
minimal  medium  (Ruby  and  Asato,  1993)  and  diluted  to 
between  103  and  10s  cells/ml  for  inoculation  of  squid. 
After  inoculation,  squid  were  rinsed  in  CSW  and  trans- 
ferred to  either  CSW  or  chloramphenicol-treated  CSW 
(as  described  below).  For  all  experiments,  positive  (in- 
fected) controls  were  exposed  to  symbiosis-competent 
bacteria  in  CSW  for  the  entire  4  days  and  negative  (un- 
infected)  controls  were  exposed  to  CSW  alone. 

Monitoring  bacterial  colonization 

Because  I ".  fischeri  is  luminous  in  the  light  organ,  suc- 
cessful colonization  of  the  organ  can  be  monitored  by 
measuring  the  bioluminescence  of  the  squid  with  a  pho- 
tomultiplier  tube  (Luminescence  Photometer,  Model 
3600.  Biospherical  Instruments.  Inc.).  For  these  mea- 
surements, individual  squid  were  kept  in  5  ml  of  seawater 
in  glass  scintillation  vials.  Seawater  in  the  vials  was 
changed  daily  throughout  the  4-day  experiments,  just  prior 
to  each  luminescence  measurement. 


SYMBIOSIS-SPECIFIC  MORPHOGENESIS 

2d  3d 


349 


4d 


Figure  2.  Time  series  of  CEA  regression  in  symbiotic  (sym)  and  aposymhiotc  (apo)  juvenile  squid  over 
4  days  (d).  Symbiotic  animals  were  inoculated  with  ~  104  cells/ml  of  I  '.fischeri  ES 1 1 4  within  6  h  of  hatching. 
The  sym-4d  panel  represents  a  fully  regressed  CEA.  Only  the  right  half  of  each  light  organ  is  shown.  Scale 
bar  =  100  fim.  Differences  in  size  reflect  individual  variation. 


Determination  of  Ciliated  Epithelial  Appendage  (CEA) 
regression 

Regression  of  the  CEA  was  determined  at  the  end  of 
4  days  for  each  experiment.  Squid  were  fixed  for  24  h  in 
seawater  containing  either  5%  formalin  or  2%  parafor- 
maldehyde/2%  glutaraldehyde.  Samples  were  subse- 
quently rinsed  twice  for  30  min  in  50  mA/  sodium  phos- 
phate buffer  with  0.45  M  NaCl  (pH  7.2),  followed  by  a 
dehydration  series  with  15%- 100%  ethanol.  Samples  were 
critical-point  dried  with  liquid  CO2,  or  desiccated  with 
hexamethyldisilazane  (Polysciences,  Inc.).  Dried  squid 
were  mounted  onto  aluminum  stubs  and  the  ventral  por- 
tions of  the  mantle  and  siphon  were  dissected  away  to 
reveal  the  juvenile  light  organs.  These  samples  were  then 
sputter  coated  with  gold  and  the  light  organs  were  ex- 
amined with  a  Cambridge  360  scanning  electron  micro- 
scope (SEM).  The  presence  or  absence  of  CEA  was  scored 
and  recorded,  and  photomicrographs  were  taken  of  rep- 
resentative samples. 


CEA  regression  of  symbiotic  juveniles  was  compared 
to  that  of  aposymbiotic  juveniles  for  4  days  following  in- 
oculation with  bacteria  (Fig.  2).  For  experiments  involving 
variable  exposure  times,  CEA  regression  at  day  4  was  di- 
vided into  five  stages  (0,  1,  2,  3,  and  4),  which  correspond 
to  the  regression  seen  at  0  (Fig.  1  A),  1,  2,  3,  and  4  days 
of  uninterrupted  symbiosis  (Fig.  2).  CEA  of  light  organs 
were  considered  regressed  if  they  were  at  or  beyond  stage 
3.  Due  to  the  high  variability  between  individual  squid, 
even  within  control  groups,  regression  is  reported  as  an 
E/C  index,  which  is  defined  as  the  percentage  of  experi- 
mental animals  with  regressed  CEA  divided  by  that  of 
the  symbiotic  controls  in  a  given  experiment  (experimen- 
tal and  control  animals  for  a  given  experiment  were  always 
from  the  same  clutch  of  eggs). 

Manipulation  of  exposure  times  and  colonization  levels 

Transient  vs.  continuous  exposure  to  V.  fischeri  strain 
ESI  14.  To  determine  first  whether  a  continuous  exposure 


350 


J.  A.  DOING  AND  M.  J.  McFALL-NGAI 


to  competent  bacteria  is  necessary  for  initiating  morpho- 
genesis of  the  light  organ,  we  used  the  bacteriostatic  an- 
tibiotic chloramphenicol  (Cm)  to  clear  the  light  organ  of 
viable  symbionts  (Fig.  3a).  Squid  were  exposed  to  one  of 
two  symbiosis-competent  strains  of  I  'ibrio  fischeri:  ESI  14, 
a  chloramphenicol-sensitive  light  organ  isolate  (Boettcher 
and  Ruby,  1990)  or  ESI  14-U2,  a  spontaneous  chloram- 
phenicol-resistant  mutant  of  ESI  14  (donated  by  J.  Graf)- 
The  inoculations  were  performed  for  two  time  periods: 
( 1 )  continuously  for  4  days,  or  (2)  for  12  h.  Those  exposed 
continuously  were  inoculated  with  I",  fischeri  and  then 
transferred  to  CSW  after  1 2  h  for  the  remainder  of  the 
4  days  (Fig.  3a,  top  bar).  These  squid  remained  infected 
for  the  duration  of  the  experiment.  Following  incubation 
with  V.  fischeri.  squid  exposed  for  only  1 2  hours  were 
transferred  to  CSW  treated  with  10  ng/ml  Cm  in  seawater 
for  the  remainder  of  the  experiment  (Fig.  3a,  second  bar). 
The  transient  time  period  of  12  h  was  chosen  because 
successful  colonization  of  the  light  organ  by  bacteria  can 
be  confirmed  by  the  appearance  of  luminescence  between 
10  and  12  h  after  exposure.  The  Cm-resistant  strain 
ESI  14-U2  was  used  as  a  control  for  any  inhibitory  phar- 
macological effects  that  Cm  may  have  on  CEA  regression. 
Squid  were  monitored  for  luminescence  before  exposure 
to  bacteria,  every  2  h  during  initial  colonization  and  every 
12  h  thereafter.  Uninfected  controls  were  exposed  to  non- 
infective  CSW  with  or  without  Cm  (Fig.  3a,  third  and 
fourth  bars)  and  monitored  for  luminescence  every  12  h. 

To  insure  that  Cm  treatment  was  effectively  clearing 
the  light  organ  of  viable  bacteria,  the  decrease  of  both 
bacteria  colony  forming  units  (CPU)  and  luminescence 
was  monitored  in  squid  treated  with  10  ng  Cm/ml  CSW 
following  exposure  to  bacteria  for  12  h. 

Variable  exposure  times  to  V.  fischeri  strain  ESI  14.  To 
determine  the  minimum  time  period  required  to  induce 
morphogenesis,  hatchling  squid  were  exposed  to  I  '.fischeri 
for  variable  lengths  of  time  (Fig.  3b).  At  time  0,  all  squid 
were  placed  in  a  single  bowl  with  of  CSW  containing  ~5 
X  103  ESI  14  cells/ml.  Groups  of  10-20  animals  were  re- 
moved from  the  bowl  at  1,4,  8,  and  12  h.  Upon  removal 
from  the  bowl  at  each  time  period,  half  of  the  squid  were 
rinsed  twice  and  transferred  to  vials  with  Cm-treated  CSW 
(Fig.  3b,  top),  while  the  other  half  were  transferred  to  vials 
with  Cm-free  CSW  (Fig.  3b.  bottom).  Groups  transferred 
to  Cm-free  CSW  became  infected  within  12  h.  Lumines- 
cence was  measured  immediately  before  and  after  expo- 
sure to  bacteria  and  once  per  day  thereafter. 

Exposure  to  other  strains  of  V.  fischeri 

To  determine  whether  colonization  of  the  light  organ 
by  the  bacteria  is  necessary  to  induce  CEA  regression, 
squid  were  exposed  to  three  noninfective  strains  of  I  . 
fischeri  (M101,  MdR12,  and  MJ1).  A  fourth  strain  of  T. 


fischeri  (MJ 1 1 ),  which  is  not  normally  associated  with  the 
E.  scolopes  light  organ  but  is  capable  of  colonization,  was 
also  tested  for  its  ability  to  induce  morphogenesis.  Fol- 
lowing the  inoculation  period,  squid  were  transferred  to 
CSW  for  the  remainder  of  the  4  days.  Possible  coloniza- 
tion of  squid  exposed  to  noninfective  strains  was  deter- 
mined by  both  luminescence  measurements  and  bacterial 
plate  counts.  Colonization  of  positive  and  negative  con- 
trols was  determined  by  luminescence  only. 

Noninfective  strains.  Strain  M101  was  produced  by 
transposon  (Mu  dl-1681)  mutagenesis  of  symbiosis-com- 
petent strain  ESI  14,  resulting  in  a  nonmotile  mutant. 
Nonmotile  mutants  of  I '.  fischeri  have  previously  been 
shown  to  be  noninfective  in  E.  scolopes  (Graf  et  al..  1994). 
Squid  were  exposed  to  ~104  M101  cells/ml  for  12  h. 
Strain  MdR12  is  a  non-symbiotic  wild  type  isolate  from 
Southern  California  coastal  seawater.  Strain  MJ1  was 
originally  isolated  from  the  light  organ  of  the  Japanese 
pinecone  fish  Monocentris  japonica,  but  has  been  in  cul- 
ture for  21  years  (Ruby  and  Nealson,  1976)  and  does  not 
infect  E.  scolopes.  Squid  were  exposed  to  ~105  cells/ml 
of  this  strain  for  24  h. 

Infective  strain.  Strain  MJ  1 1  was  isolated  from  the  light 
organ  of  M.  japonica  in  1 992  and  is  infective  to  E.  scol- 
opes. Squid  were  exposed  to  ~  1 05  MJ  1 1  cells/ml  for  1 2  h. 
This  strain  was  of  interest  because,  although  it  is  capable 
of  colonization,  bacterial  numbers  inside  the  light  organ 
reach  only  10%  of  the  levels  seen  with  ESI  14  (K.H.  Lee 
and  E.G.  Ruby,  pers.  comm.). 

Exposure  to  an  aittoinducer  mutant  strain  of  V.  fischeri 

Symbiont  bioluminescence  in  the  E.  scolopes  light  or- 
gan is  induced  via  a  well  studied  mechanism  involving 
the  secreted  I',  fischeri  molecule  autoinducer  (VAI),  a 
homoserine  lactone.  Normally  VAI  is  expressed  consti- 
tutively  at  a  low  level,  but  when  cell  densities  become 
high,  such  as  in  the  light  organ  (Boettcher  and  Ruby, 
1990),  the  build  up  of  VAI  in  the  extracellular  medium 
positively  regulates  VAI  gene  expression  and  in  turn  ac- 
tivates expression  of  the  lux  operon,  which  encodes  for 
those  genes  responsible  for  bacterial  light  production  (for 
review,  see  Dunlap  and  Greenberg,  1991).  To  determine 
whether  VAI  was  a  morphogen  of  the  squid  light  organ, 
we  used  a  mutant  strain  (3100)  of  I',  fischeri  (provided 
by  Kendall  Gray)  containing  an  insertion  in  the  autoin- 
ducer gene,  which  renders  the  cells  incapable  of  making 
autoinducer. 

One-day-old  squid  were  exposed  to  symbiosis-compe- 
tent V.  fischeri  strain  310fiortoESl  14  at  a  concentration 
of  ~103  cells/ml  for  approximately  20  h.  Because  the 
31 OS2  strain  is  nonluminous,  successful  colonization  of 
the  squid  could  not  be  monitored  with  a  photometer.  In- 
stead, at  the  end  of  4  days,  two  of  the  squid  that  had  been 


SYMBIOSIS-SPECIFIC  MORPHOGENESIS 


351 


12 


96  h 


Wll/lllllllllllllllllllllh 


\llllllllllllllllllllllllllh 


B 


EXPOSURE  TO  BACTERIA 


Cm  TREATMENT 


]     CSW 


Figure  3.  Experimental  design  for  transient  and  variable  exposure  to  bacteria,  n  =  5-20  per  experiment 
for  each  treatment  group  in  A  and  B.  At  the  end  of  4  days  (96  h),  squid  were  fixed  for  SEM  to  score 
regression.  A.  Hatchling  squid  were  exposed  to  ~  10"  cells/ml  of  ESI  14  or  ESI  14-U2  for  12  h,  at  which 
time  the  bacteria-containing  seawater  was  changed  to  CSW  (top  bar)  to  allow  the  infection  to  ensue,  or  to 
Cm-treated  seawater  (second  bar)  to  stop  the  infection  and  cure  the  light  organ.  Controls  were  exposed  to 
CSW  alone  (third  bar)  or  to  CSW  followed  by  Cm-treated  CSW  (fourth  bar).  B.  Hatchling  squid  were  exposed 
to  ~  104  cells/ml  ESI  14  for  1.  4,  8,  or  12  hours  (started  at  time  0),  at  which  time  the  bacteria-containing 
seawater  was  changed  to  Cm-treated  seawater  (top  bar)  or  to  CSW  (lower  bar). 


exposed  to  31012  were  homogenized  and  plated  to  verify 
that  they  had  been  infected.  The  rest  of  that  group  (n  =  8) 
was  scored  for  CEA  regression.  ESI  14  and  negative  con- 
trol groups  were  monitored  for  colonization  by  measuring 
luminescence  only. 

Results 

Transient  vs.  continuous  exposure  to  V.  fischeri  strain 
ESI  14 

The  number  of  viable  bacteria  in  the  light  organ  declines 
sharply  after  only  2  hours  in  Cm-treated  CSW  to  487  CPU 
(approximately  1%  of  the  initial  value),  concomitant  with 
a  decline  in  luminescence  (Fig.  4).  After  10  hours  of  Cm 
treatment,  all  of  the  squid  monitored  had  no  viable  bac- 
teria detectable  in  their  light  organs.  Additionally,  if  the 
Cm  treatment  was  removed  after  4  days  and  replaced  with 
CSW  alone,  the  light  organs  of  the  squid  did  not  become 
reinfected,  confirming  that  there  were  no  viable  J  '.fischeri 
in  the  light  organ  after  treatment  with  Cm  for  4  days. 
Squid  exposed  to  Cm  appeared  as  healthy  as  those  not 
exposed  to  Cm  and  there  was  no  adverse  effect  on  the 
ability  of  the  squid  to  infect  after  a  4-day  exposure  to  Cm; 


i.e.,  if  the  Cm  treatment  was  lifted  after  4  days  and  a  new 
inoculum  of  V.  fischeri  was  introduced  into  the  seawater 
the  squid  became  luminescent  within  24  h,  indicating  that 
they  were  still  capable  of  being  infected. 


/uuuui  — 

-60OU 

50000 

| 

•5000 

I 

40000 

| 

-4000 

30000 

1                                                   \ 

-3000 

20000 

\\ 

-2000 

10000- 

\ 

-1000 

\         *-- 

o- 

L                   "•---—                                                  A 

-o 

) 

u=o=i—  0—  0         fl         *  '  W^1^ 
)                                                            10                                                '         3 

hrs 

Figure  4.  Decrease  in  colony  forming  units  (CFU)  of  bacteria  per 
squid  (open  squares,  solid  line)  and  relative  luminescence  (in  photometer 
units)  per  squid  (closed  circles,  dashed  line)  over  time  following  Cm 
treatment.  Squid  were  exposed  to  >'  fischeri  bacteria  for  12  h  prior  to 
Cm  treatment  (h  0).  Data  points  are  the  averages  from  n  =  5  squid. 
Vertical  bars  represent  standard  deviations. 


352 


J.  A.  DOING  AND  M.  J.  McFALL-NGAI 


Figure  5.  Scanning  electron  micrographs  of  light  organs  (right  half  only)  of  4-day  old  squid  exposed 
upon  hatching  to:  (A)  CSW  for  4  days;  (B)  ESI  14  for  12  h  followed  by  treatment  with  Cm  for  3.5  days;  (C) 
ESI  14-U2  (Cm  resistant  strain)  for  12  h  followed  by  treatment  with  Cm  for  3.5  days.  See  Figure  3A  for 
experimental  design.  Scale  bar  =  100  urn. 


Squid  exposed  to  I  '.fischeri  for  12  h  showed  regression 
of  the  CEA  similar  to  that  of  squid  exposed  for  4  days 
(Fig.  5b).  Negative  control  animals  exposed  for  4  days  to 
CSW  (Fig.  5a)  or  Cm-treated  CSW  (not  shown)  showed 
no  regression  of  CEA.  Additionally,  Cm  did  not  have  an 
inhibitory  effect  on  CEA  regression,  as  evidenced  by  com- 
plete regression  of  CEA  from  squid  infected  with  the  Cm- 
resistant  strain  ESI  14-U2  and  treated  with  Cm  (Fig.  5c). 


hrs    exposed 

Figure  6.  Relative  percentage  of  light  organs  in  4-day-old  squid 
showing  stage  3  regression  of  the  CEA  (see  text).  Hatchling  squid  were 
exposed  to  ESI  14  for  1.  4.  8,  and  12  hours,  followed  by  treatment  with 
Cm  (see  Fig.  3B-for  experimental  design).  Values  are  reported  as  E/C. 
the  percentage  of  Cm-treated  squid  (experimental)  showing  regression 
divided  by  the  percentage  of  infected  controls  showing  regression.  Each 
point  represents  values  pooled  from  four  separate  experiments  (see  Table 
I).  Vertical  bars  represent  the  full  range  of  data  for  the  four  experiments. 


Variable  transient  exposure  to  V.  fischeri  strain  ESI  14 

Exposure  of  squid  to  bacteria  for  1,  4,  8,  12,  or  14  hours 
to  determine  the  minimum  time  requirement  for  induc- 
tion of  CEA  regression  revealed  that  only  those  squid  ex- 
posed to  bacteria  for  at  least  1 2  h  showed  CEA  regression 
(E/C  =  0.96)  comparable  to  that  of  continuously  exposed 
control  squid  (Fig.  6:  Table  I).  These  squid  were  visibly 
luminous  at  the  time  of  Cm  treatment.  Squid  exposed  to 
bacteria  for  1  or  4  h  were  not  luminous  and  showed  no 
CEA  regression.  Those  individuals  exposed  for  8  h  were 
only  occasionally  luminous  and  the  E/C  ratio  was  only 
0.24.  Although  some  animals  died  during  the  experiments, 
the  death  rate  was  not  greater  than  that  normally  seen  in 
animals  4  days  post-hatching  (averaging  less  than  10%) 
and  the  incidence  of  death  appeared  random  with  respect 
to  experimental  groups.  Animals  that  died  were  not  used 
in  the  calculation  of  CEA  regression  percentages.  The  level 
of  infection,  measured  by  plating  light  organ  homogenates 
after  exposure  to  bacteria,  was  significantly  higher  at  12  h 


Table  I 

Pooled  data  from  four  separate  experiments  as  described  in  Figure  3b. 
E/C  represents  ratio  ofo  E\P  (Experimental)  to  %  CONT  (Control) 


Tot.  Sample  No. 

CEA  Regression 

H 

Exposed 

EXP 

CONT 

%EXP 

%  CONT 

E/C 

1 

33 

48 

0 

77 

0 

4 

25 

43 

0 

77 

0 

8 

38 

50 

18 

76 

0.24 

12 

23 

43 

74 

77 

0.96 

14 

25 

26 

72 

96 

0.75 

SYMBIOSIS-SPECIFIC  MORPHOGENESIS 


353 


than  at  8  h  (Fig.  7).  Although  CEA  regression  was  higher 
at  12  h  than  at  14  h  (Fig.  6),  the  values  at  14  h  are  within 
the  range  of  values  for  12  h.  These  results  suggest  that  the 
minimum  exposure  time  for  complete  regression  of  the 
CEA  must  lie  between  8  and  12  h. 

Exposure  to  other  strains  of  V.  fischeri 

When  squid  were  exposed  to  the  nonmotile  strain  of 
V.  fischeri.  M101,  neither  colonization  nor  CEA  regres- 
sion was  observed,  supporting  the  above  evidence  that 
the  bacteria  must  be  within  the  light  organ  to  induce 
morphogenesis.  Additionally,  of  the  natural  isolates 
tested,  only  the  infective  strain,  MJ11,  induced  CEA 
regression  (Table  II). 

Exposure  to  an  aiiloinducer  mutant  of  V.  fischeri 

Squid  exposed  to  the  autoinducer  mutant  of  I  '.fischeri, 
31012,  were  infected  and  showed  complete  regression  of 
CEA  after  4  days  (Table  II),  thus  eliminating  the  possibility 
that  autoinducer  is  required  for  light  organ  morpho- 
genesis. 

Discussion 

The  results  of  this  study  show  that  light  organ  mor- 
phogenesis of  the  squid  Euprymna  sco/opes  in  response 
to  the  presence  of  symbiotic  bacteria  (1)  requires  only  a 
12-h  exposure  to  symbiosis-competent  bacteria;  (2)  re- 
quires colonization  of  the  light  organ  by  bacteria;  (3)  does 
not  require  I',  fischeri  autoinducer. 

The  finding  that  a  transient  exposure  to  symbiosis- 
competent  bacteria  is  sufficient  to  induce  morphogenesis 
of  the  squid  light  organ  (i.e..  the  bacteria  are  not  required 
throughout  the  4-day  morphogenetic  process)  suggests  that 


Table  11 

Observed  capability  of  various  strains  <>/  Vibrio  fischeri  to  infect 
Euprymna  scolopes  and  induce  light  organ  morphogenesis,  n  =  5 
squid  for  each  strain  and  all  live  squid  in  each  group  showed 
the  same  results 


50000  - 

40000- 
30000 

20000- 

10000- 

n- 

I  ',/:  Strain 

Source 

Phenotype      Infection     Morphogenesis 

ES114 

squid  light 

wildtype                 +                     + 

organ 

U2 

ESI  14 

Cm  resistant           +                     + 

derivative 

MIOI 

ESI  14 

motility" 

derivative 

3io« 

ESI  14 

/H.Y/"                               +                               + 

derivative 

MJ1 

fish  light  organ 

wildtype 

(1974) 

MJ1I 

fish  light  organ 

wildtype                 +                     + 

(1992) 

MdR12 

seawater 

wildtype 

isolate 

Figure  7.  Average  colony  forming  units  (CPU)  of  bacteria  per  squid 
after  8  and  1 2  h  of  exposure  to  I '.  fischeri.  Vertical  bars  represent  standard 
deviations  (n  =  5). 


the  signal  from  the  bacteria  may  trigger  an  irreversible 
cascade  of  events  that  eventually  results  in  CEA  regression. 
Signal  transduction  through  some  host  cell  surface  recep- 
tor in  the  light  organ  crypts  is  an  attractive  model  for  this 
type  of  response.  This  model  is  supported  by  evidence 
that  colonization  of  the  light  organ  crypts  is  required  to 
elicit  CEA  regression  (see  below),  and  by  the  recent  finding 
that  cell  death  and  regression  events  are  first  seen  at  the 
tips  of  the  ciliated  appendages,  several  cell  layers  away 
from  the  crypt  epithelium  adjacent  to  the  bacteria  (Mont- 
gomery and  McFall-Ngai,  1 994).  Thus,  cells  that  are  in 
direct  contact  with  the  bacteria  must  somehow  pass  the 
signal  through  several  layers  of  adjacent  host  cells  to  effect 
cell  death  in  the  ciliary  appendages,  presumably  through 
one  or  more  signal  transduction  pathways. 

The  results  of  experiments  with  various  strains  of  I', 
fischeri  indicate  that  noninfective  strains  are  not  mor- 
phogenic  and  that  the  bacteria  must  enter  the  light  organ 
to  induce  morphogenesis.  Also,  because  nonmotile  I',  fis- 
cheri are  unable  to  infect  the  light  organ,  motility  is  in- 
directly required  for  induction  of  morphogenesis.  Simi- 
larly, motility  is  required  for  infection  and  virulence  in 
many  pathogenic  bacteria,  such  as  I '.  cholerae  (Guentznel 
and  Berry,  1975;  Yancey  et  a/.,  1978),  Pseudomonas  spp. 
(Drake  and  Montie,  1988),  Helicobacter pylori  (Eaton  et 
a/..  1989;  Dunn,  1993).  and  Salmonella  trphi  (Liu  et  ai, 
1988). 

The  requirement  for  infection  suggests  that  transduc- 
tion of  the  signal  occurs  through  interactions  with  the 
light  organ  crypt  epithelial  cells,  and  not  through  inter- 
actions with  the  external  ciliated  microvillous  cells.  Unlike 
the  Rhizobium-legame  symbiosis,  in  which  a  diffusible 
morphogen  excreted  by  the  bacteria  induces  cell  division 


354 


J.  A.  DOING  AND  M.  J.   McFALL-NGAI 


and  morphological  changes  in  the  plant  without  coloni- 
zation of  the  host  by  the  symbiont  (Long,  1989;  Appel- 
baum.  1990;  Hirsch,  1992),  morphogenetic  induction  in 
the  I  'ihrio-Euprymna  symbiosis  requires  that  the  bacteria 
be  within  the  confined  space  of  the  light  organ.  Studies 
using  strain  MJ 1 1 ,  which  produces  a  colonization  con- 
sisting of  only  about  10%  of  the  typical  cell  number  yet 
induces  morphogenesis,  indicate  that  the  actual  bacterial 
volume  is  probably  not  exerting  a  physical  pressure  or 
stretching  of  the  light  organ  to  induce  morphogenesis.  It 
is  unclear  at  this  time  whether  the  signal  is  secreted  from 
the  bacteria  into  the  light  organ  crypt  lumen  or  the  signal 
is  presented  directly  on  the  surface  of  the  bacteria.  In  either 
case,  the  observed  time  window  of  between  8  and  12  hours 
necessary  to  induce  CEA  regression,  may  reflect  the  need 
for  an  accumulation  of  bacteria,  or  their  products,  to  a 
critical  density  within  the  light  organ.  If  the  morphogenic 
signal  is  secreted,  there  are  a  few  possible  scenarios:  ( 1 ) 
secretion  of  the  morphogen  is  induced  and  only  occurs 
within  the  environment  of  the  light  organ,  (2)  the  mor- 
phogen is  produced  constitutively  and  light  organ  crypts 
provide  a  barrier  to  diffusion  of  bacterial  products  such 
that  within  the  light  organ  the  signal  reaches  a  critical 
concentration  required  for  transduction,  or  (3)  the  pres- 
ence of  bacteria  within  the  light  organ  (perhaps  through 
direct  cell-cell  contact)  renders  the  host  cells  competent 
to  "accept"  the  secreted  signal  from  the  bacteria. 

Rather  than  secreted,  the  bacterial  signal  may  be  a 
molecule  presented  on  the  bacterial  cell  surface  that  in- 
teracts directly  with  a  receptor  on  the  animal  cell  mem- 
brane. Direct  interactions  via  glycan-adhesin  binding  have 
been  implicated  in  many  symbioses  and  there  is  evidence 
for  a  mannose  lectin  in  E.  scolopes:  when  squid  are  in- 
oculated with  bacteria  in  the  presence  of  mannose.  col- 
onization is  significantly  inhibited  (V.  Weis,  K.  Brennan 
and  M.  McFall-Ngai.  unpub.  data).  In  the  Rhizobium- 
legume  symbiosis,  plant  lectins  that  recognize  specific 
bacterial  surface  oligosaccharides  have  been  suggested  to 
play  a  major  role  in  attachment  and  invasion  mechanisms 
(Dazzo  and  Truchet,  1983).  Further,  in  pathogenic  as- 
sociations, bacterial  adhesins  on  pili  often  are  involved 
in  recognizing  specific  sugar  receptors  on  the  animal  cell 
surface  (Finlay  and  Falkow,  1989). 

Recently  it  has  been  shown  that  various  other  autoin- 
ducer  molecules  regulate  the  production  of  exoenzyme 
virulence  determinants  in  Pseudomonas  aeruginosa  and 
Erwinia  carolovora  (Jones  el  ai.  1993).  Also,  these  au- 
toinducers  are  structural  analogs  of  actinomycetes  A-fac- 
tor,  which  has  been  implicated  as  an  autoregulator  of  cel- 
lular differentiation  between  different  Streptomyces  spe- 
cies (Beppu,  1992).  However,  the  results  of  this  study 
indicate  that  I",  fischeri  autoinducer  is  not  required  for 
light  organ  morphogenesis.  Other  secreted  bacterial  mol- 
ecules such  as  Vibrio  spp.  endotoxins  are  known  to  in- 


teract with  animal  cells  (Lin  el  ai,  1993)  and  may  be 
potential  morphogens.  For  example,  cholera  toxin  has 
been  shown  experimentally  to  induce  metamorphosis  in 
certain  marine  larvae  (Hofmann  and  Brand,  1987).  In 
addition.  Reich  and  Schoolnik  recently  found  that  V.  fis- 
cheri carries  a  gene  homologous  to  toxR  (1994),  which 
regulates  cholera  toxin  production  in  I',  cholerae,  and 
also  synthesizes  a  cholera  toxin-like  ADP-ribosylating 
protein  (1995).  However,  while  commercially  available 
cholera  toxin  mimics  some  aspects  of  the  symbiotic  state, 
by  itself  it  does  not  cause  morphogenesis  in  E.  scolopes 
(Small  and  McFall-Ngai,  1993),  suggesting  that  if  an  en- 
dotoxin-like  molecule  is  the  squid  morphogen,  it  is  sig- 
nificantly different  from  cholera  toxin,  or  that  additional 
molecules  (perhaps  on  the  surface  of  the  bacteria)  are  also 
required.  Other  bacterial  factors  that  have  been  demon- 
strated to  affect  metamorphosis  or  morphogenesis  in  var- 
ious host  organisms  include  oligopeptides  (Hofmann  and 
Brand.  1987),  phorbol  esters  (Mullen  1985).  diacylglyc- 
erols  (Leitz  and  Muller,  1987),  and  lipo-oligosaccharides 
(Lerouge  el  a/.,  1990;  van  Brussel  el  al,  1992),  any  of 
which  may  prove  important  in  our  system. 

In  conclusion,  we  have  shown  that  a  transient  coloni- 
zation off.  scolopes  with  symbiosis-competent  V.  fischeri 
induces  morphogenesis  of  the  squid  light  organ.  Trans- 
duction of  the  morphogenic  signal  requires  the  presence 
of  the  bacteria  within  the  light  organ  for  approximately 
12  h.  Further  investigations  are  necessary  to  determine 
the  nature  of  the  bacterial  signal,  the  role  of  colonization 
in  the  generation  of  the  signal,  and  the  transduction  path- 
way within  the  host  squid. 

Acknowledgments 

We  thank  Ned  Ruby,  Mary  Montgomery,  and  Alicia 
Thompson  for  technical  advice  and  Angel  Lemus  for 
graphics  assistance.  We  thank  Jorg  Graf  and  Kendall  Gray 
for  their  generous  donations  of  mutant  I  '.fischeri  strains. 
We  also  thank  Andrea  Small,  Katie  Brennan.  Jamie  Fos- 
ter. Wes  Toller,  and  Karen  Visick  for  helpful  comments 
on  the  manuscript.  This  is  HIMB  contribution  #991.  This 
work  was  supported  by  NSF  Grant  No.  IBN  9220482  (to 
MM-N  and  EG  Ruby)  and  ONR  Grant  No.  N00014-91- 
J-1357(toMM-N). 

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Not  All  Ctenophores  Are  Bioluminescent: 
Pleurobrachia 

STEVEN  H.  D.  HADDOCK  AND  JAMES  F.  CASE 

Marine  Science  Institute,  University  of  California.  Santa  Barbara,  California  93106 


Abstract.  The  traditional  view  has  been  that  all  species 
of  the  phylum  Ctenophora  are  capable  of  producing  light. 
Our  inability  to  elicit  luminescence  from  members  of  the 
well-known  genus  Pleurobrachia.  as  well  as  a  lack  of  pub- 
lished documentation,  led  to  an  effort  to  determine 
whether  this  genus  is  truly  bioluminescent.  Physical  and 
chemical  assays  of  several  species  from  the  family  Pleu- 
robrachiidae  produced  no  evidence  of  bioluminescence 
capability,  although  all  other  species  of  Ctenophores  tested 
gave  positive  results.  Some  of  the  historical  misperception 
that  Pleurobrachia  can  produce  light  might  be  attributable 
to  confusion  with  similar  luminous  genera. 

Introduction 

Planktonic  marine  invertebrates  are  noted  for  their 
ability  to  produce  light  (Herring,  1987;  Haddock  and  Case. 
1994),  but  even  among  these  organisms,  the  phylum 
Ctenophora  is  remarkable  for  the  extent  of  biolumines- 
cence expression.  Because  there  have  been  no  systematic 
investigations,  speculation  about  the  true  extent  of  bio- 
luminescence  ability  in  Ctenophores  comes  mainly  from 
secondary  sources.  According  to  Ruppert  and  Barnes 
(1994),  "Ctenophores  are  noted  for  their  luminescence, 
which  is  characteristic  of  all  species."  Others  agree  that 
"all  Ctenophores"  (MacGintie  and  MacGintie,  1968)  or 
"probably  all  species"  (Harvey,  1940)  are  bioluminescent, 
and  Dahlgren  (1916)  goes  so  far  as  to  state  that  "all  the 
Ctenophores  have  been  known  for  a  long  time  to  be  light 
producing." 

Pleurobrachia.  perhaps  the  best-known  and  most  stud- 
ied ctenophore  genus,  has  long  been  considered  capable 
of  bioluminescence  (Gadeau  de  Kerville,  1890;  Herring, 
1987).  However  most  authors  who  mention  biolumines- 
cence in  Pleurobrachia  proceed  to  give  details  of  the  lu- 

Received  3  May  1995:  accepted  31  July  1995. 


minescent  system  of  Mnemiopsis  or  some  other  species. 
The  published  records  of  luminescent  spectra  contain  no 
measurements  from  Pleurobrachia  (Nicol,  1958;  Young, 
1981;  Herring,  1983;  Widder  el  a/..  1983;  Latz  et  a/.. 
1 988),  even  though  this  genus  is  one  of  the  most  frequently 
encountered.  Despite  "conventional  wisdom',  we  know  of 
no  credible  accounts  of  luminescence  in  the  family  Pleu- 
robrachiidae — either  in  the  genus  Pleurobrachia  or  Hor- 
miphora. 

It  is  often  difficult  to  evaluate  an  early  report  that  a 
species  is  bioluminescent.  Results  can  be  confounded  by 
the  luminescence  of  a  contaminating  organism  or  by  ex- 
ternal light  causing  reflection  or  refraction  (Herring,  1987). 
In  some  cases  the  taxonomy  of  a  group  of  organisms  has 
changed  so  much  that  it  is  not  possible  to  determine  which 
species  was  investigated  by  early  researchers.  Furthermore, 
once  an  organism  has  been  reported  as  luminous,  there 
is  considerable  resistance  to  removing  it  from  the  list  of 
luminous  species  (e.g.,  sponges).  To  an  extent  this  resis- 
tance is  understandable,  because  the  ability  to  luminesce 
may  vary  within  a  population  on  a  sexual,  ontogenic,  sea- 
sonal, or  diel  basis  (Herring,  1987).  Variation  may  also 
occur  between  subpopulations,  as  in  the  midshipman  fish, 
which  is  luminous  off  California  but  not  when  found  fur- 
ther north  (Warner  and  Case,  1980). 

With  these  caveats  in  mind,  we  have  attempted  to  rig- 
orously demonstrate  that  Pleurobrachia  is  a  notable  ex- 
ception to  the  dogma  that  all  Ctenophores  are  biolumi- 
nescent. 

Materials  and  Methods 

Various  species  of  Pleurobrachia  were  sampled  in  the 
Santa  Barbara  Channel  (P.  bachei,  throughout  the  year), 
the  Alboran  Sea  (P.  rhodopis,  spring),  the  Gulf  of  Maine 
(P.  pileus.  summer),  at  Santa  Catalina  Island,  California 
(P.  bachei.  summer),  and  at  Friday  Harbor,  Washington 


356 


NON-LUMINOUS  CTENOPHORES 


357 


FRIDAY  HARBOR 

Pleurobrachia  bachel 

Euplokamis  dunlapae 
Undescribed  cydippid 


SOUTHERN  CALIFORNIA  BIGHT 
Pleurobrachia  bachel 
Hormiphora  californensis 

Undescribed  cydippid 


GULF  OF  MAINE 

Pleurobrachia  pileus 

Euplokamis  sp. 


ALBORAN  SEA 

Pleurobrachia  rhodopis 

Euplokamis  stationis 
Undescribed  cydippids 


LOCATION  OF  SURVEY 
Non-luminous  species 

Similar  luminous  species 
ASSAYS- 


Figure  1.  Four  types  of  assays  were  conducted  to  determine  whether  ctenophores  were  bioluminescent 
or  bore  any  light-producing  chemicals.  At  each  site  where  specimens  were  collected,  we  also  found  luminous 
genera  which  could  have  been  mistaken  for  Pleurobrachia. 


(P.  bac/iei,  fall).  To  ensure  that  the  ctenophores  were  not 
prestimulated  or  damaged  during  collection,  specimens 
used  in  these  studies  were  hand-collected  in  jars  by  blue- 
water  divers,  except  at  Friday  Harbor,  where  they  were 
collected  from  the  surface  in  beakers.  Because  some  cten- 
ophores lose  their  luminescence  upon  exposure  to  light 
(Ward  and  Seliger,  1976),  specimens  were  dark-adapted 
fora  minimum  of  30  min  prior  to  experiments.  After  this 
recovery  period,  Pleurobrachia  were  subjected  to  me- 
chanical, electrical,  and  a  variety  of  chemical  stimuli  (Fig. 
1 ).  To  ensure  that  the  assay  techniques  were  effective,  we 
also  tested  the  luminescence  of  other  ctenophore  species 
found  at  the  same  locations. 

Physical  stimulation 

The  most  commonly  applied  test  for  luminescence  was 
physical  stimulation  by  a  dark-adapted  observer.  This 
technique  was  used  at  all  sites  where  ctenophores  were 
collected.  For  quantitative  tests  of  mechanical  stimulation, 
five  specimens  of  P.  pileus  collected  in  the  Gulf  of  Maine 
(northwest  Atlantic  Ocean)  were  transferred  to  filtered 
seawater.  allowed  to  dark-adapt,  and  stimulated  by  stirring 
in  a  photon-counting  chamber  for  at  least  five  seconds. 
This  test  was  repeated  three  times  with  five  or  more  P. 
bachei  collected  in  the  Santa  Barbara  Channel  (eastern 
temperate  Pacific  Ocean).  For  comparison,  luminous 
species  were  placed  in  the  same  apparatus  and  induced 
to  luminesce  by  stirring  or  brief  prodding.  Because  some 
organisms  may  be  resistant  to  physical  stimulation,  ad- 
ditional specimens  were  exposed  to  KC1,  ddH:O.  Cad:, 
and  H;O;,  which  can  bypass  normal  control  processes 
and  act  directly  on  light-producing  cells  or  chemicals 
(Herring.  1981). 


Photoprotein  extraction 

Calcium-activated  photoproteins  have  been  identified 
as  the  light-producing  agents  in  all  luminous  ctenophores 
examined  (Ward  and  Seliger,  1974;  unpub.  results).  To 
test  for  the  presence  of  active  photoproteins  in  Pleuro- 
brachia. dark-adapted  specimens  were  extracted  in  a  Ca2+- 
chelating  buffer  as  follows. 

In  the  Santa  Barbara  Channel,  five  specimens  of  Pleu- 
robrachia bachei  were  collected  at  depths  between  5  and 
20  m  on  a  blue-water  dive.  Several  small  ctenophores  from 
three  other  families  (one  Haeckelia  beehleri,  one  Beroe 
cucuinis.  and  three  I  'elamen  parallelling  were  collected 
at  the  same  time  and  used  as  positive  controls.  Specimens 
were  sorted  into  filtered  seawater  and  maintained  in  the 
dark  for  7  h  (until  2 100)  to  allow  recovery  from  potential 
photodegradation  of  their  luminescence  ability  (Ward  and 
Seliger,  1976:  Anctil  and  Shimomura,  1984)  and  to  ac- 
count for  the  possibility  of  a  diel  cycle  of  luminescence, 
which  is  present  in  some  luminous  organisms,  but  has 
never  been  reported  for  ctenophores.  These  specimens 
were  homogenized  in  200  mA/  Tris,  40  mA/  EDTA,  pH 
8.8,  and  a  400  n\  subsample  was  assayed  by  adding  100  n\ 
of360mMCaCl2. 

This  experiment  was  repeated  three  times  using  up  to 
50  P.  bachei  in  the  extraction,  once  with  P.  bachei  frozen 
directly  in  liquid  nitrogen,  and  once  using  P.  pileus  col- 
lected on  dives  in  the  Gulf  of  Maine,  with  various  local 
luminous  species  used  as  positive  controls. 

Regeneration 

To  test  the  hypothesis  that  Pleurobrachia  contains  an 
inactive  photoprotein  but  lacks  the  luciferin  necessary  to 


358 


S.  H.  D.  HADDOCK  AND  J.  F.  CASE 


Euptokamis 
stations 


o 
o 


Time  (s) 

Figure  2.  Responses  of  ctenophores  to  physical  stimulation.  Lumi- 
nous ctenophores  produced  bright  flashes  when  disturbed  (A-C),  even 
if  only  briefly  touched  (arrowhead).  In  contrast,  Pleurohrachia  pileits 
showed  no  light  emission  even  during  continuous  stirring  (D,  E).  The 
y-axis  shows  counts  per  20-ms  bin. 


produce  light,  we  attempted  to  regenerate  extracts  with 
synthesized  coelenterazine  (provided  by  O.  Shimomura), 
the  luciferin  found  in  luminous  ctenophores  and  cnidar- 
ians  (Ward  and  Cormier,  1975;  Shimomura,  1985). 

Specimens  were  homogenized  in  100  mM  Tris,  50  mA/ 
EDTA,  500  mM  NaCl,  pH  7.5,  filtered  through  a  What- 
man GF/C  glass-fiber  filter  to  remove  debris,  and  centri- 
fuged  for  30  min  at  35,000  X  g.  Photoprotein  present  in 
one  ml  of  supernatant  was  triggered  by  the  addition  of 
50  mM  CaCl2  until  no  further  light  was  produced  (typi- 
cally 250  ^1  was  sufficient,  although  no  light  was  emitted 
by  Pleurobrachia  preparations).  This  was  followed  by 
250  n\  of  200  mM  EDTA  to  chelate  the  added  Ca2+,  and 
the  solution  was  saturated  with  ammonium  sulfate  to 
precipitate  the  reacted  protein.  For  the  regeneration,  one 


ml  of  the  saturated  solution  was  centrifuged  at 
15,000  RPM  in  an  Eppendorf  minicentrifuge  for  15  min. 
The  pellet  of  precipitate  was  resuspended  in  200  ^1  of 
10  mA/Tris,  5  mM  EDTA,  500  mA/  NaCl,  and  5  mM  0- 
mercaptoethanol  (techniques  based  on  Campbell  and 
Herring,  1990).  Each  treatment  was  incubated  for  6  h  at 
4°C  with  2  n\  methanol  either  containing  coelenterazine 
or  with  no  luciferin  for  the  negative  controls.  The  light 
produced  upon  final  addition  of  CaCl2  indicated  the  extent 
of  regeneration. 

This  experiment  was  conducted  using  the  hydromedusa 
Haliscera  conica  as  a  positive  control.  We  replicated  this 
experiment  once  using  Haliscera,  the  hydroid  Obelia  sp., 
and  an  undescribed  luminous  ctenophore;  and  again  using 
the  ctenophores  Beroe  cuciimis,  I  'elamen  parallelum,  and 
Haeckelia  beehleri  with  0.1%  gelatin  present  in  the  re- 
generation solution  to  increase  the  stability  of  regenerated 
photoproteins  (Campbell  and  Herring,  1990). 

Results 

At  no  time  during  these  experiments  did  we  detect  any 
bioluminescence  produced  by  Pleurobrachia  or  by  the 
closely  related  genus  Honniphora.  Every  one  of  more  than 
forty  other  ctenophore  species  tested  produced  lumines- 
cence that  was  easily  detected  using  our  methods. 

Physical  stimulus 

Repeated  attempts  at  mechanical  stimulation  failed  to 
elicit  luminescence  from  Pleurobrachia  pileus  (Figs.  2D, 
E).  The  five  specimens  run  during  these  trials  were  neg- 
ative, as  were  ten  Pleurobrachia  bachei  collected  from  the 
Pacific  Ocean  and  run  in  an  identical  experiment  (not 


•t 


10D 


10 


10 


10 


Haeckelia  beehien 


Velamen  parallelum 


Beroe  cucumis 


.  Pleurobrachia  tjachei 

tffWWjNV^ 


0  2  4  6  8  10  12  14 

Time  (s) 

Figure  3.  Photoproteins  extracted  from  luminous  ctenophores  using 
a  calcium-chelating  buffer  can  be  triggered  to  produce  light  upon  the 
addition  of  excess  calcium.  Species  from  three  families  shown  here  il- 
lustrate typical  flashes  produced  by  extracts  of  luminous  species.  In  con- 
trast, Pleurobrachia  bachei  and  P.  pileus  showed  no  photoprotein  activity 
in  any  assays. 


NON-LUMINOUS  CTENOPHORES 


359 


shown).  Light  was  not  produced  by  Pleurohrachia  col- 
lected at  any  of  the  locations  included  in  this  study  (Fig. 
1 ).  Another  member  of  Pleurobrachiidae,  Hormiphora 
califomensis,  which  was  collected  from  the  Santa  Barbara 
Channel,  also  consistently  failed  to  produce  light.  For 
comparison,  other  luminescent  ctenophores  tested  at  the 
same  time  produced  luminescence  for  the  duration  of  the 
stirring.  Even  when  given  only  a  single  brief  stimulus, 
luminous  species  produced  bright  flashes  (Fig.  2A-C),  with 
peak  intensities  of  more  than  1.75  X  107  counts/s  (3.5 
X  105  counts  in  20ms). 

Chemical  extraction 

Assays  of  calcium-free  extracts  of  Pleurobrachia  bachei 
from  the  Santa  Barbara  Channel  (Fig.  3)  and  P.  pileus 
from  the  Gulf  of  Maine  (not  shown)  were  indistinguish- 
able from  the  background  signal.  All  extracts  of  P/euro- 
brachia  were  inert,  while  in  every  case  positive  control 
extracts  from  the  ctenophores  Haeckelia  beehleri.  Berne 
cucumis,  I'elamen  paralle/um  (Fig.  3),  Bolinopsis  infiin- 
dihuhtm.  Beroe  gracilis.  Kiyohimea  aurita.  Bathocyroe 
fosteri.  and  Bathyctena  c/n/ni.  and  from  the  hydrozoans 
Haliscera  conica  and  Obelia  sp.  (not  shown),  produced 
light  both  during  extraction  and  upon  the  addition  of 
CaCl;.,  at  intensities  up  to  2.6  X  106  counts/s. 

Photoprotein  regeneration 

Extracts  of  Pleurobrachia  bachei  incubated  with  lucif- 
erin  were  not  significantly  different  from  those  incubated 
with  methanol  only,  nor  were  they  different  from  the  neg- 
ative control  treatment,  which  contained  only  buffer  and 
luciferin  (Fig.  4).  Regeneration  was  noted  in  the  positive 
controls  treatments  of  Haliscera  conica,  Haeckelia  beeh- 
leri. and  Obelia  sp.  However  one  positive  control  replicate 
(Beroe  ciicnmis)  showed  no  luminescence  activity  after 
the  regeneration,  and  in  some  replications,  the  luminous 
species  used  as  positive  controls  (undescribed  Mertensiid. 
Velamen  parallelum)  gave  inconclusive  results,  since  re- 
sidual activity  remained  in  luminescent  extracts  which 
had  been  depleted  by  CaCl:  and  then  incubated  without 
luciferin. 


Discussion 


Past  research 


The  published  record  regarding  the  luminescence  of 
Pleurobrachia  is  sparse,  consisting  mostly  of  anecdotal 
nineteenth-century  reports.  We  have  not  found  any  pub- 
lished photographs,  spectra,  or  unequivocal  quantitative 
measurements  of  bioluminescence  from  Plewohrachia. 

Of  the  early  accounts,  the  report  of  Dahlgren  ( 1916)  is 
most  explicit  in  describing  bioluminescence  in  Pleuro- 
brachia. Although  most  of  the  text  concerns  Beroe  and 


35 
30 

§     25 

H 

X.    20 

c     15 

o 

O     10 


No  Luciferin 
Luciferin  Added 


Pleurobrachia 


Haliscera 
Specimen  Type 


Buffer  only 


Figure  4.  Photoprotein  regeneration  assays.  Even  when  incubated 
with  an  excess  of  luciferin.  extracts  of  Pleurobrachia  bachei  did  not  he- 
come  luminescent,  indicating  an  absence  of  inactive  photoprotein.  Ex- 
tracts Irom  the  hydromedusa  Haliscera  conica  show  the  regeneration 
that  typically  occurs  when  an  exhausted  photoprotein  is  combined  with 
coelenterazine. 


the  lobate  ctenophore  Mnemiopsis,  there  are  drawings  of 
Pleurobrachia  swimming  about  in  "the  lighted  and  un- 
lighted  state"  (also  reproduced  in  Nicol,  1967).  There  are 
also  drawings  of  low-power  sections  through  the  gastro- 
vascular  canal  of  a  Pleurobrachia:  one  professing  to  show 
the  "layer  of  luminous  cells  covering  ovary  and  testis," 
and  one  showing  a  closer  view  of  the  "probable  luciferine- 
secreting  cells."  Dahlgren  supposed  that  these  were  lu- 
minous cells  because  of  their  "highly-vacuolated  and 
glandular  nature."  Subsequent  work  on  the  ultrastructure 
of  the  luminous  system  of  Mnemiopsis  leidyi  has  shown 
that  these  vacuolar  cells  are  not  those  responsible  for  light 
production  (Freeman  and  Reynolds.  1973;  Anctil,  1985). 
Therefore,  the  cells  depicted  by  Dahlgren  are  not  evidence 
for  light-production  in  Pleurobrachia. 

We  have  found  only  one  quantitative  account  of  Pleu- 
rohrachia  bioluminescence.  For  this  study.  Hardy  and  Kay 
(1964)  placed  "a  large  number  of  very  small  Pleurobra- 
chia'" in  unfiltered  seawater  and  left  them  undisturbed  in 
a  light-measuring  device  to  monitor  "spontaneous"  lu- 
minescence. Their  records  show  many  brief  flashes  during 
several  hours  of  experimentation.  To  establish  that  di- 
noflagellates  in  the  seawater  were  not  producing  the 
flashes,  the  authors  sieved  the  ctenophores  from  the  con- 
tainer and  measured  the  light  again,  this  time  noting  no 
flashes.  However,  by  removing  the  ctenophores  they  also 
removed  the  stimulation  that  would  have  been  caused  by 
their  actively  beating  comb  plates.  The  authors  themselves 
noted  this  effect  in  a  later  experiment  testing  the  stimu- 
lation of  dinoflagellates  by  mysids.  The  number  and 
intensity  of  flashes  recorded  during  the  Pleurobracluu 
experiment  are  more  similar  to  the  dinoflagellate 


360 


S.  H.  D.  HADDOCK  AND  J.  F   CASE 


Figure  5.  Non-luminous  and  luminous  cydippid  ctenophores.  Reports  of  hioluminescence  from  non- 
luminous  species  like  Plciirohrachia  hacliei  (A)  and  llnninplitini  calit^rncnsis  (B)  may  he  attributed  to 
confusion  with  similar  luminous  genera.  Euplokamis  (C)  and  other  undescrihed  species  (D)  are  brightly 
luminescent  and  are  found  at  the  same  locations  as  Pleuruhrachui  (see  Fig.  1).  Scale  bars:  2  mm. 


NON-LUMINOUS  CTENOPHORES 


361 


experiments  than  to  a  Beroc  experiment  which  produced 
fewer,  but  brighter,  flashes  (Hardy  and  Kay,  1964:  figs  1, 
2,  14-16).  Because  the  authors  did  not  see  the  ctenophore 
luminesce  and  did  not  sufficiently  rule  out  the  possibility 
ofdinotlagellate  flashes,  this  account  of  Pleurobrachia  lu- 
minescence remains  unconvincing. 

Considering  that  Pleurobrachia  is  one  of  the  most 
widely  distributed  and  best-known  of  the  ctenophore  gen- 
era, it  is  remarkable  that  we  have  been  unable  to  find  any 
substantiated  reports  of  its  bioluminescence,  especially  in 
the  recent  literature. 

Unpublished  research 

The  unpublished  observations  indicating  that  Pleuro- 
brachia is  non-luminous  are  as  convincing  as  the  void  in 
the  published  literature.  In  nearly  30  years  of  observations 
on  luminous  plankton,  workers  from  this  laboratory  have 
never  encountered  a  luminescent  specimen.  Similarly, 
other  researchers  who  have  studied  bioluminescence  in 
ctenophores  from  around  the  world  have  been  unable  to 
observe  luminescence  in  this  genus  [P.  J.  Herring,  Y.  A. 
Labas  (White  Sea),  B.  H.  Robison,  E.  A.  Widder,  pers. 
comm.].  Because  these  negative  results  have  never  seen 
their  way  into  print,  apocryphal  accounts  persist. 

Results  of  our  experiments 

Because  physical  stimuli  repeatedly  failed  to  elicit  light 
from  Pleurobrachia,  we  attempted  to  determine  whether 
the  luminescent  chemicals  were  present  either  as  a  cal- 
cium-activated photoprotein,  or  as  a  luciferin-dencient 
apophotoprotein.  Extractions  in  calcium-chelating  buffers 
have  clearly  demonstrated  the  presence  of  photoproteins 
in  all  other  ctenophore  species  examined  (Ward  and 
Seliger,  1974;  Shimomura,  1985;  unpub.  results).  Based 
on  the  results  of  Tris-EDTA  extractions,  Pleurobrachia 
clearly  lacks  a  conventional  photoprotein,  and  because 
no  luminescence  was  observed  during  homogenization, 
there  is  no  evidence  that  another  mechanism  is  employed. 

At  the  chemical  level,  failure  to  detect  an  active  pho- 
toprotein could  be  due  to  the  lack  of  an  appropriate  pro- 
tein, or  to  a  lack  of  luciferin.  Based  on  the  negative  results 
of  regeneration  experiments,  it  appears  that  there  is  not 
an  apophotoprotein  present  that  merely  lacks  luciferin. 
Extracts  of  Pleurobrachia  never  became  luminous  in  any 
of  the  incubations  in  which  coelenterazine  was  supplied. 
However,  the  results  of  attempted  regenerations  were 
sometimes  ambiguous,  because  extracts  from  luminescent 
ctenophores  used  as  positive  controls  could  retain  high 
levels  of  residual  activity  even  after  treatment  with  CaCl2. 
In  Mnemiopsis  the  regeneration  of  inactive  photoproteins 
was  originally  found  to  occur  only  at  pH  9.0  (Anctil  and 
Shimomura,  1984).  but  it  is  now  thought  that  the  presence 
of  gelatin  in  the  regeneration  buffer  eliminates  this  pH 


sensitivity  (Campbell  and  Herring,  1990;  Campbell,  pers. 
comm.).  Nonetheless,  it  would  be  useful  to  repeat  these 
experiments  using  recombinant  apophotoprotein,  so  that 
discharging  and  recovering  the  positive  control  samples 
would  not  be  required. 

Although  we  have  done  most  of  our  rigorous  testing 
on  Pleurobrachia,  we  have  also  been  unable  to  find  any 
luminescence  in  mechanical  assays  of  Hormiphora,  sug- 
gesting that  this  closely  allied  genus,  which  is  abundant 
at  depths  around  1 00  meters  offthe  coast  of  southern  Cal- 
ifornia, may  also  be  unable  to  produce  light. 

Identification 

Because  historically  any  small  cydippid  was  likely  to 
be  called  Pleurobrachia.  anecdotal  accounts  of  lumines- 
cence may  be  due  to  confusion  with  similar  lesser  known 
genera  (Figs.  1,  5).  For  example,  Euplokamis  (Fig.  5C)  is 
commonly  encountered  in  the  north  Pacific,  the  Gulf  of 
Maine,  and  the  Mediterranean  Sea,  yet  this  genus  was 
grouped  in  the  family  Pleurobrachiidae  until  recent  work 
by  Mills  ( 1987).  The  luminous  species  Euplokamis  dun- 
lapae  (Mills)  found  offthe  coast  of  Washington  has  been 
alternately  described  as  "Pleurobrachia  pileus"  (Freeman, 
1977),  "P.  ?pileus"  (Kozloff,  1974),  and  other  Pleurobra- 
chia species.  (For  a  complete  list,  see  Mills,  1987.)  Prior 
to  Mills's  clarification  it  would  not  have  been  possible  to 
know  whether  a  ctenophore  that  was  seen  to  luminesce 
was  actually  Pleurobrachia.  Similarly,  the  widespread  oc- 
currence and  'pleurobrachioid'  appearance  (Fig.  5D)  of 
an  undescribed  midwater  ctenophore  (Mills  and  Harbison, 
in  prep.)  may  have  led  to  other  reports  of  luminescence 
attributed  to  Pleurobrachia.  In  light  of  recent  taxonomic 
revision  and  the  presence  of  several  luminous  genera  that 
are  easily  confused  with  Pleurobrachia.  it  is  not  difficult 
to  imagine  how  erroneous  examples  of  bioluminescence 
might  have  been  reported,  even  by  knowledgeable  re- 
searchers. 

Conclusions 

Pleurobrachia'' &  inability  to  produce  light  raises  ques- 
tions about  the  role  of  bioluminescence  for  planktonic 
organisms:  Is  this  'deficiency'  the  handicap  that  it  might 
seem,  given  the  widespread  occurrence  of  biolumines- 
cence among  marine  plankton?  If  bioluminescence  is 
serving  a  defensive  role,  it  may  not  be  important  against 
non-visual  predators  such  as  the  ctenophore  Beroe,  which 
is  known  to  prey  upon  Pleurobrachia.  Also  of  interest  is 
what  is  missing  in  Pleurobrachia  that  makes  it  unable  to 
produce  light.  Are  the  homologous  genes  present  but  in- 
active, or  are  cells  equivalent  to  photocytes  lacking  alto- 
gether? A  comparative  study  of  the  genetic  relationships 
of  ctenophores  might  help  indicate  when  the  ability  to 
bioluminesce  arose  in  this  phylum. 


362 


S    H    D    HADDOCK  AND  J.  F.  CASE 


The  Pleurobrachiidae  may  not  be  the  only  non-lumi- 
nescent group  of  ctenophores,  because  Platyctenida,  a 
small  order  of  non-planktonic  ctenophores,  has  never  been 
reported  to  be  luminescent.  However,  we  have  not  been 
able  to  assay  specimens  from  this  rarely  studied  order. 

Despite  examining  hundreds  of  specimens  of  Pleuro- 
brachia,  collected  under  ideal  conditions  at  a  variety  of 
locations  and  seasons,  we  have  never  observed  light  pro- 
duction in  the  genus,  while  over  40  other  species  of  cten- 
ophores produced  luminescence  during  similar  treatment. 
Furthermore,  we  have  found  no  substantiated  accounts 
of  luminescence  in  the  literature.  Therefore,  although  we 
cannot  say  that  no  Pleurohrachia  was  ever  luminous,  it 
is  clear  that  this  genus  is  not  generally  luminescent.  The 
burden  of  proof  should  be  shifted  to  those  who  wish  to 
show  that  Pleurobrachia  is  bioluminescent. 

Acknowledgments 

We  are  grateful  to  E.  A.  Widder  for  opportunities  to 
collect  specimens,  and  to  S.  Anderson  and  J.  McCullagh 
for  assistance  on  blue-water  dives.  O.  Shimomura  gen- 
erously provided  coelenterazine,  synthesized  by  S.  Inoue, 
which  was  used  in  photoprotein  regeneration  experiments. 

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Shimomura,  O.  1985.  Bioluminescence  in  the  sea:  photoprotein  sys- 
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Reference:  Hint.  Hull  189:  363-369.  (December.  1995) 


The  Life  of  a  Sponge  in  a  Sandy  Lagoon 

MICHA  ILAN  AND  AVIGDOR  ABELSON 

Department  of  Zoology,  Tel  Aviv  University,  Tel  Aviv  69978,  Israel 


Abstract.  Infaunal  soft-bottom  invertebrates  benefit 
from  the  presence  of  sediment,  but  sedimentation  is  po- 
tentially harmful  for  hard-bottom  dwellers.  Most  sponges 
live  on  hard  bottom,  but  on  coral  reefs  in  the  Red  Sea, 
the  species  Biemna  ehrenbergi  (Keller,  1889)  is  found  ex- 
clusively in  soft-bottom  lagoons,  usually  in  the  shallowest 
part.  This  location  is  a  sink  environment,  which  increases 
the  deposition  of  particulate  organic  matter.  Most  of  the 
sponge  body  is  covered  by  sediment,  but  the  chimney- 
like  siphons  protrude  from  the  sediment  surface.  The 
sponge  is  attached  to  the  buried  beach-rock,  which  reduces 
the  risk  of  dislodgment  during  storms.  Dye  injected  above 
and  into  the  sediment  revealed,  for  the  first  time,  a  sponge 
pumping  interstitial  water  (rich  with  particles  and  nu- 
trients) into  its  aquiferous  system.  Visual  examination  of 
plastic  replicas  of  the  aquiferous  system  and  electron  mi- 
croscopical analysis  of  sponge  tissue  revealed  that  the 
transcellular  ostia  are  mostly  located  on  the  buried  surface 
of  the  sponge.  The  oscula,  however,  are  located  on  top  of 
the  siphons;  their  elevated  position  and  their  ability  to 
close  combine  to  prevent  the  filtering  system  outflow  from 
clogging.  The  transcellular  ostia  presumably  remain  open 
due  to  cellular  mobility.  The  sponge  maintains  a  large 
population  of  bacteriocytes,  which  contains  bacteria  of 
several  different  species.  Some  of  these  bacteria  disinte- 
grate, and  may  be  consumed  by  the  sponge. 

Introduction 

Infaunal  soft-bottom  invertebrates  benefit  from  sedi- 
ment, which  provides  a  dwelling  habitat,  shelter  from 
predators,  and  sometimes  a  source  of  nutrition  (Lopez 
and  Levinton,  1987;  Watling,  1991).  In  contrast,  hard- 
bottom  dwellers  can  be  harmed  by  sediment — either  as 
a  result  of  abrasion  by  moving  particles  or  owing  to  suf- 

Received  15  March  1995;  accepted  18  June  1995. 


focation,  shading,  and  clogging  of  feeding  apparatus  by 
settled  particles  (e.g.,  Rogers,  1990). 

Sponges,  the  lowest  group  of  multicellular  organisms, 
are  common  members  of  hard-bottom  communities  (e.g., 
Schubauer  et  al..  1990;  Soest,  1993).  Most  sponges  are 
active  suspension-feeders  that  subsist  on  fine  particles  such 
as  bacterioplankton  and  dissolved  organic  matter  (Simp- 
son, 1984).  Only  a  few  sponge  species  inhabit  soft-bottom 
habitats,  usually  in  the  deep  sea  (e.g.,  Tabachnick,  1991; 
Werding  and  Sanchez,  1991),  and  information  on  their 
physiological  and  morphological  adaptation  to  this  en- 
vironment is  scarce.  These  sponge  species  may  be  endan- 
gered by  two  opposing  processes  in  their  environment. 
The  first  threat  is  complete  burial,  resulting  from  depo- 
sition of  sediment,  which  may  plug  suspension-feeding 
and  respiration  channels.  The  second  threat  is  dislodgment 
from  the  substratum,  which  can  occur  as  a  result  of  the 
erosive  force  of  moving  particles.  We  may  therefore  pos- 
tulate that  sponges  inhabiting  soft-bottom  habitats  should 
exhibit  specialized  adaptations  for  withstanding  effects  of 
cover  by  sediments  and  for  remaining  anchored  in  place. 
In  addition,  they  should  be  able  to  adjust  their  feeding 
mode  so  that  neither  burial  nor  occasional  exposure  will 
inhibit  their  ability  to  feed. 

In  the  present  study,  we  examined  the  characteristics 
of  a  typical  sediment-dwelling  sponge  species  that  enable 
it  to  survive.  In  the  course  of  many  dives  on  the  reef  flat, 
forereef  and  deep  reef  (down  to  50  m)  of  Eilat,  Red  Sea, 
and  the  backreef  lagoon  to  search  for  sediment-specific 
sponge  communities,  we  observed  that  one  species, 
Biemna  ehrenbergi  (Keller,  1889),  is  confined  to  the  la- 
goon. 

Materials  and  Methods 

Field  observations 

We  studied  Biemna  ehrenbergi  in  the  coral  reef  lagoon 
of  Eilat.  Northern  Red  Sea  (29°30'N;  34°55'E).  We  sur- 


363 


364 


M.  ILAN  AND  A.  ABELSON 


N 


O 


a, 

o 
Cu 


0 


5      10 


30    35    40    45 


Distance  from  the  shore  (m) 

Figure  1 .  Spatial  distribution  of  Biemna  ehrcnhergi  in  Eilat,  Red 
Sea,  along  a  belt  1 50  m  long  and  40-45  m  wide  in  the  inside  lagoon  of 
the  coral  reel. 


OsO4,  dehydrated,  embedded  in  Epon  812,  sectioned, 
stained  with  uranyl  acetate  and  lead  citrate,  and  viewed 
in  a  JEOL  1200-EX.  Ostia  (incurrent  openings)  diameters 
were  measured  on  photographs  taken  with  a  scanning 
electron  microscope. 

Canal  flow  circulation 

Flow  direction  through  the  sponge's  aquiferous  system 
was  recorded  by  releasing  fluorescein  dye  into  the  water 
column  near  the  sponge's  "chimneys,"  or  projecting  si- 
phons, or  by  injecting  dye  into  the  substratum  close  to 
the  sponges.  When  the  dye  was  injected  above  the  sedi- 
ment, it  was  difficult  to  detect  currents  resulting  from  the 
sponge's  activity  because  of  background  interference.  The 
sponge's  siphons  were  therefore  covered  with  a  1 -liter 
plastic  beaker,  which  was  pressed  a  few  centimeters  into 
the  sediment.  The  dye  was  then  injected  into  the  beaker, 
and  its  trajectory  was  followed  without  the  disturbance 
of  external  water  movements. 


veyed  a  belt  150  m  in  length  and  40-45  m  in  width,  re- 
cording every  sponge  within  it.  The  semi-closed  lagoon 
of  the  Eilat  reef  encompasses  beach-rocks  covered  with  a 
layer  of  sediment  of  various  thicknesses,  patches  of  coral 
heads,  and  small  knolls.  A  third  of  its  water  volume  is 
changed  each  tidal  cycle  (Lazar,  pers.  comm.).  The  effect 
of  tidal  currents  and  the  prevailing  northern  waves  is  slight, 
and  the  flow  regime  of  the  lagoon  qualifies  it  as  a  'sink', 
even  for  fine-sized  particles.  To  determine  grain  size  dis- 
tribution, cores  (8  cm  in  diameter)  of  sediment  were  taken 
adjacent  to  sponges,  from  the  surface  down  to  the  beach- 
rock.  Particles  were  differentiated  by  size,  dried  at  80°C 
for  24  h,  and  weighed. 

Architecture  of  canal  system 

Sponge  specimens  were  perfused  in  the  field  with  a 
plastic  fluid  (Batson's  17  Plastic  Replica  and  Corrosion 
Kit,  Polysciences  Inc.)  that  was  injected  by  syringe  into 
the  exhalant  openings  until  it  leaked  out  through  the  in- 
halant openings.  The  fluid  was  allowed  to  cure  for  12  h. 
then  the  specimens  were  transported  to  the  laboratory 
and  immersed  in  10%  sodium  hypochlorite  for  24  h.  The 
plastic  replicas  were  then  rinsed  in  tap  water  and  observed 
under  a  dissecting  microscope  (protocol  modified  from 
Bavestrello  el  a/.,  1988). 

Ultrastructure  analysis 

Sponge  samples  were  fixed  (2.5%  glutaraldehyde  buf- 
fered in  seawater)  for  at  least  24  h.  For  transmission  elec- 
tron microscopy  (TEM),  samples  were  post-fixed  with  1% 


Results 


Field  observations 


Biemna  ehrenbergi  sponges  were  found  exclusively 
within  the  inner  lagoon  of  the  coral  reef  in  Eilat,  Red  Sea 
(depth  0.5-1.8  m).  Despite  numerous  dives  from  the  reef 
flat  through  the  forereef  and  down  to  50  m.  no  B  ehren- 
bergi specimen  was  ever  found  in  other  areas.  A  survey 
conducted  along  150  m  of  the  lagoon  (width  40-45  m) 
revealed  56  individuals.  Sixty-six  percent  of  the  sponges 
in  this  belt  were  concentrated  5-15  m  from  the  shore  (one 
sponge  per  40.5  nr),  with  the  rest  spread  out  over  the 
lagoon  (one  sponge  per  145  m:)  (Fig.  1 ).  Sponge  locations 
within  the  lagoon  typically  had  particles  of  various  sizes, 
but  82.5%  of  the  grains  were  larger  than  500  j/m,  13.3% 


Table  I 

Distribution  of  grain  size  adjacent  to  Biemna  ehrenbergi  sponges 
(data  taken  from  5  cores) 


Grain  size  (^ml 


Average  percentage  of  total 
grain  mass  ±  SD 


X  >  2000 
2000  >  X  >  1000 
1000  >  X  >  500 
500  >  X  >  350 

350  >  X  >  21(1 
210  >  X  >  105 
105  >  X  >  74 
74  >  X  >  62 
62  >  X  >  53 
53  >  X 


28.1  ±6.4 

27.7  ±6.1 

29.4  ±  7.5 

4.4  ±  2.4 

4.0  ±  2.5 

4.1  ±  2.7 
0.8  ±  0.5 
0.3  ±0.1 
0.3  ±  0.2 
1.6  ±0.7 


SANDY  LAGOON  SPONGES 


365 


* 


Figure  2.  Bicmna  ehrenbergi  in  silu.  (A)  The  chimney-like  siphons, 
which  are  above  the  sediment  surface.  (B)  The  sponge  after  sediments 
have  been  removed.  Arrows  indicate  sediment  level. 


were  of  medium  size,  and  less  than  2%  were  smaller  than 
74  ^m  (Table  I). 

We  found  specimens  of  B.  ehrenbergi  buried  to  depths 
of  up  to  20  cm.  with  most  of  the  body  covered  with  sand 
(Fig.  2 A).  When  the  buried  part  was  uncovered  it  was 
found  to  be  attached  to  the  beach-rock  (Fig.  2B).  More- 
over, the  shape  of  the  buried  part  resembled  a  conus  with 
a  large  base  bearing  several  projections  (Fig.  2B).  Only 
these  projections — the  chimney-shaped  siphons — ex- 
truded ( 1-7  cm)  through  the  sediment  (Fig.  2A).  Oscula 
are  located  at  the  uppermost  part  of  the  siphons. 

The  sponge  is  capable  of  closing  its  oscula,  and  thus 
probably  is  able  to  avoid  intake  of  particles  during  storms. 


Observations,  using  dye  injected  above  and  into  the  sed- 
iment 10-15  cm  from  the  siphons,  revealed  that  most  of 
the  water  pumped  into  the  sponge  came  through  the  pores 
of  the  buried  surfaces;  only  a  minute  amount  was  taken 
in  by  pores  on  the  exposed  surface  of  the  siphons.  Less 
than  a  minute  after  dye  was  injected  into  the  sediment, 
it  exited  from  the  oscula  in  a  strong  flow;  but  when  injected 
above  the  sediment  surface,  only  a  small  amount  of  dye 
emerged  from  the  oscula. 

Surface  structure  and  morphology 

Most  of  the  ostia  (inhaling  openings)  are  located  on 
the  upper  surface  of  the  body  and  are  buried  in  the  sed- 
iment (Fig.  3b).  The  exposed  siphons  are  almost  devoid 
of  such  openings  (Fig.  3a).  The  average  size  of  the  oval 
pores  is  9.4  ±  5.2  ^m,  with  a  range  of  4-24.7  /urn  (Fig. 
3b).  The  ostia  are  transcellular,  composed  of  a  single  cell 
(Fig.  3b).  The  total  surface  area  of  the  buried  parts  is  ex- 
tended through  processes  that  protrude  from  the  main 
central  body. 

B.  ehrenbergi  has  many  small  excurrent  canals  that  are 
accommodated  along  a  central  larger  canal,  leading  to  the 
osculum  (Fig.  4a).  The  inhalant  canals  can  be  divided 
into  two  zones:  (a)  those  of  the  siphon-like  parts  of  the 
body,  which  extend  above  the  substratum  surface;  and 
(b)  the  canals  of  the  body  parts  that  are  buried  within  the 
sediment.  Most  of  the  sponge's  soma,  which  lies  beneath 
the  sediment  surface,  is  supported  by  numerous  small 
incurrent  canals  and  ostia  (Figs.  3b,  4b).  In  contrast,  the 
exposed  parts  of  the  body  contain  few  inhalant  canals  and 
ostia  and  a  large  excurrent  canal  (Figs.  3a,  4a). 

Large  quantities  of  various  intracellular  bacteria  con- 
fined within  bacteriocytes  are  a  prominent  feature  in  the 
mesohyl  of  B.  ehrenbergi  (Fig.  5a-e).  These  bacteria  ap- 
pear to  be  contained  in  a  single  large  vacuole  within  the 
cell  (Fig.  5e),  or  immersed  in  the  cytoplasm  without  being 
enclosed  by  a  membrane  (Fig.  5a-d).  No  phototrophic 
bacteria  were  found  (none  of  the  bacteria  appeared  to 
contain  photosynthetic  membranes).  Bacteria  within  some 
of  the  bacteriocytes  seemed  to  disintegrate  (Fig.  5b-d), 
whereas  bacteria  within  adjacent  cells  appeared  intact  and 
divided  and  grew  (Fig.  5b.  c). 


Discussion 


Sponge  distribution 


The  distribution  of  the  sediment-buried  B.  ehrenbergi 
in  Eilat  is  restricted  to  a  narrow  belt  at  the  shallower  part 
of  the  semi-closed  back-reef  lagoon  in  Eilat.  The  possibility 
of  exposure  to  a  non-oxygenated  environment  is  a  major 
problem  for  sediment-buried  sponges.  The  oxygen  level 
within  the  sediment  is  largely  dependent  on  the  tidal  and 


366 


M.  ILAN  AND  A.  ABELSON 


Figure  3.  Upper  surface  architecture  of  Biemna  ehrenbergi-  (a)  SEM  photograph  of  an  area  of  exposed 
siphon.  Scale  bar  =  100  ^m.  (b)  SEM  photograph  of  an  area  of  the  exposed  upper  surface  of  the  buried 
pans.  Scale  bar  =  10  /jm. 


subtidal  (wave)  pumps  (Riedel  ct  a/.,  1972),  as  well  as  on 
the  intensity  of  the  sponge's  pumping  activity.  These  two 
pumps  are  most  effective  in  shallowest  water,  at  the  same 
depth  as  B.  ehrenbergi.  We  therefore  suggest  that  the 
sponge's  distribution  is  restricted  to  an  area  in  which  the 
sediment  is  mostly  oxygenated.  Moreover,  the  large  sed- 
iment size  (small  particles  are  less  than  2%  of  total  grain 
mass)  also  contribute  to  the  oxygenation  of  the  sponge's 
vicinity.  A  sponge  inhabiting  a  soft-substrate,  wave-af- 
fected environment  is  also  in  danger  of  being  uncovered 
and  dislodged.  Because  closed  lagoons,  like  many  other 
sheltered  habitats,  are  'sink  environments'  in  which  the 
rate  of  sediment  deposition  is  higher  than  the  rate  of  sed- 
iment loss,  the  sponge's  chances  of  remaining  covered  are 
enhanced.  B.  ehrenbergi  also  attaches  itself  to  the  buried 
beach-rock,  thus  providing  anchorage  and  better  ability 
to  maintain  an  upright  position  following  exposure  to 
storms. 

Flow  and  particle  path 

The  morphology  of  the  canal  system  of  B.  ehrenbergi. 
the  presence  of  ostia  mostly  on  the  sponge's  buried  surface, 
and  the  observation  that  the  animal  can  pump  in  dye 
injected  into  the  sediment  and  expel  it  through  the  oscula, 
all  provide  evidence  that  this  sponge  species  receives  its 
water  supply  from  the  interstitial  reservoir.  This  is  a  first 
report  of  such  a  pattern  of  water  pumping  in  sponges. 
This  finding  revives  and  sheds  new  light  on  a  century-old 
question:  what  is  the  direction  of  water  flow  in  soft-sed- 


iment sponges  such  as  Disyringa  spp.  (Sollas,  1888;  Fry 
and  Fry,  1978;  Werding  and  Sanchez,  1991)?  On  the  basis 
of  sponge  morphology,  it  was  suggested  that  Disyringa 
spp.,  inhabiting  a  deep,  soft-bottom  environment,  pumps 
water  in  from  the  interstitial  reservoir  either  through  a 
single  large  pore  (Sollas.  1888)  or  through  several  small 
openings  buried  in  the  sediment  (Fry  and  Fry,  1978). 
Werding  and  Sanchez  (1991)  found  in  laboratory  exper- 
iments that  Oceanapia  peltata,  with  a  body  morphology 
similar  to  that  of  Disyringa  spp.,  pumps  water  in  from 
siphons  (papillae)  projecting  above  the  substrate  and  dis- 
charges it  out  into  the  sand.  This  flow  direction  is  opposite 
to  the  one  we  observed  for  B.  ehrenbergi.  The  fact  that 
sponges  are  capable  of  pumping  water  from  the  interstitial 
reservoir,  as  demonstrated  in  the  present  study,  suggests 
that  such  a  direction  of  water  movement  may  yet  be  dem- 
onstrated in  Disyringa  spp. 

Because  water  is  pumped  in  from  the  sediment,  buried 
ostia  may  be  clogged  by  sediment  particles.  To  avoid  this 
problem,  the  internal  anatomy  of  the  sponges  is  probably 
frequently  reorganized  by  continuous  cell  movements,  as 
shown  for  other  sponges  (e.g..  Bond.  1992).  Such  plasticity 
enables  the  sponges  to  open  new  ostia,  overcoming  any 
clogging.  Due  to  cell  flexibility,  a  transcellular  opening 
may  be  easily  moved  aside  or  closed,  unlike  a  system  of 
extracellular  openings,  which  requires  coordination  be- 
tween many  cells. 

The  conical  shape  of  the  chimney-like  siphon  of  B. 
ehrenbergi  gives  it  a  high  slenderness  ratio  (SR)  [in  which 


SANDY   LAGOON  SPONGES 


367 


Figure  4.  Plastic  replica  of  the  aquiferous  system  of  Bicmna  chrcn- 
bergi  (SEM  photographs),  (al  Gross  aquiferous  system.  Arrowheads  in- 
dicate the  point  at  the  surface  of  the  sediment.  Scale  bar  =  5  cm.  (b)  A 
portion  of  the  aquiferous  system  located  underneath  the  surface:  I,  high 
density  of  small  mcurrent  canals;  E,  zone  of  man>  excurrent  canals.  The 
"E"  at  the  top  of  the  picture  is  positioned  on  a  central  excurrent  canal 
that  leads  to  the  above-surface  osculum:  *  sand  particles  entrapped  within 
the  sponge. 


the  ratio  of  the  body  height  to  its  lowest  width  is  >  1  (Abel- 
son  et  al..  1993)].  This  shape  prevents  ascension  of  the 
near-bed  flow,  and  as  a  consequence  reduces  the  quantities 
of  bedload  particles  encountering  the  siphons.  In  addition 
to  being  relatively  high  above  the  sediment  surface,  the 
oscula  remain  unclogged  because  they  can  close  their  en- 
trances during  unfavorable  conditions.  This  combination 
of  morphology  and  behavior  gives  specimens  of  this  spe- 
cies the  twofold  advantage  of  being  primarily  infaunal 


organisms,  yet  having  exposure  to  the  water  column  for 
waste  discharge.  The  separation  of  sites  for  intake  and 
discharge  of  water  ensures  that  no  re-filtration  of  waste- 
water  occurs. 

Feeding  modes 

B.  t-hrenhergi  appears  to  possess  two  feeding  modes: 
one  that  is  common  to  all  sponges — filtering  small  par- 
ticles out  of  the  water  (either  above-surface  or  interstitial 
water);  and  a  second  based  on  symbiotic  intracellular 
bacteria. 

Symbiotic,  usually  heterotrophic,  bacteria  have  been 
observed  within  many  sponge  species  (e.g.,  Reiswig,  1974; 
Vacelet  and  Donadey.  1977;  Wilkinson.  1978).  Nutrient 
translocation  between  phototrophic  symbiotic  cyanobac- 
teria  and  their  sponge  host  has  been  demonstrated  (Wilk- 
inson, 1979).  In  the  sclerosponges  Ceratoporella  nicholsoni 
and  Stromatospongia  norac.  the  numerous  symbiotic 
heterotrophic  bacteria  are  spread  extracellularly  and  are 
phagocytized  in  some  parts  of  the  sponges  (Willenz  and 
Hartman.  1989).  In  Petrosiaficiformis,  as  in  B.  ehrenbergi. 
symbiotic  heterotrophic  bacteria  are  confined  intracel- 
lularly  to  bacteriocytes,  and  no  phagocytosis  and  digestion 
of  these  bacteria  is  observed  (Vacelet  and  Donadey,  1977). 
The  absence  of  a  host  membrane  around  the  symbiotic 
bacteria,  as  suggested  in  some  of  the  observed  cases,  has 
been  considered  to  indicate  a  stable  association  of  complex 
metabolic  relationships  between  the  symbiotic  partners 
(Smith.  1979;  Saffo,  1990).  It  may  be,  however,  that  even 
in  presence  of  a  vacuole.  bacteriocyte  cytoplasm  had  been 
reduced  to  a  minimum,  which  would  explain  its  virtual 
absence  in  many  electron  micrographs. 

In  the  present  study,  transmission  electron  micrographs 
provided  grounds  for  suggesting  that  B.  ehrenbergi  may 
consume  some  of  its  "bacterial  farms,"  while  leaving  other 
bacteriocytes  intact.  Thus  there  exist,  side  by  side,  some 
bacteriocytes  in  which  the  bacteria  appear  normal  and 
even  seem  to  divide,  and  others  in  which  the  bacteria 
apparently  disintegrate. 

In  a  sink  environment  (like  the  reef  lagoon),  the  content 
of  particulate  and  dissolved  organic  matter  in  the  sediment 
is  higher  than  in  the  water  column.  For  example,  dissolved 
organic  carbon  is  higher  in  interstitial  water  than  in  sea- 
water  (Krom  and  Sholkovitz,  1977).  and  dissolved  free 
amino  acids  can  be  100  times  as  concentrated  in  the  in- 
terstitial water  in  the  near-surface  sediments  as  they  are 
in  the  overlaying  seawater  (Henrichs  and  Farrington, 
1979).  We  suggest,  therefore,  that  in  the  lagoon,  B.  eh- 
renbergi and  its  symbiotic  intracellular  bacteria  may  ben- 
efit from  interstitial  water  rich  in  organic  matter  (partic- 
ulate and  dissolved),  which  the  sponge  pumps  through  its 
buried  surface. 


368 


M.  ILAN  AND  A.  ABELSON 


. 


"  w;-i~;: 
M  .      >**^~ 

'Vjct    "V*SL^->  ^  _-*•  * 

^_  ^V        jM  '       sScr  f      ^V*^*^^     ^ 


\*tf    ' 


?*?,"<**«©Ffi#5S! 


"     ' 


Figure  5.  Symbiotic  bacteria  found  in  Biemna  ehrenbergi  (TEM  photographs),  (a)  Bactenocytes — mesohyl 
cells  that  are  packed  with  a  large  number  of  intracellular  bacteria.  Scale  bar  =  2  ^m.  (b)  Partially  disintegrated 
bacteria  within  a  bacteriocyte  (B).  A  sponge  cell  is  attached  to  the  bacteriocyte,  engulfing  it  by  projections 
(arrows).  Scale  bar  =  I  ^m.  (c)  A  bacteriocyte  in  an  advanced  stage  of  bacterial  disintegration  (D),  while  in 
two  adjacent  bactenocytes  the  bacterial  population  seem  intact  and  some  bacteria  appear  to  be  dividing 
(arrowheads).  Scale  bar  =  I  pm.  (d)  A  bacteriocyte  with  proliferating  intact  bacteria  (arrowheads)  adjacent 
to  a  cell  which  resembles  a  hacteriocyte,  but  in  which  no  bacteria  are  seen.  Scale  bar  =  1  ^m.  (e)  A  bacteriocyte 
with  nucleus  (N)  and  cytoplasm,  in  which  the  bacteria  appear  to  be  enclosed  within  a  vacuole  (arrows). 
Scale  bar  =  1  jim. 


SANDY  LAGOON  SPONGES 


369 


Acknowledgments 

We  are  indebted  to  Y.  Loya  for  his  invaluable  support 
of  this  study.  V.  Delarea  helped  with  the  electron  mi- 
croscopy. An  extremely  thorough  review  by  B.  Rinkevich 
and  helpful  suggestions  from  an  anonymous  reviewer  im- 
proved the  manuscript  immensely.  We  thank  the  MBL 
at  Eilat  for  the  hospitality  and  use  of  lab  facilities. 

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Reference:  Bioi  Bull  189:  370-375.  (December,  1995) 


Interspecific  Variations  in  Adhesive  Protein  Sequences 

of  Mytilus  edulis,  M.  galloprovintialis, 

and  M.  trossulus 


KOJI  INOUE1  •*,  J.  HERBERT  WAITE2,  MAKOTO  MATSUOKA3, 
SATOSHI  ODO1,  AND  SHIGEAKI   HARAYAMA1 

^Marine  Biotechnologv  Institute.  Kamaishi  Laboratories,  Heita.  Kamaishi,  Iwate  026.  Japan; 

2Department  of  Chemistry  and  Biochemistry,  University  of  Delaware,  Newark.  Delaware  19716: 

and  ^Juneau  Center.  School  of  Fishery  and  Ocean  Sciences.  University  of  Alaska. 

Fairbanks.  Glacier  Highway.  Juneau.  Alaska  99801 


Abstract.  Variation  in  the  adhesive  protein  gene  se- 
quences of  Mytilus  edulis,  At.  galloprovintialis,  and  M. 
trossulus  collected  in  Delaware,  Kamaishi  (Japan),  and 
Alaska,  respectively,  was  analyzed  by  the  polymerase 
chain  reaction  (PCR)  using  two  sets  of  oligonucleotide 
primers.  The  first  set.  Me  1 3  and  Me  14,  was  designed  to 
amplify  the  repetitive  region.  The  length  of  the  amplified 
fragments  was  highly  variable,  even  among  samples  of  the 
same  species.  Another  set.  Me  1 5  and  Me  16,  was  designed 
to  amplify  a  part  of  the  nonrepetitive  region.  The  length 
of  the  amplified  fragments  was  uniform  in  each  species 
and  differed  interspecifically;  180,  168,  and  126  bp  for  M. 
edulis.  M.  trossulus.  and  M  galloprovincialis,  respectively. 
The  amplified  sequence  of  M.  trossulus  resembled  that  of 
M.  edulis.  Mussels  from  other  sites  were  also  examined 
by  PCR  using  Me  15  and  Me  16.  Wild  mussels  from 
Tromso  (Norway)  and  cultured  mussels  from  Brittany 
(France)  were  identified  as  M.  edulis.  Cultured  mussels 
from  the  Mediterranean  coast  of  France  and  wild  mussels 
from  Shimizu  (Japan)  were  identified  as  M.  galloprovin- 
cialis. Some  wild  mussels  from  Hiura  (Japan)  were  iden- 
tified as  a  hybrid  between  M.  galloprovincialis  and  A/. 
trossulus.  Thus,  the  length  of  this  part  (variable  region) 
of  the  sequence  is  proposed  as  a  diagnostic  marker  for 


Received  23  January  1995;  accepted  10  August  1995. 

The  partial  nucleotide  sequence  for  M  trossulux  adhesive  protein  gene 
reported  in  this  paper  has  been  submitted  to  the  GenBank/EMBL/DDBJ 
Data  Bank  with  accession  number  D50553. 

*  Address  for  correspondence:  Koji  Inoue  at  present  address:  Central 
Research  Laboratory,  Nippon  Suisan  Kaisha,  Ltd.,  559-6  Kitano, 
Hachioji,  Tokyo  192,  Japan. 


these  three  morphologically  similar  species  and  their 

hybrids. 

Introduction 

Two  types  of  polyphenolic  proteins,  foot  proteins  1  and 
2,  both  of  which  incorporate  3,4-dihydroxyphenylalanine 
(DOPA)  into  their  primary  structures,  have  been  isolated 
from  the  mussel  Mytilus  edulis.  and  characterized  (see 
Waite.  1992,  for  a  review).  Foot  protein  1  is  an  adhesive 
protein  that  contains  repeats  of  the  decapeptide  motif 
AKPSYP*P*TY*K,  where  P*  and  Y*  denote  hydroxy- 
proline  and  DOPA,  respectively,  and  the  hexapeptide 
motif  AKPTY*K(  Waite  and  Tanzer,  1981;  Waite,  1983; 
Waite  el  a/..  1985).  Foot  protein  2  is  an  epidermal  growth- 
factor-like  protein  that  forms  the  adhesive  plaque  matrix 
(Rzepecki  et  a/..  1992;  Inoue  et  a/..  1995a).  The  primary 
structure  of  the  foot  protein  1  has  been  determined  by 
cDNA  or  gene  cloning.  It  has  been  shown  that  it  consists 
of  a  relatively  short  nonrepetitive  domain  and  a  long  re- 
petitive domain.  The  repetitive  domain  contains  more 
than  70  decapeptide  repeats  and  13  or  14  hexapeptide 
repeats,  but  the  number  and  distribution  pattern  of  the 
motifs  are  variable  even  in  the  same  species  (Filpula  et 
ai.  1990:  Laursen.  1992). 

We  isolated  cDNA  encoding  foot  protein  1  from  M. 
galloprovincialis  sampled  in  Japan  and  compared  the 
predicted  amino  acid  sequence  to  those  of  A/.  edulis(lnaus 
and  Odo,  1994;  Inoue  et  ai.  1995b).  The  A/,  gallopro- 
vincialis sequence  also  consisted  of  the  nonrepetitive  and 
repetitive  domains,  but  remarkable  differences  were  ob- 
served in  both.  The  major  difference  in  the  repetitive  do- 


370 


MUSSEL  ADHESIVE  PROTEIN  GENES 


371 


M.  edulis 


Mel  5 


-Mel6 


M.  galloprovincialis 


Me  15 


Mel  3 


Mel4 


Mel6 


JL 


Mel  3- 


Mel4 


Figure  1.  Positions  ot'PCR  primers  in  the  adhesive  protein  genes  of  .\fytilis  edulix  and  M.  galloprovincialis. 
Adhesive  protein  genes  are  shown  as  cDNA  sequences.  Position  and  direction  of  primers  is  indicated  by 
arrows.  Solid  and  open  boxes  indicate  the  signal  peptide  and  the  repetitive  region,  respectively.  Dotted  boxes 
indicate  the  nonrepetitive  region,  and  the  box  with  dense  dots  indicates  the  18  ammo  acid  sequence  found 
in  the  M.  edulis  sequence  but  not  in  that  of  M.  galloprovincialis.  Solid  lines  indicate  untranslated  region. 


main  is  that  the  M.  galloprovincialis  sequence  contained 
62  decapeptide  repeats  but  no  hexapeptide  motif.  In  con- 
trast, the  nonrepetitive  domains  differ  by  a  deletion  of  18 
amino  acids  observed  in  the  M.  galloprovincialis  sequence. 
In  this  study,  we  prepared  two  sets  of  oligonucleotide 
primers  for  polymerase  chain  reaction  (PCR)  to  amplify 
the  whole  repetitive  region  and  a  part  of  the  nonrepetitive 
region.  Using  these,  we  have  analyzed  foot  protein  I  se- 
quences of  M.  edulis,  M.  galloprovincialis,  and  Af.  tros- 
sulus.  These  three  species  are  closely  related  (Gosling, 
1984;  Koehn,  1991;  Gardner,  1992;  Seed,  1992.  for  re- 
views) and  practically  indistinguishable  by  morphological 
characteristics  only.  We  report  that  the  length  of  the  frag- 
ments amplified  from  the  nonrepetitive  region  is  specific 
to  each  species,  but  the  length  of  the  repetitive  region  is 
highly  variable  even  within  the  same  species.  Thus  the 
nonrepetitive  region  can  be  used  as  a  diagnostic  marker 
for  identification  of  the  three  species. 


Materials  and  Methods 


Mussels 


M.  edulis  was  collected  at  Lewes  (Delaware,  USA);  M. 
galloprovincialis  and  M.  trossulus  were  sampled  at  Ka- 
maishi  (Iwate.  Japan)  and  Juneau  (Alaska,  USA).  All  these 
sampling  points  are  "pure  sites"  of  each  species  where 
the  other  two  species  do  not  occur  (McDonald  et  al., 
1991 ).  Wild  mussels  were  also  collected  at  Tromso  (Nor- 
way). Hiura  (Hokkaido,  Japan),  and  Shimizu  (Shizuoka, 
Japan).  Mussels  cultured  in  Brittany  (France)  and  on  the 
Mediterranean  coast  of  France  were  obtained  at  a  fish 
market  at  Ferney-Valtaire  (France). 

DNA  extraction 

A  piece  of  the  gill  from  each  mussel,  about  0.5  cm2, 
was  incubated  in  500  n\  lysis  buffer  containing  50  m.U 


Tris-HCl  (pH  7.5).  10  mAl  EDTA,  0.5%  SDS,  500  jtg/ml 
Proteinase  K  at  55°C  for  2-4  h.  Samples  were  then  ex- 
tracted twice  with  equal  volumes  of  saturated  phenol  and 
twice  with  phenol  chloroform:  isoamyl  alcohol  (24:24:1). 
The  aqueous  phase  was  precipitated  with  ethanol  and  dis- 
solved in  50  |il  TE  ( 10  mM  Tris,  1  mM  EDTA). 

PCR  amplification 

About  100  ng  of  DNA  was  dissolved  in  100  ^1  1  X  Tth 
buffer  (TOYOBO.  Japan)  containing  6  ^g  sense  primer. 
6  MB  antisense  primer,  and  200  nAf  dNTP.  After  pre- 
heating to  95°C.  1  unit  of  Tth  DNA  polymerase  (TO- 
YOBO. Japan)  was  added  and  30  cycles  of  amplification 
were  performed.  Each  cycle  consisted  of  30  s  at  94°C,  30 
s  at  56°C,  and  90  s  at  70°C.  The  sequences  of  the  primers 
were  Me  1 3,  CCA  CTT  GCA  AAG  AAG  CTG  TCA  TCT; 
Me  14,  ACA  AAC  GTT  AAA  ATG  TGT  AGT  ACA 
GTA;  Me  1 5,  CCA  GTA  TAC  AAA  CCT  GTG  AAG  A; 
Me  16,  TGT  TGT  CTT  AAT  AGO  TTT  GTA  AGA. 
Positions  of  primers  in  the  foot  protein  1  cDNA  sequence 
in  M.  galloprovincialis  are  shown  in  Figure  1 . 

Electrophoresis  of  amplified  products 

Ten  microliters  of  PCR  product  was  mixed  with  the 
loading  dye  solution  containing  bromophenol  blue  (BPB) 
and  xylene  cyanol  and  subjected  to  agarose  gel  electro- 
phoresis.  For  analysis  of  PCR  products,  4.8%  NuSieve  3: 
1  agarose  (FMC)  was  used  for  the  nonrepetitive  region, 
and  1%  LE  agarose  (FMC)  was  used  for  the  repetitive 
region.  Electrophoresis  on  a  4.8%  gel  was  continued  until 
BPB  reached  the  end  of  the  gel. 

Sequencing 

The  fragment  amplified  from  the  genome  of  M.  tros- 
sulus using  primers  Me  15  and  Me  16  was  isolated  and 


372 


K.  INDUE  ET  AL. 


123    456 


Figure  2.  Representative  results  of  amplification  of  the  repetitive 
region  of  the  adhesive  protein  gene.  Amplified  products  were  electro- 
phoresed  on  l%  agarose  gel.  Lanes  I  and  2.  Mylili\  eilitlis:  lanes  3  and 
4,  M.  irossuhts:  lanes  5  and  6,  M.  galloprovincialis.  M,  molecular  marker 
(lambda  DNA  digested  with  £«>T  1 41). 


inserted  into  the  Sma  I  site  of  pUC19.  Sequences  of  both 
strands  of  three  independent  clones  were  determined  using 
a  373A  DNA  sequencer  (Applied  Biosystems  Inc.)  and  a 
PRISM  Dyeterminator  Cycle  Sequencing  Kit  (Applied 
Biosystems  Inc.). 

Results 

Variation  in  the  repetitive  region 

The  primers  Me  1 3  and  Me  1 4  were  designed  to  amplify 
the  repetitive  region  using  the  sequences  identical  to  both 
M.  edulis  and  M.  galloprovincialis.  Since  the  sense  primer. 
Me  13.  corresponds  to  a  part  of  the  nonrepetitive  region 
and  the  antisense  primer.  Me  14,  to  a  part  of  the  3'  un- 
translated region,  the  whole  repetitive  region  is  amplified 
by  PCR.  AI.  edulis.  M.  galloprovincialis,  and  M.  trossulus 
were  collected  at  Delaware,  Kamaishi,  and  Juneau,  re- 
spectively. These  sampling  points  are  known  to  be  "pure 
sites"  at  which  no  other  species  of  the  M.  edulis  complex 
is  found  (McDonald  el  at.,  1991).  We  analyzed  8,  16,  and 
8  individuals  of  AI.  edulis,  M  galloprovincialis,  and  M. 
trossulus,  respectively,  using  primers  Me  13  and  Me  14. 
Since  the  repetitive  region  is  relatively  long  and  highly 
repetitive,  it  was  difficult  to  amplify  the  whole  repetitive 
region  if  the  template  DNA  was  insufficiently  pure  and 
long,  but  prominent  bands  were  successfully  obtained  by 
using  well-purified,  high  molecular  weight  DNA.  Typical 
results  are  shown  in  Figure  2.  Sizes  of  the  band  ranged 
from  2.2  to  2.8  kb.  The  fragments  obtained  from  M.  edulis 
were  generally  larger  than  those  of  the  other  two  species. 
The  sizes  of  bands  in  M.  trossulus  and  M.  galloprovincialis 
were  similar  but,  on  average,  the  former  were  slightly 
larger.  Many  individuals  had  two-banded  (heterozygous) 


patterns,  as  expected  for  a  highly  variable  polymorphism. 
One  sample  of  M.  galloprovincialis  exhibited  three  bands, 
which  may  be  a  naturally  occurring  triploid  or  a  mosaic 
individual  that  possesses  a  cell  lineage  having  the  differed 
length  of  foot  protein  1  gene.  It  is.  however,  also  possible 
that  the  third  band  is  a  heteroduplex  of  two  different  frag- 
ments. 

Variation  in  the  nonrepetitive  region 

Another  set  of  primers.  Me  15  and  Me  16,  was  also 
prepared  to  amplify  a  part  of  the  nonrepetitive  region 
using  sequences  perfectly  identical  between  AI.  edulis  and 
AI.  galloprovincialis  (Fig.  1).  The  size  of  the  amplified 
fragment  estimated  from  sequence  data  previously  re- 
ported (Filpula  ft  a/..  1990)  is  180  bp  in  AI.  edulis.  In  M 
galloprovincialis,  the  expected  size  is  126  bp  because  the 
sequence  of  M.  galloprovincialis  contains  a  deletion  of  18 
amino  acids  (Fig.  1;  see  also  Inoue  and  Odo,  1994).  Using 
these  primers,  8,  32,  and  16  individuals  of  M.  edulis,  M. 
galloprovincialis,  and  M.  trossulus — including  the  same 
samples  used  in  the  analysis  of  the  nonrepetitive  region — 
were  examined.  PCR  analysis  indicated  that  all  samples 
exhibited  a  single  band.  Representative  results  are  shown 
in  Figure  3.  The  position  of  the  band  was  uniform  in  each 
species  but  differed  from  species  to  species.  The  size  of 
the  amplified  fragments  of  M.  edulis  and  M.  gallopro- 
vincialis estimated  by  mobility  in  agarose  agreed  with 
those  expected.  Fragments  from  M.  trossulus  were  shorter 
than  those  of  M.  edulis  but  longer  than  those  of  M.  gallo- 
provincialis. To  determine  the  length  and  sequence  of  the 
amplified  fragment  of  M.  trossulus.  the  band  obtained 
from  one  sample  (Fig.  3,  Lane  3)  was  isolated  and  se- 


M    1 


6  M 


Figure  3.  Representative  results  of  amplification  of  the  nonrepetitive 
region  of  the  adhesive  protein  gene.  Amplified  products  were  electro- 
phoresed  on  4.8%  NuSieve  GTG  agarose  gel  (FMC).  Lanes  I  and  2. 
Mvtili.\  L'llitlfi.  lanes  3  and  4,  M.  trosxidiix:  lanes  5  and  6,  M.  gallopro- 
riiit'iuliy  M,  molecular  marker  (pUC!9  DNA  digested  with  7/<//>II). 


MUSSEL  ADHESIVE  PROTEIN  GENES 


373 


10  20  30  40  50  60 
CCAGTATACAAACCTGTGAAGACAAGTTATTCGTCACCATATAAACCACCAACATACCAA 
PVYKPVKTSYSSPYKPPTYQ 

70  80  90  100  110  120 
CCACTCAAAAAGAAACCGATGGACTATAATAGTTCTCCGCCAACATATGGATCAAAGACA 
PLKKKPMDYNSSPPTYGSKT 

130       140       150       160 
AACTATCTTGCAAAGAAGCTGTCATCTTACAAACCTATTAAGACAACA 
NYLAKKLSSYKPIKTT 

Figure  -4.     Nucleotide  and  deduced  arnino  acid  sequences  of  the  fragment  amplified  from  the  genomic 
DNA  of  Mytilis  lrossnlns  using  primers  Me  15  and  Me  16.  Underlined  sequences  were  derived  from  pnmers. 


quenced.  The  sequenced  fragment  including  the  primer 
sequences  was  168  bp,  12  bp  shorter  than  the  correspond- 
ing region  of  .I/,  eciulis  (Fig.  4).  This  difference  is  small 
but  measurable  by  mini-electrophoresis,  as  shown  in  Fig- 
ure 3.  The  nucleotide  sequence  of  the  amplified  fragment 
was  compared  with  corresponding  sequences  of  M.  cdulis 
and  M.  galloprovincialis  (Fig.  5).  Since  the  region  shown 
in  Figure  5  was  especially  variable  among  the  three  species, 
this  region  is  hereafter  referred  to  as  the  "variable  region." 
It  seems  that  variation  among  the  three  species  was  caused 
by  deletion  or  addition  of  short  sequences,  not  by  base 
substitutions. 

The  variable  region  of  mussels  cultured  in  Brittany, 
those  cultured  on  the  Mediterranean  coast  of  France, 
and  the  wild  mussels  collected  at  Tromso  and  Hiura 
were  also  examined  by  PCR  using  Me  15  and  Me  16. 


Four  individuals  were  examined  in  each  group  and  rep- 
resentative results  are  shown  in  Figure  6.  All  the  wild 
Tromso  mussels  and  all  the  cultured  Brittany  mussels 
exhibited  the  180-bp  fragment  and  were  identified  as 
M.  cdulis.  All  the  cultured  Mediterranean  mussels  and 
the  wild  mussels  from  Shimizu  exhibited  a  126-bp  frag- 
ment and  were  identified  as  M.  galloprovincialis.  These 
results  are  consistent  with  the  distribution  map  of  mus- 
sels that  was  made  by  using  allozyme  characters 
(McDonald  et  a/.,  1991).  Eight  wild  mussels  of  Hiura 
were  also  examined  using  Me  1 5  and  Me  1 6.  Two  bands, 
126  and  168  bp.  were  amplified  from  six  individuals 
(Fig.  6).  but  only  the  126-bp  band  was  amplified  from 
the  remainder  (data  not  shown).  Hiura  is  on  Hokkaido 
Island,  where  both  native  M.  trossiilus  and  introduced 
M.  gtilloprovincicilix  are  distributed.  The  mussels  that 


Mt  :    CAAGTTATTCGTCACCATATAAACCACCAACATACCAACCACTCAAAAAG 
Me  •    ***********(]************************************** 


******** 


Mt:  AAACCGATGGACTATAATAGT-  -TCTCCGCCAACATATGGATC 
Me:  ***  —  G****  ****CG*CC*ACGAAAAGT*A***  *************** 
Mg:  ---------------  CA*COACGAATAGT*A***  *************** 


Mt:    AAAGACAAACTAT-         --CTTGCAAAGAAGCTGTCA 

Me;       *************£y/\££/\** **************** 

Mg: 

Figure  5.  Comparison  of  nucleotide  sequences  of  the  variable  region  of  the  adhesive  protein  genes  of 
Mvtili*  //mwi/Kv  M.  IY/M//.S.  and  M  galloprovincialis.  Sequences  of  M.  eilulis  and  M  galloprovincialis  were 
according  to  Filpula  et  al  (1990)  and  Inoue  and  Odo  (1994),  respectively.  Asterisks  indicate  nucleotides 
identical  to  those  of  the  M.  (ri»s\»/in  sequence.  Hyphens  indicate  gaps  inserted  to  align  sequences.  Mt.  M 
trossiilus:  Me.  M  cdulis:  Mg.  M.  galloprovincialu 


374 


K.  INOUE  ET  AL 


M1   2345678  9101112M 


190- 

147- 
111- 


Figure  6.  Amplification  of  the  variable  region  of  the  adhesive  protein 
gene  of  wild  and  cultured  mussels.  Amplified  products  were  electropho- 
resed  on  4.8%  NuSieve  GTG  agarose  gel  (FMC).  Lanes  I -3,  wild  mussels 
collected  at  Tromso;  Lanes  4-6,  mussels  cultured  in  Bnttans ;  Lanes  7- 
9.  wild  mussels  collected  at  Hiura:  Lanes  10-12.  mussels  cultured  on 
the  Mediterranean  coast  of  France.  M.  molecular  marker  (pUC19  DNA 
digested  with  Hapll). 


exhibited  two  fragments  are  presumed  to  be  hybrids 
between  the  two  species. 

Discussion 

Among  the  five  species  of  the  genus  Mytilus.  M.  edulis. 
M.  galloprovincialis,  and  M.  trossulm  have  been  called 
the  "M.  edulis  complex.  "  Since  they  are  morphologically 
similar  and  shell  shape  is  often  influenced  by  local  envi- 
ronment, it  is  difficult  to  identify  these  species  by  mor- 
phological characteristics.  Recently,  allozyme  characters 
have  been  used  to  clarify  the  taxonomy  of  these  species 
(Koehn?/a/.,  1984;  McDonald  and  Koehn,  1988;Varvio 
etui,  1988;  McDonald  el  al.  1 99 1 ;  Coustau  el  at. .  1991: 
Viard  et  al..  1994).  These  characters  are  recognized  as 
reliable  markers,  but  data  for  multiple  loci  are  required 
for  accurate  identification  of  all  three  species.  Identifica- 
tion using  mitochondria!  DNA  (mtDNA)  sequences  has 
also  been  described  (Edwards  and  Skibinski,  1987;  Blot 
ft  al..  1990;  Geller  et  al..  1993,  1994).  Although  such 
attempts  were  partially  successful,  it  is  still  difficult  to  dif- 
ferentiate the  three  species  unambiguously.  In  this  study, 
we  found  that  differences  in  a  certain  "variable  region" 
of  a  sequence  in  the  nonrepetitive  domain  of  the  foot 
protein  1  agree  well  with  the  taxonomic  rank  of  species. 
It  was  also  shown  that  the  variations  can  be  attributed  to 
differences  in  the  length  of  the  fragments  amplified  by 
PCR.  Thus  the  variable  region  may  become  an  effective 
diagnostic  marker.  Because  PCR  requires  only  a  small 
amount  of  DNA  as  a  template,  the  method  may  be  used 
for  larvae  or  young  individuals  that  are  too  small  for 
analysis  by  other  methods.  In  addition,  the  PCR  system 
seems  effective  for  the  identification  of  hybrids  within  the 
Al.  edulis  complex — we  could  detect  hybrids  that  have 
the  haplotypes  of  both  M.  xalloproviticialix  and  Al.  tros- 


sulns  (Fig.  6).  This  system  may  become  a  powerful  tool 
for  studying  the  distribution  and  genetics  of  mussels,  one 
of  the  most  cosmopolitan  of  marine  animals. 

We  also  tried  to  amplify  the  adhesive  protein  gene  of 
Al.  conisciis,  a  mussel  species  native  to  Japan,  but  we 
were  unsuccessful  (data  not  shown).  The  adhesive  protein 
gene  sequence  of  M.  comscus  may  be  considerably  dif- 
ferent from  those  of  species  in  the  Al.  edulis  complex.  It 
seems  that  other  primers  are  required  for  analysis  of  mus- 
sels other  than  the  Al.  edulis  complex. 

In  contrast  to  the  nonrepetitive  domain,  the  length  of 
the  repetitive  domain  was  highly  variable,  even  in  samples 
collected  at  the  same  site.  This  result  is  consistent  with 
the  fact  that  repeat  patterns  observed  in  three  nucleotide 
sequences  encoding  foot  protein  1  of  Al.  edulis  reported 
separately  (Strausberg  et  al..  1989:  Filpula  et  al..  1990; 
Laursen,  1 992)  differ  from  one  another.  Because  the  length 
of  this  region  was  partially  overlapping  among  species,  it 
seems  inappropriate  for  use  as  a  marker  of  species.  Many 
individuals  of  the  three  species  also  have  two  or  more 
fragments  of  different  length.  These  results  suggest  that 
each  species  has  enormous  variability  in  the  patterns  of 
repetitive  sequences.  Considering  that  foot  protein  1  is  a 
key  molecule  for  adhesion  of  mussels  and  thus  is  essential 
for  their  survival,  its  extensive  diversity  is  intriguing.  We 
suggest  that  the  repetitive  domain  has  been  differentiated 
by  a  "shuffling"  of  the  repeat  pattern  as  well  as  by  the 
common  base  substitution  process  (Inoue  et  al..  1995b). 
Increased  diversity  may  have  an  important  role  in  mussel 
survival  or  evolution. 

Acknowledgments 

The  authors  express  their  sincere  thanks  to  Dr.  Shigetoh 
Miyachi  for  support  in  this  study,  to  Drs.  Shigeru  Nakao 
and  Takashi  Noda  for  valuable  advice,  to  Hiroyuki  K.a- 
wahara  for  collecting  mussels,  and  to  Sachiko  Dobashi 
for  technical  assistance.  This  work  was  performed  as  a 
part  of  the  Industrial  Science  and  Technology  Frontier 
Program  supported  by  New  Energy  and  Industrial  Tech- 
nology Development  Organization. 

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of  the  Mytilus  eilnlis  complex  in  the  North  Atlantic  region.  Mar 
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Viard,  F.,  B.  Delay,  C.  Coustau,  and  F.  Renaud.  1994.  Evolution  of 
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Waite,  J.  H.  1983.  Evidence  for  a  repeating  Dopa  and  hydroxyproline 
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Biol  Chem  258:  2911-2915. 

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Reference:  Bin/  Bull  189:  376-381).  (December,  1995) 


Flow  Cytometric  Analysis  of  Molt-Related  Changes  in 
Hemocyte  Type  in  Male  and  Female  Penaeus  japonicus 


TERESA  SEQUEIRA1,  MANUEL  VILANOyA2,  ALEXANDRE  LOBO-DA-CUNHA3. 
LUIS  BALDAIA1,  AND  MARIO  ARALA-CHAVES2 

^Laboratory  of  General  Physiology,  ^Laboratory  of  Immunology,  ^Laboratory  of  Cellular  Biology  of 
the  "Instituto  de  Ciencias  Biomedieas  Abel  Salazar.  "  Porto,  Portugal 


Abstract.  Hemocyte  cell  suspensions  obtained  from 
male  and  female  Penaeus  japonicus  were  individually 
analyzed  by  flow  cytometry  through  forward  and  side 
light-scatter  parameters.  The  hemocyte  cell  suspensions 
were  further  characterized  after  cell  sorting.  This  type  of 
cell  analysis  has  several  advantages  over  microscopy 
techniques.  After  staining  with  phenoloxidase  and  per- 
oxidase,  the  hemocytes  were  classified  into  the  three 
classic  categories  of  hyaline,  semigranular,  and  granular 
cells.  Significant  cyclic  differences  were  detected  among 
the  molting  stages  in  both  sexes.  The  hyaline  cell  pop- 
ulation was  predominant  before  and  soon  after  the  molt, 
decreasing  over  the  intermolt.  This  decrease  was.  how- 
ever, more  prolonged  in  females.  Thus,  the  hyaline  cell 
population  was  dominant  in  stages  B,  DO.  and  Dl  in 
males  and  only  in  stages  B  and  Dl  in  females.  Semi- 
granular  cells  became  predominant  in  females  during 
the  DO  stage. 

Introduction 

Most  crustaceans  molt  throughout  their  lifetimes,  and 
the  periodic  replacement  of  the  cuticle  is  intrinsically 
linked  with  their  physiology.  Although  their  exoskeleton 
forms  a  structural  and  chemical  barrier  to  parasites,  they 
still  need  an  efficient  internal  immune  system  to  deal  with 
microorganisms  that  might  enter  the  hemocoel  during 
ecdysis  or  through  wounds,  alimentary  tract,  or  gills.  This 


Received  25  April  1995:  accepted  21  September  1995. 

Address  for  correspondence:  Prof.  Mario  Arala-Chaves,  Laboratory 
of  Immunology.  Instituto  de  Ciencias  Biomedicas  Abel  Salazar,  Largo 
Prof.  Abel  Salazar.  2,  4  050  Porto,  Portugal. 

Ahhri'vialionx.  FSC,  forward  scatter;  SSC.  side  scatter;  H,  hyaline;  SG, 
semigranular;  G,  granular;  proPO,  prophenoloxidase  activating  system. 


defense  is  largely  based  on  the  activities  of  the  hemocytes 
(Soderhall  and  Cerenius,  1992).  Three  types  of  circulating 
hemocytes  can  be  distinguished  on  the  basis  of  morpho- 
logical criteria  and  different  staining  techniques  (Bauchau, 
198 1 ).  and  were  recently  found  to  have  different  functions 
(Soderhall  el  ai.  1990:  Barracco  et  at,  1991 ).  It  is  known 
that  hemocytes  are  affected  by  microorganisms  (De 
Backer.  1 96 1 ;  Bang,  1971),  but  few  data  are  available  on 
hemocyte  kinetics,  particularly  throughout  the  molt  cycle 
(Bauchau  and  Plaquet.  1973;  Tsing  et  at,  1989).  More- 
over, the  available  morphological  descriptions  are  not 
completely  satisfactory  because  they  are  based  on  fixed 
cells  or  cells  attached  to  an  artificial  substrate,  resulting 
in  a  high  percentage  of  unclassifiable  cells. 

Flow  cytometry  is  a  powerful  method  of  cell  analysis 
because  quantitative  multiparameter  measurements  on 
statistically  large  numbers  of  individual  cells  can  be  made 
without  the  necessity  to  pool  cells  from  different  individ- 
uals. Moreover,  flow  cytometry  yields  a  large  number  of 
selected  cells  in  a  relatively  short  time.  Furthermore,  au- 
tomation avoids  much  of  the  subjectivity  inherent  in  mi- 
croscopy. Therefore,  flow  cytometric  analysis  has  been 
used  in  invertebrates  to  detect  the  DNA  content  evolution 
in  nematodes  (Hoshino  et  at,  1991),  molluscs  (Elston  et 
at,  1990;  Gerard  et  at,  1994),  insects  (Marescalchi  et  at. 
1990),  and  insect  cell  lines  (Odier  et  at.  1993).  This 
method  has  also  been  used  to  evaluate  hemocyte  prolif- 
eration upon  antigenic  stimulation  in  cockroaches  (Ryan 
and  Karp.  1993)  and  to  investigate  feeding  behavior  in 
molluscs  through  measurements  of  plankton  cell  size 
(Baldwin.  1991). 

In  this  study,  we  used  flow  cytometry  to  analyze  the 
hemocyte  kinetics  of  a  cultured  shrimp,  Penaeus  japon- 
icus, throughout  its  molt  cycle. 


376 


FLOW  CYTOMETRY  OF  SHRIMP  HEMOCYTES 


377 


Materials  and  Methods 


Shrimps 


Penaeid  shrimp  for  this  study  were  bred  in  Eurodaqua, 
Algarve,  Portugal.  Males  (71  animals,  15-25#)  and  fe- 
males (83  animals,  20-30  g)  of  Penaeus  japonicus  were 
maintained  in  a  closed  system  tank  at  12-h  light/ 12-h 
dark,  3.5%  salinity,  and  20°  ±  2°C  and  were  fed  a  mixture 
of  fresh  mussels  and  squid  three  times  a  week.  All  shrimp 
were  acclimated  at  least  1  month  prior  to  use.  The  molting 
stages  were  determined  according  to  Smith  and  Dall 
(1990). 

Collection  of  hemolymph  and  preparation  of  cell 
suspensions 

From  each  individual.  0.1-0.3  ml  of  hemolymph  was 
collected  by  insertion  of  a  needle  syringe  into  the  peri- 
cardial  cavity.  The  hemolymph  was  directly  withdrawn 
into  the  syringe  containing  0. 1  ml  of  anticoagulant  buffer 
0.2  M  N-ethylmaleimide  in  3%  NaCl  at  4°C  (Martin  et 
a/..  1991).  Shrimp  were  bled  at  the  same  time  of  day  to 
avoid  possible  variations,  caused  by  endogenous  rhythm, 
in  the  hemocyte  populations.  For  cell  size  and  density 
analyses,  the  hemolymph  was  mixed  with  1  ml  of  3.5% 
NaCl.  Cells  were  washed  (spun  down  at  167  X  g  for  10 
min)  and  resuspended  in  3.5%  NaCl  supplemented  with 
5%  fetal  calf  serum.  For  cytochemistry,  the  hemolymph 
was  drawn  into  a  syringe  containing  0.5  ml  of  1%  glutar- 
aldehyde,  1%  saccharose  in  sodium  cacodylate  buffer 
(0.2  M.  pH  7.0).  The  cells  were  fixed  for  15  min  at  4°C, 
washed  once  in  the  cacodylate  buffer  and  twice  in  the 
Tris-HCl  buffer  (0.1  M,  pH  7.0)  at  167  X  g  for  10  min. 

Flow  cytometry  and  sorting 

Side-  and  forward-scatter  parameters  (SSC  and  FSC) 
were  used  for  determination  of  cell  granularity  and  cell 
size,  respectively.  SSC  and  FSC  analyses  were  conducted 
in  a  FACScan  analyzer  (Becton-Dickinson,  Mountain 
View,  CA)  with  a  Hewlett  Packard  computer  (HP900) 
equipped  with  the  LYSYS  II  analysis  program  (Becton 
Dickinson).  As  cells  pass  through  the  focused  laser  beam, 
light  is  scattered  in  all  directions.  The  amount  of  light 
scattered  at  narrow  angles  to  the  axis  (FSC  parameter)  of 
the  laser  beam  is  proportional  to  the  cell  size.  The  laser 
light  scattered  at  right  angles  (SSC  parameter)  relates  to 
the  granularity  or  interior  structure  of  the  cell.  Dead  cells 
were  excluded  by  propidium  iodide  (PI)  incorporation. 
PI  is  a  small  molecule  that  binds  to  nucleic  acids;  it  is 
very  effectively  excluded  by  cells  with  intact  cell  mem- 
branes, but  dead  cells  become  strongly  fluorescent  and 
thus  are  easily  distinguished.  At  least  10,000  cells  per 
sample  were  always  analyzed.  Cell  sorting  was  performed 


in  a  FACSsort  cell  sorter  (Becton-Dickinson).  About 
50,000  cells  of  each  population  were  analyzed  with  the 
LYSYS  II  program. 

Hemocyte  cytochemistry 

The  presence  of  the  prophenoloxidase  system  (proPO) 
in  the  hemocytes  was  determined  by  the  method  of  Hose 
et  al.  (1987),  incubating  the  cells  in  L-dopa  (dihydroxy- 
phenylalanine,  1  mg/ml  in  phosphate  buffer  0.1  M.  pH 
7.4)  for  16  h  at  room  temperature  and  then  examining 
them  by  light  microscopy.  The  hemocyte  peroxidase  ac- 
tivity was  determined  by  the  method  of  Fahimi  (1979). 
Thus  the  hemocytes  were  incubated  for  3  h  at  30°C  in 
DAB  (3.3-diaminobenzidine  tetrahydrochloride  in  Tris- 
HCl  buffer  0.1  M.  pH  7.0)  with  0.003%  H2O2.  The  cells 
were  examined  by  light  microscopy.  Control  incubations 
were  performed  with  0.01  M  sodium  azide  and  0.05  M 
triazol. 

Results 

Identification  of  hemocyte  cell  populations  by  flow 
cytometry 

As  shown  in  Figure  1 ,  three  hemocyte  cell  populations 
can  be  identified  in  individual  male  and  female  P.  japon- 
icus when  FSC  and  SSC  parameters  are  used  to  indicate 
different  cell  size  and  granularity.  Hemocytes  were  ana- 
lyzed during  stages  B,  C,  DO,  and  Dl.  After  sorting  and 
cytochemically  staining  each  population  and  analyzing  at 
least  1 50  cells  per  sample,  it  was  possible  to  identify  the 
three  basic  crustacean  cell  types.  The  cell  population  with 
low  SSC  and  FSC  parameters  was  considered  to  be  of  the 
hyaline  (H)  cell  type  because  98%  ±  2%  of  these  cells  were 
both  proPO  and  peroxidase  negative  (Fig.  2a,  b).  The  cell 
population  with  the  higher  FSC  and  usually  with  an  in- 
termediate SSC  parameter  was  considered  to  be  semi- 
granular  (SG)  because  the  cells  were  92%  ±  3%  proPO 
positive  and  85%  ±  4%  peroxidase  negative  (Fig.  2c,  d). 
The  cell  population  with  the  higher  SSC  and  with  FSC 
similar  to  H  cells  was  considered  to  be  of  the  granular  (G) 
cell  type.  Indeed,  strong  phenoloxidase  activity  was  de- 
tected in  this  cell  population,  which  was  96%  ±  4%  positive 
for  proPO  and  83%  ±  7%  positive  for  peroxidase.  Per- 
oxidase activity  was  confined  to  the  granules  and  was  ob- 
served only  in  these  cells  (Fig.  2d,  e).  Furthermore,  the 
intensity  of  this  reaction  could  be  decreased  by  incubation 
in  sodium  azide  or  triazole,  as  was  reported  by  Lanz  et 
ul.  (1993)  (data  not  shown). 

Changes  in  relative  percentages  ofH,  SG,  and  G  cells 
during  the  molting  cycle 

As  shown  in  Figure  3  and  Table  I,  marked  molt-related 
changes  of  the  pool  size  of  the  three  hemocyte  cell  pop- 


378 


T.  SEQUEIRA  KT  AL 


SSC 


Figure  I.  An  example  of  flow  eytometne  analysis  of  fresh  circulating 
hemocytes  from  Pcmu'itx  iiiponiciix  analyzed  hy  forward  scatter  (FSC). 
indicating  cell  size:  and  side  scatter  (SSC),  indicating  cell  granularity  and 
structure.  Frequency  histograms  of  FSC  (A),  SSC  (B).  and  a  dot  plot  of 
both  parameters  (C)  are  shown.  Hyaline,  semigranular.  and  granular  cell 
populations  are  designated  H,  SG.  and  G,  respectively. 


termolting  stage  in  males,  and  the  number  of  SG  cells  is 
constant  over  the  molting  and  intermolting  stages. 

Discussion 

These  flow  cytometric  results  are  in  agreement  with  the 
general  view  that  three  circulating  hemocyte  populations 
are  present  in  most  crustaceans  (Bauchau,  1981).  Thus 
one  of  the  three  sorted  cell  populations  totally  lacked 
proPO  and  was  composed  of  the  smallest  cells.  The  fea- 
tures of  this  cell  population  fit  the  H  cell  category  described 
on  the  basis  of  microscopic  techniques  (Bauchau,  1981; 
Soderhall  and  Smith.  1983;  Lanz  ct  ai.  1993).  Phenolox- 
idase  activity  was  present  in  the  other  two  cell  populations. 


f 


b 


ulations  were  observed  in  both  female  and  male  P.  ja- 
ponicus.  The  patterns  of  distribution  of  the  H.  SG,  and 
G  cell  populations  are  identical  in  both  sexes  during  molt 
stages  B  and  D 1 .  The  H  cells  are  the  most  abundant  (40%- 
44%),  followed  by  the  SG  cells  (31%-32%)  with  the  G 
cells  lowest  (25%-29%).  However,  the  percentages  un- 
derwent a  drastic  change  from  stages  B  to  C  in  cell  type 
H,  SG,  and  G  in  females,  and  in  H  and  G  cell  types  in 
males.  These  changes  are  still  present  over  the  DO  stage, 
but  the  pattern  returned  to  that  found  in  stage  B,  near 
the  end  of  the  cycle  (stage  Dl).  These  intermolting 
changes,  involving  a  decrease  in  the  percentage  of  H  cells 
and  a  subsequent  increase  in  the  percentages  of  SG  and 
G  cells,  are  more  marked  and  more  prolonged  in  females. 
Thus,  the  pattern  of  cell  distribution  observed  in  stage 
DO  is  more  similar  to  that  observed  in  stage  B  in  males 
than  that  in  females.  Moreover,  the  percentage  of  SG  and 
of  G  cells  has  increased  slightly  from  stage  B  to  C  to  the 
same  number  as  the  H  cells  in  females.  In  contrast,  only 
the  G  cell  population  is  increased  slightly  during  the  in- 


f 


.< 


Figure  2.  Light  microscopy  of  fixed  sorted  cell  populations  stained 
with  proPO  (a.  c,  e)  and  with  peroxidase  (b.  d.  f)  revealing  hyaline  (H) 
cells,  proPO  and  peroxidase  negative  (a,  b);  semigranular  (SG)  cells.  proPO 
positive  and  peroxidase  negative  (c.  d):  granular  (G)  cells,  proPO  and 
peroxidase  positive  (e.  f).  Bar  =  10  /jm. 


FLOW  CYTOMETRY  OF  SHRIMP  HEMOCYTES 


379 


FEMALES 


B 


MALES 


Figure  3.  Frequencies  of  hyaline  (H),  semigranular(SG).  and  granular 
(G)  hemocyte  cell  populations  found  in  Pemieus  japonicu\  hemolymph 
at  the  indicated  stages  of  the  molting  cycle  in  females  (A)  and  males  (B). 
Analyses  have  been  performed  with  10,000  cells  from  each  animal;  n 
indicates  the  number  of  animals  used  for  each  value,  which  is  presented 
as  the  mean  and  1  standard  deviation. 


Cells  of  one  of  these  populations  were  classified  as  G  cells 
because  their  peroxidase  activity  was  like  that  previously 
observed  in  similar  cells  in  Procambarus  clarki  (Lanz  et 
at..  1993).  When  examined  by  light  microscopy,  the  G 
cells  appear  slightly  larger  than  the  SG  cells,  but  our  flow 
cytometric  results  indicate  that  this  may  not  be  the  case. 


This  discrepancy  might  result  from  analyzing  fresh  cell 
suspensions  instead  of  the  fixed  hemocyte  smears  used  in 
light  microscopy. 

This  study  provides  some  evidence  that  the  hemocyte 
cell  populations  of  P.  iaponicux  exhibit  sex-related  vari- 
ations associated  with  the  molt  cycle.  In  both  sexes,  the 
relative  percentages  of  H,  SG,  and  G  cells  are  identical 
after  (stage  B)  and  before  (stage  Dl )  ecdysis.  Similar  results 
were  reported  by  Bauchau  and  Plaquet  (1973).  However, 
that  study,  which  relied  on  morphological  observations 
of  hemocyte  smears,  was  not  directly  comparable  because 
SG  and  G  cells  could  not  be  identified  and  were  thus 
counted  together,  and  because  the  samples  were  not  iden- 
tified by  sex.  More  recently,  Tsing  et  al.  (1989)  reported 
that  no  significant  changes  of  the  hemocyte  cell  popula- 
tions occurred  in  P.  japonicus  during  the  molting  stages. 
However,  this  study,  like  the  one  by  Bauchau  and  Plaquet 
( 1973),  pooled  male  and  female  samples  and  was  based 
on  smear  observations;  furthermore,  more  than  50%  of 
the  hemocytes  could  not  be  identified.  It  seems,  then,  that 
flow  cytometric  analysis  is  a  better  and  more  reliable 
method  for  studying  hemocyte  variations  than  is  the  tra- 
ditional technique  of  morphological  observation  of  smears 
(Bauchau  and  Plaquet,  1973:  Tsing  et  al..  1989). 

Molt-related  changes  in  hemocyte  populations  were  also 
demonstrated  in  insects  (Crossley,  1965;  Jones,  1967; 
Hinks  and  Arnold,  1977).  The  information  about  the  role 
of  molting  hormones  in  these  animals  is  not  yet  fully  un- 
derstood, but  20-hydroxyecdysone  is  known  to  induce  a 
significant  increase  in  the  percentage  of  circulating 
phagocytic  cells  in  Calliphora  erythrocephala  (Crossley, 
1968).  Although  insects  have  somewhat  different  hemo- 
cyte types,  the  SG  and  G  hemocytes,  which  may  be 
phagocytic  in  crustaceans  (Hose  et  al.,  1990),  also  in- 
creased in  P.  japonicux  coincidentally  with  elevated  ec- 
dysteroid  liters  described  in  several  crustaceans  (Baldaia 
cial.,  1 984;  Roudy-Cuzin  et  al.,  1989).  Tsing  et  al.  (1989) 
also  observed  an  increase  in  total  hemocyte  count  during 
these  molt  stages.  Ecdysteroid  liters  are  higher  and  increase 
progressively  from  stages  C  to  Dl  in  females.  The  rise  of 
this  hormone  is  considerably  smaller  in  males  and  it  is 
confined  to  Ihe  DO  slage  (Baldaia  el  al..  1984).  Quanli- 


I  able  I 

Abundance  of  hemocyte  types  throughout  the  molting  cycle  <>/  Penaeus  japonicus 


H 


SG 


Females 
Males 


B**  >  C  =  DO**  <  Dl  =  B 
B*  >  C  =  DO  =  Dl  =  B 


DO*  >  C*  >  B  =  Dl 
B  =  C  =  DO  =  Dl 


C  =  DO*  >  B  =  Dl 
C  =  DO*  >  B  =  Dl 


Statistical  analysis  by  paired  Student's  t  test  of  the  differences  found  in  the  hemocyte  cell  populations  throughout  the  indicated  molting  stages. 
Differences  are  classified  as  nonsignificant  at  P  >  0.05  (  =  );  significant  at  P  <  0.05  (*);  and  highly  significant  at  P  <  0.01  (**). 


380 


T.  SEQUEIRA  ET  AL 


tative  differences  in  ecdysteroids  were  also  reported  be- 
tween the  two  sexes  (Baldaia  el  al..  1984;  Roudy-Cuzin 
el  al,  1989);  these  may  explain  the  sex-related  differences 
reported  here. 

Flow  cytometry  is  standardly  used  in  mammalian  he- 
matology  because  it  has  large  advantages  over  light  mi- 
croscopy. The  present  report  indicates  that  this  tool  can 
also  be  used  advantageously  in  the  study  of  invertebrate 
cells  such  as  hemocyte  populations.  In  the  future,  such 
study  may  not  only  focus  on  cell  size  and  granularity,  but 
may  also  include  more  detailed  analysis — for  example, 
the  detection  of  special  cell  markers  using  immunofluo- 
rescence-specific  antibodies  or  the  investigation  of  cell  ac- 
tivation under  various  immunological  stimuli. 

Acknowledgments 

Teresa  Sequeira  was  supported  by  a  grant  from  Junta 
Nacional  de  Investigacao  Cientifica  e  Tecnologica,  Por- 
tugal. 

Literature  Cited 

Baldaia,    L.,    P.    Porcheron,    J.    Ooimbra,    and    P.    Cassier.    198-4. 

Ecdysteroids  in  the  shrimp  Palaemon  .scrra/u.s:  relation  with  molt 
cycle.  Gen.  Comp  Endocrinol  55:  437-443. 

Baldwin,  B.  S.  1991.  Ingestion  of  natural  plankton  by  oyster  larvae: 
the  relative  importance  of  different  cell  size  fractions.  Am  Zoo/.  31: 
7A. 

Bang,  F.  B.  1971.  Transmissible  disease,  probably  viral  in  origin,  af- 
fecting the  amoebocytes  of  the  European  shore  crab  Carcimis  maenas. 
Infect.  Immun.  3:  6 1 7-623. 

Barracco,  M.  A.,  B.  Duvic,  and  K.  Soderhall.  1991.  The  /M.3-glucan- 
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crayfish  granular  cells.  Cell  Tissue  Rex  266:  491-417. 

Bauchau,  A.  G.,  and  J.  C.  Plaquet.  1973.  Variation  du  nombre  des 
hemocytes  chez  les  crustaces  brachyoures.  Cruslaceana  24:  2 1 5-223. 

Bauchau,  A.  G.  1981 .  Crustaceans.  Pp.  386-420  in  Invertebrate  Blood 
Cells  Vol.  2.  Academic  Press,  New  York. 

Crossley,  A.  C.  1965.  Transformations  in  the  abdominal  muscles  of 
the  blue  blow-fly  Calliphora  erythrocephala  (Meig)  during  meta- 
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Crossley,  A.  C.  1968.  The  fine-structure  and  mechanism  of  break  down 
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Elston,  R.  A.,  A.  S.  Drum,  and  S.  K.  Allen,  Jr.  1990.  Progressive  de- 
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Fahimi,  H.  D.  1979.  An  assessment  of  the  DAB  methods  for  cyto- 
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Gerard,  A.,  Y.  Naciri.  J.  M.  Peignon,  C.  Ledu,  P.  Phelipot,  C.  Noiret, 

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Lanz,  H.,  V.  Tsutsumi,  and  H.  Arechiga.  1993.  Morphological  and 
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Marescalchi,  O.,  V.  Scali,  and  M.  Zucotti.  1990.  Genome  size  in  pa- 
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southeastern  Sicily:  A  flow  cytometric  analysis.  Genome  33:  789- 
793. 

Martin,  G.  G.,  J.  E.  Hose,  S.  Omori,  C.  Chong,  T.  Hoodbhoy,  and  N. 
McKrell.  1991 .  Localization  and  roles  of  coagulogen  and  transglu- 
taminase  in  hemolymph  coagulation  in  decapod  crustaceans.  Comp. 
Biochem  Phvsiol  100B:  517-522. 

Odier,  F.,  P.  Vago,  J.  M.  Quiot,  G.  Devauchelle,  and  J.  P.  Bureau.  1993. 
Determination  of  DNA  in  densovirus-infected  invertebrate  cell  line 
by  flow  cytometry.  J  Invenebr.  Pathol.  62:  252-256. 

Ryan,  N.  A.,  and  R.  D.  Karp.  1993.  Stimulation  of  hemocyte  prolif- 
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Roudy-Cuzin,  J.,  C.  Strambi,  A.  Strambi,  and  J.-P.  Delbecque.  1989. 
Hemolymph  ecdysteroids  and  molt  cycle  in  males  and  females  of 
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the  MI-ME  X  organ  of  the  eyestalk.  Gen.  Comp.  Endocrinol  74:  96- 
109. 

Smith,  D.  M.,  and  \V.  Dall.  1990.  Moult  staging  in  the  tiger  prawn 
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Soderhall,  K.,  and  V.  J.  Smith.  1983.  Separation  of  the  haemocyte 
populations  of  Carciiui.s  maenas  and  other  marine  decapods,  and 
prophenoloxidase  distribution.  Dev.  Comp.  Immunol.  1:  229-239. 

Soderhall,  K.,  A.  Aspan,  and  B.  Duvic.  1990.  The  proPO-system  and 
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Soderhall,  K.,  and  L.  Cerenius.  1992.  Crustacean  immunity.  Aniui 
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Tsing,  A.,  J.-M.  Arcier,  and  M.  Brehelin.  1989.  Hemocytes  of  penaeid 
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grams.  J.  Invencbr.  Pathol.  53:  64-67. 


INDEX 


A  settlement  bioassay  assessing  the  response  of  soft  shell  clam  larvae  to 
sediments  from  various  sites  in  Massachusetts  Bay,  240 

A  short  story  ofaequorin,  1 

A  transient  exposure  to  symbiosis-competent  bacteria  induces  light  organ 
morphogenesis  in  the  host  squid.  347 

ABELSON,  AVIGDOR,  see  Micha  Man,  363 

Accumulation  and  retention  of  dimethylsulfoniopropionate  by  bivalve 
molluscs:  high  and  nonnormal  variation.  233 

Accumulation  of  dimethylsulfoniopropionate  in  Geitkensia  Jemima  de- 
pends on  trophic  interactions.  235 

acetylcholine,  207 

Acet\  Icholine-induced  Ca2+  flux  across  the  sarcolemma  of  an  echinoderm 
smooth  muscle.  207 

ACHBERGER,  E.  C,  see  H.  Silverman.  308 

acid  secretion,  219 

Acropora  tormosa,  288 

adhesive  protein,  370 

aequonn.  I 

aequonn  luminescence  in  giant  axon,  208 

AHERN,  JENNY,  JULIE  LYONS,  JAMES  MCCLELLAND,  and  IVAN  VALIELA, 
Invertebrate  response  to  nutnent-induced  changes  in  macrophyte 
assemblages  in  Waquoit  Bay.  241 

AHERN.  JENNY,  see  Julie  Lyons,  255 

AIMES,  RONALD  T.,  JAMES  P.  QUIGLEY,  SNEHASIKTA  SWARNAKAR, 
DUDLEY  K.  STRICKLAND,  and  PETER  B.  ARMSTRONG,  Preliminary 
investigations  on  the  scavenger  receptors  of  the  amebocyte  of  the 
American  horseshoe  crab,  Limulus polyphemus,  225 

ALDERMAN.  DERRICK,  BRIAN  S.  BALSIS,  Ism  D.  BUFFAM.  ROBERT  H. 
GARRITT.  CHARLES  S.  HOPKINSON.  JR..  and  JOSEPH  J.  VALLINO, 
Pelagic  metabolism  in  the  Parker  River/Plum  Island  Sound  estuanne 
system.  250 

ALDERMAN.  DERRICK  W.  M..  see  Brian  Balsis,  252 

amoeba.  198 

amphipod,  244 

Anaphase  spindle  dynamics  under  D:O-enhanced  microtubule  poly- 
merization, 204 

Annual  Report  of  the  Marine  Biological  Laboratory,  Rl 

Antarctic  Polar  Frontal  Zone,  77 

aquatic  pollution,  196 

arachidonic  acid.  203 

ARALA-CHAVES,  MARIO,  see  Teresa  Sequeira.  376 

ARMSTRONG.  PETER  B..  see  Ronald  T.  Aimes,  225;  Snehasikta  Swar- 
nakar.  226 

ascidian,  36 

ATEMA,  J..  see  T.  R.  Consi.  231 

ATEMA.  JELLE,  see  Kevin  Dittmer.  232 

autotrophy  and  heterotrophy.  250 

AVILA,  CONXITA,  and  ALAN  M.  KUZIRIAN,  Natural  diets  for  1/,'i/m-,- 
senda  crassicornis  mariculture,  237 

Axial  specification  in  a  basal  member  of  the  spiralian  clade:  lineal  rela- 
tionships of  the  first  four  cells  to  larval  body  plan  in  the  polyclad 
turbellarian  Hophplana  iin/i/ilniu.  194 


B 


bacterial  culture.  91 

BALDAIA,  Luis,  see  Teresa  Sequeira,  376 


BALSIS,  BRIAN,  DERRICK  W.  M.  ALDERMAN,  ISHI  D.  BUFFAM,  ROBERT 
H.  GARRITT.  CHARLES  S.  HOPKINSON,  JR.,  and  JOSEPH  J.  VALLINO, 
Total  system  metabolism  of  the  Plum  Island  Sound  estuarine  system, 
252 

BALSIS.  BRIAN  R.,  see  Derrick  Alderman,  250 

BARKER,  M.  F..  see  Maeve  S.  Kelly,  91 

BARLOW,  R.  B..  see  C.  L.  Passaglia.  213 

BARLOW.  ROBERT  B..  see  Estela  V.  O'Brien,  212 

BATTELLE.  B.-A..  see  G.  H.  Renmnger,  69 

behavior.  120.  128,  272 

Behavioral  control  of  swash-nding  in  the  clam  Donax  variahilis,  120 

Behavioral  responses  ofConcholepas  concholepas  (Bruguiere,  1 789)  larvae 
to  natural  and  artificial  settlement  cues  and  microbial  films,  272 

BEHR,  PETER  J..  see  Chaka  Drake,  243 

benthic  invertebrates,  245 

benthos,  49 

Bioassay  and  preliminary  characterization  of  ovigerous-hair  stripping 
substance  (OHSS)  in  hatch  water  of  crab  larvae,  175 

biological  variation,  233 

biolummescence,  1,  263,  356 

biomechamcs,  120,  128,  138 

BITTNER,  G.  D.,  see  C.  S.  Eddleman,  218 

BITTNER,  GEORGE  D..  see  Harvey  M.  Fishman,  208 

Bleaching  patterns  of  four  species  of  Caribbean  reef  corals.  298 

BOHRER.  TRAVIS,  AMOS  WRIGHT,  JENNIFER  HAUXWELL,  and  IVAN 
VALIELA,  Effect  of  epiphyte  biomass  on  growth  rate  ofZoslera  ma- 
rina in  estuaries  subject  to  different  nutrient  loading,  260 

BOHRER,  TRAVIS,  see  Amos  Wright,  26 1 

Bolitaenidae,  I  1 3 

BOLONGARO-CREVENNA,  ANDREA,  see  Carlos  Rosas,  168 

Bolryllus  schlosseri,  36 

BOYER,  BARBARA  C..  see  Jonathan  Q.  Henry,  194 

Branchiopoda,  22 

BRANDAN,  ENRIQUE,  see  Sebastian  R.  Rodriguez.  272 

BRAZIK,  DAVID  C.,  and  ROBERT  A.  BULLIS,  The  effect  of  temperature 
on  the  relationship  between  a  ciliated  protozoan,  Trichodina  cot- 
tidarum,  and  the  longhorn  sculpin,  Myoxocephalus  octodecemspi- 
nosus,  239 

BUFFAM,  ISHI  D.,  see  DERRICK  ALDERMAN,  250;  Brian  Balsis,  252 

BULLIS,  ROBERT  A.,  see  David  C.  Brazik,  239 

BUMANN,  DIRK,  Localization  of  digestion  activities  in  the  sea  anemone 
Haliplanella  luciac,  236 

BURGER,  MAX  M.,  see  William  J.  Kuhns,  223 

butter  clams.  229 


Ca2+  ionophore,  201 
caged  cyclic-AMP.  198 
calcein  acetoxymethyl  (AM)  ester.  218 
calcium,  200,  203,  207,  209 
flux,  228 

injury  current,  208 
movement  into  severed  axon,  208 
probe,  1 

CALLAWAY,  DAVID  W.,  IVAN  VALIELA.  KENNETH  FOREMAN,  and  LORI 
SOUCY,  Effects  of  nitrogen  loading  and  salt  marsh  habitat  on  gross 
primary  production  and  chlorophyll  a  in  estuaries  of  Waquoit  Bay. 
254 


381 


382 


INDEX  TO  VOLUME   189 


CAMPOS.  ELISEO  O.,  see  Sebastian  R.  Rodriguez.  272 

CAREFOOT.  THOMAS  H.,  and  DEBORAH  A.  DONOVAN,  Functional  sig- 
nificance of  varices  in  the  muricid  gastropod  Ceraloslmna  folialum, 
59 

CASE,  JAMES  F..  see  Kellie  J.  Heischer.  263 

catalase  and  microtubules.  222 

CCAMLR  (Commission  for  the  Conservation  of  Antarctic  Marine  Living 
Resources).  77 

cell 

division,  204 
fate,  190 
lineage,  192.  194 

cephalopod,  77,  1 13 

Cephalopod  predation  facilitated  by  dinotlagellate  luminescence,  263 

Cephalopods  occupy  the  ecological  niche  of  epipelagic  fish  in  the  Antarctic 
Polar  Frontal  Zone.  77 

Ceralostimm  fohatum.  59 

CHADWICK-FURMAN,  NANETTE  E.,  and  IRVING  L.  WEISSMAN,  Life  his- 
tories and  senescence  ofBotryllits  schlosseri  (Chordata,  Ascidiacea) 
in  Monterey  Bay.  36 

CHAMBERLAIN,  S.  C,  see  G.  H.  Renninger,  69 

CHAPLIN.  SUE  ANN.  CATHERINE  HUNTER  MACGREGOR,  IVAN  VALIELA, 
KENNETH  FOREMAN,  and  LORI  Soucv,  The  effect  of  residential 
and  forested  watershed  land  cover  on  nutrient  loading  to  Hamblm 
and  Jehu  Ponds,  Waquoit  Bay,  Massachusetts,  247 

CHAPLIN,  SUE  ANN,  see  Catherine  Hunter  MacGregor,  248 

Characterization  and  use  of  isolated  toadtish  hepatocytes  for  studies  of 
heme  synthesis  and  utilization.  227 

CHAVEZ.  PAMELA,  see  Sebastian  R.  Rodriguez,  272 

chemoreception,  69 

chemosensory  responses,  69 

chemosensory  sensilla,  69 

Chemotaxis.  aggregation  behavior,  and  foot  formation  in  Dictyostelium 
discoideum  amoeba  controlled  by  microbeam  uncaging  of  cyclic- 
AMP.  198 

chimerism.  106 

chlorophyll  a,  250 

Cladophora  vagabunda,  244 
effects  on  predation.  243 

clam,  120,  128,  138 

cleavage  furrow.  20 1 

Cnideria.  280 

Coexistence  and  possible  parasitism  of  somatic  and  germ  cell  lines  in 
chimeras  of  the  colonial  urochordate  Botryllus  schlosseri,  106 

colonial  ascidian,  29 

coloration,  288 

Commission  for  the  Conservation  of  Antarctic  Marine  Living  Resources 
(CCAMLR),  77 

cone  photoreceptors,  220 

CONSI,  T.  R.,  F.  GRASSO,  D.  MOUNTAIN,  and  J.  ATEMA,  Explorations 
of  turbulent  odor  plumes  with  an  autonomous  underwater  robot. 
231 

contaminated  sediments,  240 

copepod,  42 

coquina,  120,  128 

coral  bleaching,  298 

CORNELL,  NEAL  W.,  MARK  E.  HAHN,  and  HOLLY  A.  MARTIN,  Char- 
acterization and  use  of  isolated  toadfish  hepatocytes  for  studies  of 
heme  synthesis  and  utilization,  227 

cortex,  215 

crab  larvae,  175 

crayfish.  340 

critical  daylength  modified  by  temperature,  42 

crustacean  175.  376 

ctenophore,  356 

cuticle,  29 

cytochalasin  B,  29 

cytochrome  P450.  227 

cytoskeletal  modifications  induced  by  injury.  216 


D 


DACEY,  JOHN  W.  H.,  see  Richard  W.  Hill,  233;  Bradley  A.  White,  235 

DEEGAN,  LINDA  A.,  see  Matthew  C.  Preisser,  242 

DEMAREST,  JEFFERY  R.,  and  JAMES  L.  M.  MORGAN.  Effect  of  pH  buffers 
on  proton  secretion  from  gastric  oxyntic  cells  measured  with  vi- 
brating ion-selective  microelectrodcs,  219 

development.  200,  20 1 .  347 

DEVLIN,  C.  LEAH,  and  PETER  J.  S.  SMITH,  Acetylcholine-induced  Ca2t 
flux  across  the  sarcolemma  of  an  echinoderm  smooth  muscle,  207 

Diagonal  development:  establishment  of  the  anal  axis  in  the  ctenophore 
Mncnuiipsis  Icitlyi,  190 

diapause  timing.  42 

Diaplomiis  xangitineux,  42 

Dicyenni.  81 

DIETZ,  T.  H.,  see  H.  Silverman.  308 

Differences  in  benthic  invertebrate  assemblages  in  two  estuaries  in  Wa- 
quoit Bay  receiving  disparate  nutrient  loads,  245 

digestion  physiology.  236 

dimethyl  sulfide  (DMS).  235 

dimethylsulfoniopropionate.  233 

dinotlagellate.  263 

directional  hearing,  21  I 

Discrimination  among  wave-generated  sounds  by  a  swash-riding  clam. 
128 

dissolved  organic  matter,  256 

disturbance,  49 

DITTMER,  KEVIN,  FRANK  GRASSO,  and  JELLE  ATEMA,  Effects  of  varying 
plume  turbulence  on  temporal  concentration  signals  available  to 
orienting  lobsters.  232 

DMS  (dimethylsulfide).  235 

DNA,  370 

ISSribosomal,  81 

DODGE,  F.  A.,  see  C.  L.  Passaglia,  2 1 3 

dogfish  lens.  222 

Dogfish  (Atiistelwi  ctuiit)  lens  catalase  reduces  H2O^-induced  opacifica- 
tion,  222 

DOING,  JUDITH  A.,  and  MARGARET  J.  MCFALL-NAGAI.  A  transient 
exposure  to  symbiosis-competent  bacteria  induces  light  organ  mor- 
phogenesis in  the  host  squid.  347 

DOM  transport.  1 59 

Donax,  120.  128.  138 

DONOVAN.  DEBORAH  A.,  see  Thomas  H.  Carefoot.  59 

dopamine,  340 

dorsoventral  axis.  192 

dorsoventral  polarity,  194 

DOVE,  SOPHIE  G..  MISAKI  TAKABAYASHI.  and  OVE  HOEGH-GULDBERG, 
Isolation  and  partial  characterization  of  the  pink  and  blue  pigments 
of  pocilloporid  and  acroporid  corals,  288 

DRAKE,  CHAKA,  PETER  J.  BEHR.  and  IVAN  VALIELA,  Effect  of  algal 
cover  on  size-selective  predation  of  Gammants  mucronatus  by  the 
striped  killifish.  Fundulus  majalis,  243 

Drcisseiw  polymorpha,  320 

DUNCAN,  JILL,  see  Peter  P.  Fong,  320 


Echinaracnius  parma,  203 

ecological  niche,  77 

ecology,  49.  246 

EDDLEMAN,  C.  S..  C.  M.  GODELL,  H.  M.  FISHMAN,  M.  TYTELL,  and 

G.  D.  BITTNER,  Fluorescent  labeling  of  the  glial  sheath  of  giant 

nerve  fibers.  2 1 8 
EDDS-WALTON,  PEGGY  L..  and  RICHARD  FAY,  Regional  differences  in 

directional  response  properties  of  afferents  along  the  saccule  of  the 

toadfish  (Opsanus  tan).  2  I  1 
Effect  of  algal  cover  on  size-selective  predation  ofGammarus  mummatiis 

by  the  striped  killifish,  b'undulus  maialis.  243 
Effect  of  changing  plant  morphology  on  invertebrate  susceptibility  to 

predation  in  eelgrass  beds.  242 


INDIA    10  VOl  UMF    1S9 


383 


Effect  of  epiphyte  hiomass  on  growth  rate  of  /.n\icra  martini  in  estuaries 
subject  to  different  nutrient  loading.  260 

Effect  of  exogenous  heat  shock  protein  (hsp70)  on  neuronal  calcium 
lluv  2m 

Effect  of  macroalgal  species  and  nitrogen-loading  rates  on  colonisation 
of  macroalgae  b\  herbivorous  amphipods.  244 

Etl'ect  of  nutrient  enrichment  on  phytoplankton  growth  in  Waquoit  Bav. 
Massachusetts.  258 

Effect  of  pH  buffers  on  proton  secretion  from  gastric  oxyntic  cells  mea- 
sured with  \ibrating  ion-selective  microelectrodes.  219 

Effects  ot  land  use  on  the  degradability  of  dissolved  organic  matter  in 
three  watersheds  of  the  Plum  Island  Sound  Estuary.  256 

Effects  ot  nitrogen  loading  and  salt  marsh  habitat  on  gross  primary  pro- 
duction and  chlorophyll  u  in  estuaries  of  Waquoit  Bay.  254 

Effects  of  varying  plume  turbulence  on  temporal  concentration  signals 
available  to  orienting  lobsters.  232 

egg  actuation.  13 

electron  microscopy  of  transected  axon.  216 

Eledonella  /'vxinaca.  1 13 

ELLERS.  OLAF.  Behavioral  control  of  swash-riding  in  the  clam  Dana\ 
\'aruihih\.  120;  Discrimination  among  wave-generated  sounds  by 
a  svvash-nding  clam.  128:  Form  and  motion  of  f)i»ni.\  \'uruihili.\  in 
ttow.  138 

ELLIOTT,  CAROL  F..  see  Jane  A.  Westfall.  280 

embryo,  200 

embryonic  polarity.  197 

ENCORE.  288 

energetics.  1 59 

enrichment  experiment,  257 

environmental  studies.  246 

ephemeral  pools.  22 

epipelagic  fish.  77 

epiphyte,  260,  261 

Escherichia  coli.  308 

ESCOBAR,  ELVA,  see  Carlos  Rosas,  168 

estuary.  241,  242.  243.  244.  245,  247,  248,  250.  252.  254.  255.  256.  258. 
260.  261,  262 

Euprymna.  263.  347 

eutrophication.  241,  255 

evolution  ol  development.  194 

Existence  ot  three  mechanisms  for  blocking  polyspermy  in  oocytes  of 
the  mussel  Mylilus  edulis.  330 

Explorations  of  turbulent  odor  plumes  with  an  autonomous  underwater 
robot.  231 


FASZEWSKI,  ELLEN,  see  Joseph  G.  Kunkel,  197 

FA>  ,  RICHARD,  see  Peggy  L.  Edds-Walton,  21 1 

feeding.  363 

fertilization.  320.  330 

filter  feeding.  308 

fine  structure  of  spermatozoa.  6 

Fine  structure  of  spermatozoa  of  the  hagfish  Eplairctus  burden (Agnatha). 
6 

FINGERMAN.  MILTON,  see  Rachakonda  Sarojini.  340 

fish  ear.  2 1  I 

fisheries  management.  246 

FISHMAN,  H.  M.,  see  J.  Metuzals.  216:  C.  S.  Eddleman.  218 

FISHMAN,  HARVEY  M.,  TODD  L.  K.RAUSE,  ANDREW  L.  MILLER,  and 
GEORGE  D.  BITTNER.  Retardation  of  the  spread  of  extracellular 
Ca2+  into  transected,  unsealed  squid  giant  axons.  208 

FITT.  WILLIAM  K..  and  M.  E.  WARNER,  Bleaching  patterns  of  four  species 
of  Caribbean  reef  corals.  298 

FLEISCHER,  K.ELLIE  J.,  and  JAMES  F.  CASE,  Cephalopod  predation  fa- 
cilitated by  dinoflagellate  luminescence,  263 

Flow  cytometric  analysis  of  molt-related  changes  in  hemocyte  type  in 
male  and  female  Pcnacux  jupimicu*.  376 

flow-cytometry,  376 

Fluorescent  labeling  of  the  glial  sheath  of  giant  ner\e  fibers.  218 


Flutter-like  response  in  visual  cortex  of  the  semi-isolated  turtle  brain. 
215 

Foliar  release  of  ammonium  and  dissolved  organic  nitrogen  by  Spuniini 
altemiflora,  262 

FONG,  PFTFR  P..  K.EIICHIRO  KVOZHKA,  JILL  DUNCAN.  STACY  RYN- 
KOWSKI,  DANIEL  MEKASHA.  and  JEFFREY  RAM.  The  effect  of  sa- 
linity and  temperature  on  spawning  and  fertilization  in  the  zebra 
mussel  Dn'ivii'nu  polymorpha  (Pallas)  from  North  America,  320 

foot  protein.  370 

FOREMAN,  KENNETH,  see  Rafael  Sarda,  245;  Sue  Ann  Chaplin.  247: 
David  W.  Callaway.  254;  Cecelia  C.  Sheridan,  258 

Form  and  motion  of  DUIKIX  variahilix  in  flow.  138 

frequency  tuning.  213 

freshwater  bivalve,  308 

fringing  salt  marsh,  255 

FUKUI,  YOSHIO,  and  SHINYA  INOLIE,  Chemotaxis,  aggregation  behavior, 
and  foot  formation  in  Dictyostelium  dixandcum  amoeba  controlled 
by  microbeam  uncaging  of  cyclic-AMP,  198 

functional  morphology.  59 

Functional  significance  of  varices  in  the  muricid  gastropod  Ceralitxioma 
lolialitm.  59 

t'undutus  majali'i.  243 

FURUYA.  HIDETAKA,  see  Tomoe  Fialavama.  81 


GARRITT.  ROBERT  H.,  see  Derrick  Alderman.  250;  Brian  Balsis,  252 

gastric  cavity,  236 

GAXIOLA.  GABRIELA,  see  Carlos  Rosas,  168 

GERSHON,  HARRIET,  see  Zeev  Pancer,  106 

GLAS,  PATRICIA  S.,  JEFFREY  D.  GREEN,  and  JOHN  W.  LYNN,  Oxidase 

activity  associated  with  the  elevation  of  the  penaeoid  shnmp  hatching 

envelope.  13 

GLEESON,  R.  A.,  see  G.  H.  Renninger.  69 
glial  cytosolic  marker.  2 1 S 
glial  layer  thickness,  218 
GODELL,  C.  M.,  see  C.  S.  Eddleman,  218 
Gracilaria  likrahiae.  244 
graphical  model.  42 
GRASSO.  F..  see  T.  R.  Consi.  231 
GRASSO,  FRANK,  see  Kevin  Dittmer.  232 
GREEN.  JEFFREY  D.,  see  Patricia  S.  Glas,  13 
GRIMMELIKHUIJZEN.  CORNELIS  J.  P.,  see  Jane  A.  Westfall.  280 
groundwater.  247.  248 
growth.  36 
Growth  of  epiphytes  on  /mlci\i  iinirnni  in  estuaries  subject  to  different 

nutrient  loading.  261 

H 

HVK+-ATPase,  219 

HADDOCK,  STEVEN  H.  D..  and  JAMES  F.  Case.  Not  all  ctenophores  are 
bioluminescent:  Plciirohrtichia.  356 

hagfish.  6 

HAHN,  MARK  E.,  see  Neal  W.  Cornell,  227 

HAIRSTON,  NELSON  G.,  JR.,  and  COLLEEN  M.  KEARNS,  The  interaction 
of  photopenod  and  temperature  in  diapause  timing:  a  copepod  ex- 
ample. 42 

HARAYAMA,  SHIGEAKI.  see  Koji  Inoue.  370 

HAROSI,  FERENC  I.,  see  Inigo  Novales  Flamarique.  220 

HARTLEY,  WILLIAM,  see  Merle  Mizell,  196 

hatch  water,  1 75 

hatching  envelope.  13 

HAUXWELL,  JENNIFER,  see  Nicole  Martinez,  244;  Travis  Bohrer,  260: 
Amos  Wright,  261 

heat  shock  protein,  209 

heavy  water,  204 

HECK,  DIANA  E..  see  Walter  Troll.  200 

heme  synthesis,  227 

hemocvte.  376 


384 


INDEX  TO  VOLUME   189 


HENRY,  JONATHAN  Q.,  and  MARK  Q.  MARTINDALE,  The  experimental 
alteration  of  cell  lineages  in  the  nemertean  Cerebratulus  /acteus: 
implications  for  the  precocious  establishment  of  embryonic  axial 
properties,  192 

HENRY,  JONATHAN  Q.,  MARK  Q.  MARTINDALE,  and  BARBARA  C. 
BOYER,  Axial  specification  in  a  basal  member  of  the  spiralian  clade: 
lineal  relationships  of  the  first  four  cells  to  larval  body  plan  in  the 
polyclad  turbellarian  Hop/opium/  iiu/uiHna,  194 

HENRY,  JONATHAN  Q..  see  Mark  Q.  Martindale,  190 

hermatypic,  288 

Hermissenda,  237 

HERZOG,  E.  D..  see  G.  H.  Renninger,  69 

hexachlorobenzene  heart  defects,  1 96 

HILL,  DAVID  K.  D.,  see  Richard  W.  Hill,  233 

HILL,  RICHARD  W..  JOHN  W.  H.  DACEY,  DAVID  K.  D.  HILL,  JUDITH 
E.  MCDOWELL,  and  DALE  F.  LEAVITT,  Accumulation  and  retention 
of  dimethylsulfoniopropionate  by  bivalve  molluscs:  high  and  non- 
normal  variation.  233 

HILL.  RICHARD  W.,  see  Bradley  A.  White,  235 

HIROSE,  EUICHI,  and  TERUHISA  ISHII,  Microfilament  contraction  pro- 
motes rounding  of  tunic  slides:  an  integumentary  defense  system 
in  the  colonial  ascidian  Aplidimn  yama:ii.  29 

HOBBIE.  JOHN  E.,  see  Amy  G.  Uhlenhopp.  256 

HoEGH-GuLDBERG,  OVE,  see  Sophie  G.  Dove,  288 

HOPKINSON,  CHARLES  S..  JR..  see  Derrick  Alderman.  250;  Brian  Balsis. 
252 

horseshoe  crab.  2 1 3 

hydrothermal  vents,  69 

5-hydroxytryptamine,  320 


Identification  of  calcium  flux  in  single  preimplantation  mouse  embryos 

with  the  calcium-sensitive  vibrating  probe.  200 
ILAN,  MICHA,  and  AVIGDOR  ABELSON,  The  life  of  a  sponge  in  a  sandy 

lagoon,  363 

immunocytochemistry,  280 
Immunohistochemical  localization  of  saxitoxin  in  the  siphon  epithelium 

of  the  butter  clam.  S<i.\uii>nn<.\  gigunleiis,  229 
immunohistochemical  staining.  229 
Impact  on  marine  species  of  New  England  recreational  fishing  policies. 

246 
//;  wivi  effects  of  dopamine  and  dopaminergic  antagonists  on  testicular 

maturation  in  the  red  swamp  crayfish.  Procambarus  clarkii.  340 
induction  by  poly-chlorinated  biphenyl,  227 
INESTROSA,  NIBALDO  C.,  see  Sebastian  R.  Rodriguez,  272 
Inhibitors  of  protein  phosphatascs  (okadic  acid  and  tautomycin)  block 

sea  urchin  development.  201 
INOUE,  KOJI,  J.  HERBERT  WAITE,  MAKOTO  MATSUOKA,  SATOSHI  ODO, 

and  SHIGEAKI  HARAYAMA,  Interspecific  variations  in  adhesive 

protein  sequences  of  Aty/ilm  aliili-i.  M  galloprovincialis,  and  At. 

trossiiln.'i,  370 

INOUE,  SHINYA,  see  Yoshio  Fukui,  19X;  Mira  Krendel,  204 
intermittent  burst,  148 
Interspecific  variations  in  adhesive  protein  sequences  of  Mytilus  edulis. 

At.  galloprovincialis,  and  At.  trossulus,  370 
interstitial  water,  363 
intnnsic  response.  148 
intrinsic  signals,  212 
invertebrate.  241,  242 

immunology,  106 

Invertebrate  response  to  nutrient-induced  changes  in  macrophyte  as- 
semblages in  Waquoit  Bay,  241 
ionic  currents,  197 

ISHII,  TERUHISA,  see  Euichi  Hirose,  29 
Isolation  and  partial  characterization  of  the  pink  and  blue  pigments  of 

pocillopond  and  acroporid  corals.  288 


JAECKLE,  WILLIAM  B..  Transport  and  metabolism  of  alanine  and  palmitic 
acid  by  field-collected  larvae  of  Tedania  ignis  (Porifera,  Demo- 
spongiae):  estimated  consequences  of  limited  label  translocation, 
159 

Japanese  medaka  sentinel  embryos,  196 

jellyfish,  1 

JINKS.  R.  N..  see  G.  H.  Renninger,  69 

K 

KAPLAN,  ILENE  M.,  see  Jonathan  S.  O'Neil,  246 

KASS,  L.,  see  G.  H.  Renninger,  69 

KATAYAMA,  TOMOE,  HIROSHI  WADA,  HIDETAKA  FURUYA,  NORIYUKI 
SATOH,  and  MASAMICHI  YAMAMOTO,  Phylogenetic  position  of  the 
dicyemid  mesozoa  inferred  from  18S  rDNA  sequences,  81 

K.EARNS,  COLLEEN  M..  see  Nelson  G.  Hairston.  Jr.,  42 

K.EEFE,  DAVID,  JOHN  PEPPERELL,  PAULO  RINAUDO,  JOSEPH  KUNKEL, 
and  PETER  SMITH,  Identification  of  calcium  flux  in  single  preim- 
plantation mouse  embryos  with  the  calcium-sensitive  vibrating 
probe.  200 

KELLY,  MAEVE  S..  M.  F.  BARKER,  J.  DOUGLAS  MCKENZIE.  and  JAN 
POWELL.  The  incidence  and  morphology  of  subcuticular  bacteria 
in  the  echinoderm  fauna  of  New  Zealand,  91 

KRAUSE,  TODD  L.,  see  Harvey  M.  Fishman,  208 

KRENDEL,  MIRA,  and  SHINYA  INOLIE,  Anaphase  spindle  dynamics  under 
DiO-enhanced  microtubule  polymerization,  204 

KUHNS,  WILLIAM  J.,  MAX  M.  BURGER,  and  GRADIMIR  MISEVIC,  Sul- 
fotransferase  activities  in  the  marine  sponge  Alicnicinnn  pro/item 
correlation  with  sulfated  glycan  adhesive  structures,  223 

KUNKEL,  JOSEPH,  see  David  Keefe,  200 

KUNKEL,  JOSEPH  G.,  and  ELLEN  FASZEWESKI,  Pattern  of  potassium  ion 
and  proton  currents  in  the  ovariole  of  the  cockroach,  Periplaneta 
amcnciina,  indicates  future  embryonic  polarity,  197 

KUZIRIAN,  ALAN  M.,  see  Conxita  Avila,  237 

KYOZUKA.  KEIICHIRO,  see  Peter  P.  Fong.  320 


lability.  256 

Land  cover  effects  on  inorganic  nutrients  in  groundwater  entering  es- 

tuanes  of  Waquoit  Bay.  Massachusetts.  248 
LAND,  S.  C.,  and  P.  J   S.  SMITH,  Suppression  of  Ca2+  flux  during  the 

transition  to  anoxia  in  turtle  hepatocytes  revealed  by  a  noninvasive 

Ca2+-selective  vibrating  probe,  228 
land  use,  256 
larva.  159 

LASKIN,  JEFFREY  D.,  see  Walter  Troll,  200 
LDL-receptor-related  protein  (LRP),  225 
LEAVITT,  D.,  see  M.  L.  Wintermyer,  240 
LEAVITT,  DALE  F.,  see  Richard  W.  Hill,  233 
lectin.  226 
leukotriene  B4,  203 
Leukotnene  B4  induces  release  of  calcium  from  endomembrane  stores 

in  wi'ii  in  eggs  and  second  cell  blastomeres  of  the  sand  dollar  Echi- 

naruciinit  purnui,  203 
Life  histories  and  senescence  of  Bolryllits  schlosserie  (Chordata,  Asci- 

diacea)  in  Monterey  Bay.  36 
life  history.  36 

light  transmission  properties  offish  cone  photoreceptors.  220 
Lixia.  148 
limulin.  226 
Limulu\.  2  1  3 

optic  nerve,  2 1 2 

l.inntliui  is  tuned  into  its  visual  environment,  213 
LINDSAY,  SARA  M..  see  Sarah  A.  Woodin,  49 
loading  to  estuaries.  247.  248 
LOBO-DA-CLINHA,  ALEXANDRE,  see  Teresa  Sequeira,  376 


INDEX  TO  VOLUME   189 


385 


lobster.  232 

Localization  of  digestion  activities  in  the  sea  anemone  ILilipkiiii'lla  liiaac, 
236 

LYNN,  J.  W.,  see  H.  Silverman.  308 

LYNN.  JOHN  W.,  see  Patricia  S.  Glas,  13 

LYONS.  JULIE.  JENNY  AHERN.  JAMES  MCCLELLAND,  and  IVAN  VALIELA, 
Macrophyte  abundances  in  Waquoit  Bay  estuaries  subject  to  dif- 
ferent nutrient  loads  and  the  potential  role  of  fringing  salt  marsh 
in  groundwater  nitrogen  interception.  255 

LYONS,  JULIE,  see  Jenny  Ahern.  241 

M 

MACGREGOR.  CATHERINE  HUNTER,  see  Sue  Ann  Chaplin.  247 

MACGREGOR.  CATHERINE  HUNTER,  SUE  ANN  CHAPLIN,  and  IVAN 
VALIELA.  Land  cover  effects  on  inorganic  nutrients  in  groundwater 
entering  estuaries  of  Waquoit  Bay.  Massachusetts,  248 

macrofauna,  49 

«;-macroglobulin.  225.  226 

macrophyte.  241 
abundance.  255 

Macrophyte  abundances  in  Waquoit  Bay  estuaries  subject  to  different 
nutrient  loads  and  the  potential  role  of  fringing  salt  marsh  in 
groundwater  nitrogen  interception.  255 

MAMMAR.  KATHERINE.  see  Peter  J.  S.  Smith.  209 

manculture.  237 

Mart/alia  hyadesi.  77 

MARTIN.  HOLLY  A.,  see  Neal  W.  Cornell,  227 

MARTINDALE,  MARK  Q..  and  JONATHAN  Q.  HENRY,  Diagonal  devel- 
opment: establishment  of  the  anal  axis  in  the  ctenophore  Mne- 
/mo/n/s  Iculyi.  190 

MARTINDALE,  MARK  Q.,  see  Jonathan  Q.  Henry,  192.  194 

MARTINEZ,  NICOLE,  JENNIFER  HALIXWELL.  and  IVAN  VALIELA,  Effect 
of  macroalgal  species  and  nitrogen-loading  rates  on  colonization  of 
macroalgae  by  herbivorous  amphipods,  244 

MATSUOKA.  MAKOTO,  see  Koji  Inoue,  370 

MCCLELLAND,  JAMES,  see  Jenny  Ahern.  241:  Julie  Lyons.  255 

MCDOWELL.  J..  see  M.  L.  Wintermyer.  240 

MCDOWELL.  JUDITH  E..  see  Richard  W.  Hill.  233 

McFALL-NGAi.  MARGARET  J..  see  Judith  A.  Doino.  347 

McKENZiE.  J.  DOUGLAS,  see  Maeve  S.  Kelly.  91 

MEKASHA.  DANIEL,  see  Peter  P.  Fong,  320 

MELCHIOR,  RALPH,  see  Snehasikta  Swarnakar.  226 

mesenteries.  236 

mesozoa.  81 

metabolic  suppression.  228 

metabolism.  159,  168,  252 

METUZALS.  J..  H.  M.  FISHMAN,  and  I.  A.  ROBB.  The  neurofilamentous 
network-smooth  endoplasmic  reticulum  complex  in  transected 
squid  giant  axon.  2 1 6 

Microsaona  sponge  sulfotransferases.  223 

Microfilament  contraction  promotes  rounding  of  tunic  slides:  an  inte- 
gumentary defense  system  in  the  colonial  ascidian  Aplidntm  \WIMZII. 
29 

microtubules.  204.  206 

Mid-Atlantic  Ridge.  69 

migration.  120.  128 

MILLER.  ANDREW  L..  see  Harvey  M.  Fishman.  208 

MISEVIC.  GRADIMIR.  see  Wiliam  J.  Kuhns.  223 

mitosis.  203 

MIZELL.  MERLE.  ERIC  ROMIG,  WILLIAM  HARTLEY,  and  ARUNTHA- 
VARANI  THIYAGARAJAH,  Sex  on  the  brain  but  the  heart  is  not  really 
in  it:  developmental  heart  defects  associated  with  aquatic  pollution 
and  microinjcction  of  hexachlorobenzene  into  the  Japanese  medaka 
embryo.  196 

molecular  phylogeny,  81 

mollusc.  233 
larvae.  272 

molt.  376 

Monterey  Bay.  36 


MORGAN,  JAMES  L.  M..  see  Jeffery  R.  Demarest.  219 

MORISAWA.  MASAAKI,  see  Tatsuru  Togo.  330 

MORISAWA,  SACHIKO,  Fine  structure  of  spermatozoa  of  the  hagtish  £'/>- 
uiti'i'tim  hurgeri  (Agnatha).  6 

morphology.  36 

Morphology  and  physiology  of  the  thoracic  and  abdominal  stretch  re- 
ceptors of  the  isopod  crustacean  Ligia  exotica,  148 

MOUNTAIN.  D..  see  T.  R.  Consi.  231 

Muricidae,  59 

mussel.  235.  370 

Mva  arenaria.  240 

Mytilus,  370 


N 


N-cell.  148 

NAGABHUSHANAM,  RACHAKONDA.  see  Rachakonda  Sarojini.  340 

Natural  diets  for  Hermissenda  crassicornis  mariculture,  237 

Nemenea.  192 

neuromuscular,  280 

neuron,  209 

neuropeptide.  280 

New  Zealand  echinoderms.  9 1 

niche  divergence.  1 1 3 

NIIDA.  AKIYOSHI.  YOSHIKO  TAKATSUKI.  and  TSUNEO  YAMAGUCHI. 
Morphology  and  physiology  ot  the  thoracic  and  abdominal  stretch 
receptors  of  the  isopod  crustacean  Ligia  exotica,  148 

nitrogen 

concentration.  247,  248 

loading.  254 

release  (leachates).  262 

Not  all  ctenophores  are  bioluminescent:  Pleurobrachia,  356 

NOVALES  FLAMARIQUE,  Iftioo,  RUDOLF  OLDENBOURG,  and  FERENC 
I.  HAROSI.  Transmission  of  polarized  light  through  sunnsh  double 
cones  reveals  minute  optical  anisotropies,  220 

nudibranchs,  237 

nutrient  enrichment.  245,  258 

nutrient  limitation.  257 

Nutrient  limitation  of  phytoplankton  growth  in  Waquoit  Bay.  Massa- 
chusetts. 257 

nutrient-loading.  260.  261 

nutrition.  168 


o 


O'BRIEN.  ESTELA  V..  and  ROBERT  B.  BARLOW,  Optical  imaging  of  in- 
trinsic signals  from  the  Limn/us  optic  nerve,  2 1 2 
O'NEIL,  JONATHAN  S..  and  ILENE  M.  KAPLAN,  Impact  on  marine  species 

of  New  England  recreational  fishing  policies.  246 
ODO.  SATOSHI,  see  Koji  Inoue,  370 
OHSS  (ovigerous-hair  stripping  substance).  175 
OLDENBOLIRG.  RUDOLF,  see  Phong  Tran.  206:  Inigo  Novales  Flamarique, 

220 

C/iHjmn  ran.  2  1 1 
optical  imaging.  212 

Optical  imaging  of  intrinsic  signals  from  the  Limulus  optic  nerve.  212 
orientation.  138.  231.  232 
OSANAI,  KENZI,  see  Tatsuru  Togo.  330 
oscillation.  2 1 5 

ovigerous-hair  stripping  substance  (OHSS),  175 
oxidase.  13 
Oxidase  activity  associated  with  the  elevation  of  the  penaeoid  shrimp 

hatching  envelope,  13 
oxygen 

consumption.  22 

sensitivity.  22 

oxyntic  cells.  219 


386 


INDEX  TO  VOLUME   189 


PANCER,  ZEEV,  HARRIET  GERSHON,  and  BARLICH  RINKEVICH.  Coex- 
istence and  possible  parasitism  of  somatic  and  germ  cell  lines  in 
chimeras  of  the  colonial  urochordate  Bnlryllux  .vc/i/o.v.vm,  106 

PASSAGLIA,  C.  L..  F.  A.  DODGE,  and  R.  B.  BARLOW.  Linntlns  is  tuned 
into  its  visual  environment,  213 

pattern  formation,  190,  197 

Pattern  of  potassium  ion  and  proton  currents  in  the  ovanole  of  the  cock- 
roach, Pcnplaneta  americana,  indicates  future  embryonic  polarity, 
197 

PCR,  370 

pelagic  metabolism,  250 

Pelagic  metabolism  in  the  Parker  River/Plum  Island  Sound  estuarine 
system,  250 

penaeoid,  13 

Penaeus  iiipoiiuiix.  376 

PCHM'UX  xclilcnix,  168 

PEPPERELL,  JOHN,  see  David  Keefe,  200 

peroxidase,  I  3 

phosphatase,  201 

photoperiod,  42 

photoprotein.  I,  356 

Phylogenetic  position  of  the  dicyemid  mesozoa  inferred  from  1 8S  rDN  A 
sequences,  81 

physiology.  159.  168 

phytoplankton,  254.  258 
growth,  257 

pigment,  288 

plant  morphology.  242 

Pleurobrachia,  356 

Plum  Island  Sound.  252 

Pocillopora  damicornis,  288 

pocilloponn.  288 

polarized  light 

detection  in  fish.  220 
microscopy.  206 

policy.  246 

polysperm>  block,  330 

Porifera.  159 

POWELL,  JAN,  see  Maeve  S.  Kelly.  41 

PRECHTL,  JAMES  C..  Flutter-like  response  in  visual  cortex  of  the  semi- 
isolated  turtle  brain,  215 

predation.  242 

PREISSER,  MATTHEW  C..  and  LINDA  A.  DEIGAN,  Effect  of  changing 
plant  morphology  on  invertebrate  susceptibility  to  predation  in  eel- 
grass  beds.  242 

Preliminary  investigations  on  the  scavenger  receptors  of  the  amebocyte 
of  the  American  horseshoe  crab.  l.timilii\  polvphcniiix.  225 

primary  production.  254 

Procambanis  clarkn.  340 

Process-specific  recruitment  cues  in  marine  sedimentary  systems.  49 

protease  clearance,  225 

protein  kinases.  201 

protein  phosphates,  201 
i\.  263 


Q 

quantification,  91 

Quantifying  single  and  bundled  microtubules  with  the  polarized  light 

microscope,  206 
QuiGLEY,  JAMES  P..  see  Ronald  T.  Aimes.  225;  Snehasikta  Swarnakar. 

226 


RAFFERTY.  NANCY  S..  see  Seymour  Zigman.  222 
RAM,  JEFFREY,  see  Peter  P.  Fong.  320 
recruitment,  49 


Regional  differences  in  directional  response  properties  of  afferents  along 

the  saccule  of  the  toadfish  (Opxanux  tail).  21  1 
Regulation  of  the  plasma  cytolytic  pathway  of  Limulux  polyphemiis  by 

«2-macroglobulin,  226 
RENNINGER,  G.  H.,  L.  KASS.  R.  A.  GLEESON,  C.  L.  VAN  DOVER,  B.-A. 

BAITELLE.  R.  N.  JINKS,  E.  D.  HERZOG,  and  S.  C.  CHAMBERLAIN, 

Sulfide  as  a  chemical  stimulus  for  deep-sea  hydrothermal  vent 

shnmp,  69 

reproduction,  36.  320.  330 
retardancc.  206 
Retardation  of  the  spread  of  extracellular  Ca:+  into  transected,  unsealed 

squid  giant  axons.  208 

Reversible  regression  of  cytokinesis  induced  by  Ca2+  ionophore,  201 
RIETSMA.  CAROL,  see  Cheryl  Ann  Wolfe,  262 
RINAUDO.  PALILO.  see  David  Keefe.  200 
RINKEVICH.  BARUCH,  see  Zeev  Pancer.  91 
RIQUELME,  CARLOS,  see  Sebastian  R.  Rodriguez.  272 
ROBB.  I.  A.,  see  J.  Metuzals.  216 
robot.  231 
RODHOLISE,  PAUL  G.,  and  MARTIN  G.  WHITE,  Cephalopods  occupy  the 

ecological  niche  of  epipelagic  fish  in  the  Antarctic  Polar  Frontal 

Zone.  77 
RODRIGUEZ.  SEBASTIAN  R..  CARLOS  RIQUELME,  ELISEO  O.  CAMPOS, 

PAMELA  CHAVEZ.  ENRIQUE  BRANDAN,  and  NIBALDO  C.  INES- 

i  ROSA,  Behavioral  responses  ofConcholepas  concAo/epos(Bruguiere, 

1 789)  larvae  to  natural  and  artificial  settlement  cues  and  microbial 

films.  272 

ROEGIERS.  FABRICE.  see  Keisuke  Suzuki.  201 
Role  of  digestive  gland  in  the  energetic  metabolism  of  Pcnucux  setiferus, 

168 

ROMIG,  ERIC,  see  Merle  Mizell.  19h 
ROSAS,  CARLOS,  ANDREA  BOLONGARO-CREVENNA,  ADOLFO  SANCHEZ, 

GABRIELA  GAXIOLA,  Luis  SOTO,  and  ELVA  ESCOBAR,  Role  of 

digestive  gland  in  the  energetic  metabolism  of  Pcnucux  setiferus, 

168 
RYNKOWSKI,  STACY,  see  Peter  P.  Fong,  320 


saccule.  2 1 1 

SAIGUSA,  MASAYUKI,  Bioassay  and  preliminary  characterization  of  ovi- 
gerous-hair  stripping  substance  (OHSS)  in  hatch  water  of  crab  larvae, 
175 

salinity.  320 

SALMON.  E.  D..  see  Phong  Tran,  206 

salt  marsh.  235,  262 

SANCHEZ.  ADOLFO,  see  Carlos  Rosas,  168 

sand  dollar.  203 

SARDV  RAFAEL.  KENNETH  FOREMAN,  and  IVAN  VALIELA,  Differences 
in  benthic  invertebrate  assemblages  in  two  estuaries  in  Waquoit 
Bay  receiving  disparate  nutrient  loads,  245 

SAROJINI,  RACHAKONDA,  RACHAKONDA  NAGABHUSHANAM,  and  MIL- 
TON  FINGERMAN.  lii  wr«  effects  of  dopamine  and  dopaminergic 
antagonists  on  testicular  maturation  in  the  red  swamp  crayfish.  Pro- 
cunihurux  clurkii.  340 

SATOH,  NORIYLIKI,  see  Tomoe  Katayama,  81 

saxiloxin,  229 

SAYI  AR.  KELLEY  L..  see  Jane  A.  Westfall,  280 

SCHOLNICK.  DAVID  A..  Sensitivity  of  metabolic  rate,  growth,  and  fe- 
cundity of  tadpole  shrimp  Triopx  longicaudatus  to  environmental 
variation,  22 

SCHULTZ,  MARK,  see  Seymour  Zigman.  222 

scleractinian.  288 

sculpin,  239 

sea  anemone,  280 

sea  urchin,  201 

seasonal  phenology,  42 

scgmcntal  mobility.  148 

self-nonself  recognition.  106 

senescence.  36 


INDEX  TO  VOLUME   189 


387 


Sensitivity  of  metabolic  rate,  growth,  and  fecundity  of  tadpole  shrimp 
Triops  longicaudatta  to  environmental  variation.  22 

SEQUEIRA.  TERESA.  MANUEL  VILANOVA,  ALEXANDRE  LOBO-DA- 
CUNHA,  Luis  BALDAIA.  and  MARIO  ARALA-CHAVES.  Flow  cyto- 
metric  analysis  of  molt-related  changes  in  hemocyte  type  in  male 
and  female  /Y/wt'/i.v  ;<;/><  wniv,  376 

Scruittipniu  /nvm.  288 

serotonin.  320 

settlement,  240.  272 

settlement  cue.  49 

severed  giant  axons  of  squid.  216 

Sex  on  the  brain  but  the  heart  is  not  really  in  it:  developmental  heart 
defects  associated  with  aquatic  pollution  and  microinjection  of 
hexachlorobenzene  into  the  Japanese  medaka  embryo.  146 

sexual  dimorphism,  113 

Sexual  dimorphism  and  niche  divergence  in  a  mid-water  octopod  (Ceph- 
alopoda: Bolitaenidae),  113 

SHERIDAN,  CECELIA  C..  IVAN  VALIELA,  KENNETH  FOREMAN,  and  LORI 
A.  SOUCY,  Effect  of  nutrient  enrichment  on  phytoplankton  growth 
in  Waquoit  Ba\,  Massachusetts,  258 

SHIMOMURA,  OSAMU.  A  short  story  of  aequonn.  1 

shrimp.  13 

shrimp  sulndes.  69 

SILVER.  ROBERT  B..  Leukotriene  B4  induces  release  of  calcium  from 
endomembrane  stores  in  vivo  in  eggs  and  second  cell  blastomeres 
of  the  sand  dollar  Echinaracnius  parma,  203 

SlLVERMAN.   H..   E.   C.   ACHBERGER,   J.   W.   LYNN,   and   T.    H.    DlETZ. 

Filtration  and  utilization  of  laboratory-cultured  bacteria  by  Dreis- 

sena  [>ol\'ini>rpha.  Curhiciila  ftuminea,  and  Carunculina  texasensis, 

308 

size-selective  predation,  243 
slowly  adapting  stretch  receptor.  148 
SMITH.  P.  J.  S..  see  S.  C.  Land.  228 
SMITH.  PETER  J.  S..  (CATHERINE  HAMMAR.  and  MICHAEL  TYTELL,  Effect 

of  exogenous  heat  shock  protein  (hsp70)  on  neuronal  calcium  flux. 

209 

SMITH.  PETER  J.  S.,  see  C.  Leah  Devlin.  207 
SMITH.  PETER,  see  David  Keefe.  200 
SMOLOWITZ,  ROXANNA  M.,  Immunohistochemical  localization  of  sax- 

itoxin  in  the  siphon  epithelium  of  the  butter  clam.  Sa.\u/«niiix  gi- 

ganletis,  229 
smooth  muscle,  207 
soft  bottom.  363 

SOTO,  Luis,  see  Carlos  Rosas,  168 

SOUCY,  LORI  A.,  see  David  W.  Callaway,  254:  Cecelia  C.  Sheridan.  258 
SOUCY.  LORI,  see  Sue  Ann  Chaplin.  247 
sound.  128 

Spariina  alternijlora,  262 
spawning.  320 
spermatozoa.  6 
sponge.  363 
squid  predation.  263 
stress.  298 

stretch  receptor  of  Isopoda.  148 
STRICKLAND.  DUDLEY  K...  see  Ronald  T.  Aimes.  225 
Stylopora  pislillala,  288 
subcuticular  bacteria.  91 
SUEOKA,  ElSABORO.  see  Walter  Troll.  201 
SLIEOKA,  NAOKO,  see  Walter  Troll.  201 
Sullide  as  a  chemical  stimulus  for  deep-sea  hydrothermal  vent  shrimp, 

69 

sulfotransferase  activities  in  sponge.  223 
Sulfotransferase  activities  in  the  marine  sponge  Micrwioiiu  prnlih'i\i 

correlation  with  sulfated  glycan  adhesive  structures,  223 
sulfotransferase  enzyme  activities,  223 
Suppression  of  Ca2+  flux  during  the  transition  to  anoxia  in  turtle  hepa- 

tocytes  revealed  by  a  noninvasive  Ca2+-selective  vibrating  probe, 

228 
SUZUKI,  KEISLIKE,  FABRICE  ROEGIERS,  PHONG  TRAN.  and  SHINYA 

INOUE.  Reversible  regression  of  cytokinesis  induced  by  Ca:+  ion- 

ophore.  201 


SWARNAKAR,  SNEHAS1KTA.  RALPH  MELCHIOR.  JAMES  P.  QUIGLEY,  and 

PETER  B.  ARMSTRONG.  Regulation  of  the  plasma  cytolytic  pathway 
of  Limulus  polyphcmus  by  «;-macroglobulin.  226 

SWARNAKAR,  SNEHASIKTA.  see  Ronald  T.  Aimes.  225 

swash,  138 

swash  riding.  120.  128 

symbiosis.  91.  347 

symbiotic  bacteria.  363 

symmetry  properties.  190 

synapses.  280 


TAKABAYASHI.  MISAKI,  see  Sophie  G.  Dove,  288 

TAKATSUKI,  YOSHIKO.  see  Akiyoshi  Niida,  148 

Tedania  ignis,  159 

temperature  sensitivity.  22 

testicular  maturation.  340 

The  effect  of  residential  and  forested  watershed  land  cover  on  nutrient 

loading  to  Hamblin  and  Jehu  Ponds.  Waquoit  Bay.  Massachusetts. 

247 
The  effect  of  salinity  and  temperature  on  spawning  and  fertilization  in 

the  zebra  mussel  Dreisscnu  polymorpha  (Pallas)  from  North  Amer- 
ica, 320 
The  effect  of  temperature  on  the  relationship  between  a  ciliated  protozoan. 

Tnchodina  cottiduruni.  and  the  longhorn  sculpin.  Mymoivphalus 

octodecemspinosus,  239 
The  experimental  alteration  of  cell  lineages  in  the  nemertean  Cerebralulns 

lacteus:  implications  for  the  precocious  establishment  of  embryonic 

axial  properties.  192 
The  incidence  and  morphology  of  subcuticular  bacteria  in  the  echinoderm 

fauna  of  New  Zealand,  9 1 
The  interaction  of  photoperiod  and  temperature  in  diapause  timing:  a 

copepod  example.  42 

The  life  of  a  sponge  in  a  sandy  lagoon,  363 
The  neurofilamentous  network-smooth  endoplasmic  reticulum  complex 

in  transected  squid  giant  axon.  216 
thermal  stress.  239 

THIYAGARAJAH,  ARUNTHAVARANI,  see  Merle  Mizell,  196 
toadnsh.  211 
TOGO.  TATSURU,  KENZI  OSANAI.  and  MASAAKI  MORISAWA.  Existence 

of  three  mechanisms  for  blocking  polyspermy  in  oocytes  of  the 

mussel  Myliliis  edit/is.  330 

TOMASKY.  GABRIELLE.  and  IVAN  VALIELA.  Nutrient  limitation  of  phy- 
toplankton growth  in  Waquoit  Bay,  Massachusetts,  257 
Total  system  metabolism  of  the  Plum  Island  Sound  estuarine  system. 

252 
TRAN.  PHONG,  E.  D.  SALMON,  and  RUDOLF  OLDENBOURG,  Quantifying 

single  and  bundled  microtubules  with  the  polarized  light  microscope. 

206 

TRAN,  PHONG,  see  Keisuke  Suzuki.  201 
Transmission  of  polarized  light  through  sunnsh  double  cones  reveals 

minute  optical  anisotropies.  220 
Transport  and  metabolism  of  alanine  and  palmitic  acid  by  field-collected 

larvae  of  Tcdunui  i.vnix  (Porifera.  Demospongiae):  estimated  con- 
sequences of  limited  label  translocation.  159 
Tnchodina,  239 
TROLL.  WALTER,  NAOKO  SUEOKA.  EISABORO  SUEOKA,  JEFFREY  D. 

LASKIN,  and  DIANA  E.  HECK.  Inhibitors  of  protein  phosphatases 

(okadic  acid  and  tautomycin)  block  sea  urchin  development.  201 
tunic  cell,  29 
tunicate.  106 
turbulence.  231.  232 
TYTELL,  M.,  see  C.  S.  Eddleman.  218 
TYTELL,  MICHAEL,  see  Peter  J.  S.  Smith,  209 

U 

UHLENHOPP.  AMY  G..  JOHN  E.  HOBBIE,  and  JOSEPH  J.  VALLINO.  Effects 
of  land  use  on  the  degradability  of  dissolved  organic  matter  in  three 
watersheds  of  the  Plum  Island  Sound  Estuarv.  256 


388 


INDEX  TO  VOLUME   189 


Ultrastructural  localization  of  Antho-RWamides  1  and  II  at  neuromus- 
cular  synapses  in  the  gastrodcrmis  and  oral  sphincter  muscle  of  the 
sea  anemone  Calliaclix  paraxilica.  280 

ultrastructure.  9 1 ,  280 

unionid,  308 

Urochordata,  29 

UV  microbeam,  198 

UV-damage.  222 


VALIELA,  IVAN,  see  Jenny  Ahern  241;  Chaka  Drake.  243:  Nicole  Mar- 
tinez. 244;  Rafael  Sarda.  245;  Sue  Ann  Chaplin.  247;  Catherine 
Hunter  MacGregor.  248;  David  W.  Callaway,  254;  Julie  Lyons, 
255;  Gabnelle  Tomasky,  257;  Cecelia  C.  Sheridan,  258;  Travis 
Bohrer,  260;  Amos  Wright,  261;  Cheryl  Ann  Wolfe,  262 

VALLINO,  JOSEPH  J.,  see  Derrick  Alderman,  250;  Brian  Balsis.  252;  Amy 
G.  Uhlenhopp,  256 

VAN  DOVER,  C.  L..  see  G.  H.  Renninger.  69 

varix,  59 

vibrating  probe,  228 

VILANOVA,  MANUEL,  see  Teresa  Sequeira.  376 

vision.  2 1 3 

visual,  215 

VoiGHT,  JANET  R..  Sexual  dimorphism  and  niche  divergence  in  a  mid- 
water  octopod  (Cephalopoda:  Bolitaenidael.  1  13 


W 


WADA,  HIROSHI.  see  Tomoe  Katayama,  81 

WAITE,  HERBERT  J.,  see  Koji  Inoue.  370 

Waquoit  Bay,  258 

WARNER,  M.  E.,  see  William  K.  Fin,  298 

wave,  120,  128.  138 

waveguide  properties  offish  cone  photoreceptors,  220 

WEISSMAN,  IRVING  L.,  see  Nanette  E.  Chadwick-Furman,  36 


WESTFALL,  JANE  A..  K.ELLEY  L.  SAYYAR,  CAROL  F.  ELLIOTT,  and 
CORNELIS  J.  P.  GRIMMELIKHUIJZEN,  Ultrastructural  localization 
of  Antho-RWamides  I  and  II  at  neuromuscular  synapses  in  the 
gastrodermis  and  oral  sphincter  muscle  of  the  sea  anemone  Calliat'tis 
paraxitica,  280 

WETHEY,  DAVID  S.,  See  Sarah  A.  Woodin.  49 

WHITE,  BRADLEY  A.,  RICHARD  W.  HILL,  and  JOHN  W.  H.  DACEY, 
Accumulation  of  dimethylsulfoniopropionate  in  (jiiikiiixia  tlt-missa 
depends  on  trophic  interactions.  235 

WHITE,  MARTIN  G..  see  Paul  G.  Rodhouse.  77 

WINTERMYER,  M.  L.,  D.  LEAViTT,  and  J.  MCDOWELL,  A  settlement 
bioassay  assessing  the  response  of  soft  shell  clam  larvae  to  sediments 
from  various  sites  in  Massachusetts  Bay,  240 

WOLFE,  CHERYL  ANN,  CAROL  RIETSMA,  and  IVAN  VALIELA,  Foliar 
release  of  ammonium  and  dissolved  organic  nitrogen  by  Spuninu 
ulk-niillnra,  262 

WOODIN,  SARAH  A..  SARA  M.  LINDSAY,  and  DAVID  S.  WETHEY,  Process- 
specific  recruitment  cues  in  marine  sedimentary  systems,  49 

WRIGHT.  AMOS,  see  Travis  Bohrer,  260 

WRIGHT.  AMOS,  TRAVIS  BOHRER.  JENNIFER  HAUXWELL,  and  IVAN 
VALIELA,  Growth  of  epiphytes  on  Zostera  marina  in  estuaries  subject 
to  different  nutrient  loading.  261 


YAMAGUCHI,  TSLINEO.  see  Akiyoshi  Niida,  148 
YAMAMOTO.  MASAMICHI,  see  Tomoe  Katayama,  81 


zebra  mussel,  308,  320 

ZIGMAN,  SEYMOUR,  NANCY  S.  RAFFERTY,  and  MARK  SCHULTZ,  Dogfish 

(Musk-lux  aunx)  lens  catalase  reduces  H;O2-induced  opacification. 

222 

zooxanthcllac.  29X 
/iixlcru  manna,  260 


CONTENTS 


NEUROBIOLOGY  AND  BEHAVIOR 

Fleischer,  Kellie  J.,  and  James  F.  Case 

Cephalopocl  predation  facilitated  by  dinoflagellate 
luminescence 263 

Rodriguez,  Sebastian  K...  Carlos  Riquelme,  Eliseo  O. 

Campos,  Pamela  Chavez,  Enrique  Brandan,  and  Ni- 

baldo  C.  Inestrosa 

Behavioral  responses  of  Cmirhiilcjiii\  concholepas 
(Bruguiere,  1789)  larvae  to  natural  and  artificial 
settlement  cues  and  microbial  films 272 

Westfall,  Jane  A.,  Kelley  L.  Sayyar,  Carol  F.  Elliott, 

and  Cornells  J.  P.  Grimmelikhuijzen 

Ultrastructural  localization  of  Antho-RWamides  I 
and  II  at  neuromuscular  synapses  in  the  gastro- 
drrmisand  oral  sphincter  muscle  of  the  sea  anemone 
Caliunti\  /a/ KI \ilii a  280 

PHYSIOLOGY 

Dove,  Sophie  G.,  Misaki  Takabayashi,  and  Ove 
Hoegh-Guldberg 

Isolation  and  partial  characterization  <>l  the  pink 
and  blue  pigments  of  pocilloporid  and  acroporid 
corals 288 

Fitt,  W.  K.,  and  M.  E.  Warner 

Bleaching  patterns  of  four  species  of  Caribbean  reef 
corals 298 

Silverman,  H.,  E.  C.  Achberger,  J.  W.  Lynn,  and 

T.  H.  Dietz 

Filtration  and  utilization  of  laboratory-cultured 
bacteria  by  A>irmr/jr;  pohmorpha,  C.i»l>i<  nlu  fluminea, 
and  Carunculina  tc\tiv>i\i\  308 

DEVELOPMENT  AND  REPRODUCTION 

Fong,  Peter  P.,  Keiichiro  Kyozuka,  Jill  Duncan, 
Stacy  Rynkowski,  Daniel  Mekasha,  and  Jeffrey  L. 
Ram 

The  effect  of  salinity  and  temperature  on  spawning 
and  fertilization  in  the  /ebra  mussel  Dm^i mi  [itily- 
i/Kn/i/ui  (Pallas)  from  North  America  320 


Togo,  Tatsuru,  Kenzi  Osanai,  and  Masaaki  Mori- 
sawa 

Existence  of  three  mechanisms  for  blocking  poly- 
spermy  in  oocytes  of  the  mussel  M\t/lu\  iv/»/n  .  .  .  330 

Sarojini,   Rachakonda,    Rachakonda   Nagabhusha- 

nam,  and  Milton  Fingerman 

In  vivo  effects  of  dopamine  and  dopaminergic  an- 
tagonists on  testicular  maturation  in  the  red  swamp 
crayfish,  Prucnmbanis  rliirku  340 


SYMBIOSIS 

Doino,  Judith  A.,  and  Margaret  J.  McFall-Ngai 

A  transient  exposure  to  symbiosis-competent  bac- 
teria induces  light  organ  morphogenesis  in  the  host 
squid  347 


ECOLOGY  AND  EVOLUTION 

Haddock,  Steven  H.  D.,  and  James  F.  Case 

Not  all  ctenophores  are  bioluminescent:  Pli-nrnbrti- 

chiti    356 

Ilan,  Micha,  and  Avigdor  Abelson 

The  life  of  a  sponge  in  a  sandy  lagoon     363 

Inoue,  Koji,  J.  Herbert  Waite,  Makoto  Matsuoka, 

Satoshi  Odo,  and  Shigeaki  Harayama 

Interspecific  variations  in  adhesive  protein  se- 
(|uences  of  M\tilu.<i  i'tlitli\.  M  galloprovincialis,  and 
.\/.  tr/i^iihn  370 


CELL  BIOLOGY 

Sequeira,  Teresa,  Manuel  Vilanova,  Alexandre 
Lobo-da-Cunha,  Luis  Baldaia,  and  Mario  Arala- 
Chaves 

Flow  cytometric  analysis  of  molt-related  changes  in 
hemocyte  type  in  male  and  female  Pfiim-m  /njiui/n  i<\      376 

Index  for  Volume  189  381 


MBL  WHOI    LIBRARY 


UH    1BET    t.