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Comparative morphology and functional anatomy of the digestive tract of the copepods Tigriopus californicus… McGroarty, James Roy 1985

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COMPARATIVE MORPHOLOGY AND FUNCTIONAL ANATOMY OF THE DIGESTIVE TRACT OF THE COPEPODS T i g r i o p u s c a l i f o r n i c u s AND Calanus  plumchrus.  A LIGHT AND ELECTRON MICROSCOPE STODY By ROY JAMES McGROARTY B . S c * The O n i v e r s i t y o f B r i t i s h Columbia,, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE f/2'." '.• in THE FACULTY OF GRADUATE STUDIES Department o f Zoology  We a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA  \ F e b r u a r y , 1985 ©  Roy James McGroarty, 1985  In  presenting  degree  this  at the  thesis  in  partial  fulfilment  of  University of  British  Columbia,  I agree  freely available for reference and study. copying  of  department  this or  thesis by  publication of this  for scholarly  his  or  thesis  her  c^. \  T  t>0  e  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  C V t  -\ I  I further agree  purposes  may  representatives.  It  be is  requirements  for  an  .  advanced  that the Library shall make it that permission  for extensive  granted  head  by the  understood  that  for financial gain shall not be allowed without  permission.  Department of  the  of  my  copying  or  my written  (ii) Abstract A s t u d y of the d i g e s t i v e t r a c t o f the c o p e p o d s cali fornicus techniques  and  of  Calanus  light  cal ifornicus,  the  and  plumchrus  electron  foregut  was  carried  microscopy.  contains  a  Tigriopus out  In  curved  T i g r iopus  cuticle  e s o p h a g u s w h i c h e x t e n d s from the v e n t r a l mouth to the o f the a n t e r i o r m i d g u t and portion  of  the  digestive  spherical  midgut  midgut,  3.  anterior  and  A  m i d g u t caecum.  caecum  tract  located  posterior  posterior  The  consists  of  There  hindgut  regions  In C a l a n u s p l u m c h r u s , the  foregut  lined  junction  noncuticulized :  1.  2.  An  anteriorly,  midgut.  using  are  A  single  anterior  cuticulized  ending  in  a  dorsal  c o n s i s t s of a  cuticle  anus.  lined  esophagus  junction  of  the  noncuticulized of  :  1.  A  extending midgut  and  portion  single  midgut  from the  of  the  ventral  midgut  the  mouth  diverticulum.  digestive  diverticulum,  tract  2.  A  ending  is  a  i n an In  long  abruptly  and of  the  T i g r i opus  position  type  of  were  c a l i f o r n i c u s , four  midgut  that  function.  lined  hindgut  cell  types  could  from s u c h u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s  i n the  digestive  organelles,  vesiculation,  cells  cuticle  The  anus.  be d i s t i n g u i s h e d and as  narrowing  the  consists  i s d i v i s i b l e on t h e b a s i s of e p i t h e l i a l c e l l t y p e and There  to  and  lipid electron  determined.  Cell  t r a c t , abundance, p o s i t i o n ,  content, density, type  presence  and  functions is  an  for  type the  embryonic  (iii) 'stem'  cell.  entiates Cell in  when  lipid for  cells  ' 2 '  type  the  I t functions  of  absorption.  midgut  i s also  major  function. the abundance  or  M  1  ' 3 '  ,  cell.  of  functions  2.  The  lipid  cell  but  i s important  cells  midgut  Cell  Cell  'R'  type  midgut  plumchrus,  type  regions,  a transport  coel .  Cell  similar i s a  glandular  type and  to  and  3 .  mainly  anterior  i s not i t s  examination  of the d i g e s t i v e 1.  made:  The  midgut  nutrients.  in nutrient The  in  of electron  from  absorption  posterior  cell  i n the midgut  function 'D'  I t s developed  between  i s found  large region  observed vacuolated of  could  diverticulum  midgut  the c e l l  I t has  for cell  the midgut.  Cell  distincell.  1  and p o s t e r i o r basal  surfaces  a n d t h e haemoregion  of  an u l t r a s t r u c t u r e  type  absorptive  be  'stem  i n the glandular  i s absorptive.  that  types  undifferentiated  is absorptive.  suggest  midgut  role  i n the  of digested  a role  five  'E' i s an  found  f  a  absorptive.  Calanus  guished.  functions  plays  regions  were  in secretion.  are mainly In  plays  and  absorption  type  conclusions  differ-  in secretion.  The p r e s e n c e  i n the absorption  anterior  cell  only  the u l t r a s t r u c t u r e i n the various  caecum  lost  and  i s absorptive,  found  that  cell  It also  type  suggests  the following  'B'  proteins.  type  tract,  the  away  secretory  an a b s o r p t i v e  vesicles  From  a  Cell  Cell  dense  of  a r e worn  i s mainly  synthesis  lipids.  as a r e p l a c e m e n t  'R' .  cell type  Cell  found 'F'  type  i n the  functions  (iv) in  the  synthesis In  and  Calanus  s e c r e t i o n o f d i g e s t i v e enzymes.  plumchrus,  specialized  for  transport  metabolites  of  the  the  absorption to  the  midgut of  region  In  the  middle  to  the  glandular  specialized are mainly In be with  used the  specialized  s e c t i o n of  for  region,  the is  secretion.  for  nutrients The  pinocytotic  area  The  is and  anterior  It includes a vacuolated  midgut, an  digested  haemocoel .  midgut r e g i o n s are m a i n l y a b s o r p t i v e . glandular  diverticulum  adjacent of  absorption. and  posterior  epithelial  p o s t e r i o r midgut  cells regions  absorptive. T igr iopus  to  cali fornicus  determine  cell  type  observed u l t r a s t r u c t u r e .  biological function  in  markers  can  correlation  (v) TABLE OF CONTENTS Abstract TABLE OF CONTENTS L I S T OF FIGURES ACKNOWLEDGEMENTS  ii v v i i xi  Introduction Foregut Midgut Midgut S t r u c t u r e s Hindgut Endocytos i s (i) pinocytosis (ii) phagocytosis (iii) f l u i d phase p i n o c y t o s i s (iv) receptor mediated p i n o c y t o s i s  1 2 4 6 9 11 11 12 13 13  M a t e r i a l s and Methods Light Microscopy (a) Tigr iopus c a l i f o r n i c u s (b) Calanus plumchrus Electron Microscopy (a) Tigr iopus c a l i f o r n i c u s (b) Calanus plumchrus (c) f l u i d phase e n d o c y t o s i s (d) receptor mediated endocytosis  17  Results Tigr iopus cal i f ornicus Esophagus M i d g u t Caecum A n t e r i o r Midgut P o s t e r i o r Midgut Hindgut  23 23 23 24 26 28 30  Calanus plumchrus Esophagus Midgut Hindgut  17 18 19 20 20 21  30 31 31 36  R e c e p t o r M e d i a t e d E n d o c y t o s i s ( T . c a l i f o r n i c u s ) ...37 F l u i d Phase E n d o c y t o s i s (T. c a l i f o r n i c u s ) 38 Discussion Foregut Midgut C e l l Types M i d g u t Caecum and M i d g u t D i v e r t i c u l u m Midgut R e g i o n a l S p e c i a l i z a t i o n s Hindgut P e r i o t r o p h i c membrane  40 40 43 43 50 52 .56 58  (vi) Endocytosis f l u i d phase adsorpt ive Key f o r F i g u r e s FIGURES References  61 61 64 69 7  0  93  (vii) LIST Figure  1.  Sagittal  Figure  2.  Light micrograph T_. c a l i f o r n i c u s  Figure  Figures  3.  OF  FIGURES  s e c t i o n o f T.  5.  6.  E l e c t r o n micrograph  Figure  7.  Esophageal  Figure  8.  L i g h t micrograph  Figure  10.  Figure Figure Figure Figures Figure  70  70  of the esophagus cuticle  of the midgut  E l e c t r o n micrograph  the  13. C e l l t y p e midgut 14  and 15. midgut  '3'  caecum  i n T_. c a l i f o r n i c u s  72  o f the m i d g u t  72  caecum  Figure  18.  type  '4'  73 74  i n the a n t e r i o r  Light micrograph  of the a n t e r i o r 75  of the c e l l  types found  i n the  midgut  L i g h t micrograph  75 of the a n t e r i o r midgut  L i g h t m i c r o g r a p h o f the p o s t e r i o r segment of the a n t e r i o r midgut 19. E l e c t r o n m i c r o g r a p h o f t h e a n t e r i o r midgut 20.  73  74  16. M i c r o g r a p h  17.  and  71 72  T r a n s v e r s e s e c t i o n t h r o u g h the m i d g u t caecum 11. E l e c t r o n m i c r o g r a p h o f t h e t r a n s i t i o n from m i d g u t caecum t o a n t e r i o r m i d g u t 12. M i c r o g r a p h o f c e l l t y p e '3' i n t h e a n t e r i o r midgut  Figure  Figure  esophagus  caecum  associated  a n t e r i o r midgut  anterior  Figure  the  T r a n s v e r s e s e c t i o n through  Figure  9.  of 70  e s o p h a g u s and m i d g u t  and  70  o f t h e mouth p a r t s  Transverse s e c t i o n through o f T. c a l i f o r n i c u s  4 and  Figure  californicus  ' B r i d g e ' shaped c e l l p o s t e r i o r midgut  type  '2' o f  76  76 76  the 77  (viii) Figure Figures  21.  I n v a g i n a t i o n s of the b a s a l i n the p o s t e r i o r midgut  22 and  23.  plasma  membrane 77  L i g h t m i c r o g r a p h s of the p o s t e r i o r  midgut Figure  24  Figures  25 and 26. T r a n s v e r s e s e c t i o n s of the anterior hindgut 27. E l e c t r o n m i c r o g r a p h o f t h e c e l l s o f t h e p o s t e r i o r midgut  Figure Figure  Figure Figure  Cuboidal  78 cells  of the p o s t e r i o r midgut  79 79  28. M i c r o g r a p h o f t h e c u t i c l e lined lumen o f t h e c e l l s o f t h e p o s t e r i o r hindgut  80  29. T y p i c a l banded c u t i c l e h i n d g u t n e a r t h e anus  80  30. S a g i t t a l  of the p o s t e r i o r  s e c t i o n of the d i g e s t i v e  tract  of Calanus plumchrus Figure  78  .81  31. L i g h t m i c r o g r a p h o f t h e e s o p h a g u s  32. E l e c t r o n m i c r o g r a p h o f t h e e s o p h a g e a l cuticle F i g u r e s 33 and 34. L i g h t m i c r o g r a p h s of the  81  Figure  midgut 35.  Figure  36. M i c r o g r a p h o f t h e a p i c a l p o r t i o n o f 'R' c e l l s 37. H i g h power view o f t h e l u m i n a l microvilli  82  38. R' c e l l s region  82  Figure Figure Figure Figure  1  cells  81  Figure  Figure  'R'  81  of the midgut  diverticulum  82  82  of the p o s t e r i o r midgut  39. T r a n s i t i o n c e l l s between t h e m i d g u t d i v e r t i c u l u m and t h e m i d g u t  82  40. L i g h t m i c r o g r a p h o f t h e m i d g u t i n C. p l u m c h r u s  82  41. M i c r o g r a p h o f t h e g l a n d u l a r region  valve  midgut 83  (ix) Figure  Figure  Figure  Figures  Figures  44.  45.  46.  47  49  Micrograph of the a p i c a l portions of the c e l l s i n the g l a n d u l a r region  83  Apical of the  84  v e s i c l e s and 'F cell  H i g h power the midgut and 'F'  microvilli  1  l i g h t micrograph valve  48. Transverse cells  to  51.  of  the  84  sections  of 84  micrographs  p o s t e r i o r midgut  Figure  53.  Transverse  section  of  the  anterior  54.  hindgut region Transverse section hindgut region  of  the  posterior  Figure  Figures  55.  56.  57  Micrograph of the o f C. p l u m c h r u s Low p o w e r posterior and  58.  lining  the  Anterior  Figure  60.  Cell  61.  caecum Micrograph  '2'  85  cell  of  of  the 85  the  cuticle  T.  85  californicus  86  midgut  types  '2'  and  '3'  a n t e r i o r midgut midgut  Figure  63.  Colloidal  gold  66.  cuticle  86 of  Posterior  Figure  hindgut  i n the  62.  64  85  of  Figure  Figures  85  posterior hindgut  midgut  type  the  hindgut  85  Micrographs  59.  in  the  l i g h t micrograph hindgut  Figure  Figure  of  ..84  52.  Figure  micrograph  region  Figure  Figure  Light  Electron  of  of  T.  86 californicus  particles  in  the  lumen and 65. Gold c o l l o i d s associated w i t h t h e p l a s m a membrane o f c e l l type ' 3 ' Colloidal posterior  gold i n the midgut  lumen  87  of  87  88  the 88  (x) Figure  Figure  67.  68.  M i c r o g r a p h of the a n t e r i o r midgut  Figure  69.  70.  of  the 89  Gold p a r t i c l e s i n the of c e l l type 2 1  Figure  cells  ctyoplasm 89  1  Electron micrograph midgut  of  Electron micrograph  of  the  anterior 90  the  midgut  caecum  90  Figure  71.  HRP  i n the  cytoplasm  Figure  72.  HRP  i n the  midgut  caecum  Figure  73.  Micrograph  of  found  Figure  74. M i c r o g r a p h  Figure  75.  midgut  caecum of  Micrograph the midgut midgut  HRP  of  cell  type  '3'  91 91  in  the 91  a n t e r i o r midgut  of the caecum  .....92  j u n c t i o n between and t h e a n t e r i o r 92  (xi) Acknowledgements I wish  t o e x p r e s s my g r a t i t u d e  Bisalputra.  He  i n t r o d u c e d me  t o my  supervisor  to the f i e l d  of  D r . T.  electron  m i c r o s c o p y and p r o v i d e d v a l u a b l e s u g g e s t i o n s and i n t r e p r e t a t i o n s throughout  this  study.  Special  thanks  Dr.  A.G. L e w i s  for h i s suggestions  Dr.  A.B. A c t o n  for h i s assistance  ideas I  on my  wish  friends  project  t o thank K. B e l l ,  L. B o r t o l a m i o l suggestions. Without  and s u p p o r t  D r . E . de  are extended  and and  support,  Harven.  I am  for their  For  i t s conclusion, indebted  H. C h r i s t e n s e n , G. B o o t s m a ,  and S. Ramjohn  and t o  friendship.  through  to  support  R.  t o my Leung,  and h e l p f u l  To Jane Van S i c k l e I e x t e n d my d e e p e s t g r a t i t u d e .  her courage,  persistence,  and v a l u a b l e  assistance  w i t h t h i s m a n u s c r i p t , t h e p r o j e c t would n o t have been c o m p l e t e d . Summer  support  t o D r . T. B i s a l p u t r a .  was  supplied  by a NSERC  grant  awarded  -1Introduction: Few  studies,  at the l i g h t  or e l e c t r o n  microscope  l e v e l s , have examined t h e c o m p a r a t i v e m o r p h o l o g y and f u n c t i o n a l anatomy  of the d i g e s t i v e  tract  of copepods.  This  study  w i l l compare t h e d i g e s t i v e t r a c t m o r p h o l o g i e s o f a h a r p a c t i c o i d copepod T i g r i o p u s c a l i f o r n i c u s and a c a l a n o i d copepod plumchrus.  I t i s intended  of  the gut morphology  is  a study  tract  and  copepods.  understanding Also  included,  of the  digestive  of T i g r iopus c a l i f o r n i c u s .  those  their  of marine  the  o f t h e e n d o c y t o t i c mechanisms  Comparisons to  to further  Calanus  of other  similarities  with  of the digestive arthropods w i l l and d i f f e r e n c e s .  the study  californicus,  some  insight  f o r the morphology  and  cells.  There  is a  and t h e i r  be i n c l u d e d  to  Through such  o f e n d o c y t o t i c mechanisms  suggested their  tracts  great  c a n be g i v e n  illustrate comparison,  in Tigriopus  to the f u n c t i o n s  of the d i g e s t i v e  deal  cells  tract  of c o n f l i c t i n g  regions  terminology  i n t h e l i t e r a t u r e r e g a r d i n g some o f t h e a r e a s o f t h e a r t h r o p o d digestive  tract.  No  clear  convention  o f t h e terms d i g e s t i v e d i v e r t i c u l a ,  exists  f o r t h e use  g l a n d s , c a e c a , or h e p a t o -  p a n c r e a s . S u l l i v a n (1977) d e s c r i b e d t h e caecum as a d i v e r t i c u l u m of  the d i g e s t i v e  tract  since  a diverticulum  was  term d e f i n e d as a b l i n d - e n d i n g t u b u l a r or s a c - l i k e  a  general  outpushing  from a c a v i t y t h a t i s n o t e x t r e m e l y b r a n c h e d .  That t e r m i n o l o g y  will  diverticula  be  upheld  here  since  the d i g e s t i v e  are  -2-  not  branched  glands  or  like  those  described  of  a  crustacean  a f o r e g u t , m i d g u t , and ventral origin to  have  from  A.  anus. and  other  arthropods  as  hepatopancreas.  There are three d i s t i n c t components  in  The  tubular  d i g e s t i v e system.  a hindgut  foregut  the m i d g u t  r e g i o n s t h a t are the  and  They  include  a p o s t e r i o r or  postero-  hindgut  are  of  i s of e n d o d e r m a l o r i g i n .  outgrowths,  the midgut.  with  (McLaughlin  principle  glands  and/or  ectodermal I t i s common  caeca  arising  1983).  Foregut In c r u s t a c e a n s t h e r e i s a c o n s i d e r a b l e f o r e g u t d i v e r s i t y .  It  has  to  a  been  variously described  complex  multichambered  as  a  simple  structure.  passageway  Previous  on c o p e p o d s have shown t h a t t h e f o r e g u t i s a s i m p l e In  the  copeped  Epilabidocera and so  (Park  T i g r i opus  Calanus  (Marshall  1966), Centropages  (Sullivan  and  and  (Arnaud  Bisalputra,  1980)  esophagus. Orr,  1955),  e t a l , 1978), i t has  been  described. Other  of  genera  studies  the  genera  cuticulized 1961)  authors  (Briggs  Paranthessius  chamber  while  1977) as  a  others  described short  the  esophagus  (Bresciani  and  d e s c r i b e d the f o r e g u t of a p a r a s i t i c s p e c i e s ,  steenstrupi,  as  an  elongated  tube  and  large  foregut with  a  Lutzen,  Saccoposis  muscularized  stomach. In t h e b a r n a c l e a l i m e n t a r y t r a c t t h e f o r e g u t i s d e s c r i b e d  -3-  as  cuticle  hameri; the  lined  in  (Rainbow  and  pharynx, The  tions.  the  both  Walker,  esophagus,  foregut  of  Posterior  the  Also and  grinding  to  i n the mysid posterior It  the  is  the  to  prevent  that  of  series  esophagus  the  of  Hinton  and  standing  of  Corey,  activity the  (1976) p r o p o s e d  an  the  blowfly  in  the  esophageal  absorption  evidence  of  decapods  fat  food.  1972).  esophagus,  1980). influence are  formed  and  function  1955).  (Naylor,  highly  complex.  chambers  is a  chambers  function  Pillai,  ampullae  There  i s extemely  Deep  are  short,  (Maynard  the  important  processes. model  regina.  portion  in  they  and  lined  muscular  and  Dando,  1979).  (1924) s t a t e d but  that and  anterior  explanatory  Phormia  of  plates. these  anterior  articulation  is  and  digestive  in  Yonge  to  an  differences  isopods,  chamber  posterior  anterior  are  specializa-  (Mauchline,  dietary In  Balanus portions;  i s a short muscular  regurgitation  calcified  Foregut  to  the  three  further  (Nath  chambers.  development.  of  has  been suggested  there  likely  foregut  infoldings  1974;  foregut,  and  ventriculus.  particles,  esophageal/foregut  The  a  food  back  the  with  esophagus  I t has  anterior  the  1977)  and  the  and  foregut  at  by  of  balanoides  malacostracans  a p o s t e r i o r chamber. in  Balanus  of  Thomson  and  of  foregut  underHailing  peristalsis  activity  occurs  foregut. the  foregut  cockroach,  absorption.  the  Peristaltic  the  that  to  Vonk  was  Eisner  (1960)  impermeable  (1955)  found  found  evidence  -4-  for  the  secretory  mucous  and  of  the  gut.  B.  Midgut As  is  function  possibly  was  noted  amylase  for  the  also highly v a r i a b l e .  into the  three  parts.  anterior  Their caeca.  The  and  feces  the  of  foregut.  He  found  were  secreted  in  this  foregut,  and  is  the  the  the  that region  crustacean  Kennedy primary  anterior  midmesenteral formation  the  midgut  In D a p h n i a , the m i d g u t i s d i v i s i b l e  Schultz  portion  description  of  region  is  (1976) site  of  portion the  state  site  that  absorption.  includes of  the  digestion,  i n c a r r i e d out p o s t e r i o r l y .  In c o p e p o d s o f the genus C a l a n u s , the m i d g u t i s a n t e r i o r l y expanded, thoracic  narrowing s o m i t e s and  (Marshall the  Orr,  and  level  continuing  of  the  t o the  195.5; H a l l b e r g  d i s t i n g u i s h three  Bisalputra,  Tigriopus been cell  (1980),  midgut  found  c a l i forn icus.  described  for  Two  caligoid  the  four e p i t h e l i a l  calanoid  Centropages  Considerable the  midgut  cell  has  or  Hirche,  only  H a l l b e r g and  two  i n the  epithelial copepods  third somites  1980).  d i v i s i o n s while  In  Hirche,  Sullivan  harpacticoid,  cell  (Lewis  types  have  1961),  four  ( S u l l i v a n and B i s a l p u t r a ,  t y p e s have been d i s t i n g u i s h e d  (Arnaud  et  differentiation  epithelium  second  l a s t abdominal  and  types i n T i g r i o p u s c a l i f o r n i c u s  1980) , and  of  the  c a l a n o i d s , Arnaud e t a l . , (1978) and  (1980),  in  and  at  been  a l . , 1978). of  the  correlated  fine with  structure season,  -5sex,  and s t a g e  of development.  Fahrenbach  ( H a l l b e r g and H i r c h e ,  (1962) d e s c r i b e d  valve  and  Sullivan  tight  constriction  and  a centrally  Bisalputra,  located  (1980),  at the a n t e r i o r  1980). midgut  described  and p o s t e r i o r  a  midgut  j unction. In is  the isopods  termed  the midgut  of ectodermal that  i s wholely  region  studies  that  the woodlouse  t h e r e f o r e a midgut. data a  small  are  segment  and t h a t  between  i ti s  and  i s an hindgut  by  Goodrich  the midgut i s e x t r e m e l y exists  i n the  and S c h u l t z  (1969)  i s mainly  endodermal  (1973) s t a t e s t h a t i n isopods  the foregut  and  previous  there  i s only  and t h e h i n d g u t  that  the remains of the midgut. In  Pike  decapods  (1947)  practically Barker for  But H o l d i c h  was m i s i n t e r p r e t e d  Studies  Schmitz  gut  that  the midgut  controversy  midgut.  of the gut  (1968) s t a t e d  the foregut  isopods  Considerable  show  Jones  isopods  in nature.  i n these  o f t h e isopod  stated  1965).  between  glandular  or a b s e n t .  results  terrestrial  (1939) i n d i c a t e t h a t small  though  (Stromberg,  and  small  the central portion  even  origin  i n marine  extremely and  only  considerable  reported absent.  and G i b s o n  that  midgut  the midgut  state  that  was  very  exists. short  or  In t h e e u r o p e a n l o b s t e r Homarus gammarus (1977)  state  that  5/6 o f t h e p o s t g a s t r i c i n t e s t i n a l Fahrenbach  variation  (1962),  i n most  and  Holdich  crustaceans,  the midgut  accounts  tract. and M a y e r s ,  a periotrophic  (1975) membrane  -6-  is  actively  C.  Midgut  s e c r e t e d by t h e m i d g u t  Structures  Numerous  midgut  structures  been d e s c r i b e d by many a u t h o r s . work has been a v a i l a b l e the  cells.  caeca  to date  of the crustaceans  Very  little  The  by  Sullivan  (1977)  of the midgut,  terminology  been a p p l i e d w i t h c o n s i s t e n c y by t h e a u t h o r s . developed  ultrastructural  on t h e g l a n d s  or the d i v e r t i c u l a .  equates  has not  The t e r m i n o l o g y  the midgut  caecum  to t h a t of a d i v e r t i c u l u m i n T i g r i o p u s c a l i f o r n i c u s . et  a l .,  (1977)  decapods because  should  authors,  be  that  n o t be  i t does  established  that  state  not f u n c t i o n  Gibson  and B a r k e r  referred  structures  dorsal has  pockets  been  used  midgut w a l l . For of  or p a n c r e a s .  and d u c t - l i k e  this  Other  by  stating  structures  carbohydrate metabolism  have  been  t h e term  should  demonstrated  midgut  and d i g e s t i v e  i n these g l a n d u l a r  and G i b s o n ,  caecum  1978).  i s used  for small  o f growth from t h e m i d g u t .  Midgut  to r e f e r  pouches  (McLaughlin,  the purpose  the midgut  hepatopancreas  to t h e c o n v e n t i o n s  dispute  ( M c L a u g h l i n , 1983, B a r k e r  Normally  of  hepatopancreas.  In many d e c a p o d s , secretions  (1979)  tubular  t o as a  liver  Phillips  diverticula  t o as a  according  f o r t h e mammalian  t h e complex  enzyme  the glandular  referred  have  to b l i s t e r - l i k e  diverticula from  the  1983).  of t h i s  o f T i g r i opus  study,  the a n t e r i o r  c a l i fornicus  will  be  outgrowth referred  -7to  as  the midgut  outgrowth  of  the midgut  to  are  state  caeca  term  1  and  that  since  an  the  anterior  will  be  called  of  digestive 1  of  with  'midgut'  to the midgut  the  in  small  the  Daphnia  diverticula  the midgut.  diverticulum or  that  They  anterior  the  term  go  on  midgut  digestive  is preferred  over  the  caeca.  hepatopancreas  in Tigriopus  has  californicus  similar  (Sullivan,  and f u n c t i o n s i n f o o d a b s o r p t i o n and p u r i n e m e t a b o l i s m ,  (Vonk,  1960)  d i g e s t i v e enzyme s e c r e t i o n , s t o r a g e a t g l y c o g e n ,  l i p i d s and m i n e r a l s . 1969;  Loizzi A  a  state  pair  analogue  Malacostracans  functions  and  anterior  invaginations  hepatopancreas  1977)  an  (1976)  Malacostraca i s guestionable,  or  In  and  of Calanus plumchrus  Kennedy  exists  simple  of  caecum  1977)  diverticulum.  there  that  (Sullivan,  the midgut  Schultz pulex  caecum  1968;  single  land icus  anterior and  Woodhouse, S t a n i e r  anterior  harpacticoid  single  ( H a r t e n s t e i n , 1964;  caecum  copepods.  Ong  outgrowth  termed  was  Loizzi  and  Griffin,  reported and  midgut  1968).  for  Lake,  of the midgut  i t the  and P e t e r s o n ,  calanoid  (1970)  found  of Calanus  helgo-  diverticulum.  Arnaud  e t a l . , (1978) f o u n d a s i n g l e a n t e r i o r caecum w i t h no a s s o c i a t e d glands cell  i n Centropages  categories  described  high  (E,R,F,B,D)  and  identified  i n the midgut  f o r t h e m a l a c o s t r a c a n s and  of decapods. and  typicus  The  'R'  microvilli,  cells the  had  'F'  five  similar  i n the  had  to  those  hepatopancreas  smooth e n d o p l a s m i c  cells  principle  rough  reticulum  endoplasmic  -8reticulum  and s h o r t  staining,  t h e 'B' c e l l s  were s l e n d e r  with  Sullivan spherical three  and of  type  could  cells  vacuolated  cytoplasm  caecum  ' 1 ' , was  and r a r e (1980),  in Tigriopus  a t the base  three  and was  rough Cell  invaginations, and  randomly  was  due  Dilated cell  anterior  californicus  i n which  (cell  lipid  dispersed  Golgi  bodies  associated  '3' w i t h  droplets.  smaller  transparent electron  rough  were  also  and  I t had d i s p e r s e d  found  basal  microvilli, cytoplasm  reticulum. abundant  lamellate  rough  nuclear  v e s i c l e s and '4', was  rough  vesicles  i n the a p i c a l  found  endoplasmic  G o l g i , abundant  other  mainly  regular  The most  type  some  transparent  sized  type,  and l e s s d i l a t e d  in clusters  mictochondria,  various  cell  epithelium  The d e n s e  invaginations,  v e s i c l e s , and many  density  and  luminal  found.  1,2,3).  ribosomes,  endoplasmic  abundant  fourth  i n the m i d g u t .  reticulum,  and  bodies A  droplets,  basal  found  t o have  mitochondria.  reticulum, Golgi  found  types  of the gut  reticulum  '2' was  to ribosomes  endoplasmic  only  type  cells  an  by abundant  endoplasmic  apical  was t y p e  lipid  characterized  darkly  organelles.  found  be d i s t i n g u i s h e d  found  were  and t h e 'E'  n e v e r r e a c h e d t h e lumen. I t was n o r m a l l y  vesicles.  of  electron varying  portions  of  cell. Baccari  the  were  t h e 'D'  and B i s a l p u t r a  types  mitochondria,  the  light  midgut  cell  Cell  microvilli,  digestive  and  Renaud-Morant  system  (1974)  found  of the mystacocarid  no c a e c a i n  Derocheilocaris  -9remanei. in  As many  as  seven  some b a l a n o m o r p h s . Diversity  among  (McLaughlin,  abounds  gland  there  i s present  (1972)  found  lobes  been  found  1983). and m i d g u t  In t h e l e p t o s t r a c a n s ,  glands single  i n N e b a l i o p s i s but i n other  genera  of caeca  (McLaughlin,  mysids  have  a  a r e one or two p a i r s  glandular  caeca  i n the caeca  the malacostracans.  midgut  or  midgut  with  and two or t h r e e 1983).  multilobed  Nath  glands  and  glands Pillai,  accompanied  by one or two c a e c a . Both  midgut  decapods. of  decapods  1977, and  glands  have  (Pillai,  on  glands been  1960; D a l l , In d e c a p o d s ,  either  side  are normally  described  for a  1967; B a r k e r these  found i n  glands  number  and  Gibson,  are  lobular  of the gut at the junction  of  f o r e g u t and m i d g u t .  Hindgut In  ending  crustaceans  there  i s normally  i n a m u s c u l a r i z e d anus.  Sullivan of  and  1978, 1 9 7 9 ) . found  the  D.  Midgut  caeca  and B i s a l p u t r a ,  a striated cuticle.  Ci rripeds, regions.  anterior  (1980),  described  i s characterized muscular  sphincter  (McLaughlin,  1983).  Rainbow  and W a l k e r  (1977)  state  to a h i g h l y  folded  muscular  leads  hindgut  In t h e r e g i o n o f t h e anus,  a n a l chamber.  midgut  short  the arrangement  In some c r u s t a c e a n s such as Balanomorph  the hindgut An  a  by  two  and a  that  distinct posterior  the p o s t e r i o r  hindgut  region  -10that  acts  wide  anal  found  as a  sphincter.  chamber  and  a  t o be h i s t o l o g i c a l l y  P o s t e r i o r l y , i t extends slit-like similar  anus.  The h i n d g u t  i n isopods  of the d i g e s t i v e t r a c t .  a n t e r i o r and p o s t e r i o r p o r t i o n s s e p a r a t e d  with  the a n t e r i o r p o r t i o n s Vernon  hindgut of  two chambered  structure  with  the l a r g e deep  isopod  infoldings  the a n t e r i o r w a l l s . Fox  (1952),  i n many C r u s t a c e a .  described  o f the r e c t a l  action  as a n a t u r a l  but  acted  was p r o b a b l y  the anal  role  o f water  intake  He d i s c u s s e d t h e pumping and a n t i p e r i s t a l t i c  contraction  or  by a s p h i n c t e r  distensible.  e t a l . , (1974) a l s o d e s c r i b e d  as a  represents  I t c a n be d i v i d e d  into  being  was  to the f o r e g u t .  J o n e s (1968) s t a t e d t h a t t h e h i n d g u t a large proportion  to a  muscles enema  not involved  and s u g g e s t e d  that  i n the d e f e c a t i o n  i n osmoregulation,  this  process  respiration  the maintenance of t u r g o r . In  the decapods,  of  the hindgut.  in  Golathea  such  as  Pike  species  Pagurus,  confusion  exists  (1947) , d e s c r i b e d  where  as  the hindgut  over a  the  long  i n others  (Jackson,  was  t o be  found  extent hindgut 1913),  extremely  short.  was  In  the d i g e s t i v e  found  t o be v e r y  system short,  pulex,  cuticulized,  structure  to the foregut.  controlled  by d i l a t o r m u s c l e s .  Sullivan  o f Dapnia  There  and B i s a l p u t r a  and  i s a small  the  similar  terminal  ( S c h u l t z and Kennedy, (1980),  hindgut  described  the  in anus  1976). hindgut  -11of  Tigr iopus  with  c a l i fornicus,  a c u t i c u l i z e d luminal  tions.They  suggested  as  an  surface  that  epithelial  lined  and e x a g g e r a t e d  the hindgut  could  copepod  S. P a r k  Epilabidocera  surrounded ridges three  by  a  similar pairs  have  been  longitudinal  anal  segment  has  with i t .  absorption  suggested.  I n t h e brown  of dissacharides, (Edson  wasp,  lipids,  and V i n s o n ,  Microplitis and  triolein  1977).  Endocytosis  (1983), cules  pinch  as  i s a process  and  portions  be  associated  epithelia  a r t h r o p o d s s e v e r a l f u n c t i o n s have been s u g g e s t e d  Endocytosis,  the  muscles  The  absorption.  lined  containing  the esophagus.  in  of the calanoid  as c u t i c l e  membrane  the c u t i c u l i z e d hindgut.  croceipses,  E.  basement to  the hindgut  amphitrites  of d i l a t o r  In o t h e r for  (1966) d e s c r i b e d  invagina-  function  a o s m o r e g u l a t i o n , w a t e r t r a n s p o r t , a n d some n u t r i e n t T.  chamber  the  ingested subdivided  intracellular The  two m a j o r  I t i s often  Bruce  ingestion  membrane  material. into  by  are progressively  plasma  o f f , forming  tosis.  of c e l l u l a r  particles of  reviewed  classified  by  whereby  et a l . ,  macromole-  enclosed  which  in  invaginate,  vesicles  process  types:  Alberts  of  which  the s i z e  of  then  contain  endocytosis  Pinocytosis  small  can  and Phagocythe v e s i c l e s  formed. (i)  Pinocytosis Fluid  and/or  solute  ingestion  i s usually  referred  -12-  to  as p i n o c y t o s i s  1983;  called  ingestion  pinocytotic  dissolved There  solutes  containing process receptors  of f l u i d  mediated  (ii)  and/or phase  recycling.  bound  vesicles ingestion  1983;  mediated  which  vesicles  arise  does  not  from  a  involve  membrane  vesicles arise  containing  from  receptor  endocytosis.  or c e l l - e a t i n g , involves  p a r t i c l e s such often  referred  i s usually  function,  process. but  solutes  phase  Phagocytosis  large  that  fluid  pinocytotic  Pinocytotic  t o t h e plasma  Phagocytosis of  pinocytosis  fluid  t o t h e i r membranes.  and r e c e p t o r  Those  This  vesicles  cellular  of pinocytosis;  dissolved  1931).  by s m a l l  contain  (endocytosis)  et al . ,  and i s r e l a t e d t o t h e s e c r e t o r y p r o c e s s and i n v o l v e d  membrane  molecules  They  types  (endocytosis). fluid  1 9 6 9 ; Lewis,  or m o l e c u l e s bound  a r e two main  pinocytosis  (B. A l b e r t s  i s characterized  vesicles.  or b u l k u p t a k e p i n o c y t o s i s  in  drinking  Steinman, 1 9 8 3 ; C a s l e y - S m i t h ,  type of c e l l u l a r  and  or c e l l  and  as m i c r o o r g a n i s m s t o as v a c u o l e s .  the domain  i s not considered  P h a g o c y t o s i s may a l s o  i t i s an Steinman,  uptake  of c e l l s  of large  involve  a  v i a much Large  1969).  larger particle  specialized for routine  a receptor  particles.  1 9 8 3 ; Casley-Smith,  the i n g e s t i o n  cellular mediation  (Alberts  et a l . ,  -13(iii)  Fluid The  in  a  fluid  number  brief to  Phase P i n o c y t o s i s ( E n d o c y t o s i s )  of  plasma  include  an  molecules  the  such  as  Horseradish  with  and  b i o c h e m i c a l l y , and tration  and  al.,  1983) .  (iv)  Receptor  binding  to  Mediated  a  some  involves does  and  of  plasma  Pincocytosis  type  colloidal  gold  tend  largely  t o be  clathrin. deliver  coated  marker  visualized  after  diaminobenzidine. 1973;  Cornell  with  time  the  et  made up  a  ligand then  to  the  which  membrane  lattice  lysosomes  recycle  t o t h e plasmalemma.  the  concenet  (Endocytois)  receptor,  These of  al. ,  (Alberts  work  or  other  clathrin  and  ligand  are  often  clustering  coated  regions  of  protein  the  I t i s assumed t h a t t h e p i n c h e d - o f f c o a t e d  compartments, back  pits.  (HRP)  valuable  d i f f u s e l y d i s t r i b u t e d , f o l l o w e d by 1 i g a n d - r e c e p t o r in  bind  this  pinocytosis involves  membrane  the  The enzyme can be q u a n t i f i e d  continually  mediated  complexes  not  peroxidase  is a be  visualized  i t s uptake i n c r e a s e s l i n e a r l y with  increases  Receptor  can  Fahimi,  Graham and K a r n o u s k y , 1966) .  be  markers  peroxidase  vesicles  Herzog  that  horseradish  hydrogen p e r o x i d e  1983;  solute  ferritin, as  can  common method  Cytological  such  pinocytotic  (Steinman,  most  events  impermeable  enzymes  dextrans.  ingestion  1971;  to  The  membrane.  preparations,  because  endocytotic  ways.  exposure  the  and  phase  vesicles  intracellular the  ( G o l d s t e i n e t a l . , 1979).  receptors  -14Recent may  not  recycle  cytoplasmic (1981), the  u l t r a s t r u e t u r a l work but  remains  suggests  attached  to  s i d e o f the plasma membrane.  suggest  ligands  that  once  instead  bound  in  of  the  that  the  pits  P a s t a n and  recycling coated  on  the  Willingham  the  pits,  clathrin  clathrin,  bud  to  form  an u n c o a t e d r e g i o n or c r y p t i c p i t i n t h e i n v a g i n a t i n g membrane. Uncoated  vesicles  invagination  from  clathrin  coat.  somes' .  The  of  the  binding  were  without  the  the  could  termed  their  the  plasmalemma these  coated  pit  and  be  agglutinate  red  group  of  known  in  the  ' receptoas  further  part  ligand  recycling. that  plant  The  term  lectin  the  via  remain  cells.  various  to a g g l u t i n a t e  unique  clathrin  blood  from  for  pits  opening  vesicles  then  been  Today,  cryptic  an  uncoated  of  i t has  proteins  or  available  requirement  'lectins'.  these  would  1880's,  ability  This  from  call  plasmalemma  sugar-binding of  formed  They  empty  Since extracts  are  cell  agglutinins  sources  refers  to  regardless  cells.  proteins  has  provided  biologists  w i t h a p o w e r f u l t o o l t o s t u d y many c e l l u l a r p r o c e s s e s i n c l u d i n g endocytosis. particular with  Since  lectins  carbohydrate  identical  sugar  have  a  specificity  structure,  compositions  even  can  be  toward  a  oligosaccharides  i s o l a t e d and  ident-  ified. Certain while others require  that  lectins recognize the  bind  to  residues  only galactose  sugar  be  in  the  of mannose or  residues. terminal  Some  glucose lectins  non-reducing  -15position charide  while  others  to sugars w i t h i n  an o l i g o s a c -  chain.  Since  virtually  glycoconjugates can  can bind  be used  a l lbiological  (glycoproteins  to study  the c e l l  and  membranes  contain  glycol ipids) ,  surface  features  lectins  of a l l l i v i n g  organisms. Wheat  Germ  Agglutinin  (WGA)  is a  w e i g h t p r o t e i n t h a t has two i d e n t i c a l sugar  f o r WGA  chitobiose WGA  containing  which  has a l s o  Lis,  acid  been  reported acid,  residues.  to o l i g o -  N-acetylglucosamine  to i n t e r a c t to  (Monsigny  The r e c e p t o r  I t can b i n d  t o many membrane  and  molecular  some  or  glycoproteins.  with  glycolipids,  glycoproteins v i a  e t a l . , 1980;  Sharon  and  1972) . Colloidal  marker  easily  with  Due  with  to t h e i r  detected.  lectins  Specific  cytochemical  for transmission  opacity  electron  to the electrons  markers  a v a r i e t y of macromolecules  erger,  have  been  including lectins.  they  obtained (Horisb-  1979). The  lectins  conjugation  provides  mediated The light  g o l d has r e c e n t l y been used as a  i n conjunction  microscopy. are  terminal  a r e common  N-acetylneuraminic sialic  subunits.  i s N-acetylglucosamine.  saccharides  36,000  of c o l l o i d a l  gold to the s u g a r - s p e c i f i c  the v e h i c l e to study  the events of r e c e p t o r  endocytosis. purpose  of  this  study  and e l e c t r o n m i c r o s c o p e  was  levels  to d e s c r i b e the d i g e s t i v e  at the tract  -16-  of  two c o p e p o d s ; T i g r i o p u s c a l i f o r n i c u s and C a l a n u s  Also  included i s a characterization  cytosis  i n the d i g e s t i v e  From  the  tract  descriptions  of the p r o c e s s  of T r i g r i o p u s  provided,  and  From studies  r e g i o n and this  of the  processes  cell  type w i l l  comparative  functional  in other  be  of  comparisons  relationships  description  anatomy o f t h e s e c o p e p o d s , be  with  apparent.  morphological  crustacecea w i l l  endo-  californicus.  o t h e r a r t h r o p o d s , i t i s hoped t h a t t h e f u n c t i o n a l between g u t  plumchrus.  better  and  similar  understood.  _17Materials  and  Methods:  Tigr iopus  cali fornicus  a c u l t u r e maintained  by  U.B.C.  were m a i n t a i n e d  sea  The  copepods  water,  in  laboratory fish  changed the  culture  under  food  was  to  culture  continued  f e e d i n g was  no  collection  from  depth  of  200m  to  Zoology  in enriched  room  artificial  temperature  the  and  months. out  Regular  in  the  tropical  water  was  cleaning  of  Once  a  good  i n the c u l t u r e c o n t a i n e r s ,  necessary.  specimens 1983  sea  bi-weekly.  from  Department,  source  longer  i n June  obtained  'Tetramin'  established  plumchrus  single  Lewis,  at  carried  was  were  conditions.  three  d e b r i s was  bacterial  a  jars,  nutrient  two  Calanus  A. G.  fluorescent  the  every  bottom  Dr.  specimens  were  obtained  using a plankton  s u r f a c e i n the  a  net  dragged  of  Georgia  Straight  i n an a r e a a d j a c e n t t o t h e F r a s e r R i v e r plume.  from  (Fra-1 sampling  station).  A.  Specimen P r e p a r a t i o n  (i)  Light  Microscopy  (a) T i g r i o p u s c a l i f o r n i c u s Specimen out in  i n the 5%  (PBS) in  following  manner:  glutaraldehyde a t pH  buffer  five  preparation for l i g h t  hours.  7.4. and  The  post  The  in  A  0.2M  two  specimens  in  hour  phosphate  specimens  fixed  microscopy  2%  were  were  then  primary  carried fixation  buffered  washed  osmium  was  for  washed  several a in  saline times  minimum the  of  buffer  -18several  times  acetate  and  followed  in d i s t i l l e d  specimens  were  embedded  in Polybed  Ft.  After  in a  812  staining further  graded  embedding  in  (  uranyl  washing,  the  series  and  methanol  media  5%  Polysciences,  Warrington, Pennsylvania).  sections a  blocks  cut using  were  glass  P o r t e r - B l u m MT-1  onto to  bulk  water.  dehydrated  Specimen  on  by  distilled  a distilled  After  water, water  drying,  toludine  blue  plus and  1%  or  and  the  loop,  a cleaned  sections  Borax  using  a  1.0  micron  diamond were  and  knife floated  transferred  microscope  were  for several  photographed  -  Sections  in a  on  0.5  a Diatome  microtome.  droplet  heat  observed  knives  collected  a  were  trimmed  stained  slide.  with  1%  seconds.  Sections  polaroid  equipped  Zeiss photomicroscope.  (b)  Calanus  plumchrus  Specimen out  in  the  primary  preparation  following  fixation  saline  buffer  buffer  then post  After uranyl the  several acetate  specimens  manner  i n 4%  (PBS)  washings,  7.4.  i n 1% the  in d i s t i l l e d were  :  A  m i c r o s c o p y was one  osmium  water.  dehydrated  in a  Warrington, Pennsylvania).  were  tetroxide  specimens  were  After  washed  stained  in  hour. in  5%  in buffer,  methanol  media  hour  phosphate  f o r one  washing  graded  carried  one-half  i n 0.2M  Specimens  and embedded i n Epon or S p u r r ' s embedding Ft.  and  glutaraldehyde  a t pH  fixed  for light  series  (Polysciences,  -19Specimen cut  using a  with  either  were  floated  placed heat  Porter-Blum glass,  1%  borax  seconds. equipped  (ii)  MT-1  trimmed  water,  o f water  sections  or  Electron  or  1-2 a  micron  sections  Reichert  1%  collected  with  Ultratome  were  stained  methylene  blue and  with  plus  1%  1%  loop,  and  slide.  After  toludine  blue  borax  photographed  Sections  a  on a c l e a n e d m i c r o s c o p e  S e c t i o n s were o b s e r v e d Zeiss  and  microtome,  distilled  on a d r o p l e t the  were  d i a m o n d , or t u n g s t e n - c a r b i d e k n i v e s .  onto  drying,  plus  blocks  for  several  with a  polaroid  photomicroscope.  Microscopy  (a) T i g r i o p u s  californicus  Specimen p r o p a r a t i o n f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y was  carried  hour  out  primary  i n the  fixation  1%  osmium  in  the  dark.  The  pH  7.4  f o r at  least  fixation After  for  finally least  30%,  50%, in  day  manner.  mixture  0.2M  were washed  tetroxide  a  in a  70%,  6%  t o the r e l a t i v e infiltration  pH  of  one-half  saline  times  was  (PBS)  f o l l o w e d by  a  for one-half 7.4,  hour)  series;  1/2  and  propylene  100%,  oxide  carried  Pennsylvania)  out  post hour.  1/2  f o r 90%  and  in buffer,  i m p e r m e a b i l i t y of the  period  (Polysciences,Ft. Warrington,  was  methanol  each  bath  and  gluteraldehyde  several  (PBS,  1 hour  one  buffered  This  graded  prolonged  A  in buffer  in buffer  and  of  phosphate  one-half hour.  dehydrated  Due  a  specimens  washings  placed  a 5-7  in  in  osmium  were  2 hours.  barrier, 812  1%  several  specimens each  tetroxide  in  following  the hour and  for at cuticle  i n Polybed  embedding  media.  -20Polymerization  was  The  carried  out  polymerized  a t 60  blocks  degrees C  were  f o r 24  trimmed  hours.  then  sectioned  u s i n g a R e i c h e r t 0MU3 U l t r a m i c r o t o m e or a P o r t e r - B l u m MT-1 crotome w i t h  a D u p o n t ' or a D i a t o m e ' diamond k n i f e . x  were mounted mesh  uncoated  solution by  of  (b)  300  Calanus  mesh  copper  uranyl  10 m i n u t e s  The  >  on  grids.  acetate  in lead  Formvar  coated  They  were  i n methanol  citrate  copper  for  (Reynolds,  grids  stained 30  ultrami-  in  sections or  on  300  a  saturated  minutes  followed  1963).  plumchrus Specimen p r e p a r a t i o n f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y  was  carried  fixation (PBS) the  7.4.  a  in  the  following  glutaraldehyde After were  in  several  post  manner: 0. 2 M  in  1%  one-hour  phosphate  washings  fixed  A  in  buffered  buffer  osmium  primary  (1/2  saline hour),  tetroxide  for  one  A g a i n , a f t e r r e p e a t e d w a s h i n g s , the s p e c i m e n s were d e h y d r a t e d graded in  methanol  propylene  Spurr's  lvania) 60  5%  specimens  bath or  in  pH  hour. in  out  oxide.  embedding media  for  degrees  2  days.  (as  above)  Infiltration  was  followed  by  carried  out  a  (Polysciences, Ft. Warrington,  Polymerization  occurred  in  24  1  hour  in  Epon  Pennsy-  hours  at  C. Trimming,  procedure described  (c) F l u i d  series  sectioning for Tigriopus  phase E n d o c y t o s i s  and  staining  followed  the  same  californicus.  (Tigriopus  californicus)  Specimen p r e p a r a t i o n f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y  -21was c a r r i e d with  ( 0 . 5 mg/ml)  intervals by  o u t i n t h e f o l l o w i n g manner:  ( l  fixation  phosphate hours.  f  i n 6%  g1uteraldehyde  buffered  Several  saline  then  incubated  o f PBS  (H202) f ° o n e - q u a r t e r r  Control  with were  with  hour.  for Tigriopus  horseradish peroxidase.  saline  peroxide is  again  (d)  Receptor  described between Co.)  f o r one-half  3,3'  hour.  -Diaminobenzidine  ml 1% h y d r o g e n  are identical  carried  peroxide  hour.  to  o u t i n two  another  peroxidase  those  ways.  group o f copepods (as above),  fixed  t e t r o x i d e i n phosphate b u f f e r e d  and t h e n  incubated  with  0.2 ml  The r e m a i n d e r  endocytosis  colloids  phosphate  1%  of the  hydrogen procedure  gold  (Tigriopus californicus)  (15-20mm) were  by H o r i s b e r g e r  colloidal  one-half  to that p r e v i o u s l y d e s c r i b e d .  mediated  Gold  tetroxide in  f o r one and  (PBS) pH 7.4 c o n t a i n i n g o n l y  identical  followed  as a b o v e , b u t were n o t i n c u b a t e d  horseradish  f o r one-quarter  was  californicus.  Secondly,  (PBS) pH 7.4, washed,  buffered  0.2  were  i n 6% g l u t a r a l d e h y d e and 1% osmium saline  osmium  followed  procedures  experiments  with  This  time  The p o s t f i x a t i o n i n osmium t e t r o x i d e ,  t h e c o p e p o d s were p r e p a r e d  incubated  7.4  5.0 mg  containing  and embedding  p r e v i o u s l y mentioned  First,  in buffer  incubated  (HRP) i n 7 g r a d e d  a n d 1%  (PBS) pH  washings  i n 20mls  dehydration  peroxidase  2, 5, 10, 20, 30, 45 m i n u t e s ) .  S p e c i m e n s were (DAB)  horseradish  Copepods were  (1979).  and wheat  i n t h e f o l l o w i n g manner:  prepared  Conjugation  Germ  using  was c a r r i e d o u t  Agglutinin  18mls o f WGA  t h e method  (WGA)  (0.1mg/ml)  (Sigma  was added  -22dropwise of  t o 90mls  10 m i n u t e s  (PEG)  (18mls)  5 minutes. minutes  of the c o l l o i d a l  while was  constantly  added  slowly  The m i x t u r e  at 4 degrees  C.  c o n j u g a t e was r e s u s p e n d e d A primary three  incubation  stages  was  Control with  this  conjugate  for Tigriopus  experiments  conjugate  10 mg/ml  and  t h e n p r o c e s s e d as above.  collected  300,  or a P h i l l i p s  X g  f o r 30  and t h e  was  carried  out i n  t h e n p r o c e s s e d i n t h e manner  californicus.  were  carried  o u t by  for transmission and p l a c e d  g r i d s were c a r b o n c o a t e d f o r s t a b i l i t y The s p e c i m e n s  glycol  volume i n F2 m e d i a .  N-acetyl-D-glucosamine  In a l l t h e e x p e r i m e n t s  period  and m i x e d f o r  a t 15,000  i n 1/4 i t s o r i g i n a l  with  sections  a  Polyethylene  to the s o l u t i o n centrifuged  over  The s u p e r n a t a n t was d i s c a r d e d  the  those  solution  stirring.  (5, 15, and 30 m i n u t e s )  previously described  scopy,  gold  prior  on  incubating (15 m i n u t e s )  electron uncoated  microcopper  to viewing.  were o b s e r v e d u s i n g a Z e i s s EM9S, P h i l l i p s  400 t r a n s m i s s i o n  electron  microscope.  -23Results: A.  Tigriopus A  californicus  longitudinal  californicus higher in  is  section  shown  magnification  figure  tract  2.  can  of  From  be  the  of  in  an  its  the  adult  entirety  in  oral/mandibular  morphology  subdivided  copepod  into  the  esophogus,  a n t e r i o r m i d g u t , p o s t e r i o r m i d g u t , and  figure  region  observed,  the  Tigr iopus 1.  i s shown digestive  midgut  anterior  A  and  caecum,  posterior  hindgut.  Esophagus The segment it  esophagus l i e s of  curves  posterior  the  cephalothorax.  anteriorly bend t o  and  located  esophagus  region  of  a  cross  caecum. and An a  midgut  the  to  section  electron lumen  by  dense  by  a  caecum.  dorsally  to  the  through  dorsal  the  the  ability  micrograph by  epithelial  an  of  the  area  cells  c i r c u l a r muscle  layer  is  in  times  (5-10  um  tall)  and  N  thick).  at 5  45  show  the  midgut  H'  shaped  of  feeding. 6)  cuticle and  anterior  area  (figure  distinct urn  centrally  and  typically  esophagus  (6-17  4  slight  anterior  the  mandibular  distend  of  from  a  the  the  Figures  esophagus to  of  shows  esophagus  opening,  with  junction 3  anterior  ventral  through  plane.  its  bounded  cut  transverse  of  its  Figure  in a section  lumen  reflects  extends  cephalothorax  the  The  and  From  i t s o p e n i n g a t the  midgut  degrees  i n a c e n t r a l p o s i t i o n i n the  shows followed  surrounded The  cuticle  -24lining at  the  esophagus  l e a s t two  um)  layers.  epicuticle  inner  (figure  layer  0.2  um  thick.  in  the  inner  is  An  of  on  dense  the  medium  Variations most  appears  electron  found  appears  7)  and  and  be  made  thin  luminal  and  density  thickness  appears  to  be  up  of  ( 0.02-0.03  surface  electron  in c u t i c l e  layer  to  the  and  about  are  found  less  electron  dense. The  epithelial  cells  have  deep  invaginations  plasma membrane a t t h e b a s a l  and c u t i c u l a r r e g i o n s .  containing  t o be  with  a  nuclei  prominent  show round  (0.2-0.5  um)  reticulum  are found.  In  secretory  Midgut  nucleolus.  few  cristae  and  material  there  i s often  shaped  Circular mitochondria some  rough  Ribosomes a r e abundant esophagus  Sections  to i r r e g u l a r  i n the c y t o p l a s m .  are signs  found  but  endoplasmic  of  no  digestion.  evidence  f u n c t i o n s were f o u n d i n t h e c u t i c l e l i n e d  parasagittal  of the midgut The m i d g u t that  lies  from  the  towards  and  round  the  of  esophagus.  Caecum A  cells  with  the d o r s a l  Semidigested  them  of  (figure  caecum and a s m a l l  caecum above  anterior  of  the a n t e r i o r columnar  microvilli.  8)  shows  a  the  to  of  the  esophagus. and  the  of the copepod.  w i t h many  Some  the  esophagus  portion  portion  area of the a n t e r i o r  ( f i g u r e 9) i s a s p h e r i c a l chamber  and  junction  are  section  cells  having  junctions  midgut.  (40-45um) It  extends  anterior The  epithelial  apical  between  midgut  vesicles  the  cells  -25of  this  cavity  structures. have  The c e l l s  some b a s a l  lamina. in  are furrowed vary  regions  c l o s e a s s o c i a t i o n with  found  appears  sections  through  which  vacuole-  form  3-17  are ringed  the b a s a l  caecum.  or p o s s i b l y  caecum  um  and  c a n be  seen  lamina.  Figures  the midgut  like  by t h e b a s a l  l o n g i t u d i n a l muscle  t o be t h r e e  i n the midgut  large  i n height  invaginations  I n many  There  and h a v e  9  four  and  10  illustrating  cell  types  are  cross  these  cell  , types. Cell or  four  type  cells  that  epithelium. with  '1' i s u s u a l l y  cells  cells  usually  nucleolus.  do n o t r e a c h  i n c l u s t e r s of  l i eat the basal  These  a prominent  seen  portion  have  some s m a l l Cell  The  t h e lumen.  cytoplasm  Golgi  type  There  appears  bodies  contained  of  these  t o be a random  loose  ribosomes  tend  were  invaginations  and r o u g h  t o be v e r y  found  endoplasmic  dilated.  and  centrally  large  cytoplasmic  matter.  They  includes  reticulum.  Numerous  i n the cytoplasm.  This  is  The  exocytotic suggestive  In a d d i t i o n to the e x o c y t o t i c  o f opaque v e s i c l e s , some c e l l s  particulate  and a  I t has a v e r y dense c y t o p l a s m w h i c h  of a m a i n l y s e c r e t o r y f u n c t i o n . function  nucleus  and r o u g h e n d o p l a s m i c  contains  '2' has b a s a l  mitochondria  vesicles  central  s p h e r i c a l v e s i c l e s (0.2 um).  located nucleus. random  of the gut  The a p i c a l p o r t i o n s  d i s t r i b u t i o n of G o l g i bodies, mitochondria reticulum.  a  three  masses  a r e found  i n the midgut with  electron  mainly  caecum dense  i n the a p i c a l  -26one-half the  of the c e l l  luminal  and t e n d  surface.  The  cytoplasmic  to t h a t d e s c r i b e d  forcell  opaque  has g r e a t l y  was  vesicles  found  between  mainly  figure  cellular  In  many  appear  i s of  into  m i d g u t caecum.  are  found  distributed  some  fusing  This  cell  seen  midgut. for substantial  I t appears  that  type.  of the midgut  caecum  contents.  abundant  cell  type  randomly  small  sections  and  the nucleus.  the Golgi  i n the  sometimes basally  reticulum  Mitochondria  bodies  are  less  B o t h e l e c t r o n opaque and e l e c t r o n  vesicles these  rough e n d o p l a s m i c  with  this  or h o l o c r i n e  i s a round n u c l e u s most o f t e n Lamellate  type  a r e found  small  opaque  i n the  vesicles  cytoplasm. c a n be  seen  t o t h e p l a s m a membrane.  Anterior The midgut  i s similar  The basement membrane i s i n v a g i n a t e d ,  d i l a t e d and q u i t e numerous.  In  cellular  c l o s e l y associated  translucent  cells  '3' i s t h e most  to c e n t r a l l y l o c a t e d .  towards  and i n t h e t r a n s i t i o n  t h e lumen.  their  q u i t e d e e p l y , and t h e r e  is  c a n be  the dense  t o be e x t r u d i n g type  caecum  the macro-apocr ine  sections,  Cell  diminished.  evidence  secretions  secretion  density  caecum and t h e a n t e r i o r  8,  in size  t y p e '2' b u t t h e number o f e l e c t r o n  i n the midgut  the midgut  In  to increase  Midgut anterior  caecum  of p e r e i o p o d s .  midgut  and ends  begins  at the junction  a t the p o s i t i o n  The e p i t h e l i a  of the  of the t h i r d  pair  i s made up o f p s e u d o s t r a t i f i e d  -27cuboidal  cells  to  25  with  um  into  at  their  tend  um  the  be  c e l l s had  cytoplasmic  an  As but  fourth  there  the  most  is  gut.  It  mitochondria,  some  the  small 14,  type  gap  i n the  reticulum  midgut type  and  greater  of  has  1  joined and  portion  of  epithelial and  three  scattered cell  types  in  their  continuity  '4',  can  luminal  rough  16  the  to  junctions  the  5  12).  type  and  the  up  are  anterior  caecum,  from  cells  cells  sections,  be  are  (figure  cross  anterior  seen  microvilli,  endoplasmic  dense v e s i c l e s  15  '1'  a  (figure type,  Cell  in  this  abundant  reticulum  and  13).  sections  through  midgut.  is ultrastructurally  identical  to  those  caecum.  '2'  has  has  an  abundance  vesicles  electron density.  of  containing  They a l s o c o n t a i n o t h e r  rough  endoplasmic  p a r t i c l e s of  high  l i p i d - l i k e electron  droplets. Cell  reticulum  in  in  height  i n most  prominent  midgut  cell  Cell  The  the  anterior portion  opaque  by  In  in  epithelial  convoluted  in  Figures  seen  The  midgut.  abundance o f  the  invaginations  densities  of  basal  range  round n u c l e i w i t h a prominent n u c l e o l u s  present,  region  that  surfaces  highly  chromatin.  A  11)  cytoplasm.  anterior  are  basal  luminal  to  the  (figure  type and  presence the  '3' still  of  anterior  has  a r e d u c e d amount o f rough e n d o p l a s m i c  has  some  apical  lipid  portion,  but  small  dilated  droplets were  could  reduced  Golgi still in  bodies. be  number  seen or  -28absent  i n the  posterior  Towards the  cells  14  tend  um) . of  the  convoluted  in  posterior  to  Figure  portion  of  the  In  is  a  the of  the  is  In  seen  Numerous  midgut,  shorter  of  the  figure  with  midgut.  anterior  and  section  midgut.  muscle.  anterior  convoluted  cross  lamina  o f many  of  segment  less  anterior  basal  apices  be  17  longitudinal the  portion  19,  (5  posterior the  highly  i t s associated  vesicles  to  can  band  be  seen  cells.  the p o s t e r i o r  segment  of the a n t e r i o r  midgut  (figure  18) t h e e p i t h e l i a l c e l l s become c o n v o l u t e d and f o r m a v a l v e - l i k e structure  at  Posterior  Midgut  The cavity  and  same  in  shape.  and  The  The  figure  between  as  those  of  are  basal  20, cell  rimmed type  neighbouring  at  and  vesicles  can  be  seen  in  i s  their  has  more  highly  and  gap  less  (figure  junctions. structure  electron I n some  association  the  convoluted  as a b r i d g e  vesicles.  of  are  cuboidal  muscle  by  body  junction cells  shorter  prominent  close  the the  and  apices seen  the  segment  longitudinal  p a r e n t and e l e c t r o n opaque l u m i n a l the  midgut,  slightly  of  At  convoluted  ' 2 ' c a n be  cells  centre  posterior  lamina  with  midgut.  tissues.  the  microvilli  are  the  posterior  less  The  in  body  are  associated  cells  lies  by  and  They  packed.  closely  21). In  height  the p o s t e r i o r  midgut  anterior  midgut.  densely  to  i s surrounded  the  anterior  entrance  posterior  between the  the  trans-  sections, with  the  -29plasma base  membrane  and o c c a s i o n a l l y  of the m i c r o v i l l i .  through The  the a n t e r i o r  posterior  Figure  portion  midgut  open  to the lumen  at the  i s a cross  section  22,  of  the p o s t e r i o r  i n the middle  portion  midgut.  (figure  23)  shows t h e c u b o i d a l shaped e p i t h e l i u m w i t h n u c l e i and p r o m i n e n t nucleoli. have  an  shows are  The  almost  these  just  i n height  beneath  '2',  uniform  invaginations  secretory and  average  low c u b o i d a l  reduced  lamina  cells  these  cytoplasmic  i s markedly  '3' a r e p r e s e n t  as  some a  with  rough  few  Cell It  type  i s often  type were  '3'.  Mitochondria  present  electron  are  found  Cell ations The  with  a  type  density.  i n the c e l l s '3' i n t h i s  interdigitating  microvilli  The p r o p o r t i o n reduced. type  Cell  of a p i c a l types  ' l ,  ' 1 ' i s much  more  of the midgut.  1  Mitochondria  are present  as  vesicles  well  a r e seen  cores.  'bridge'  and  but the c e l l  extreme also  forming  The  24  layer  '2' i s more e v i d e n t  seen  Figure  l o n g i t u d i n a l muscle  Occasionally  e l e c t r o n dense c e n t r a l  and  basal  reticulum  bodies.  i n height  are several  but c e l l  endoplasmic  Golgi  There  invaginations.  vesicles  um  cells.  and d e n s i t y . and a t h i c k  5.0  density.  epithelial  common i n t h e a n t e r i o r p o r t i o n s and  about  rough  i s mainly Electron  in this  midgut  adjacent  to c e l l s  endoplasmic  of  reticulum  characterized dense  region.  by i t s  apical vesicles  of the p o s t e r i o r midgut. region folds  has b a s a l  lamina  i n the plasma  invagin-  membrane.  r o u g h e n d o p l a s m i c r e t i c u l u m i s found m a i n l y i n a s s o c i a t i o n  -30with are  the  plasma  present.  citernae. are  membrane.  Vesicles  The  Mitochondria  are  often  microvilli  l o o s e l y packed  are  (figure  seen  and  Golgi  budding  shorter  (0.5  bodies  o f f the -  0.7  Golgi  um)  and  27).  Hindgut Two In  cross  the a n t e r i o r  compressed cuticle  sections hindgut  and  lined  somewhat  furrows.  or G o l g i  The 25)  The  is  less  of  most  cuticle Near is  dense  than  membrane lined,  the  anus,  typical  of  and the  bound furrowed  epithelial  of  cells  are  26.  numerous  are  squamous  endoplasmic  of  The  cells  Figure the  is striated  cuticle  hindgut  furrowed.  epithelial  region  outer  There  anterior  organelles.  the  and  are elongated,  i f any, rough  the  midgut  cuticle  25  seen.  greatly  the  figure  the c e l l s  V e r y few,  portion  lined  in  wedge-shaped.  b o d i e s were  posterior  is cuticle  shown  ( f i g u r e 26)  and have c i r c u l a r n u c l e i . reticulum  are  lining  cytoplasm and  28  which  devoid  shows  posterior (figure  (figure  the  hindgut. 29)  which  protects  the  copepod.  B. C a l a n u s A of  Plumchrus  longitudinal  an a d u l t  Calanus  section  Plumchrus  through  the  digestive  i s shown i n f i g u r e  30.  tract Morph-  o l o g i c a l l y , t h e g u t i s d i v i d e d i n t o f o u r r e g i o n s ; the e s o p h a g u s , midgut d i v e r t i c u l u m ,  m i d g u t , and  the  hindgut.  -31Esophagus The  esophagus  of Calanus  plumchrus  (figures  30  and  31) i s an i r r e g u l a r l y i n v a g i n a t e t u b e t h a t bends a n t e r i o d o r s a l l y from  i t s ventral  opening.  I t becomes  ovoid  or  round  a p p e a r a n c e and bends p o s t e r i o d o r s a l l y i n i t s d o r s a l There and  i s an  epithelial  s u r r o u n d e d by  bands  of  an  circular  layer  inner  bounded  by  a  muscles.  It  is cuticle  32) w i t h a t h i c k c u t i c l e i n i t s v e n t r a l r e g i o n to  about  region,  1 um  i t opens  and m i d g u t lined  rapidly  at  the  this  midgut  As  p r e v e n t s the  into  dorsal  to the midgut  diverticulum.  ridge  directly  and  i n i t s most  opening  diverticulum  In  at the j u n c t i o n  the  cells  (figure  i t s dorsal  of the midgut a  from  The  cuticle spilling  cuticle  lining  develop m i c r o v i l l i  and  narrowing  the midgut,  diverticulum. and  layer  (5 um)  esophageal contents  midgut  lamina  lined  segments.  i t enters  segments.  basal  l o n g i t u d i n a l muscle  in  on  the  their  thins midgut luminal  surfaces.  Midgut The canal. midgut is  On  the  forms  dorsal  diverticulum  wide  and  ventrally In  midgut  side  largest of  i s formed.  sac-like.  part  of  i t s anterior  the end,  At i t s beginning,  I t narrows  and  becomes  alimentary a  conical  the midgut displaced  as i t p r o c e e d s p o s t e r i o r l y , t h e n i t bends d o r s a l l y .  the m i d d l e r e g i o n  position  the  and  of the copepod,  proceeds  backwards  the midgut to  the  has a d o r s a l  junction  of  the  -32hindgut.  The  luminal lumen  shape  basis  of  midgut  'V  their  a  slightly  In  the  lated.  which  Upon the  the  at  the  of  the  cells  the  front  layer  of  on  non-  the wide  sac-  epithelial but  on  anterior  the  entering  appearance, portion  In  uniform  lining  takes  i s non-vacuolated. midgut  which  very  cells  similar  to  lose the  their  there  i s a  are highly  vacuolated  epithelial  of  the  midgut  and  midgut  junction  of  the  midgut  and  the  lining  t a k e s on a h i g h l y  form  a midgut  The is  dorsally,  defined  starts  junction  to  be  linings.  cells.  of e p i t h e l i a l  triangular  vacuo-  Once t h e m i d g u t bends d o r s a l l y and p r o c e e d s p o s t e r i o r l y ,  epithelial  look  can  i s a  widest  region  cell  midgut,  a  34).  there  different  central  glandular  the  has  i t bends  midgut  portion  opening,  midgut as  (figure  epithelial  of  the  and  the  epithelial  portion  on  the  of  diverticulum  vacuolated  of  33)  shaped  regions  esophageal  like  part  (figure  becomes Four  the  widest  made  of  type.  These  Arnaud  et  located nuclear nuclei.  cells  nuclei  are  1978). with  envelope They  lining  compressed  a l.,  found  diverticulum. the  appearance  and  at  the  At  the  epithelial  and  constricts  valve.  e p i t h e l i a l  up  lining  hindgut,  convoluted  appearance  have  or an  of  the  columnar  midgut  cells  of  diverticulum a  single  the  'R *  cells  (nomenclature  The  'R'  cells  haves  heterochromatin lying continuously invaginated  dispersed  a  cell from  centrally  adjacent  to the  throughout  and u n d u l a t e d b a s a l  the  lamina.  -33Some m u s c l e vary  i s seen  i n height  from  a t the b a s a l 40 t o 70  In t h e a p i c a l r e g i o n s  lamina  of these c e l l s ,  (7 - 12 um t a l l ) .  is  layer.  have  dense  constricted  numerous  bases.  with  In t h i s  translucent  vesicles.  vesicles  a r e found  and o f t e n  The to  basal  the h i g h l y  membrane The  lamina  breaks  highly  convoluted  plasma  basal  regions  cristae  were 'R'  seen. cells  a high open  bodies  Figure  developed  membrane. lamina  with  view  to the luminal  midgut,  35  rough  illustrates  There where  adjacent a r e some  t h e plasma  i s packed i n  In many s e c t i o n s  endoplasmic  a  section  diverticulum  cells.  reticulum  through  and f i g u r e Note  the  37 shows  the v e s i c l e s  surface.  the e p i t h e l i a l shape.  of the midgut cells  take  diverticulum  on a  The b o u n d a r y  h i g h l y d e v e l o p e d and t h e m i t o c h o n d r i a structures.  dense  electron  and  membrane  of the a p i c a l s u r f a c e .  the j u n c t i o n  columnar  abundant  the m y o e p i t h e l i a l  plasma  and some  of the midgut  power  At  more  Golgi  and  (figure 36).  i t s i n t e r d i g i t a t i n g f o l d s with mitochondria. frequent  contained  vesicles containing  i s highly  contact  the m i c r o v i l l i  few medium e l e c t r o n  i n the basal  i s in close  cell  a r e a l s o seen  convoluted  intermittent  A  there are t i g h t l y  region,  tubular  electron  dense p a r t i c u l a t e matter  cells  Beneath the m i c r o v i l l i  The a p i c a l  mitochondria  The  um.  compressed m i c r o v i l l i an e l e c t r o n  region.  The n u c l e i a r e p r o m i n e n t  less  muscle  densely layer  appear as l a r g e and c e n t r a l l y  and t h e packed i s more dilated located.  -34The  b a s a l p l a s m a membrane i n v a g i n a t i o n s a r e s l i g h t l y  The  apical  surface  has numerous  reduced.  v e s i c l e s present  (figure  39) . In the  the middle  midgut  region  diverticulum  glandular.epithelial There in  this  highly are  The  section  luminal  cytoplasm  and a n t e r i o r are found.  t o be  three  with  surface  greatly can  distends  be seen  gressive  formation  its  luminal be  occurs  associated  regions  and b e t w e e n  with  an  cell  extusions  adjacent  extremely  were  large vacuolar  with  There  some  the progressive  cells.  A  90  seen  apparatus.  um)  which  inclusions  types.  uptake  along tends  i n the  few G o l g i  pro-  vacuolar  cell  t h e rough e n d o p l a s m i c  sometimes  basal  reticulum  membrane  The  and numerous  The l a r g e  endoplasmic  'B'  nucleoli.  are several  'B'  this cells  ( 1 - 3 um) .  (50 -  the o l d e s t  the plasma  a r e found i n a s s o c i a t i o n w i t h Whole  vacuole  development.  Rough  with  density  to  vacuolated  and p r o m i n e n t  surface.  through  surface.  large  found  type  Numerous p i n o c y t o t i c  i n 'B' c e l l  is consistant  Its  to  the c e l l .  types  'R'  some m i t o c h o n d r i a  large  i n the a p i c a l  stages  apparatus  is a  to  microvilli  electron  p l a s m a membrane c o n v o l u t i o n s , There  similar  nuclei  has s h o r t  cell  to  segment,  In the t r a n s i t i o n  region,  basal  i s o f medium  vesicles.  different  cells  In t h i s  posterior  to the d o r s a l  cells  region,  found.  a r e found  the midgut,  of the midgut.  vacuolated  still  cells  appears  of  basal bodies  reticulum.  i n 'B' c e l l s  with  -35-  A between  dense many  vesicles  staining large  and l o n g  nucleoplasm,  'D'  cell  vacuolated  'B  microvilli  numerous  1  i s seen  adjacent  cells.  I t has  (7 - 12 um) .  apical  t o and  abundant  I t has a dense  mitochondria,  some  endoplasmic  r e t i c u l u m , G o l g i b o d i e s and a c o n v o l u t e d b a s a l p l a s m a membrane. It  i s very  found  similar  a  stained  are less  basal  light  'E' c e l l s  abundant  nuculeus  chromatin  than  with  matrix  in their  was  In  observed  the p o s t e r i o r  in transition  type  c e l l types. takes  The  cell  type  with  a light  apical  invaginated  borders.  with  a  endoplasmic Golgi  bodies  41 t o 4 4 ) . midgut  region,  areas  adjacent  t h e 'F'  exclusive  They  and  long  of  cell  other  have  granular  apical  They  have nuclei  , and p a t c h y  and  tubular  rough  vesicles,  dilated  Golgi  scattered mitochondria.  a t the base  appearance.  elongated  a prominent n u c l e o l u s  r e t i c u l u m , many  i s seen  and c o n v o l u t e d  to p s e u d o s t r a t i f i e d .  microvilli  and numerous  layer  rough  have  contrasted  mitochondria  Some  (figures  i n most  nucleoplasm,  heterochromatin.  dense  have  the vacuolated  are columnar  shaped  bodies,  They  and  They  As t h e lumen c o n s t r i c t s and bends p o s t e r i o d o r s a l l y ,  club  endoplasmic  cells.  p o r t i o n of the middle  and  on a more  cells  'B*  i n many s e c t i o n s  nucleoplasm  at the c e l l  with  i s prominent  or  apices.  were r a r e o r n o t o b s e r v e d  it  t o t h e ' R"  are present  'D*  light  and n u c l e o l u s .  reticulum  and  respects  i n the midgut d i v e r t i c u l u m . Lightly  and  i n many  An  electron  of the m i c r o v i l l i .  The  -36microvilli and  bases  38  the p o s t e r i o r the  the  'R'  basal  less  with  size  'R'  the  are  type  cells  45,  47  are  found  in  identical  d i v e r t i c u l u m except  less  muscular  that  They a r e a l m o s t  midgut  much  a dense  are  convoluted band.  The  reduced  and  the  basal  the  junction  of  the  midgut  cells  become  (30  um)  densely  basal  .  lamina  base A  of  compressed  and  and  that  they  number  lamina  cells  fewer  club  of  are  apical  evaginations  forms  (figures  40  elongate  at and  and and  shaped with  greatly  to  reduced  organelles  the plasma can  The  membrane  be  seen  at  51).  junction  46).  the  microvilli.  bands  49  the  hindgut,  cytoplasmic  muscular  (figures  valve  into  shaped  continuous  Distinct  these  hindgut  are  apical  is thick  midgut  cuboidal  There  packed  invaginations.  and  (figures  compact.  epithelial  and  of  regions  Near  the  constricted  segment o f the m i d g u t .  mitochondria  in  shows  cells  associated  are  highly  48) . Figure  to  are  The  structures.  of  cells  The  lumen  the are  midgut tightly  is severely  contr icted.  Hindgut At in a  the  midgut/hindgut  the  prominent  brush  border  regions,  the  triangular or  cuticle  epithelial  junction, ridges  lining. lining  are In  is  the  cell  virtually the  anterior  cuboidal  surfaces free  of  hindgut  shaped  with  -37large Deep  central  invaginations  project cell as  nuclei  between  height  and  of  the  the  posteriorly.  hindgut.  Figures  to  posterior  portions  the  gut by  lies  in  the  connective  portion flattened have  lined  elaborate  mitochondria  the a  are  and  anus,  the  the  distinct  Receptor  the  Mediated  Control of no  the  groups. in  After colloidal  cells  cuticle  Endocytosis  be the  of  that  laterally  of  posterior  the  in  and  They  Numerous  large  the  cuticular 57).  flatter,  Near  and  appearance  protecting  the  number.  (figure  in  the  braced  with  and  the  progressions  Note  appearance.  layer  the  narrows  ( f i g u r e 55)  reduced  similar  surface  circle  show  is  cuticle  smaller  cuticle  of  and  muscle  become  could  56  associated  digestive  gold  bands  copepod.  are  shaped  banding  A l l areas  several  thick  micrographs,  copepod  colloidal  a  outer  the  and  luminal  abruptly  invaginations  closely  layered  54  chromatin.  two-thirds  hindgut  segment  star  cells  e s o p h a g u s and  of  to  Muscular  52,  The  by  epithelial  an  surface  dorsal  tissue.  are  The  patchy  lined  sometimes  53).  entire  or  cuticle  cells,  (figure  i t progresses  scattered  the  have to  the  copepod.  (Tigriopus c a l i f o r n i c u s ) figures  tract. found  59 In  to  62,  show  a l l sections  i n the  cells  digestive  tract  of  the  were  areas viewed,  control examined  copepods. 5 gold  minutes  incubation  particles  could  be  followed found  by in  fixation, the  lumen.  -38-  Luminal  colloidal  gold  was  also  found  i n the c o n t r o l  groups  b u t was n e v e r f o u n d bound t o t h e plasma membrane or i n t e r n a l i z e d ( f i g u r e s 6 3 and 66) . illustrating marker  figures  gold  Note  epithelial  cells.  of c e l l  20  um)  of  be  after  the  Both  anterior  colloidal  gold  region  Colloidal  to  junction  are high  incubation, the  (figure  power  was  of  that  (figure  68)  shows  A high  the  the  micrographs  of the midgut  gold  67).  plasma  between  found  t y p e '2' i n t h e m i d d l e t o p o s t e r i o r  midgut  region  minutes  adhered  gap  sections  midgut.  15  seen  prominant  the a n t e r i o r  caecum in  the  portion  power m i c r o g r a p h  cytoplasmic  colloidal  particles.  Fluid  Phase  Endocytosis (Tigriopus  Control experiment  be  included  experiments  showed  a specific  could  3,3'  65,  could  of  was  -  i n the j u n c t i o n  cytoplasm of c e l l  gold  (15  and  the  '3'  type  the  64  particles  membrane.  and  size  used. In  the  the  These a r e h i g h and low power m i c r o g r a p h s  that  the  response.  seen  after  f o r the  No  specificity  micrographs  the a n t e r i o r midgut Peroxidase  uptake  viewing  D i aminobenz idene)  Control  f o r the  did  (figures and m i d g u t  positive  californicus) fluid  endocytosis  of h o r s e r a d i s h p e r o x i d a s e  endogenous the of  phase  sections.  the  not 69  process  have and  caecum  activity  peroxidase  was  Those (those  positive 70)  show  activity controls lacking  inclusions. regions  of  respectively. found  to  occur  only  -39in  cell  midgut.  type  '3'  Figure  in 71  the  midgut  caecum  illustrates  membrane bound p e r o x i d a s e  a  positive  high  uptake  endocytotic granular  seen  in  the  vesicles  1  to  are  and  or p a r t i c u l a t e  Within be  products  10  can  i n the  of  the  cytoplasm  i n membrane  d i s t i n g u i s h e d by  bound  the  incubations, peroxidase  vesicles.  peripheral portions  t h e membrane l i n i n g  view  dark  appearance. minute  in endocytotic  anterior  Horseradish peroxidase  sequestered be  the  power  granules  o f c e l l t y p e '3' i n t h e a n t e r i o r m i d g u t . positive  and  (figure  of  Initially, these  i t is  vesicles  could found  adjacent  to  74).  As i n c u b a t i o n t i m e i n c r e a s e s , t h e s e e n d o c y t o t i c v e s i c l e s become more c o n c e n t r a t e d w i t h p e r o x i d a s e p o s i t i v e matter.  Within  a  dense  cental  (20-40  15 area  minutes)  minutes, (figure  produced  the  vesicles  75).  Longer  vesicles  which  are  activity  was  seen  i n the c e l l s  seen  to  incubation appeared  a l m o s t h o m o g e n e o u s l y d e n s e ( f i g u r e s 72 and 73) . positive  particulate  of type  No  dark  have times and  peroxidase  '2'.  -40Discussion: Examination californicus is  and  consistent  of  Calanus with  results  presented  by  Sullivan  (1975),  T i g r i opus  studies  this to  be  an  midgut  and  found  the  great those  their  and  found (1980)  morphological  tract  similarity other  those  Bisalputra  previous  shape  described.  with  digestive  of  Tigr iopus  that  consistent  No  of  previously  Sullivan  on  a  with  of  exists  Calanus between  authors  pertaining  cuticulized  esophagus,  copepods.  Tigriopus a  are  tracts  reveals  copepods  here  However,  description  related  plumchrus  cali fornicus.  could  plumchrus.  digestive  many  The  for  the  c a l i f ornicus  caecum,  anterior  and  anterior  has  and  a posterior  a  posterior  hindgut  midgut  ending  in  a  regions,  cuticulized  anus. Calanus midgut  plumchrus  diverticulum,  narrowing  hindgut  a  ending  Areas of r e g i o n a l  has  and with  of  the  differences reference  gut in  to  the  cell  cuticulized  differentiated  esophagus,  midgut,  in a c u t i c u l i z e d  and  a  a  long  anus.  s p e c i a l i z a t i o n occur i n both copepods.  These s t r u c t r u a l d i f f e r e n c e s functions  a  are  correlated  regions.  the  Morphological  ultrastructure t y p e and  to  will  functional  particular  similarities be  discussed  significance.  Foregut The  characteristics  of  the  foregut  in  both  copepods  -41are  remarkably  consistent (1924),  similar.  with  those  described  descriptions  These found  similar  of  1935;  Marshall  (1962) the  in  and  described  by  foregut  1955;  foregut  the  foregut  Fahrenbach musculature icoids.  i n the  The  and  region  musculature  esophagus,  i t s contractions  important  in  the  initial  (Dakin,  calanoid  1908;  1966).  Lowe,  Fahrenbach observed  cystoecus,  while  Lewis  the  is  in  while  those  Lang  of  Yonge  crayfish  features  also  to  of a c a l i g o i d  (1962),  the  Park,  similar  harpacticoid, Diarthrodes  described  for  are  arthropods.  many a u t h o r s  Orr,  a  structures  other  features  comparable  c o p e p o d s were d e s c r i b e d  foregut  (1961)  copepod. (1948),  esophagus  involved  and  in  in  movement  crushing  of  described  similar  i n other  harpact-  the of  the  shape food,  of  the  and  is  ingested  food  particles. Nunez  and  in  the  of  digestive  esophagus  suggested from  Crawford  the  of  a  millipede.  enzymes  that  the  (1976) d e s c r i b e d  occurs  in  Little the  initial or  no  synthesis  esophagus,  d i g e s t i v e enzymes p a s s  digestion  i n t o the  so  i t is  esophagus  midgut.  The  foregut  in both T i g r iopus  californicus  and  Calanus  p l u m c h r u s a p p e a r s to be a s i m p l e e s o p h a g u s . T h i s i s c o n s i s t a n t with  the copepod g e n e r a  pages,  (Arnaud  Bisalputra,  et  ( E p i l a b i d o c e r a , (Park,  a l , 1978),  1980)).  In  both  and  Tigriopus,  organisms,  s u r r o u n d s the l i n i n g o f the e s o p h a g u s .  a  1966),  Centro-  (Sullivan thick  and  cuticle  Its thickness decreases  -42as  the esophagus extends d o r s a l l y .  crustaceans,  no  secretion  the  dorsal in  cuticle  some  the  on  evidence  foregut  observed  probably  account  arthropods one  in  two  of  of  an  the foregut  Eucarids  1947 ;  i n the foregut.  isopod,  food  the  differences  and  influence  1983). short  Several esophagus  I n some,  by  than  setae  are  material.  lateral  ampullae  1955). complexity  decapods.  Maynard  i s crushed  in  found  complex  Dietary  a  seen  (1955)  i s more  only  be  foregut.  the observations  having  or the  occurs  t h e back r e g u r g i t a t i o n o f food  foregut  and  Eisner  (McLaughlin,  that  can  absorption  or C a l a n u s .  observed  (Naylor,  Extreme  impermeable  1977).  foregut  chambers  Evidence  athropods  of  especially  f o r absorption  i n the cockroach  many  the  which prevent  Idotea,  Pike,  Agarwal,  f o r many  were  or  present In  and  found  cholesterol  in Tigriopus  development  and  entirely  where  (Joshi  be  esophagus.  for f a t absorption  The  the  can  ventral  i s not  insects  foregut  that  evidence  In a r t h r o p o d s ,  and  has  been  (Mauchline  Dando ,  1974 ;  observed  and  i n the  Fisher,  Hinton  and  1969; Corey,  1979). In up  i t s simplest  the food  particles  structures  where  and  out  carry  appear the  that  foregut  form, and  the c e l l s lipid  carries  out  foregut  functions  a c t as a  passageway  actively  secrete  and  i n T i g r iopus  the  nutrient  a  similar  to the  break midgut  d i g e s t i v e enzymes  absorption.  cali fornicus  to  and C a l a n u s  function  and  It  would  plumchrus does  not  -43actively  digest into  In basal that  the  foregut  invaginations have  radical  elasticity  or a b s o r b  in  of  in  m a t e r i a l from  T i g r i opus  the  area  connection  with  lumen.  C a l i f o r n i c u s there  opposite  interdigitating  the  the  muscle  folds.  the  This  muscular  are  cells  suggests  activities.  The lumen of t h e f o r e g u t can be g r e a t l y d i s t e n d e d t o accommodate large  good  particles.  stretched muscle  during  the  lined  contraction  cells  feeding  layers contract  cuticle  and  The  to  serves  to  break  t o move them t h r o u g h  process  crush  esophagus.  the  lining  the  This up  the and  good  the  the  in  stretching  ingested  l e n g t h of  are  surrounding  particles  rhythmic  the  esophagus  food  the and  particles  esophagus.  Midgut In T i g r i o p u s c a l i f o r n i c u s into  three d i s t i n c t  midgut,  and  the  frequency In  two  main  with the  A.  further five  Cell In  plumchrus  regions,  cell  the  subdivided  epithelial  the  midgut  cell  ultrastructure  to the f u n c t i o n . midgut  can  be  divided  d i v e r t i c u l u m and  subdivisions based types  Four  i n the t h r e e r e g i o n s t h e i r  varied according  Calanus  be  t h e m i d g u t caecum, the a n t e r i o r  p o s t e r i o r midgut.  t y p e s were f o u n d and and  regions;  the m i d g u t can  on  the  the  into  midgut,  ul trastructure  of  present.  Types T i g r i opus  cali fornicus cell  type  ' 1 '  was  found  -44occupying  a basal  caecum.  I t s morphological  it  i s not a c t i v e  functions that in  have  many  as been  basal  and  probably  (1971),  shed  lamina  near  not a c t i v e  or replacement  cell  type  Cell to  those c e l l s In  lasmic is  type  cell  in nutrient  portion  from  observed cells.  undulate  De  clustered  Priester in  'cell  of d i f f e r e n t i a t i o n  ribosomes,  were  Little  and o n l y  a few  Basal invaginations  of d i f f e r e n t i a t i o n . c a l i fornicus  d e s c r i b e d by De P r i e s t e r '1' abundant  r e t i c u l u m or G o l g i  suggestive  probably  of the e p i t h e l i u m .  , many  that  f o r those  less  exchange.  cells  '1' i n T i g r i o p u s  type  cell  i s much  s m a l l m i t o c h o n d r i a were u s u a l l y o b s e r v e d . a sign  suggest  of u n d i f f e r e n t i a t e d  s e q u e n t i a l stages  reticulum  are probably  the midgut  or a b s o r p t i o n , b u t  regenerative  the b a s a l  endoplasmic  characteristics  are t y p i c a l  of t h i s  i n which  and  i n the e x t r u s i v e processes  They  described  nests'  i n the midgut  in secretion precursor  cells.  The  found  a  position  i n the f l y .  ribosomes  and no  o f some s y n t h e t i c  i s similar  with  luminal  activity  little  surface  endopcontact  but without  export  the c e l l . In  similar  Calanus  in function  plumchrus, to c e l l  and  amphipods, t h e 'E' c e l l s  and  found  Schultz,  t h e 'E' c e l l s  type  '1'.  are probably  In d e c a p o d s ,  isopods,  have been d e s c r i b e d as e m b r y o n i c  i n the hepatopancreatic  epithelium  (Loizzi,  1971;  1976) .  In C e n t r o p a g e s  t y p i c u s , Arnaud e t a l . , ( 1 9 7 8 ) , d e s c r i b e d  -45th e  'E'  cell  as  organelles.  slender  They  do  with  however,  and have s l e n d e r m i c r o v i l l i . is  present  but  were r a r e or  Golgi  two  genera  cells.  and  reach  californicus.  and  Hirche,  '2' It  (1980),  and  described  termed  was  found  is usually  Golgi  bodies,  secretory vesicles, Golgi  (Fawcett,  and  bodies  From  the  sectioning, of  few  surface  i n many  cells  similar  cells  them as  in  observed.  midgut  of  T i g r iopus  darkly stained endoplasmic  mitochondria  i t is likely  are  were  the  more  undifferentiated  and  t o be  indicative  of  than  reticulum,  various  highly  active  the  sized  synthetic. organelles  1969) .  Endocytotic '2'.  and  luminal  mitochondria  s u r r o u n d i n g c e l l s , w i t h abundant rough  Dilated  the  calanoids, mitochondria  type  ribosomes,  cytoplasm  Some rough e n d o p l a s m i c r e t i c u l u m  bodies  of C a l a n u s  In b o t h Cell  light  unseen.  Hallberg in  a  was  microscopy  cell  its cellular  activity  type  in a l l c e l l s  observed,  '2'  contents  seen  was  into  found the  and to  of  through  type  serial  undergo e x t r u s i o n  lumen.  in  the  anterior  p o r t i o n s o f the g u t i t ' s shape i s c o m p r e s s e d by t h e n e i g h b o u r i n g cells  and  narrow type '2', A  '2'  is aften  luminal were  varied  observed.  mostly  face  in  surface.  p r o g r e s s i o n of  luminal  seen  was  the  Many Those  i n t h e number shape  from  observed.  a  shape  of  different cells and  a  wedge  with  a  shapes  of  cell  identified  as  type  size  of t h e i r  large  b a s a l wedge  This  shape  vesicles. to  a  broad  progression  was  -46accompanied entiated and  by  the  greater  extrusions cell  was  i n t o the  became,  the  the  lumen.  larger  proportion  and  The  was  more  differ-  i t s luminal  size  of  the  face  vesicles  present. Many Together o f an  lipid  with  the  absorptive Again,  and  synthesis.  The  '2'  the  In  basal  microvilli,  described  isopod  al.,  (Jones  (1978)  'F'  enzyme  similar  decapods,  the  is  likely  the  to  cell  type.  suggestive  the  'F'  to et  cells in  cells  are  short,  similar  secreting.  described  The  secretory  a l , 1969)  Procambarus  which  clark ii the  'F'  of  protein  cell  carry  stocky,  type  or  out  and  in  club  cells  shaped dilated  many m i t o c h o -  structure  are  the  cuboidal  reticulum,  to  believed of  described  pluchra,  (Loizzi,  cell  ultimate  digestion.  morphology  Eurydice  it  of C a l i g o i d  columnar  endoplasmic  are  of  cells  cellular  'F'  cells  decapods  the  be  progression  metabolism.  dark  for extra  rough  cell  numerous s e c r e t o r y g r a n u l e s ,  F  is  The  of  in cellular  c h a r a c t e r i z e d by  These  cell  this  invaginations  plumchrus,  ndria.  digestive  this  characteristic  described  abundant  bodies,  cells  lamina  i t s role  functions. are  is  are  secretory c e l l s  shape and  Golgi  to  Calanus  secretory  secretion  (1961),  c o p e p o d s as  in  observed,  observation  organelles  adds s u p p o r t Lewis  observed  function.  differentiation, Its  were  endocytosis  from  function.  in  droplets  and  1971).  i n Centropages  to  the be  the  'F'  in  the  in  the  Arnaud  et  typicus.  -47They as  found  well  the  i t t o be s e c r e t o r y  as by  by a m a c r o a p o c r i n e  a microapocr ine  microvilli  (first  process  described  by  from  process  the t i p s  Kurosumi  in  of  1961).  T h i s d u a l s e c r e t o r y p r o c e s s may be t h e r e s u l t o f a p a r t i c u l a r l y intense  nutritional  In found has  T igr iopus  throughout  luminal  rough  a  a n d many  the a p i c a l  smooth  in this  portions  endoplasmic  the major f u n c t i o n o f c e l l  for of the  the metabolism basal  Cell in the  ingested  type  the a n t e r i o r lumen  and  cell  type  cell  have  The  apical  lipid  that  some  undilated  mitochondria  and  ultrastructure  Golgi  abundant  The  a transport  of is  apparatus  high  degree  function  between  and t h e h a e m o c o e l .  californicus  epithelial  microvilli.  bodies.  There  electron  i s suggestive  material  an i n t r a c e l l u l a r mechanism  i s only  of  dense  lipid  found  cells  extend  There  are minor  b a s a l l a m i n a l i n v a g i n a t i o n s , some r o u g h e n d o p l a s m i c and  i s not  absorption  The s y n t h e t i c  and m e t a b o l i z e d ,  midgut.  amount  the presence  material.  '4' i n T i g r i o p u s  Golgi,  secretion  and  and  lamellate  numerous  The  suggest  to provide  suggests  that  '3'.  t h e lumen  intermediate  type.  of absorbed  invagination  products  The  suggest  reticulum  '3' i s commonly  contains  mitochondria,  of t h i s  i s l i m i t e d and f u n c t i o n s  type  I t reaches  vesicles.  organelles  function  cell  The c y t o p l a s m  reticulum,  the s y n t h e t i c  in  the midgut.  endoplasmic  major  cali fornicus,  microvilli.  ribosomes, of  phase.  are  to  reticulum, cytoplasmic  vesicles.  The  absorption.  The  -48p o l a r i t y o f the o r g a n e l l e s w i t h G o l g i b o d i e s , r o u g h e n d o p l a s m i c reticulum,  and  vesicles  suggests that c e l l  type  in '4'  the  apical  does not  portions  of  participate in  the  cell  endocrine  secretion. In were  Calanus  cells  features. the  absorptive  cell  an  plasma  have  several  characteristic  membrane, m i t o c h o n d r i a ,  reticulum.  absorptive  the  intestinal  and  L a k e , 1970)  This  role.  morphological  diverticulum and  are  exhibited  in  other  and  a  well  are  of  similar  analogous  suggestive to  to midgut c e l l s  associated  with  absorption.  cells  in type  'R'.  The  with  large  numbers  evidence  that  of m e t a b o l i t e s 'R' cells They and  observed include  Witkus,  cells  in  also in  the  1971),  Carcinus  glycocolyx  plasma  cells  are  was  membrane  observed  in  invaginations  mitochondria involved  absorptive  in  corroborates the  transfer  haemolymph. have  the S  no  associated  'R'  t o the  cells  but  basal of  (Ong  in Mytilicola  (1971) d e s c r i b e d a g l y c o c o l y x a s s o c i a t e d w i t h  cell  in  They l a c k c e r t a i n c h a r a c t e r i s t i c s  crustaceans  crayfish,  cells  Calanus. h e l g o l a n d i o u s  Loizzi  the  developed  ultrastructure is  They  i n t e s t i n a l i s ( D u r f o r t , 1971) .  the  types  They have l o n g l u m i n a l m i c r o v i l l i , deep i n v a g i n a t i o n s  endoplasmic of  three  observed. 'R'  of  plumchrus,  similarities  digestive  cells L  in  cells  maenas  to  tract  Oniscous in  Atya  (Stanier  of  absorptive  Malacostracans.  asellus  (Van et  the  Weel,  (Clifford 1955),  a l . , 1968),  and  R in  -49the  crayfish  (Loizzi,  Another is  have  basal  or  have  near  cytoplasm  abundant  bodies  final  invaginations vesicles.  invaginated.  'B' c e l l s  which  compress  absorptive  cell  distinct  o f the a p i c a l  There  activity.  type  cells.  The  adjacent or  'D' to  partially  found  i n the midgut  I t has s h o r t m i c r o v i l l i ,  plasma  i s the appearance  membrane, of i n t e n s e  and  particulate  pinocytotic  matter  and  of p a r t i a l l y  a r e most digested  likely material  I n many s e c t i o n s , a d e v e l o p m e n t o f t h e l a r g e  apparatus  was  gressive.  The v a c u o l e s  vesicles  numerous  The p i n o c y t o t i c i n c l u s i o n s d i d not c o n t a i n  the absorption  seen.  The d e v e l o p m e n t  dense b o d i e s  described  that  apparatus  initiation. some  'B' c e l l s  of the vacuolar the coalescense  w i t h i n the vacuole  suggested  of  any  involved from t h e vacuolar i s pro-  a r i s e from a f u s i o n o f t h e p i n o c y t o t i c  and t h e e l e m e n t s  al.,(1978),  In  and t h e  o f t h e 'R'  a r e found  X  et  are present  and  of Calanus plumchrus i s the B ' c e l l .  lumen.  cell.  them.  The  in  It  smooth v e s i c u l a r  to those  i s highly  cell.  t o t h e 'R'  microvilli  the v a c u o l a r  surround  i s t h e 'D*  respects  similar  membrane  luminal  type  with  Golgi  are l a r g e l y  plasma  cell  i n many  reticulum.  mitochondria  cells  similar  a dense  endoplasmic  The  absorptive  remarkably  They  1971).  autophagic  sections,  complex. of  Arnaud  cytoplasmic  i n C e n t r o p h a g e s t y p i c u s . They  activity  would  the contents  allow  of  the vacuolar  large  vacuolar  -50'B'  c e l l s were s e e n e x t r u d i n g  ingested In  material  the l a t t e r  extrude could  fuses  stages  this  'B'  The p i n o c y t o t i c  i s digested cell  material  i n the  development, into  the digested  reabsorbed  by  the m i d g u t  t h e 'R'  (Arnaud  Vacuolar plumchrus,  and  been  like  found  Mysids,  them  and many  Jacobs,  is  i n the posterior  those  observed  et al.,(1974) described (1969)  where i t  probably  p o r t i o n of  e t a l . , 1978).  cells  have  cells  In Centropages  l i b e r a t e d material  cells  vacuole.  the  t h e lumen  t h e n be r e a b s o r b e d e l s e w h e r e i n t h e g u t .  typicus,  and  and  of  digested  i n t o t h e lumen.  o f the midgut  i n many  of Calanus  crustaceans.  Raymont  them i n c o p e p o d s , S c h m i t z and S c h u l t z ,  i n isopods,  Nath  examples were  1930; D a l l ,  and  found  Pillai  (1973), i n  i n the decapods  1967; S t a n i e r  (Hirsh  e t a l . , 1968;  Loizzi,  1971) . In  newborn  rats  have a c t i v e p r o t e i n bodies  that  pinocytotic released cell  pinocytosis  a r e lysosomal. material  i n Calanus  and e x t r u d e d  cells  o f dense  a r e formed  and t h e  i n t h e phagosomes This  and  is similar  t o 'B'  f o r the d e s t i n a t i o n  of the  material.  largest cells  californicus  Phagosomes  except  intestinal  and t h e p r o d u c t i o n  system.  B. M i d g u t Caecum and M i d g u t The  1968) some  i s digested  t o the lymphatic  action  digested  (Morris,  a r e found  Diverticulum  i n the d i g e s t i v e t r a c t i n the midgut  caecum.  of Tigriopus Many  of the  -51epithelial basal type as  lamina  have  as  region  basal  evaginations  invaginations.  '3' i s t h e most  well  the  cells  some  Of  abundant.  cell  type  of the midgut  the c e l l  Cell  '1'.  adds  associated types  type  The  '2*  long  evidence  with  present,  is  present  microvilli  to the r o l e  in  of the  m i d g u t caecum i n a b s o r p t i o n . Cell  types  '2' and '3' o f t e n  contained  lipid  S c h u l t z and Kennedy (1976) s u g g e s t e d t h a t t h e c e l l s lipid  droplets  i n Dapnia  a r e t h e major  sites  droplets. containing  of n u t r i e n t  uptake. Cell  type  absorptive  and  that the  midgut  derived  that  serves  cell  type  function In lined  both  by  a  secretory  '3' i s found  of the midgut Calanus only  (1935),  described  a  similar  finmarchicus.  the midgut t h e 'R'  that  found  region  and was  polarized  i n the  indicates function. the  Since  activity. diverticulum  cell.  diverticulum  In c a l i g o i d s ,  that  absorptive  is  I t forms  i s non-vacuo1ated.  midgut  of  Kennedy  '3' c e l l s  numbers,  i s t h e major  type;  both  caeca.  and a b s o r p t i v e  plumchrus,  layer  pulex  '2' and  i n greater  has  (1960)  Schultz  cali fornicus  caecum  one c e l l  epithelial  Calanus  type  of Tigriopus  uniform  in  of both  vonk  i n Dapnia  and d i g e s t i v e  caecum  t o a n y one  elements.  secretion  presence  caecum  functions.  not r e s t r i c t e d  the a n t e r i o r midgut The  it  was  endodermally found  i n the midgut  secretory  secretion  (1976) to  '2' f o u n d  a  Lowe  structure  the s e c r e t o r y  'B'  -52cell  i s found  regions  primarily  (Lewis,  The alized  in  the  a n t e r i o r midgut  absorption  of  nutrients.  C.  of  the  p l a s m a membrane and  indicate i t s transport  Midgut Regional  lined  T i g r i opus  californicus,  by  epithelial  cells  Cell  '3', type  with  '1'  of absorbed  the  The  i s more  abundant  for  cells  This  It  coupled  '4 ,  is  1  projects  region  anterior  than  especially  o f the in  midgut.  midgut.  the  replacement  and  midgut  requirement  In  cell  type  of  the  of  the  cells  nutrient absorption.  found  into  density  the  I t s major  exclusively lumen  and  function  in  has  A  fourth  the  cell  anterior  dense  appears  indicates  the  type,  midgut.  vesicles  to be  a  in  its  absorption  lipids. In  the  with  degree  cytoplasm. of  the  types,  is  '3' more l i p i d d r o p l e t s were o b s e r v e d i n the c y t o p l a s m .  greater type  lining  midgut  invaginations  here  T h i s would s u g g e s t a g r e a t e r  '2' and  mitoch-  nutrients.  anterior  cell  to be more dense i n t h i s  caecum. the  numerous b a s a l  luminal  of the b a s a l r e g i o n s .  tend  evaginations,  Specializations  In  evaginations type  role  is speci-  Its ultrastructure  i s c o n s i s t a n t w i t h a b s o r p t i v e c e l l s and i t s b a s a l infoldings  foregut  1961) .  midgut d i v e r i t c u l u m of Calanus plumchrus  for  ondria  and  th  midgut  lipids,  brown are  shrimp,  thought  nutrient  to  Pomaeus function  absorption,  aztecus, in  the  osmoregulation,  the  cells  of  absorption  of  periotrophic  -53membrane f o r m a t i o n In  and s e c r e t i o n  Artemia,  (Talbot  a brachiopod,  correlated  the a p i c a l  and  associated  mitochondria  et al.,1972).  Hootman  basal  cell  and C o n t e  (1974),  amplifications  to a b s o r p t i v e  and  with  osmoregulatory  functions. In  Calanus  plumchrus,  the a n t e r i o r  midgut, not i n c l u d i n g the d i v e r t i c u l u m , cells but  i n the a n t e r i o r  progressively  vacuolated.  The  lead  portion  cells  adjacent  are  cells  of  a  of  i s found.  which  this  major  absorption in  is lined  function  the  with  'B'  absorptive  cells  cells  In lining cells.  secretory  The  'D' 1  small  stem  'B'  1  have  cells  The  cell of  vacuolar  number  cells.  region  cells  midgut  'R'  of  'E'  majority  types.  The  the midgut  is  activity  occurring  c h a r a c t e r i s t i c s of  but they are d i f f i c u l t  In C a r c i n u s maenus , G e o r g i ( 1 9 6 9 ) , i n t e r p r e t e d young regions  cells. of the midgut,  non-vacuolated  cells  cells.  a  and  diverticulum  the a b s o r p t i v e  pinocytotic  s i m i l a r to R  the middle  These  'D'  the a n t e r i o r  as b e i n g  i s again  by  abundant  cells.  t o c h a r a c t e r i ze . similar  of  from  It contains  i s highly  cells.  the c e n t r a l  are u n d i f f e r e n t i a t e d  region  which  to the midgut  transition  the d i v e r t i c u l u m ,  apparatus cells  short  non-vacuolated,  region  have an a p p e a r a n c e s i m i l a r t h e t h e 'R' After  o f the  i s l i n e d by e p i t h e l i a l  which  to a glandular  portion  have  and  made  the up  of  epithelial ' F'  an u l t r a s t r u c t u r e c o n s i s t e n t  The m i d d l e p o r t i o n  type with  of the midgut i n Calanus  -54plumchrus  i s t h e p r o b a b l e s i t e o f d i g e s t i v e enzyme s e c r e t i o n .  In has  T i g r iopus  cali fornicus  an e p i t h e l i a l  cells.  Cell  region  cell  type  lining  '1'  of the midgut  of  and  both  present.  Presence  lipid  absorption  i s occurring.  Often  type  they  neighbouring be  Tigriopus and  from  cali fornicus  secretion.  '2',  the s e c r e t o r y  The  one  The  'R'  in  midgut  characteristic  the midgut  in this  invaginations  the  c h a r a c t e r i s t i c s of  evaginations,  dual  plasma  the  densely midgut. with  secretions  role  observed region  the a n t e r i o r  that  structures  the greater  functions  '3' a r e  could  midgut  in  of  absorption  proportion in cell  type  appears  midgut  of  t o be  regions.  in digestive  enzyme  of the midgut o f Calanus plumchrus  epithelial  diverticulum,  basal  basal  of  posterior  of t h i s  region  and  reduced  and  absorption.  p o s t e r i o r regions  cells  a  with  than  and n u t r i e n t  The  sections,  plays  function  importance  cells  The  in this  '3' i n d i c a t e s  'bridge-like'  the s e c r e t i o n s  posterior  secretion  has  '1' and  '2'  i n the p o s t e r i o r  '2'.  Together  type  greater  type  type  region shaped  prominent  types  Many  I n many  cell  cell  of  forming  cells.  observed  of c e l l  midgut  cuboidal  more  cell  '2' a r e p r e s e n t  a r e seen  smaller  is slightly  still  staining  the p o s t e r i o r  cell  type;  type  are s i m i l a r to those but t h e i r  organelle  a r e n o t as e l a b o r a t e . absorptive  membrane  less mitochondria,  cell  and  described structure  They  types,  invaginations,  'R'.  but  reduced  a r e bounded  retain have basal  by a more  -55distinct  muscle  metabolites important cells  layer.  appears,  in this  The  from  region  of the p o s t e r i o r  hindgut  a r e much  microvilli membrane thick  basal  lamina.  region  near  The  shaped.  numerous.  endoplasmic  reduced.  capacity  absorptive  the m i d g u t  or  Calanus  plumchrus  that  Neither  have  absorptive,  crayfish  absorptive absorption a l .,  cells  midgut  cells  between  was  by  i s much  mitochondria  i t s absorptive  1968)  whereas  caligoids,  Tigriopus  regions  and  californicus  'R'  to occur  according  cells.  i n 'R'  the major  function  (1964)  cells  termed Iron  cells  to Schultz absorb  (Lewis,  and B u r n e t t  (1971)  and 'F' c e l l s  (1928),  and t h e haemocoel  embryonic  Loizzi  of  i n the hepatop-  Tigriopus  Davis  secretory  suggested  occur  the c e l l s  i n the c r a y f i s h  i s absorption In  and  plasma  lined  a h e p a t o p a n c r e a s . Dorman  hepatopancreas.  absorb copper In  and  i n the p o s t e r i o r  the hepatopancreas of c r a y f i s h .  the  luminal  reticulum  absorptive,  to c e l l s  arthropods.  t h e exchange  described  et  cells  are analogous  o f many  described in  still  The  i s diminished.  The  ancreas  Although  of the  The b a s a l  reduced  less  epithelial  the j u n c t i o n  and c u b o i d a l  are g r e a t l y  digested  t o be  The  l e s s d e v e l o p e d and t h e number o f G o l g i b o d i e s are  of  the ultrastructure,  and l e s s  invaginations  transfer  of the midgut.  smaller  are shorter  basal  in the  and f a t (Stanier  (1976),  'R'  iron. of the p o s t e r i o r  1961).  cali fornicus,  the p o s t e r i o r  midgut,  -56-  like  the  hepatopancreas,  secrete  digestive  posterior secrete cell There  are  the  discreet  out  absorbs  and  of  these  cell  Such  can  be  found  between  junctions  are  referred  allows  variety  of  small  acids, nucleotides  and  v i t a m i n s ) to be  either the  D.  macromolecules (Aberts  the  having  a  Tigr iopus  type of  size  between  molecules  (proteins,  to  epithelial  the of  junctions  epithelial  the  a  digestive  communicating  the  coupled  (sugars,  ions,  shared while  nucleic  acids  cells amino  excluding  and  polysacch-  a l . / 1983). of  septate  junctions  c a l i f o r n i c u s or  could  Calanus  be  seen  plumchrus  in  after  f i x a t i o n used.  Hindgut  and  is  et  evidence  The  a  each  of  regions  pore  No  also  regionalized.  of a d j a c e n t most  the  aride) .  are  types  a p i c a l regions  because  their  The  but  c a l i f o r n i c u s many examples o f gap  junctions a  that  plumchrus  nutrients,  functions  of  types  nutrients.  Calanus  absorb  t h r o u g h o u t the n o n - c u t i c u l i z e d  tract.  cell  function.  They  cells  and  segments  seen between the  cells.  epithelial  midgut  enzymes  carrying  In T i g r i o p u s be  of  digestive  particular  can  enzymes  regions  types  has  a n t e r i o r hindgut regions  Calanus  midgut highly  piumchrus  valve.  The  convoluted.  are  of T i g r i o p u s  separated  epithelial The  lumen  from  lining is  californicus  their at  midgut  this  constricted  by  junction and  in  -57some  sections, The  fornicus and  at  i s not  cells  of  are lined times  but  by a t h i n  t h e number  cuticle.  that  Vernon  et  material  of  Tigriopus  The lumen  synthetic  occur  nutrients  in was  the  the  anterior  Osmoregulation and  Kennedy,  1976)  form  described  of  pellets  peristatic water  and s a l t  Tolman in  the gut  from  and was  used  of  the cockroach  to  anterior  by a  or  linked  this  lack  convoluted  similar  to  i n the the  of this  or  found  that  more  into  1977) .  (Schultz by  anti-  evidence  water  of  the  some  of  hindgut.  ions.  presence  ions  of  region  i n t a k e o f water  found  often  Some a b s o r p t i o n  i n the shrimp  one  were  and V i n s o n ,  (1967)  (1976)  pump  regulation The  ATPase cell  energy could  cavities  hindgut. no  muscle  portions of  transport  Dall  excretion  Steele  actively  Little  Ion  to the anal  the mitochondria,  be  not  energy  a  Absorption  (Edson  may b e a f u n c t i o n  contractions.  uptake  hindgut.  i n t h e wasp  due  does  isopod.  in  observed  The  a high  observed i n t h i s region of the d i g e s t i v e t r a c t . may  cells.  activity  suggest  al.,(1974)  cali-  i s convoluted  the e p i t h e l i a l  of mitochondria  in a terrestrial  Fecal  hindgut  separates  suggests  requirement. hindgut  the anterior  almost  ul t r a s t r u c t u r e occur,  apparent.  and  was  the hindgut  and ends  in a  associated  of Tigr iopus  osmoregulation  of musculature. lumen  found  would  The thick  be  posterior cuticle  with  the  cali fornicus. fascilitated hindgut lined  has  anus.  -58In C a l a n u s p l u m c h r u s ,  t h e h i n d g u t i s a l o n g and s t e a d i l y  narrowing  tube.  Posterior  spherical  chamber  of c u t i c l e  a  furrowed  gut  lumen  narrows  fecal basal  were  often  most  circling  pellet  lies  adjacent  down  i n those  the hindgut  with  cells.  The  segment  cells  near  serve  i t sconsiderable length.  The  muscle  but i s reduced  of  Developed  i n the hindgut.  by a p r o m i n e n t  would  i t is a  cells  i n the d o r s a l  observed  of the hindgut,  i s not apparent  muscles  epithelial  between  s u r f a c e s a r e bounded  throughout and  and  valve,  I t ends a t a t h i c k c u t i c l e l i n e d anus.  pellets cell  lined  extending  abruptly  the copepod.  t o the midgut  layer  posteriorly  the anus. t o move  These  the  The p r e s e n c e  fecal  of l a r g e  m i t o c h o n d r i a i n the e p i t h e l i a l c e l l s suggest t h a t o s m o r e g u l a t i o n and  i o n a b s o r p t i o n may be o c c u r r i n g  i n the p o s t e r i o r  hindgut  regions.  Peritrophic In  Membrane  some  sections  Calanus  plumchrus  unding  the f e c a l  is  seen  o f both  Tigr iopus  cal i fornicus  a p e r i t r o p h i c membrane was o b s e r v e d pellet.  i n the midgut  and  In both i s most  obvious  surrounding the  material  Gauld  (1975) r e p o r t e d t h a t t h e p e r i t r o p h i c membrane  midgut.  In  membrane  was  portions  surro-  t h e membrane  fecal  the f e c a l p e l l e t s  i n the p o s t e r i o r  copepods,  and  o f the  midgut.  surrounds  i n C a l a n o i d s and i s s e c r e t e d by t h e p o s t e r i o r  Isopods,  Holdich  s y n t h e s i z e d by  (1975)  suggested  the midgut  caecum  that  the  cells.  In  -59insects,  components  produced  i n part  midgut,  and a  of  the p e r i t r o p h i c  by c e l l s  second  part  membrane  i n the a n t e r i o r by c e l l s  may  portion  i n the foregut  be  of the (Smith,  1968) .  In D a p h n i a , t h e f o r e g u t forms t h e p e r i t r o p h i c membrane.  Burgos  and G u t i e r r e z  membrane  (1976)  i s the g l y c o c a l y x  t o , the m i c r o v i l l i . enzymes.  and C o n t e  shrimp,  glycocalyx  produce  the p e r i t r o p h i c  et  in a protein  flow  both  of m a t e r i a l  the d i g e s t i v e  is  accomplished  of  the e p i t h e l i a l  activity.  with,  and  The  matrix  muscle  and C a l a n u s  plumchrus  the gut i s very  the c o n t i n u a l  matter  contractions  contraction  i n conjunction  and f o r c e In  expansion and  i t through  the midgut  diverticulum)  serve  this  and  important mastication  and  with  i s extremely  antiperistaltic  expansion  the muscular  interdigitating  to grind  folds  particles  the p a r t i c u l a t e  the esophagus.  and t h e a n t e r i o r process  (Clarke  1976).  o f t h e plasma membrane expand t o accommodate t h e f o o d and  i s the  of chitinous  In t h e e s o p h a g u s  invaginations  i n the  the m i c r o v i l l i  and c o n s i s t s  californicus  cells  that  membrane  and p o l y s a c c h a r i d e  throughout  adjacent  forhydrolytic  propose  membrane.  process.  by  Basal  the p e r i t r o p h i c  v e s i c l e s between  and Kennedy,  Tigriopus  to  (1974)  of c u t i c l e  a l . , 1977; S c h u l t z In  the  and/or  equivalent  microfibrils  associated  that  I t may a c t as a s u b s t r a t e  Hootman  endodermal  reported  regions  of continual  important  (caecum  and  contraction  and  to d i g e s t i o n .  contractions  along  Peristaltic  the length  of the  - 6 0 -  digestive  tract  allow  the food  particles  t o mix w i t h  d i g e s t i v e enzymes and t o be p r e s e n t e d  to the s p e c i f i c  of  occurs.  t h e g u t where n u t r i e n t a b s o r p t i o n In  for  Tigr iopus  digestion  muscle back  layer  and s e c r e t i o n acts  and f o r t h  ingested  food  Californicus  comes  into  the e p i t h e l i a l  midgut  contact  cycle  that  cells  surface  has t h e a b i l i t y  to force  the caecum.  s e c r e t e d by t h e s p e c i a l i z e d with  o f enzymes.  a s a pump  within  t h e caecum  t h e d i g e s t i v e enzymes  cells.  in a  The  rhythmic  t h e caecum. digestive  and a b s o r p t i v e c e l l s a r e r e g i o n a l l y s p e c i a l i z e d .  The s e c r e t i o n  The  rhythmic  enzymes material The to  occurs  contractions  within  necessary  of rhythmic  the s e c r e t o r y  d i g e s t i v e enzymes  i s very  contact  since  of  contractions  a c t i o n , the  of the absorptive  In C a l a n u s p l u m c h r u s t h i s p r o c e s s tract  this  material  and a l s o comes i n t o  with  site  I t s surrounding  t o expand and c o n t r a c t  i s i n conjunction  regions  as a  the food  By  with  acts  the  mainly  of the gut serve  the d i g e s t i v e  tract  t o t h e d i v e r t i c u l u m where  diverticulum  can also  f u r t h e r mix t h e f o o d  i n the p o s t e r i o r midgut. t o move t h e  a n d t o move  some a b s o r p t i o n  contract  and a c t l i k e  and enzymes and move them  the food occurs. a  pump  throughout  the g u t . In b o t h c o p e p o d s t h i s c o n t i n u a l c h u r n i n g a c t i o n p r o v i d e s a of  mechanism  to ensure  adequate  the r e l e a s e d n u t r i e n t s .  degestion  and  absorption  -61Endocytosis Endocytosis extracellular membrane. give of  material  within  Ultimately,  rise  t o an  vesicle  the  i s the process  mode  internalization  an i n v a g i n a t i o n  the invagination  independent  formed  of  i s named  of endocytosis  cytoplasmic according  involved  of  o f the plasma  i s thought  vesicle.  The  to the c e l l (Besterman  to type  t y p e and  and  Low,  1983) . In  this  were s t u d i e d Pinocytosis is called  study, using  two t y p e s  (endocytosis)  e s t a b l i s h e d b i o l o g i c a l marker m e t h o d o l o g y .  of substance  fluid  of p i n o c y t o s i s  entering  phase p i n o c y t o s i s .  with  the f l u i d  Pinocytosis  of substances  bound t o t h e v e s i c l e membrane i s c a l l e d a d s o r p t i v e Adsorptive tosis of  pinocytosis  of markers  ligands  the  cell  bound  mediated surface  i s further  subdivided  non-specifically,  v i a binding  membrane  (receptor  (Besterman and Low, 1983; S i l v e r s t e i n Tigriopus  californicus  (HRP)  was used t o s t u d y f l u i d p h a s e e n d o c y t o s i s  study  receptor  Fluid  Phase  agglutinin  mediated  (WGA)  pinocytosis  receptors  on  pinocytosis)  a c t i v i t y was s t u d i e d  two b i o l o g i c a l m a r k e r s .  germ  the pinoc-  e t a l . , 1977).  using  gold/wheat  and  mediated  endocytotic Soluble  pinocytosis.  into  to s p e c i f i c  content  horseradish  peroxidase  and a c o l l o i d a l  conjugate  was  used  to  endocytosis.  Endocytosis  In T i g r i o p u s c a l i f o r n i c u s , s o l u b l e h o r s e r a d i s h  peroxidase  -62appears in  t o be  cytoplasmic  caecum  and The  is  absorbed vesicles  (Alberts  peroxidase as  be  made  of  forming  1973;  After vesicles matter  were  mainly  portions Due  dense  with  a  of the midgut  phase  marker  the marker  plant  i n the  glycoprotein.  a cytochemical found  the  that  The  osmium  diami-  activity  product.  i n the presence  technique  3,3'  enzyme  reaction  peroxide.  minutes in  in  DAB  of  served  o f low  concen-  reaction  products  can  tetroxide  (Herzog  and  i n c u b a t i o n , the peroxidase  cell  the  resulted  vesicles.  found  of  They  localize  substrate  dense  five  found  peroxidase  cells  found  Rennke e t a l . , 1979).  incubations the  a  hydrogen  electron  Fahimi,  activity.  could  a hydrogen-donor  trations  I t was  of a f l u i d  i s  (1966) d e v e l o p e d  (DAB) by  accumulation  peroxidase  peroxidase  nobenzidine  '3'  '3'.  e t a l . , 1983).  Graham and K a r n o v s k y observe  type  the c o n c e n t r a t i o n  Horseradish  to  type  midgut.  intracellular to  in cell  within  the anterior  proportional  medium  only  The  type  periphery  throughout  the  and  the  rapid  the  digestive  the  extreme  postmortem tract,  show  dense  the  matter times  dense  cytoplasm  particulate  vesicles.  dense  incubation  homogenously  of the e p i t h e l i a l to  of  in scattered  longest  positive  '3'  positive  Longer throughout  resulted  vesicles.  including  in They  the  basal  cells. impermeability degeneration  l i t t l e  work  has  of  of  the  the c e l l s  been  cuticle lining  completed  to  -63date  on  the  electron  microscopic  Tigriopus  californicus.  with  absorptive  the  the  electron In  microscope  horseradish  The very and  factors  poorly  of  A  role  induction  ionic  signal  cell  for  cycle  factors  that  type  at  soluble '3'  is  pinocytosis  cells,  the  are  for  calcium  has  includes  the the  and  and  been  are  magnitude  variable  an  and  increased  calcium and  p o t e n t i a l l y harmful.  Degredation most  of  common  the  cell  the  solute  form  of  fluid  in  the  the  It  This  pinocyamoeba. membrane  has  cytosol of  and have  phase  plasma  network.  in  1980)  in  influx.  (Prusch,  internalized from  al .,  contraction  filament  vesiculation  et  a l . , 1980)  increased  calcium  arrangement  et  described  a  material  as  (Davies  regulate  the  the  cell  phase  (Berlin  extruded  be  of  effects  selectively  to  dealing  described.  mammalian  marker  contractile  invagination  cell  of  tract  uptake  in  fluid  as  process  that  associated  The  In  the  permeability,  suggested a  only  observed  on  the  described  tosis.  gut  found  digestive  the  ultrastructure  the  charge  stage  been  the  be  the  dependent.  The the  found  controlling  of  could  of  cali fornicus,  understood.  direction  cell-type  The  the  of  level.  peroxidase with  literature  mechanisms  T i g r i opus  consistent  No  analysis  a  been  provides membrane  results  in  1980). Amoeba  i f  i t  is  proteus  may  be  perceived  by  macromolecules  intracellular  appears  modification  -64and  likely  i n v o l v e s the  P i n o c y t o s i s may of  the  this  plasma  lysosome  also  membrane,  (Poole  et  a l . , 1980).  s e r v e as a pathway f o r t h e  lipids  and/or  proteins.  turnover However,  i s p r o b a b l y o n l y a segment of the f u n c t i o n s of p i n o c y t o s i s . In  Tigr iopus  c a lifornicus,  the  use  of  a  horseradish  p e r o x i d a s e marker p r o v i d e d evidence i n support of the a b s o r p t i v e function  of  cell  type  Adsorptive In were of  the  '2'  adhered  '3'.  found  midgut.  In  particles  the  were  to  and  The  in cell  (Tigriopus  cali fornicus,  microvilli  and  were  Endocytosis  T igriopus  observed  '3'.  the  majority type  '3'  also  the  of of  anterior  gold  plasma  within  the  colloids  in  the  (17  membrane cytoplasm  gold  the midgut  midgut  found  californicus)  at of  -  20um)  the  base  cell  types  particles  observed  caecum  anterior  regions,  and  numerous  cytoplasm  of  gold  cell  type  '2' . Receptor binding he  to  mediated  membrane  1igand-recepter  and  Low,  the  receptors complexes  involves  followed into  by  coated  the  ligand  clustering pits  of  (Besterman  1983) .  Researches that  endoyctosis  regions  u s i n g mammalian of  the  plasma  cells  membrane  cytoplasmic  surfaces  with  a  1978;  Lindon  and  1983;  Bretscher  It  been  has  Roth,  have p r o v i d e d are  protein,  g e n e r a l l y assumed  clathrin  and  that  coated  evidence on  their  (Pearse,  Pearse,1984).  the  coated  vesicles  -65pinch  o f f and d e l i v e r  intracellular coated  the l i g a n d  compartments  and t h e n  v e s i c l e s and r e c e p t o r s  (Pearse,  back  recycle  or  other  the c l a t h e r i n  to the plasma  membrane  1980) .  Willingham lasts,  to lysosomes  these  and P a s t a n  coated  (1980) s u g g e s t e d  v e s i c l e s remain  membrane and t h e l i g a n d - r e c e p t o r s vesicles  from  an o p e n i n g  plasma membrane.  that  attached  in fibrob-  to the plasma  are t r a n s f e r r e d to uncoated  i n the c l a t h e r i n coat  or  adjacent  These u n c o a t e d v e s i c l e s a r e termed  'recepto-  somes'. Monsigny wheat on  germ  Agglutinin  the c e l l  used  described very  used  range  to gold germ  were  types  The  microscopy. of p r o t e i n s  under a p p r o p r i a t e  marker  receptors  readily visualized Horisberger  that  c a n be  (1979) adsorbed  conditions. conjugate  was  for endocytosis.  that  types The  1  results  consistent  '2' and '3'.  showed  of  T i g r i o p u s c a l i f o r n i c u s , no c o a t e d p i t s or r e c e p t o s o m e s  be s e e n .  probes  for glycoprotein  a g g l u t i n i n / c o l o i d a l gold  as a b i o l o g i c a l  could  the s p e c i f i c i t y  i s one o f t h e most  in electron  wheat  I n  cell  gold  a wide  tightly A  (WGA)  described  surface.  Colloidal markers  e t a l (1980)  with  Sections  the absorption  observed  using  t h e gold/WGA  the u l t r a s t r u c t u r e of of the e n t i r e o f gold/WGA  digestive  was  cell tract  specific for  2 ' and 3 ' .  use  1  of  this  biological  marker  r e i n f o r c e s the  -66conclusions that  made from  absorption  midgut.  The  remains  to be  the  the  are  to  results  lining  study  be  a  anterior  internalization  process  the to  of  be  For  monoclonal and  by  increasing  of  conjugated the  receptor The  the  to  problems  cuticle  of  benefit  of  to  improve  on  others  or  mediated the  its transit  Also,  raised  mediated  a  the  transit  soluble dextrans  colloidal  be  degeneration  the  may  time  or  of  the  they  ferritin.  concentration  use  try  peroxidase  endocytosis  time  against gold  might  horseradish  absorptive  receptor  antibody  to  impermeability  possible  of  of  gold.  research.  proved  mortem  pinocytosis  study  colloidal  of  relative  t r a c t or by s e l e c t i n g a s e r i e s o f d i f f e r e n t with  mechanisms  presented.  use  and  this  overcome.  phase  the  area  r a p i d post  the  marker  endocytotic  The  concentration  improved  and  exciting  i t might  increase  biological  shows  the  cali fornicus  and  fluid  the  m i g h t employ The  are  problems  thereby  also  tract  t h a t have been  increase  and  the  absorptive  researchers,  To  of  t e c h n i c a l problem.  difficult  the  the  T i gr i opus  cuticle  future  u l t r a s t r u c t u r e and  i n the m i d g u t caecum and  nature  of  in  challenging  observed  elucidated.  digestive  However,  of  occurs  exact  Studies in  the  in  the  lectins an  of  the  digestive conjugated  intermediate  lectin  amplify  might  the  of  choice  senstivity  event. rapid  impermeability  post may  mortem be  degeneration  decreased  i f , at  and the  -67inital or  time of f i x a t i o n ,  posterior  allow  rapid  many  be  Enzymes  portions  fixation  employed that  be employed  a c a r e f u l d i s s e c t i o n of the a n t e r i o r of  t o enhance  copepod  penetration.  that  soften  the  have  the  a  are  Also,  faster  invertebrate  the p e n e t r a t i o n  carried  other  out  fixatives  penetration cuticle  o f the  to  time.  might  also  fixative.  In summary, t h e d i g e s t i v e t r a c t o f T i g r i o p u s c a l i f o r n i c u s is  composed  of  a  c u t i c u l a r i z e d esophagus,  a midgut  caecum,  an a n t e r i o r and p o s t e r i o r m i d g u t , and an a n t e r i o r and p o s t e r i o r hindgut. The of  a  digestive  In  Calanus  limited  copepods, functions  digestion  the d i g e s t i v e  the  midgut  californicus  midgut  observed enzymes  caecum  has c e l l s  esophagus  to draw  f o r w a r d by a n t i p e r i s t a l t i c The  a  Calanus  specialized  consists  diverticulum,  the  and  a  food  into  i n the d o r s a l secreted  i t s associated the g u t .  esophagus  i n the midgut  The  results  and  moved  contractions. and  anterior  midgut  of  s p e c i a l i z e d for absorption  The a n t e r i o r r e g i o n s have a g r e a t e r a b s o r p t i v e In  plumchrus,  for absorption.  the The  midgut  Tigriopus and  secre-  capacity.  diveritiulum is  anterior  a r e h i g h v a c u o l i z e d and m a i n l y a b s o r p t i v e . occur  plumchrus  m i d g u t , and a h i n d g u t .  both  musculature  tion.  of  c u t i c u l a r i z e d esophagus,  diversified  from  tract  midgut  Secretory  i n the p o r t i o n of the midgut p o s t e r i o r to the  regions functions  vacuolated  -68-  r e g i o n and t h e p o s t e r i o r end o f the m i d g u t has c e l l s s p e c i a l i z e d for  absorption. The  hindgut  specialized  of both  copepods  i s a c u t i c u l i z e d chamber  f o r t h e movement o f t h e f e c a l p e l l e t  and p o s s i b l y  osmoregulation. The Tigriopus  overall  cali fornicus  to  that  observed  in  the gut serves  through serve gut  t o move  of the d i g e s t i v e and C a l a n u s  i n other  arthropods.  t o move  the d i g e s t i v e  tract.  the ingested  and  d i s t r i b u t i o n probably food  copepods also tract.  ingested  as w e l l  are found.  affect  Sex  food  of  both  i s similar  Peristaltic  Antiperistaltic  material  specialities  tract  piumchrus  the ingested  s p e c i a l i z e d f o r absorption Regional  of  shape  activity  posteriorly contractions  to the regions  of the  and s e c r e t i o n . and d i f f e r e n c e s  relate  t o t h e abundance  as t o t h e h a b i t a t and  the morphological  in cell  seasonal structure  and  type type  i n which t h e  variations  would  of the d i g e s t i v e  -69Key  for figures  A AH AM Bl c E fm g H L M m MC Md MD MdV mvb MV N No PH PM pm RER SER " Gj V  Vac  -  anus a n t e r i o r hindgut a n t e r i o r midgut basal lamina cuticle esophagus fecal material Golgi bodies hindgut lumen muscle mitochondria m i d g u t caecum midgut midgut d i v e r t i c u l u m midgut v a l v e m u l t i - v e s i c u l a r bodies microvilli nucleus nucleolus p o s t e r i o r hindgut p o s t e r i o r midgut p e r i o t r o p h i c membrane rough e n d o p l a s m i c reticulum smooth e n d o p l a s m i c reticulum gap j u n c t i o n vesicle vacuole  1 2 3 4  cell cell cell cell  B D E F R  'B' D' 'E' 'F" 'R 1  1  type 1 type 2 type 3 type 4 cell cell cell cell cell  type type type type type  -70L i g h t m i c r o g r a p h i l l u s t r a t i n g the r e g i o n s of t h e d i g e s t i v e t r a c t i n T i g r i o p u s c a l i f o r n i c u s (sagittal section). L i g h t m i c r o g r a p h showing t h e o r a l and m a n d i b u l a r regions. The e s o p h a g u s E s o p h a g u s and californicus.  s u r r o u n d e d by midgut  muscle.  caecum  of  Tigriopus  A h i g h e r power l i g h t m i c r o g r a p h s h o w i n g the c e l l s of the m i d g u t caecum and t h e 'H' shaped e s o p h a g u s .  70 a  -71An e l e c t r o n m i c r o g r a p h o f t h e e s o p h a g u s in T i g r i o p u s c a l i f o r n i c u s . Note the b a s a l p l a s m a membrane invaginations, the m u s c l e l a t e r , and t h e t y p i c a l c u t i c l e l i n e d 'H' s h a p e .  71a  -72Figure  7.  A h i g h e r power view o f t h e c u t i c l e the e s o p h a g u s .  lining  Figure  8.  L i g h t micrograph of a p a r a s a g i t t a l s e c t i o n t h r o u g h t h e m i d g u t c a e c u m and a n t e r i o r midgut. Note the e x t r u s t i o n of c e l l c o n t e n t s i n t h e m i d g u t caecum.  Figure  9.  A low power e l e c t r o n m i c r o g r a p h o f a c r o s s s e c t i o n through the midgut caecum in Tigr iopus c a l i f o r n i c u s .  72 a  -73Figures  10  and 11. Two low power e l e c t r o n m i c r o g r a p h s of the e p i t h e l i a l l i n i n g of the midgut caecum. Note the a p i c a l lipid vesicles found i n many c e l l s .  73a  -74Figure  12.  A h i g h power e l e c t r o n m i c r o g r a p h o f e p i t h e l i a l cells i n the a n t e r i o r midgut. Note the d e n s e l y p a c k e d l u m i n a l m i c r o v i l l i and t h e p r o m i n a n t gap j u n c t i o n .  Figure  13.  Cell t y p e '4' (bottom r i g h t ) i s seen i n t h i s e l e c t r o n m i c r o g r a p h of the a n t e r i o r midgut. Note the p r e s e n c e of electron dense v e s i c l e s .  74a  -75Figures  Figure  14  and 15. L i g h t m i c r o g r a p h s of the cells of the a n t e r i o r midgut. Note t h e p r e s e n c e o f d a r k l y s t a i n i n g c e l l t y p e *2'.  16.  An e l e c t r o n m i c r o g r a p h of the anterior midgut. The c e l l s a r e more compressed t h a n t h o s e seen i n t h e m i d g u t caecum.  75a  -76Figures  Figure  17  and 18. L i g h t m i c r o g r a p h s o f the a n t e r i o r midgut. The lumen varies considerably in shape d e p e n d i n g upon c o n t r a c t i o n or the p r e s e n c e o f f o o d p a r t i c l e s .  19.  An e l e c t r o n m i c r o g r a p h o f t h e a n t e r i o r midgut i n T i g r i opus c a l i f o r n i c u s . Lipid v e s i c l e s are abundant. Note the basal muscle bands. I n many c e l l s , the G o l g i bodies are quite d i l a t e d .  76a  -77-  Figure  20.  An e l e c t r o n m i c r o g r a p h o f t h e a n t e r i o r p o r t i o n of the p o s t e r i o r m i d g u t . Note the d e n s e l y s t a i n e d 'bridge' shaped cell type ' 2 ' .  Figure  21.  An e l e c t r o n m i c r o g r a p h o f t h e p o s t e r i o r midgut. The b a s a l l a m i n a a p p e a r s c o n t i n u o u s w i t h t h e plasma membrane i n v a g i n a t i o n s .  -78-  Figures  Figure  22 and 23. L i g h t micrographs of the p o s t e r i o r midgut of T i g r i o p u s californicus. The c e l l s l i n i n g t h i s p a r t of the gut appear much l e s s c o n v o l u t e d a n d more c u b o i d a l i n shape. T h i s area o f the gut l i e s c e n t r a l l y i n t h e body c a v i t y . 24.  An e l e c t r o n m i c r o g r a p h o f some c e l l s i n the p o s t e r i o r m i d g u t . The m u s c l e bands a s s o c i a t e d w i t h t h e b a s a l lamina a r e w e l l developed. Note t h e d e c r e a s e i n t h e d e n s i t y o f t h e m i c r o v i l l i and t h e c u b o i d a l shape of the c e l l s .  78a  -79-  and 2 6 . Two l i g h t m i c r o g r a p h i c v i e w s ( c r o s s sections) through the anterior hindgut in Tigr iopus c a l i f o r n i c u s . The l u m e n i s cuticulized and f u r r o w e d . In f i g u r e 2 6 i t a p p e a r s as t i g h t l y c o n s t r i c t e d . This e l e c t r o n m i c r o g r a p h shows t h a t t h e c e l l s i n the p o s t e r i o r p o r t i o n of the p o s t e r i o r m i d g u t a r e low and c u b o i d a l shaped. The basal cell surfaces are invaginated and the a p i c a l m i t o c h o n d r i a are reduced i n s i z e and number.  79 a  -80Figure  28.  The cuticle lined f u r r o w e d lumen o f t h e p o s t e r i o r h i n d g u t i s shown i n t h i s e l e c t r o n micrograph.  Figure  29.  The cuticle i n t h e r e g i o n of the anus. I t t a k e s on t h e t y p i c a l b a n d e d appearance o f t h e copepod p r o t e c t i v e c u t i c l e .  80 a  -81Figure  30.  Sagittal section through t r a c t of Calanus plumchrus.  the  digestive  Figure  31.  L i g h t micrograph of the c u t i c l e agus.  Figure  32,  An e l e c t r o n m i c r o g r a p h o f t h e c u t i c l e the r e g i o n of the esophagus Note prominant muscle.  Figure  33  The a n t e r i o r p o r t i o n o f t h e m i d g u t . Note the p r e s e n c e of l a r g e vacuolated cells s u r r o u n d e d by d a r k e r s t a i n i n g e p i t h e l i a .  Figure  34  In t h e c e n t r e o f t h e v a c u o l a r r e g i o n , t h e lume n i s o f t e n s e e n t o be U' shaped. In C a l a n u s p l u m c h r u s , t h e e p i t h e l i a l cells l i n i n g the gut a r e compressed. There are l a r g e numbers o f v a c u o l a t e d cells.  lined  1  esophfrom the  81a  -82Figures  35 t o 37. 'R' c e l l s o f t h e m i d g u t d i v e r t i c u l u m . Note t h e v e s i c l e s p r e s e n t n e a r t h e m i c r o v i 1 1 i . In ( f i g u r e 35) t h e s e c e l l s , t h e b a s a l p l a s m a membrane i s h i g h l y i n v a g i n a t e and t h e b a s a l c e l l portions evaginate. In f i g u r e 3 7 , v e s i c l e s can be s e e n open t o t h e c y t o p l a s m (arrow h e a d ) .  Figure  38.  'R' t y p e c e l l f o u n d i n t h e p o s t e r i o r r e g i o n of the midgut. They a r e r e m a r k a b l y s i m i l a r i n a p p e a r a n c e t o t h e 'R' c e l l s o f t h e m i d g u t diverticulum. The b a s a l p o r t i o n s a r e l e s s i n v a g i n a t e d and t h e m i t o c h o n d r i a l e s s d e n s e l y packed.  Figure  39.  An e l e c t r o n m i c r o g r a p h o f an 'R' t y p e c e l l i n t h e t r a n s i t i o n between m i d g u t d i v e r t i c u l u m and m i d g u t .  Figure  40.  A l i g h t m i c r o g r a p h o f the midgut valve Note the between t h e m i d g u t a n d h i n d g u t . c o m p r e s s e d a r r a n g e m e n t o f t h e ep i t h e l i a1 cells.  82 a  -83Figure  41.  The g l a n d u l a r r e g i o n o f t h e m i d g u t o f C a l a n u s p l u m c h r u s i s shown. Note the presence o f 'B' 'D' and E " t y p e c e l l s 1  Figure  42.  The l a r g e v a c u o l e o f t h e 'B' c e l l i s s e e n in this e l e c t r o n m i c r o g r a p h . Note the numerous p i n o c y o t o i c i n c l u s i o n s on i t s luminal surface.  Figure  43.  B a s a l p o r t i o n of the c e l l s i n the g l a n d u l a r r e g i o n of the midgut. Note t h e b a s a l n u c l e u s o f t h e v a c u o l a t e d "B cells. 1  Figure  44  i n the g l a n d u l a r midgut •E a n d 'D' c e l l s The 'E' c e l l s a r e much l e s s d e n s e region. and o n l y l i g h t l y s t a i n e d . 1  83a  -84Figure  45.  'F' c e l l type i n the midgut of Calanus p 1 u m c h r us a d j a c e n t and p o s t e r i o r to the g l a n d u l a r midgut r e g i o n . The microvilli are t y p i c a l l y club-shaped.  Figure  46.  A h i g h power l i g h t m i c r o g r a p h v a l v e s e e n i n f i g u r e 40.  of  the  midgut  Figures  47  and 48. S e c t i o n s of the s e c r e t o r y 'F' t y p e c e l l s i n the m i d g u t . Note the d i l a t e d G o l g i b o d i e s and the g r a n u l a r rough e n d o p l a s m i c reticulum.  Figures  49  t o 51. I n t h e p o s t e r i o r p o r t i o n o f the m i d g u t near the m i d g u t v a l v e . The e p i t h e l i a l c e l l s a r e s h o r t e r and have a w e l l d e v e l o p e d b a s a l l a m i n a c o n t i n u o u s w i t h p l a s m a membrane invaginations. Organelles were s c a r c e . The m i c r o v i l l i r e t a i n t h e i r ' c l u b ' s h a p e .  84 a  -85-  Figure  52.  A light micrograph of the middle of the h i n d g u t . The lumen tends a star shaped appearance and is 1ined.  Figure  53.  A section through the anterior portion of the hindgut is seen. Note the deep luminal invaginations and the prominent nuclei.  Figure  54.  A low power m i c r o g r a p h of the posterior p o r t i o n of the hindgut i n Calanus plumchrus. Note i t s d o r s a l p o s i t i o n . It is considerably reduced i n s i z e as compared with figure 53.  Figure  55.  This e l e c t r o n m i c r o g r a p h shows t h e cuticle lining the luminal invagination of the c e l l s i n the p o s t e r i o r p o r t i o n of the h i n d g u t .  Figure  56.  A transverse s e c t i o n showing h i n d g u t a n d some m u s c u l a t u r e .  Figures  57  the  portion to have cuticle  posterior  and 58. Two electron micrographs of the c u t i c l e lining of the p o s t e r i o r hindgut c e l l s near the r e g i o n of the anus. M i t o c h o n d r i a were f o u n d i n t h e h i n d g u t e p i t h e l i a .  85a  -86Figure  59.  C o n t r o l m i c r o g r a p h of c o l l o i d a l g o l d a b s o r p t i o n i n the a n t e r i o r m i d g u t of T i g r i o p u s c a l i f o r n icus .  Figure  60.  C o n t r o l micrograph of c o l l o i d a l gold a b s o r p t i o n i n the r e g i o n o f the m i d g u t c a e c u m . Note d e n s e l y s t a i n i n g c e l l type '2'.  Figure  61.  C o n t r o l m i c r o g r a p h of the a n t e r i o r in T i g r i o p u s c a l i f o r n i c u s . Cell '2' and '3' a r e s e e n .  midgut types  86 a  -87Figure  62.  A r e g i o n of the p o s t e r i o r midgut. t h e p r e s e n c e o f a m u l t i v e s i c u l a r body in the 'bridge' shaped cell type C o l l o i d a l g o l d / WGA c o n t r o l .  Figure  63.  L u m i n a l g o l d c o l l o i d s a r e seen a f t e r minutes i n c u b a t i o n . The c e l l s u r f a c e b e l o n g s t o c e l l t y p e '3'.  Note (mvb) '2'. 5 viewed  87a  -88-  Figures  Figure  64  and 65. A f t e r 15 m i n u t e i n c u b a t i o n s , the g o l d c o l l o i d s a r e seen a d h e r e d t o t h e p l a s m a membrane o f c e l l t y p e '3'. Note t h e p r e s e n c e of t h e gap j u n c t i o n .  66.  C o l l o i d a l g o l d p a r t i c l e s i n t h e lumen o f the d i g e s t i v e t r a c t i n t h e r e g i o n of the posterior midgut. Arrows i n d i c a t e the 17 t o 20 um c o l l o i d s .  88a  -89Figure  67.  An e l e c t r o n m i c r o g r a p h of the p o s t e r i o r p o r t i o n o f the a n t e r i o r midgut i n T i g r i o p u s californicus. G o l d p a r t i c l e s were f o u n d i n t h e c y t o p l a s m o f c e l l t y p e '2'.  Figure  68.  A high power e l e c t r o n m i c r o g r a p h of the r e g i o n shown by t h e a s t e r i x i n f i g u r e 6 7 . Numerous g o l d p a r t i c l e s c a n be o b s e r v e d .  89 a  -90F i g u r e 69.  Control micrograph f o r horseradish peroxidase a b s o r p t i o n i n the a n t e r i o r midgut of T i g r i o p u s cali fornicus.  Figure  C o n t r o l micrograph of a s e c t i o n through t h e m i d g u t caecum. No p e r o x i d a s e a c t i v i t y c o u l d be s e e n .  70.  90a  -91F i g u r e 71.  A h i g h power e l e c t r o n m i c r o g r a p h of cell t y p e '3' i n t h e a n t e r i o r m i d g u t a s s e e n . Densely s t a i n i n g peroxidase p o s i t i v e v e s i c l e s bound i n a membrane c a n be seen ( a r r o w s ) .  Figure  72.  A section of c e l l t y p e '3' i n t h e m i d g u t caecum. At the arrows, v e s i c l e s c o n t a i n i n g positive horseradish peroxidase reaction p r o d u c t c a n be s e e n . They a r e r e a d i l y distinguishable from o t h e r dense v e s i c l e s in the cytoplasm.  Figure  73.  Membrane bound v e s i c l e s wi t h p o s i t i v e p e r o x i d a s e r e a c t i o n p r o d u c t c a n be seen i n t h e c y t o p l a s m o f c e l l t y p e '3' i n t h e m i d g u t caecum.  91a  -92Figure  74.  A f t e r a 1 t o 10 m i n u t e i n c u b a t i o n , v e s i c l e s containing horseradish peroxidase can be seen i n the c y t o p l a s m of c e l l type '3'. With s h o r t incubations, the peroxidase r e a c t i o n p r o d u c t i s found m a i n l y i n the periphery of the v e s i c l e s (see a r r o w s ) . T h i s i s a s e c t i o n o f the a n t e r i o r m i d g u t .  Figure  75.  The arrows i n d i c a t e peroxidase containing v e s i c l e s a f t e r a 15 m i n u t e incubation. The c e n t r a l p o r t i o n s o f the v e s i c l e s a p p e a r denser than that observed with s h o r t i n c u b a tions. 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