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UBC Theses and Dissertations

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 Tigriopus 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.Sc* The Oniversity of 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'." '.• i n THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA \ February, 1985 © Roy James McGroarty, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Tc^.t>0\e The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date C V t -\ I . DE-6(3/81) ( i i ) A b s t r a c t A study of the d i g e s t i v e t r a c t 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 C a l a n u s plumchrus was c a r r i e d out using t echniques of l i g h t and e l e c t r o n microscopy. In T i g r iopus  c a l i f o r n i c u s , the foregut c o n t a i n s a curved c u t i c l e l i n e d esophagus which extends from the v e n t r a l mouth to the j u n c t i o n of the a n t e r i o r midgut and midgut caecum. The n o n c u t i c u l i z e d p o r t i o n of the d i g e s t i v e t r a c t c o n s i s t s of : 1. A s i n g l e s p h e r i c a l midgut caecum l o c a t e d a n t e r i o r l y , 2. An a n t e r i o r m i d g u t , 3. A p o s t e r i o r m i d g ut. There a r e c u t i c u l i z e d a n t e r i o r and p o s t e r i o r hindgut regions ending i n a d o r s a l anus. In Calanus plumchrus, the foregut c o n s i s t s of a c u t i c l e l i n e d esophagus e x t e n d i n g from the v e n t r a l mouth to the j u n c t i o n of the midgut and the midgut d i v e r t i c u l u m . The n o n c u t i c u l i z e d p o r t i o n of the d i g e s t i v e t r a c t c o n s i s t s of : 1. A s i n g l e midgut d i v e r t i c u l u m , 2. A midgut t h a t i s d i v i s i b l e on the b a s i s of e p i t h e l i a l c e l l type and f u n c t i o n . There i s a long a b r u p t l y narrowing c u t i c l e l i n e d h i n d g u t ending i n an anus. In T i g r i opus c a l i f o r n i c u s , f o u r c e l l t y p e s c o u l d be d i s t i n g u i s h e d and from such 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 as the p o s i t i o n i n the d i g e s t i v e t r a c t , abundance, p o s i t i o n , and type of o r g a n e l l e s , l i p i d c o n t e n t , p r e s e n c e and type of v e s i c u l a t i o n , and e l e c t r o n d e n s i t y , f u n c t i o n s f o r the c e l l s were d e t e r m i n e d . C e l l type i s an e m b r y o n i c ( i i i ) 'stem' c e l l . I t f u n c t i o n s as a r e p l a c e m e n t c e l l and d i f f e r -e n t i a t e s when c e l l s a r e worn away o r l o s t i n s e c r e t i o n . C e l l t y p e ' 2 ' i s m a i n l y a s e c r e t o r y c e l l a n d f u n c t i o n s i n t h e s y n t h e s i s o f p r o t e i n s . I t a l s o p l a y s a r o l e i n l i p i d a b s o r p t i o n . C e l l t y p e ' 3 ' i s a b s o r p t i v e , m a i n l y f o r l i p i d s . C e l l t y p e M 1 , f o u n d o n l y i n t h e a n t e r i o r m i d g u t i s a l s o an a b s o r p t i v e c e l l . The p r e s e n c e o f e l e c t r o n d e n s e v e s i c l e s s u g g e s t s t h a t l i p i d a b s o r p t i o n i s not i t s major f u n c t i o n . From t h e abundance o f c e l l t y p e and from e x a m i n a t i o n o f t h e u l t r a s t r u c t u r e i n t h e v a r i o u s r e g i o n s o f t h e d i g e s t i v e t r a c t , t h e f o l l o w i n g c o n c l u s i o n s were made: 1 . The m i d g u t caecum f u n c t i o n s i n t h e a b s o r p t i o n o f d i g e s t e d n u t r i e n t s . 2 . The a n t e r i o r m i d g u t p l a y s a r o l e i n n u t r i e n t a b s o r p t i o n b u t i s i m p o r t a n t i n s e c r e t i o n . 3 . The p o s t e r i o r m i d g u t c e l l s a r e m a i n l y a b s o r p t i v e . In C a l a n u s p l u m c h r u s , f i v e c e l l t y p e s c o u l d be d i s t i n -g u i s h e d . C e l l t y p e 'E' i s an u n d i f f e r e n t i a t e d 'stem 1 c e l l . C e l l t y p e 'R'f f o u n d i n t h e m i d g u t d i v e r t i c u l u m and p o s t e r i o r m i d g u t r e g i o n s , i s a b s o r p t i v e . I t s d e v e l o p e d b a s a l s u r f a c e s s u g g e s t a t r a n s p o r t f u n c t i o n between t h e c e l l and t h e haemo-c o e l . C e l l t y p e 'D' i s fo u n d i n t h e g l a n d u l a r r e g i o n o f th e m i d g u t and i s a b s o r p t i v e . I t h a s an u l t r a s t r u c t u r e s i m i l a r t o t h a t o b s e r v e d f o r c e l l t y p e 'R' . C e l l t y p e 'B' i s a l a r g e v a c u o l a t e d a b s o r p t i v e c e l l f o u n d i n t h e 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 . C e l l t y p e 'F' f u n c t i o n s (iv) i n the s y n t h e s i s and s e c r e t i o n of d i g e s t i v e enzymes. In C a l a n u s p l u m c h r u s , the midgut d i v e r t i c u l u m i s s p e c i a l i z e d f o r the a b s o r p t i o n of d i g e s t e d n u t r i e n t s and t r a n s p o r t of m e t a b o l i t e s to the haemocoel . The a n t e r i o r midgut regions are mainly a b s o r p t i v e . I t i n c l u d e s a vacuolated g l a n d u l a r region s p e c i a l i z e d f o r p i n o c y t o t i c a b s o r p t i o n . In the middle s e c t i o n of the midgut, adjacent and p o s t e r i o r to the g l a n d u l a r r e g i o n , i s an a r e a of e p i t h e l i a l c e l l s s p e c i a l i z e d f o r s e c r e t i o n . The p o s t e r i o r midgut regions are mainly a b s o r p t i v e . In T i g r i o p u s c a l i f o r n i c u s b i o l o g i c a l markers can be used to d e t e r m i n e c e l l t ype f u n c t i o n i n c o r r e l a t i o n with the observed u l t r a s t r u c t u r e . (v) TABLE OF CONTENTS A b s t r a c t i i TABLE OF CONTENTS v LIST OF FIGURES v i i ACKNOWLEDGEMENTS x i I n t r o d u c t i o n 1 Foregut 2 Midgut 4 Midgut S t r u c t u r e s 6 Hindgut 9 Endocytos i s 11 (i) p i n o c y t o s i s 11 ( i i ) phagocytosis 12 ( i i i ) f l u i d phase p i n o c y t o s i s 13 (iv) r e c eptor mediated p i n o c y t o s i s 13 M a t e r i a l s and Methods 17 Li g h t Microscopy (a) T i g r iopus c a l i f o r n i c u s 17 (b) Calanus plumchrus 18 E l e c t r o n Microscopy (a) T i g r iopus c a l i f o r n i c u s 19 (b) Calanus plumchrus 20 (c) f l u i d phase endocytosis 20 (d) r e ceptor mediated endocytosis 21 Resul t s 23 T i g r iopus c a l i f o r n i c u s 23 Esophagus 23 Midgut Caecum 24 A n t e r i o r Midgut 26 P o s t e r i o r Midgut 28 Hindgut 30 Calanus plumchrus 30 Esophagus 31 Midgut 31 Hindgut 36 Receptor Mediated Endocytosis (T. c a l i f o r n i c u s ) ...37 F l u i d Phase Endocytosis (T. c a l i f o r n i c u s ) 38 D i s c u s s i o n 40 Foregut 40 Midgut 43 C e l l Types 43 Midgut Caecum and Midgut D i v e r t i c u l u m 50 Midgut Regional S p e c i a l i z a t i o n s 52 Hindgut .56 P e r i o t r o p h i c membrane 58 (vi) Endocytosis 61 f l u i d phase 61 adsorpt ive 64 Key f o r F i g u r e s 69 FIGURES 7 0 References 9 3 ( v i i ) LIST OF FIGURES F i g u r e 1. S a g i t t a l s e c t i o n of T. c a l i f o r n i c u s 70 F i g u r e 2. L i g h t micrograph of the mouth p a r t s of T_. c a l i f o r n i c u s 70 F i g u r e 3. Transverse s e c t i o n through the esophagus of T. c a l i f o r n i c u s 70 F i g u r e s 4 and 5. Transverse s e c t i o n through the esophagus and midgut caecum 70 F i g u r e 6. E l e c t r o n micrograph of the esophagus 71 F i g u r e 7. Esophageal a s s o c i a t e d c u t i c l e 72 F i g u r e 8. L i g h t micrograph of the midgut caecum and a n t e r i o r midgut i n T_. c a l i f o r n i c u s 72 F i g u r e 9. E l e c t r o n micrograph of the midgut caecum 72 F i g u r e 10. Transverse s e c t i o n through the midgut caecum 73 F i g u r e 11. E l e c t r o n micrograph of the t r a n s i t i o n from midgut caecum to a n t e r i o r midgut 73 F i g u r e 12. Micrograph of c e l l type '3' i n the a n t e r i o r midgut 74 F i g u r e 13. C e l l type '3' and type '4' i n the a n t e r i o r midgut 74 F i g u r e s 14 and 15. L i g h t micrograph of the a n t e r i o r midgut 75 F i g u r e 16. Micrograph of the c e l l types found i n the a n t e r i o r midgut 75 F i g u r e 17. L i g h t micrograph of the a n t e r i o r midgut 76 F i g u r e 18. L i g h t micrograph of the p o s t e r i o r segment of the a n t e r i o r midgut 76 Figure 19. E l e c t r o n micrograph of the a n t e r i o r midgut 76 Fi g u r e 20. 'Bridge' shaped c e l l type '2' of the p o s t e r i o r midgut 77 ( v i i i ) F i g u r e 21. I n v a g i n a t i o n s of the b a s a l plasma membrane in the p o s t e r i o r midgut 77 F i g u r e s 22 and 23. L i g h t micrographs of the p o s t e r i o r midgut 78 F i g u r e 24 Cuboidal c e l l s of the p o s t e r i o r midgut 78 F i g u r e s 25 and 26. Transverse s e c t i o n s of the a n t e r i o r hindgut 79 F i g u r e 27. E l e c t r o n micrograph of the c e l l s of the p o s t e r i o r midgut 79 F i g u r e 28. Micrograph of the c u t i c l e l i n e d lumen of the c e l l s of the p o s t e r i o r hindgut 80 Fig u r e 29. T y p i c a l banded c u t i c l e of the p o s t e r i o r hindgut near the anus 80 F i g u r e 30. S a g i t t a l s e c t i o n of the d i g e s t i v e t r a c t of Calanus plumchrus .81 F i g u r e 31. L i g h t micrograph of the esophagus 81 Fig u r e 32. E l e c t r o n micrograph of the esophageal c u t i c l e 81 F i g u r e s 33 and 34. L i g h t micrographs of the midgut 81 F i g u r e 35. 'R' c e l l s of the midgut d i v e r t i c u l u m 82 F i g u r e 36. Micrograph of the a p i c a l p o r t i o n of 'R' c e l l s 82 F i g u r e 37. High power view of the luminal m i c r o v i l l i 82 F i g u r e 38. 1R' c e l l s of the p o s t e r i o r midgut region 82 F i g u r e 39. T r a n s i t i o n c e l l s between the midgut d i v e r t i c u l u m and the midgut 82 F i g u r e 40. L i g h t micrograph of the midgut valve i n C. plumchrus 82 F i g u r e 41. Micrograph of the g l a n d u l a r midgut region 83 ( i x ) F i g u r e 44. 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 s o f t h e c e l l s i n t h e g l a n d u l a r r e g i o n 83 F i g u r e 45. A p i c a l v e s i c l e s and m i c r o v i l l i o f t h e ' F 1 c e l l 84 F i g u r e 46. H i g h power 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 v a l v e 84 F i g u r e s 47 and 48. T r a n s v e r s e s e c t i o n s o f 'F' c e l l s 84 F i g u r e s 49 to 51. E l e c t r o n m i c r o g r a p h s o f t h e p o s t e r i o r m i d g u t r e g i o n ..84 F i g u r e 52. L i g h t m i c r o g r a p h o f t h e h i n d g u t 85 F i g u r e 53. T r a n s v e r s e s e c t i o n o f t h e a n t e r i o r h i n d g u t r e g i o n 85 F i g u r e 54. T r a n s v e r s e s e c t i o n o f t h e p o s t e r i o r h i n d g u t r e g i o n 85 F i g u r e 55. M i c r o g r a p h o f t h e h i n d g u t c u t i c l e o f C. p l u m c h r u s 85 F i g u r e 56. Low power l i g h t m i c r o g r a p h o f t h e p o s t e r i o r h i n d g u t 85 F i g u r e s 57 and 58. M i c r o g r a p h s o f t h e c u t i c l e l i n i n g t h e p o s t e r i o r h i n d g u t 85 F i g u r e 59. A n t e r i o r m i d g u t o f T. c a l i f o r n i c u s 86 F i g u r e 60. C e l l t y p e '2' i n t h e m i d g u t caecum 86 F i g u r e 61. M i c r o g r a p h o f c e l l t y p e s '2' and '3' i n t h e a n t e r i o r m i d g u t 86 F i g u r e 62. P o s t e r i o r m i d g u t o f T. c a l i f o r n i c u s 87 F i g u r e 63. 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 87 F i g u r e s 64 and 65. G o l d c o l l o i d s a s s o c i a t e d w i t h t h e plasma membrane o f c e l l t y p e ' 3 ' 88 F i g u r e 66. C o l l o i d a l g o l d i n t h e lumen o f t h e p o s t e r i o r m i d g u t 88 (x) F i g u r e 67. 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 a n t e r i o r m i d g u t 89 F i g u r e 68. G o l d p a r t i c l e s i n t h e c t y o p l a s m o f c e l l t y p e 1 2 1 89 F i g u r e 69. 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 m i d g u t 90 F i g u r e 70. E l e c t r o n m i c r o g r a p h o f t h e m i d g u t caecum 90 F i g u r e 71. HRP 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' 91 F i g u r e 72. HRP i n t h e m i d g u t caecum 91 F i g u r e 73. M i c r o g r a p h o f HRP f o u n d i n t h e m i d g u t caecum 91 F i g u r e 74. M i c r o g r a p h o f a n t e r i o r m i d g u t .....92 F i g u r e 75. M i c r o g r a p h o f t h e j u n c t i o n between t h e m i d g u t caecum and t h e a n t e r i o r m i d g u t 92 (xi) Acknowledgements I wish to express my g r a t i t u d e to my s u p e r v i s o r Dr. T. B i s a l p u t r a . He i n t r o d u c e d me to the f i e l d o f e l e c t r o n microscopy and provided v a l u a b l e suggestions and i n t r e p r e t a t i o n s t h r o u g h o u t t h i s s t u d y . S p e c i a l thanks a r e extended to Dr. A.G. Lewis f o r h i s s u g g e s t i o n s and s u p p o r t , and to D r . A.B. Acton f o r h i s a s s i s t a n c e and f r i e n d s h i p . For ideas on my p r o j e c t and s u p p o r t t h r o u g h i t s c o n c l u s i o n , I wish t o thank Dr. E. de Harven. I am i n d e b t e d to my f r i e n d s K. B e l l , H. C h r i s t e n s e n , G. Bootsma, R. L e u n g , L. B o r t o l a m i o l and S. Ramjohn f o r t h e i r support and h e l p f u l s u g g e s t i o n s . To Jane Van S i c k l e I extend my deepest g r a t i t u d e . W i t h o u t her c o u r a g e , p e r s i s t e n c e , and v a l u a b l e a s s i s t a n c e with t h i s manuscript, the p r o j e c t would not have been completed. Summer support was s u p p l i e d by a NSERC grant awarded to Dr. T. B i s a l p u t r a . -1-I n t r o d u c t i o n : Few s t u d i e s , a t the l i g h t or e l e c t r o n m i c r o s c o p e l e v e l s , have examined the comparative morphology and f u n c t i o n a l anatomy of the d i g e s t i v e t r a c t o f copepods. This study w i l l compare the d i g e s t i v e t r a c t morphologies of 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 Calanus  p l u m c h r u s . I t i s i n t e n d e d to f u r t h e r the understanding of the gut morphology of marine copepods. Al s o i n c l u d e d , i s a study of the e n d o c y t o t i c mechanisms of the d i g e s t i v e t r a c t of T i g r iopus c a l i f o r n i c u s . Comparisons of the d i g e s t i v e t r a c t s and t h e i r c e l l s to those of other arthropods w i l l be in c l u d e d to i l l u s t r a t e t h e i r s i m i l a r i t i e s and d i f f e r e n c e s . Through such comparison, and w i t h the stud y of e n d o c y t o t i c mechanisms i n T i g r i o p u s  c a l i f o r n i c u s , some i n s i g h t can be given to the f u n c t i o n s suggested f o r the morphology of the d i g e s t i v e t r a c t regions and t h e i r c e l l s . T h e r e i s a g r e a t d e a l o f c o n f l i c t i n g t e r m i n o l o g y i n the l i t e r a t u r e regarding some of the areas of the arthropod d i g e s t i v e t r a c t . No c l e a r convention e x i s t s f o r the use of the terms d i g e s t i v e d i v e r t i c u l a , glands, caeca, or hepato-pancreas. S u l l i v a n (1977) d e s c r i b e d the 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 t r a c t s i n c e a d i v e r t i c u l u m was a g e n e r a l 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 outpushing from a c a v i t y that i s not extremely branched. That terminology w i l l be u p h e l d here s i n c e the d i g e s t i v e d i v e r t i c u l a are -2-not branched l i k e those d e s c r i b e d i n o t h e r a r t h r o p o d s as glands or hepatopancreas. There are three d i s t i n c t regions that are the p r i n c i p l e components of a crustacean d i g e s t i v e system. They i n c l u d e a f o r e g u t , midgut, and a hindgut with a p o s t e r i o r or p o s t e r o -v e n t r a l anus. The foregut and h i n d g u t are of e c t o d e r m a l o r i g i n and the midgut i s of endodermal o r i g i n . I t i s common to have t u b u l a r o u t g r o w t h s , g l a n d s and/or caeca a r i s i n g from the midgut. (McLaughlin 1983). A. Foregut In crustaceans there i s a c o n s i d e r a b l e foregut d i v e r s i t y . I t has been v a r i o u s l y d e s c r i b e d as a s i m p l e passageway to a complex m u l t i c h a m b e r e d s t r u c t u r e . Previous s t u d i e s on copepods have shown that the f o r e g u t i s a simple esophagus. In the copeped genera C a l a n u s ( M a r s h a l l and Orr, 1955), E p i l a b i d o c e r a (Park 1966), Centropages (Arnaud et a l , 1978), and T i g r i opus ( S u l l i v a n and B i s a l p u t r a , 1980) i t has been so d e s c r i b e d . Other a u t h o r s ( B r i g g s 1977) d e s c r i b e d the f o r e g u t of the g e n e r a P a r a n t h e s s i u s as a s h o r t esophagus with a c u t i c u l i z e d chamber w h i l e o t h e r s ( B r e s c i a n i and L u t z e n , 1961) d e s c r i b e d the foregut of a p a r a s i t i c s p e c i e s , Saccoposis  s t e e n s t r u p i , as an e l o n g a t e d tube and l a r g e muscularized stomach. In the barnacle alimentary t r a c t the foregut i s d e s c r i b e d - 3 -a s c u t i c l e l i n e d i n b o t h B a l a n u s b a l a n o i d e s and B a l a n u s  h a m e r i ; ( R a i n b o w and W a l k e r , 1977) w i t h t h r e e p o r t i o n s ; t h e p h a r y n x , t h e e s o p h a g u s , and t h e v e n t r i c u l u s . The f o r e g u t o f m a l a c o s t r a c a n s has f u r t h e r s p e c i a l i z a -t i o n s . P o s t e r i o r t o t h e e s o p h a g u s a r e an a n t e r i o r and a p o s t e r i o r chamber. I t has been s u g g e s t e d t h a t t h e y f u n c t i o n i n t h e g r i n d i n g o f f o o d p a r t i c l e s , (Nath and P i l l a i , 1972). A l s o i n t h e m y s i d f o r e g u t , t h e r e i s a s h o r t m u s c u l a r e s o p h a g u s , and p o s t e r i o r and a n t e r i o r chambers. ( M a u c h l i n e , 1980). I t i s l i k e l y t h a t t h e d i e t a r y d i f f e r e n c e s i n f l u e n c e t h e f o r e g u t d e v e l o p m e n t . In i s o p o d s , a m p u l l a e a r e formed a t t h e e s o p h a g e a l / f o r e g u t chamber a r t i c u l a t i o n and f u n c t i o n t o p r e v e n t t h e back r e g u r g i t a t i o n o f f o o d . ( N a y l o r , 1955). T h e f o r e g u t o f d e c a p o d s i s h i g h l y c o m p l e x . D e e p i n f o l d i n g s o f t h e p o s t e r i o r and a n t e r i o r chambers a r e l i n e d by a s e r i e s o f c a l c i f i e d p l a t e s . T h e r e i s a s h o r t , m u s c u l a r e s o p h a g u s a n t e r i o r t o t h e s e chambers (Maynard and Dando, 1974; H i n t o n and C o r e y , 1979). F o r e g u t a c t i v i t y i s e x t e m e l y i m p o r t a n t t o t h e u n d e r -s t a n d i n g o f t h e d i g e s t i v e p r o c e s s e s . Thomson and H a i l i n g (1976) p r o p o s e d an e x p l a n a t o r y model o f f o r e g u t p e r i s t a l s i s i n t h e b l o w f l y Phormia r e g i n a . P e r i s t a l t i c a c t i v i t y o c c u r s i n t h e e s o p h a g e a l p o r t i o n o f t h e f o r e g u t . Yonge (1924) s t a t e d t h a t t h e f o r e g u t was impermeable to a b s o r p t i o n b u t i n t h e c o c k r o a c h , E i s n e r (1955) f o u n d e v i d e n c e o f f a t a b s o r p t i o n . Vonk (1960) f o u n d e v i d e n c e - 4 -f o r the s e c r e t o r y f u n c t i o n of the f o r e g u t . He found t h a t mucous and p o s s i b l y amylase were secreted i n t h i s region of the gut. B. Midgut As was noted f o r the f o r e g u t , the crustacean midgut i s a l s o h i g h l y v a r i a b l e . In Daphnia, the midgut i s d i v i s i b l e i n t o three p a r t s . Schultz and Kennedy (1976) s t a t e t h a t the a n t e r i o r p o r t i o n i s the p r i m a r y s i t e of a b s o r p t i o n . T h e i r d e s c r i p t i o n of the a n t e r i o r p o r t i o n i n c l u d e s the c a e c a . The midmesenteral region i s the s i t e of d i g e s t i o n , and the feces formation i n c a r r i e d out p o s t e r i o r l y . In copepods of the genus Calanus, the midgut i s a n t e r i o r l y expanded, narrowing at the l e v e l of the second or t h i r d t h o r a c i c somites and c o n t i n u i n g to the l a s t abdominal somites (Marshall and Orr, 195.5; H a l l b e r g and H i r c h e , 1980). In the c a l a n o i d s , Arnaud et a l . , (1978) and H a l l b e r g and H i r c h e , (1980), d i s t i n g u i s h three midgut d i v i s i o n s w h i l e S u l l i v a n and B i s a l p u t r a , (1980), found o n l y two i n the h a r p a c t i c o i d , T i g r i o p u s c a l i f o r n i c u s . Two e p i t h e l i a l c e l l types have been d e s c r i b e d f o r c a l i g o i d copepods (Lewis 1961), four c e l l types i n T i g r i o p u s c a l i f o r n i c u s ( S u l l i v a n and B i s a l p u t r a , 1980) , and four e p i t h e l i a l c e l l types have been d i s t i n g u i s h e d i n the c a l a n o i d Centropages (Arnaud et a l . , 1978). C o n s i d e r a b l e d i f f e r e n t i a t i o n of the f i n e s t r u c t u r e of the midgut e p i t h e l i u m has been c o r r e l a t e d w i t h s e a s o n , -5-sex, and stage of development. (Ha l l b e r g and Hir c h e , 1980). Fahrenbach (1962) d e s c r i b e d a c e n t r a l l y l o c a t e d midgut v a l v e and S u l l i v a n and B i s a l p u t r a , (1980), d e s c r i b e d a t i g h t c o n s t r i c t i o n a t the a n t e r i o r and p o s t e r i o r midgut j u n c t i o n . In the i s o p o d s o n l y the c e n t r a l p o r t i o n of the gut i s termed the midgut even though r e s u l t s show t h a t i t i s of ectodermal o r i g i n (Stromberg, 1965). Jones (1968) s t a t e d that i n marine and t e r r e s t r i a l i s o p o d s the midgut i s an e x t r e m e l y s m a l l r e g i o n between the f o r e g u t and h i n d g u t and i s wholely g l a n d u l a r i n n a t u r e . S t u d i e s by G o o d r i c h (1939) i n d i c a t e that i n these isopods the midgut i s extremely small or absent. C o n s i d e r a b l e c o n t r o v e r s y e x i s t s i n the s t u d i e s o f the isopod midgut. Schmitz and Sc h u l t z (1969) s t a t e d t h a t the woodlouse gut i s m a i n l y endodermal and t h e r e f o r e a midgut. But Ho l d i c h (1973) s t a t e s that previous data was m i s i n t e r p r e t e d and that i n isopods t h e r e i s o n l y a s m a l l segment between the foregut and the hindgut that are the remains of the midgut. In decapods c o n s i d e r a b l e midgut v a r i a t i o n e x i s t s . P i k e (1947) r e p o r t e d t h a t the midgut was v e r y s h o r t or p r a c t i c a l l y absent. In the european l o b s t e r Homarus gammarus Barker and Gibs o n (1977) s t a t e t h a t the midgut a c c o u n t s for 5/6 of the p o s t g a s t r i c i n t e s t i n a l t r a c t . F a h r e n b a c h (1962), and H o l d i c h and Mayers, (1975) s t a t e t h a t i n most c r u s t a c e a n s , a p e r i o t r o p h i c membrane - 6 -i s a c t i v e l y s e c r e t e d by the midgut c e l l s . C. Midgut S t r u c t u r e s Numerous midgut s t r u c t u r e s o f the crustaceans have been d e s c r i b e d by many authors. Very l i t t l e u l t r a s t r u c t u r a l work has been a v a i l a b l e to date on the glands of the midgut, the c a e c a or the d i v e r t i c u l a . The t e r m i n o l o g y has not been a p p l i e d with c o n s i s t e n c y by the authors. The terminology d e v e l o p e d by S u l l i v a n (1977) equates the midgut caecum to that 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 . P h i l l i p s e t a l ., (1977) s t a t e t h a t the g l a n d u l a r d i v e r t i c u l a o f decapods s h o u l d not be r e f e r r e d to as a h e p a t o p a n c r e a s because i t does not f u n c t i o n according to the c o n v e n t i o n s e s t a b l i s h e d f o r the mammalian l i v e r or p a n c r e a s . Other authors, Gibson and Barker (1979) d i s p u t e t h i s by s t a t i n g t h a t the complex t u b u l a r and d u c t - l i k e s t r u c t u r e s should be r e f e r r e d to as a hepatopancreas. In many decapods, carbohydrate metabolism and d i g e s t i v e enzyme s e c r e t i o n s have been demonstrated i n these g l a n d u l a r s t r u c t u r e s (McLaughlin, 1983, Barker and Gibson, 1978). Normally the term midgut caecum i s used f o r s m a l l d o r s a l pockets of growth from the midgut. Midgut d i v e r t i c u l a has been used to r e f e r to b l i s t e r - l i k e pouches from the midgut w a l l . (McLaughlin, 1983). For the purpose of t h i s study, the a n t e r i o r outgrowth of the midgut of T i g r i opus c a l i f o r n i c u s w i l l be r e f e r r e d -7-to as the midgut caecum ( S u l l i v a n , 1977) and the a n t e r i o r outgrowth of the midgut of Calanus plumchrus w i l l be c a l l e d the midgut d i v e r t i c u l u m . S c h u l t z and Kennedy (1976) s t a t e t h a t i n Daphnia  p u l e x t h e r e e x i s t s an a n t e r i o r p a i r of small d i v e r t i c u l a t h a t a r e s i m p l e i n v a g i n a t i o n s of the midgut. They go on to s t a t e that s i n c e an analogue w i t h the a n t e r i o r midgut c a e c a of M a l a c o s t r a c a i s g u e s t i o n a b l e , the term d i g e s t i v e caecum or d i g e s t i v e d i v e r t i c u l u m i s p r e f e r r e d over the term 1hepatopancreas 1 or 'midgut' caeca. In M a l a c o s t r a c a n s the h e p a t o p a n c r e a s has s i m i l a r f u n c t i o n s to the midgut i n T i g r i o p u s c a l i f o r n i c u s ( S u l l i v a n , 1977) and f u n c t i o n s i n food a b s o r p t i o n and purine metabolism, (Vonk, 1960) d i g e s t i v e enzyme s e c r e t i o n , storage at glycogen, l i p i d s and m i n e r a l s . ( H a r t e n s t e i n , 1964; L o i z z i and Peterson, 1969; L o i z z i 1968; Woodhouse, S t a n i e r and G r i f f i n , 1968). A s i n g l e a n t e r i o r caecum was r e p o r t e d f o r c a l a n o i d and h a r p a c t i c o i d copepods. Ong and Lake, (1970) found a s i n g l e a n t e r i o r outgrowth of the midgut of Calanus helgo- l a n d i c u s and termed i t the midgut d i v e r t i c u l u m . Arnaud et a l . , (1978) found a s i n g l e a n t e r i o r caecum with no a s s o c i a t e d glands i n Centropages t y p i c u s and i d e n t i f i e d f i v e p r i n c i p l e c e l l c a t e g o r i e s (E,R,F,B,D) i n the midgut s i m i l a r to those d e s c r i b e d f o r the malacostracans and i n the hepatopancreas of decapods. The 'R' c e l l s had smooth endoplasmic r e t i c u l u m and high m i c r o v i l l i , the 'F' c e l l s had rough e n d o p l a s m i c -8-r e t i c u l u m and short m i c r o v i l l i , the 'D' c e l l s were d a r k l y s t a i n i n g , the 'B' c e l l s were vacuolated and the 'E' c e l l s were slender with l i g h t cytoplasm and rare o r g a n e l l e s . S u l l i v a n and B i s a l p u t r a (1980), found an a n t e r i o r s p h e r i c a l midgut caecum i n T i g r i o p u s c a l i f o r n i c u s i n which three c e l l types could be d i s t i n g u i s h e d ( c e l l types 1,2,3). C e l l type '1', was found at the base of the gut e p i t h e l i u m and never reached the lumen. I t was normally found i n c l u s t e r s of three and was c h a r a c t e r i z e d by abundant ribosomes, some mitochondria, rough endoplasmic r e t i c u l u m and t r a n s p a r e n t v e s i c l e s . C e l l type '2' was found to have r e g u l a r basal i n v a g i n a t i o n s , a p i c a l l i p i d d r o p l e t s , l u m i n a l m i c r o v i l l i , and randomly d i s p e r s e d m i t o c h o n d r i a . The dense cytoplasm was due to rib o s o m e s and rough e n d o p l a s m i c r e t i c u l u m . D i l a t e d G o l g i b o d i e s were a l s o found. The most abundant c e l l was type '3' with basal i n v a g i n a t i o n s , l a m e l l a t e rough e n d o p l a s m i c r e t i c u l u m , abundant m i c t o c h o n d r i a , n u c l e a r a s s o c i a t e d G o l g i b o d i e s and v a r i o u s s i z e d v e s i c l e s and l i p i d d r o p l e t s . A f o u r t h c e l l type, type '4', was found only i n the midgut. I t had d i s p e r s e d rough e n d o p l a s m i c r e t i c u l u m , smaller and l e s s d i l a t e d G o l g i , abundant e l e c t r o n t r a n s p a r e n t v e s i c l e s , and many o t h e r v e s i c l e s of v a r y i n g e l e c t r o n d e n s i t y found m a i n l y i n the a p i c a l p o r t i o n s of the c e l l . B a c c a r i and Renaud-Morant (1974) found no caeca i n the d i g e s t i v e system of the m y s t a c o c a r i d D e r o c h e i l o c a r i s -9-r e m a n e i . As many as seven midgut c a e c a have been found i n some balanomorphs. (McLaughlin, 1983). D i v e r s i t y abounds i n the ca e c a and midgut g l a n d s among the m a l a c o s t r a c a n s . In the l e p t o s t r a c a n s , a s i n g l e midgut gland i s present i n N e b a l i o p s i s but i n other genera there are one or two p a i r s of caeca and two or three glands or g l a n d u l a r l o b e s ( M c L a u g h l i n , 1983). Nath and P i l l a i , (1972) found m y s i d s w i t h m u l t i l o b e d g l a n d s accompanied by one or two caeca. Both midgut c a e c a and g l a n d s are normally found i n decapods. Midgut glands have been d e s c r i b e d f o r a number of decapods ( P i l l a i , 1960; D a l l , 1967; Barker and Gibson, 1977, 1978, 1979). In decapods, these glands a r e l o b u l a r and found on e i t h e r s i d e o f the gut a t the j u n c t i o n of the foregut and midgut. D. Hindgut In c r u s t a c e a n s t h e r e i s n o r m a l l y a s h o r t h i n d g u t ending i n a muscularized anus. In the re g i o n of the anus, S u l l i v a n and B i s a l p u t r a , (1980), d e s c r i b e d the arrangement of a s t r i a t e d c u t i c l e . In some crustaceans such as Balanomorph  C i r r i p e d s , the h i n d g u t i s c h a r a c t e r i z e d by two d i s t i n c t r e g i o n s . An a n t e r i o r m u s c u l a r s p h i n c t e r and a p o s t e r i o r anal chamber. (McLaughlin, 1983). Rainbow and Walker (1977) s t a t e that the p o s t e r i o r midgut leads to a h i g h l y f o l d e d m u s c u l a r h i n d g u t r e g i o n -10-that acts as a s p h i n c t e r . P o s t e r i o r l y , i t extends to a wide a n a l chamber and a s l i t - l i k e anus. The hindgut was found to be h i s t o l o g i c a l l y s i m i l a r to the f o r e g u t . Jones (1968) s t a t e d that the hindgut i n isopods represents a l a r g e p r o p o r t i o n of the d i g e s t i v e t r a c t . I t can be d i v i d e d i n t o 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 separated by a s p h i n c t e r with the a n t e r i o r p o r t i o n s being d i s t e n s i b l e . Vernon et a l . , (1974) a l s o d e s c r i b e d the l a r g e isopod hindgut as a two chambered s t r u c t u r e with deep i n f o l d i n g s of the a n t e r i o r w a l l s . Fox (1952), d e s c r i b e d the anal r o l e of water intake in many Crustacea. He d i s c u s s e d the pumping and a n t i p e r i s t a l t i c c o n t r a c t i o n o f the r e c t a l muscles and suggested that t h i s a c t i o n acted as a n a t u r a l enema i n the d e f e c a t i o n p r o c e s s but was probably not involved i n osmoregulation, r e s p i r a t i o n or the maintenance of t u r g o r . In the decapods, c o n f u s i o n e x i s t s over the e x t e n t of the h i n d g u t . P i k e (1947) , d e s c r i b e d a long h i n d g u t i n G o l a t h e a s p e c i e s where as i n o t h e r s (Jackson, 1913), such as Pagurus, the h i n d g u t was found to be e x t r e m e l y s h o r t . In the d i g e s t i v e system of Dapnia pulex, the hindgut was found to be v e r y s h o r t , c u t i c u l i z e d , and s i m i l a r i n s t r u c t u r e to the fo r e g u t . There i s a small t e r m i n a l anus c o n t r o l l e d by d i l a t o r muscles. (Schultz and Kennedy, 1976). S u l l i v a n and B i s a l p u t r a (1980), d e s c r i b e d the hindgut -11-o f T i g r i o p u s c a l i f o r n i c u s , as an e p i t h e l i a l l i n e d chamber w i t h a c u t i c u l i z e d l u m i n a l s u r f a c e and e x a g g e r a t e d i n v a g i n a -t i o n s . T h e y s u g g e s t e d t h a t t h e h i n d g u t c o u l d f u n c t i o n i n a o s m o r e g u l a t i o n , water t r a n s p o r t , and some n u t r i e n t a b s o r p t i o n . T. S. Park (1966) d e s c r i b e d t h e h i n d g u t o f t h e c a l a n o i d c o pepod E p i l a b i d o c e r a a m p h i t r i t e s as c u t i c l e l i n e d e p i t h e l i a s u r r o u n d e d by a basement membrane c o n t a i n i n g l o n g i t u d i n a l r i d g e s s i m i l a r t o t h e e s o p h a g u s . The a n a l s e g m e n t h a s t h r e e p a i r s o f d i l a t o r m u s c l e s a s s o c i a t e d w i t h i t . In o t h e r 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 f o r t h e c u t i c u l i z e d h i n d g u t . In t h e brown wasp, M i c r o p l i t i s  c r o c e i p s e s , a b s o r p t i o n o f d i s s a c h a r i d e s , l i p i d s , and t r i o l e i n have been s u g g e s t e d . (Edson and V i n s o n , 1977). E. E n d o c y t o s i s E n d o c y t o s i s , a s r e v i e w e d b y B r u c e A l b e r t s e t a l . , (1983), i s a p r o c e s s o f c e l l u l a r i n g e s t i o n whereby macromole-c u l e s and p a r t i c l e s a r e p r o g r e s s i v e l y e n c l o s e d i n s m a l l p o r t i o n s o f t h e p l a s m a membrane w h i c h i n v a g i n a t e , t h e n p i n c h o f f , f o r m i n g i n t r a c e l l u l a r v e s i c l e s w h i c h c o n t a i n t h e i n g e s t e d m a t e r i a l . T he p r o c e s s o f e n d o c y t o s i s c a n be s u b d i v i d e d i n t o two major t y p e s : P i n o c y t o s i s and Phagocy-t o s i s . I t i s o f t e n c l a s s i f i e d by t h e s i z e o f t h e v e s i c l e s f o r m e d . ( i ) P i n o c y t o s i s F l u i d a n d / o r s o l u t e i n g e s t i o n i s u s u a l l y r e f e r r e d - 1 2 -to as p i n o c y t o s i s or c e l l d r i n k i n g (B. A l b e r t s e t a l . , 1 9 8 3 ; Steinman, 1 9 8 3 ; Casley-Smith, 1 9 6 9 ; Lewis, 1 9 3 1 ) . This type of c e l l u l a r i n g e s t i o n i s c h a r a c t e r i z e d by small v e s i c l e s c a l l e d p i n o c y t o t i c v e s i c l e s . They c o n t a i n c e l l u l a r f l u i d and d i s s o l v e d s o l u t e s or molecules bound to t h e i r membranes. There are two main types of p i n o c y t o s i s ; f l u i d phase or bulk uptake p i n o c y t o s i s (endocytosis) and receptor mediated p i n o c y t o s i s ( e n d o c y t o s i s ) . Those p i n o c y t o t i c v e s i c l e s c o n t a i n i n g f l u i d and/or d i s s o l v e d s o l u t e s a r i s e from a p r o c e s s of f l u i d phase p i n o c y t o s i s which does not i n v o l v e r e c e p t o r s and i s r e l a t e d to the s e c r e t o r y process and i n v o l v e d i n membrane r e c y c l i n g . P i n o c y t o t i c v e s i c l e s c o n t a i n i n g molecules bound to the plasma membrane a r i s e from receptor mediated e n d o c y t o s i s . ( i i ) Phagocytosis P h a g o c y t o s i s or c e l l - e a t i n g , i n v o l v e s the i n g e s t i o n of l a r g e p a r t i c l e s such as microorganisms v i a much l a r g e r v e s i c l e s o f t e n r e f e r r e d to as v a c u o l e s . L a r g e p a r t i c l e i n g e s t i o n i s u s u a l l y the domain of c e l l s s p e c i a l i z e d f o r t h a t f u n c t i o n , and i s not c o n s i d e r e d a r o u t i n e c e l l u l a r p r o c e s s . Phagocytosis may a l s o i n v o l v e a receptor mediation but i t i s an uptake of l a r g e p a r t i c l e s . ( A l b e r t s et a l . , 1 9 8 3 ; Steinman, 1 9 8 3 ; Casley-Smith, 1 9 6 9 ) . -13-( i i i ) F l u i d Phase P i n o c y t o s i s (Endocytosis) The f l u i d phase e n d o c y t o t i c events can be v i s u a l i z e d i n a number of ways. The most common method i n v o l v e s the b r i e f exposure to an impermeable s o l u t e that does not bind to the plasma membrane. C y t o l o g i c a l markers of t h i s type i n c l u d e m o l e c u l e s such as f e r r i t i n , some c o l l o i d a l gold p r e p a r a t i o n s , enzymes such as h o r s e r a d i s h peroxidase (HRP) and dextrans. Horseradish peroxidase i s a v a l u a b l e marker because the p i n o c y t o t i c v e s i c l e s can be v i s u a l i z e d a f t e r i n g e s t i o n with hydrogen peroxide and diaminobenzidine. (Steinman, 1983; Herzog and Fahimi, 1973; C o r n e l l e t a l . , 1971; Graham and Karnousky, 1966) . The enzyme can be q u a n t i f i e d b i o c h e m i c a l l y , and i t s uptake i n c r e a s e s l i n e a r l y with concen-t r a t i o n and i n c r e a s e s c o n t i n u a l l y w i t h time ( A l b e r t s et a l . , 1983) . (iv) Receptor Mediated P i n c o c y t o s i s (Endocytois) R e c e p t o r m e d i a t e d p i n o c y t o s i s i n v o l v e s the l i g a n d b i n d i n g t o a plasma membrane r e c e p t o r , which a r e o f t e n d i f f u s e l y d i s t r i b u t e d , followed by 1igand-receptor c l u s t e r i n g complexes i n c o a t e d p i t s . These membrane coated regions tend to be l a r g e l y made up of a l a t t i c e work of the p r o t e i n c l a t h r i n . I t i s assumed that the p i n c h e d - o f f coated v e s i c l e s d e l i v e r the l i g a n d to lysosomes or o t h e r i n t r a c e l l u l a r compartments, then r e c y c l e the c l a t h r i n and the r e c e p t o r s back to the plasmalemma. ( G o l d s t e i n et a l . , 1979). -14-Recent u l t r a s t r u e t u r a l work s u g g e s t s that c l a t h r i n may not r e c y c l e but remains a t t a c h e d to the p i t s on the cytoplasmic s i d e of the plasma membrane. Pastan and Willingham (1981), suggest t h a t i n s t e a d of r e c y c l i n g the c l a t h r i n , the l i g a n d s once bound i n the c o a t e d p i t s , bud to form an uncoated r e g i o n or c r y p t i c p i t i n the i n v a g i n a t i n g membrane. Uncoated v e s i c l e s a r e formed from these c r y p t i c p i t s v i a i n v a g i n a t i o n from the plasmalemma or an o p e n i n g i n the c l a t h r i n c o a t . They c a l l these uncoated v e s i c l e s ' recepto-somes' . The empty c o a t e d p i t would then remain as p a r t of the plasmalemma and be a v a i l a b l e f o r f u r t h e r l i g a n d b i n d i n g without the requirement of c l a t h r i n r e c y c l i n g . S i n c e the 1880's, i t has been known that p l a n t c e l l e x t r a c t s could a g g l u t i n a t e red blood c e l l s . The a g g l u t i n i n s were termed ' l e c t i n s ' . Today, the term l e c t i n r e f e r s to s u g a r - b i n d i n g p r o t e i n s from v a r i o u s s o u r c e s r e g a r d l e s s of t h e i r a b i l i t y to a g g l u t i n a t e c e l l s . T h i s unique group of p r o t e i n s has provided b i o l o g i s t s with a powerful t o o l to study many c e l l u l a r processes i n c l u d i n g e n d o c y t o s i s . S i n c e l e c t i n s have a s p e c i f i c i t y toward a p a r t i c u l a r c a r b o h y d r a t e s t r u c t u r e , even o l i g o s a c c h a r i d e s with i d e n t i c a l sugar compositions can be i s o l a t e d and i d e n t -i f i e d . C e r t a i n l e c t i n s bind to r e s i d u e s of mannose or glucose while others recognize only g a l a c t o s e r e s i d u e s . Some l e c t i n s r e q u i r e t h a t the sugar be i n the t e r m i n a l n o n - r e d u c i n g -15-p o s i t i o n while others can bind to sugars w i t h i n an o l i g o s a c -c h a r i d e c h a i n . S i n c e v i r t u a l l y a l l b i o l o g i c a l membranes c o n t a i n glycoconjugates ( g l y c o p r o t e i n s and g l y c o l i p i d s ) , l e c t i n s can be used to study the c e l l s u r f a c e f e a t u r e s of a l l l i v i n g organisms. Wheat Germ A g g l u t i n i n (WGA) i s a 36,000 m o l e c u l a r weight p r o t e i n that has two i d e n t i c a l s u b u n i t s . The receptor sugar f o r WGA i s N-acetylglucosamine. I t can bind to o l i g o -s a c c h a r i d e s c o n t a i n i n g t e r m i n a l N - a c e t y l g l u c o s a m i n e o r c h i t o b i o s e which are common to many membrane g l y c o p r o t e i n s . WGA has a l s o been reported to i n t e r a c t w i t h g l y c o l i p i d s , N - a c e t y l n e u r a m i n i c a c i d , and to some g l y c o p r o t e i n s v i a s i a l i c a c i d r e s i d u e s . (Monsigny et a l . , 1980; Sharon and L i s , 1972) . C o l l o i d a l gold has r e c e n t l y been used as a cytochemical marker i n c o n j u n c t i o n with l e c t i n s 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 . Due to t h e i r o p a c i t y to the e l e c t r o n s they are e a s i l y d e t e c t e d . S p e c i f i c markers have been o b t a i n e d with a v a r i e t y of macromolecules i n c l u d i n g l e c t i n s . (Horisb-e r g e r , 1979). The c o n j u g a t i o n 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 l e c t i n s provides the v e h i c l e to study the events of receptor mediated e n d o c y t o s i s . The p u r p o s e of t h i s s t u d y was to d e s c r i b e a t the l i g h t and e l e c t r o n microscope l e v e l s the d i g e s t i v e t r a c t -16-of two 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. Also i n c l u d e d i s a c h a r a c t e r i z a t i o n of the process of endo-c y t o s i s i n the d i g e s t i v e t r a c t of T r i g r i o p u s c a l i f o r n i c u s . From the d e s c r i p t i o n s p r o v i d e d , and comparisons with other arthropods, i t i s hoped that the f u n c t i o n a l r e l a t i o n s h i p s between gut region and c e l l type w i l l be apparent. From t h i s c o m p a r a t i v e m o r p h o l o g i c a l d e s c r i p t i o n and s t u d i e s of the f u n c t i o n a l anatomy of these copepods, s i m i l a r processes i n other c r u s t a c e c e a w i l l be b e t t e r understood. _17-M a t e r i a l s and Methods: T i g r i o p u s c a l i f o r n i c u s specimens were obtained from a c u l t u r e maintained by Dr. A. G. Lewis, Zoology Department, U.B.C. The copepods were maintained i n enriched a r t i f i c i a l sea water, i n c u l t u r e j a r s , a t room t e m p e r a t u r e i n the l a b o r a t o r y under f l u o r e s c e n t c o n d i t i o n s . 'Tetramin' t r o p i c a l f i s h food was the n u t r i e n t s o u r c e and the sea water was changed e v e r y two to t h r e e months. R e g u l a r c l e a n i n g of the bottom d e b r i s was c a r r i e d out bi-weekly. Once a good b a c t e r i a l c u l t u r e was e s t a b l i s h e d i n the c u l t u r e c o n t a i n e r s , continued feeding was no longer necessary. C a l a n u s plumchrus specimens were o b t a i n e d from a s i n g l e c o l l e c t i o n i n June 1983 using a plankton net dragged from a depth of 200m to s u r f a c e i n the S t r a i g h t of Georgia i n an area adjacent to the F r a s e r River plume. (Fra-1 sampling s t a t i o n ) . A. Specimen P r e p a r a t i o n (i) L i g h t Microscopy (a) T i g r i o p u s c a l i f o r n i c u s Specimen p r e p a r a t i o n for l i g h t microscopy was c a r r i e d out i n the f o l l o w i n g manner: A two hour primary f i x a t i o n i n 5% g l u t a r a l d e h y d e i n 0.2M phosphate b u f f e r e d s a l i n e (PBS) at pH 7.4. The specimens were washed s e v e r a l times i n b u f f e r and p o s t f i x e d i n 2% osmium f o r a minimum of f i v e hours. The specimens were then washed i n the b u f f e r -18-s e v e r a l times and followed by b u l k s t a i n i n g i n 5% u r a n y l a c e t a t e i n d i s t i l l e d w a t e r . A f t e r f u r t h e r washing, the specimens were dehydrated i n a graded methanol s e r i e s and embedded i n P o l y b e d 812 embedding media ( P o l y s c i e n c e s , F t . Warrington, P e n n s y l v a n i a ) . Specimen b l o c k s were trimmed and 0.5 - 1.0 micron s e c t i o n s cut using g l a s s knives or a Diatome diamond k n i f e on a P o r t e r - B l u m MT-1 microtome. S e c t i o n s were f l o a t e d onto d i s t i l l e d water, c o l l e c t e d i n a loop, and t r a n s f e r r e d to a d i s t i l l e d water d r o p l e t on a cleaned microscope s l i d e . A f t e r a heat d r y i n g , the s e c t i o n s were s t a i n e d w i t h 1% t o l u d i n e blue p l u s 1% Borax f o r s e v e r a l seconds. Sec t i o n s were observed and photographed u s i n g a p o l a r o i d e q u ipped Z e i s s photomicroscope. (b) Calanus plumchrus Specimen p r e p a r a t i o n f o r l i g h t microscopy was c a r r i e d out i n the f o l l o w i n g manner : A one and o n e - h a l f hour p r i m a r y f i x a t i o n i n 4% g l u t a r a l d e h y d e i n 0.2M phosphate s a l i n e b u f f e r (PBS) at pH 7.4. Specimens were washed i n b u f f e r then post f i x e d i n 1% osmium t e t r o x i d e f o r one hour. A f t e r s e v e r a l washings, the specimens were s t a i n e d i n 5% ur a n y l a c e t a t e i n d i s t i l l e d water. A f t e r washing i n b u f f e r , the specimens were dehydrated i n a graded methanol s e r i e s and embedded i n Epon or Spurr's embedding media ( P o l y s c i e n c e s , F t . Warrington, P e n n s y l v a n i a ) . -19-Specimen b l o c k s were trimmed and 1-2 micron s e c t i o n s cut using a Porter-Blum MT-1 microtome, or a R e i c h e r t Ultratome with e i t h e r g l a s s , diamond, or tungsten-carbide k n i v e s . S e c t i o n s were f l o a t e d onto d i s t i l l e d water, c o l l e c t e d w i t h a l o o p , and placed on a d r o p l e t of water on a cleaned microscope s l i d e . A f t e r heat d r y i n g , the s e c t i o n s were s t a i n e d w i t h 1% t o l u d i n e b l u e p l u s 1% borax or 1% m e t h y l e n e b l u e p l u s 1% borax f o r s e v e r a l seconds. S e c t i o n s were observed and photographed with a p o l a r o i d equipped Zeiss photomicroscope. ( i i ) E l e c t r o n Microscopy (a) T i g r i o p u s c a l i f o r n i c u s 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 microscopy was c a r r i e d out i n the f o l l o w i n g manner. A one and o n e - h a l f hour p r i m a r y f i x a t i o n i n a m i x t u r e of 6% g l u t e r a l d e h y d e and 1% osmium t e t r o x i d e i n 0.2M phosphate b u f f e r e d s a l i n e (PBS) i n the dark. The specimens were washed s e v e r a l times i n b u f f e r , pH 7.4 f o r at l e a s t one-half hour. T h i s was followed by a post f i x a t i o n i n 1% osmium t e t r o x i d e i n b u f f e r f o r one-half hour. A f t e r s e v e r a l washings i n b u f f e r (PBS, pH 7.4, 1/2 hour) the specimens were dehydrated i n a graded methanol s e r i e s ; 1/2 hour each f o r 30%, 50%, and 70%, 1 hour each f o r 90% and 100%, and f i n a l l y p l a c e d i n a p r o l o n g e d b a t h of propylene oxide f o r at l e a s t 2 hours. Due to the r e l a t i v e impermeability of the c u t i c l e b a r r i e r , a 5-7 day i n f i l t r a t i o n p e r i o d was c a r r i e d out i n Polybed 812 ( P o l y s c i e n c e s , F t . Warrington, Pennsylvania) embedding media. -20-P o l y m e r i z a t i o n was c a r r i e d out at 60 degrees C f o r 24 hours. The p o l y m e r i z e d b l o c k s were trimmed then s e c t i o n e d using a R e i c h e r t 0MU3 Ultramicrotome or a Porter-Blum MT-1 u l t r a m i -crotome with a xDupont' or a >Diatome' diamond k n i f e . The s e c t i o n s were mounted on 300 mesh Formvar coated copper g r i d s or on 300 mesh u n c o a t e d copper g r i d s . They were s t a i n e d i n a s a t u r a t e d s o l u t i o n of u r a n y l acetate i n methanol for 30 minutes f o l l o w e d by 10 minutes in lead c i t r a t e (Reynolds, 1963). (b) Calanus 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 microscopy was c a r r i e d out i n the f o l l o w i n g manner: A one-hour p r i m a r y f i x a t i o n i n 5% g l u t a r a l d e h y d e i n 0. 2M phosphate b u f f e r e d s a l i n e (PBS) pH 7.4. A f t e r s e v e r a l washings i n b u f f e r (1/2 h o u r ) , the specimens were p o s t f i x e d i n 1% osmium t e t r o x i d e f o r one hour. Again, a f t e r repeated washings , the specimens were dehydrated i n a graded methanol s e r i e s (as above) f o l l o w e d by a 1 hour bath i n propylene oxide. I n f i l t r a t i o n was c a r r i e d out i n Epon or Spurr's embedding media ( P o l y s c i e n c e s , F t . Warrington, Pennsy-l v a n i a ) f o r 2 d a y s . P o l y m e r i z a t i o n o c c u r r e d i n 2 4 hours a t 60 degrees C. Trimming, s e c t i o n i n g and s t a i n i n g followed the same procedure d e s c r i b e d f o r T i g r i o p u s c a l i f o r n i c u s . (c) F l u i d phase Endocytosis ( T i g r i o p u s c a l i f o r n i c u s ) 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 microscopy -21-was c a r r i e d out i n the f o l l o w i n g manner: Copepods were incubated w i t h (0.5 mg/ml) h o r s e r a d i s h peroxidase (HRP) i n 7 graded time i n t e r v a l s ( l f 2, 5, 10, 20, 30, 45 minutes). T h i s was followed by f i x a t i o n i n 6% g 1 u t e r a l d e h y d e and 1% osmium t e t r o x i d e i n phosphate b u f f e r e d s a l i n e (PBS) pH 7.4 f o r one and o n e - h a l f h o u r s . S e v e r a l washings i n b u f f e r followed f o r one-half hour. Specimens were then incubated with 5.0 mg 3,3' -Diaminobenzidine (DAB) i n 20mls of PBS c o n t a i n i n g 0.2 ml 1% hydrogen peroxide (H202) f ° r one-quarter hour. The post f i x a t i o n i n osmium t e t r o x i d e , d e h y d r a t i o n and embedding p r o c e d u r e s a re i d e n t i c a l to th o s e p r e v i o u s l y mentioned f o r T i g r i o p u s c a l i f o r n i c u s . C o n t r o l e x p e r i m e n t s were c a r r i e d out i n two ways. F i r s t , the copepods were prepared as above, but were not incubated with h o r s e r a d i s h peroxidase. Secondly, another group of copepods were i n c u b a t e d w i t h h o r s e r a d i s h p e r o x i d a s e (as above), f i x e d i n 6% g l u t a r a l d e h y d e and 1% osmium t e t r o x i d e i n phosphate b u f f e r e d s a l i n e (PBS) pH 7.4, washed, and then incubated with phosphate b u f f e r e d s a l i n e (PBS) pH 7.4 c o n t a i n i n g only 0.2 ml 1% hydrogen p e r o x i d e f o r one-quarter hour. The remainder of the procedure i s again i d e n t i c a l to that p r e v i o u s l y d e s c r i b e d . (d) Receptor mediated endocytosis ( T i g r i o p u s c a l i f o r n i c u s ) Gold c o l l o i d s (15-20mm) were prepared using the method d e s c r i b e d by H o r i s b e r g e r (1979). Conjugation was c a r r i e d out between c o l l o i d a l gold and wheat Germ A g g l u t i n i n (WGA) (Sigma Co.) i n the f o l l o w i n g manner: 18mls of WGA (0.1mg/ml) was added -22-dropwise to 90mls of the c o l l o i d a l gold s o l u t i o n over a p e r i o d of 10 mi n u t e s w h i l e c o n s t a n t l y s t i r r i n g . P o lyethylene g l y c o l (PEG) (18mls) was added s l o w l y to the s o l u t i o n and mixed f o r 5 m i n u t e s . The m i x t u r e was c e n t r i f u g e d at 15,000 X g f o r 30 minutes at 4 degrees C. The supernatant was d i s c a r d e d and the conjugate was resuspended i n 1/4 i t s o r i g i n a l volume i n F2 media. A p r i m a r y i n c u b a t i o n w i t h t h i s c o n j u g a t e was c a r r i e d out i n three stages (5, 15, and 30 minutes) then processed i n the manner p r e v i o u s l y d e s c r i b e d f o r T i g r i o p u s c a l i f o r n i c u s . C o n t r o l e x p e r i m e n t s were c a r r i e d out by i n c u b a t i n g the conjugate with 10 mg/ml N-acetyl-D-glucosamine (15 minutes) and then processed as above. In a l l the experiments f o r tr a n s m i s s i o n e l e c t r o n micro-scopy, those s e c t i o n s c o l l e c t e d and p l a c e d on unco a t e d copper g r i d s were carbon coated f o r s t a b i l i t y p r i o r to viewing. The specimens were observed using a Z e i s s EM9S, P h i l l i p s 300, or a P h i l l i p s 400 t r a n s m i s s i o n e l e c t r o n microscope. -23-R e s u l t s : A. T i g r i o p u s c a l i f o r n i c u s A l o n g i t u d i n a l s e c t i o n of an a d u l t copepod T i g r iopus  c a l i f o r n i c u s i s shown i n i t s e n t i r e t y i n f i g u r e 1. A higher m a g n i f i c a t i o n of the oral/mandibular region i s shown in f i g u r e 2. From the morphology observed, the d i g e s t i v e t r a c t can be subdivided i n t o the esophogus, midgut caecum, a n t e r i o r midgut, p o s t e r i o r midgut, and a n t e r i o r and p o s t e r i o r hindgut. Esophagus The esophagus l i e s i n a c e n t r a l p o s i t i o n i n the a n t e r i o r segment of the c e p h a l o t h o r a x . From i t s v e n t r a l o p e n i n g , i t c u r v e s a n t e r i o r l y and e x t e n d s d o r s a l l y with a s l i g h t p o s t e r i o r bend to i t s opening at the j u n c t i o n of the a n t e r i o r midgut and midgut caecum. F i g u r e 3 shows the c e n t r a l l y l o c a t e d esophagus i n a s e c t i o n cut through from the a n t e r i o r r e g i o n of the c e p h a l o t h o r a x to the mandibular area at 45 d e g r e e s to the t r a n s v e r s e p l a n e . F i g u r e s 4 and 5 show a c r o s s s e c t i o n through the d o r s a l esophagus and the midgut caecum. The lumen of the esophagus i s t y p i c a l l y NH' shaped and r e f l e c t s i t s a b i l i t y to d i s t e n d i n times of f e e d i n g . An e l e c t r o n micrograph of the esophagus ( f i g u r e 6) shows a lumen bounded by an a r e a of d i s t i n c t c u t i c l e followed by dense e p i t h e l i a l c e l l s (6-17 urn t a l l ) and s u r r o u n d e d by a c i r c u l a r muscle l a y e r (5-10 um t h i c k ) . The c u t i c l e -24-l i n i n g the esophagus ( f i g u r e 7) appears to be made up of at l e a s t two l a y e r s . An e l e c t r o n dense and t h i n ( 0.02-0.03 um) e p i c u t i c l e i s found on the l u m i n a l s u r f a c e and the i n n e r l a y e r a ppears of medium e l e c t r o n d e n s i t y and about 0.2 um t h i c k . V a r i a t i o n s i n c u t i c l e t h i c k n e s s a r e found i n the i n n e r most l a y e r and a p p e a r s to be l e s s e l e c t r o n dense. The e p i t h e l i a l c e l l s have deep i n v a g i n a t i o n s of the plasma membrane at the b a s a l and c u t i c u l a r r e g i o n s . S e c t i o n s c o n t a i n i n g n u c l e i show them to be round to i r r e g u l a r shaped with a prominent round n u c l e o l u s . C i r c u l a r m i t o c h o n d r i a (0.2-0.5 um) w i t h few c r i s t a e and some rough endoplasmic r e t i c u l u m are found. Ribosomes are abundant i n the cytoplasm. In the d o r s a l esophagus there are signs of d i g e s t i o n . Semidigested m a t e r i a l i s o f t e n found but no e v i d e n c e of s e c r e t o r y f u n c t i o n s were found i n the c u t i c l e l i n e d esophagus. Midgut Caecum A p a r a s a g i t t a l s e c t i o n ( f i g u r e 8) shows a p o r t i o n of the midgut caecum and a small area of the a n t e r i o r midgut. The midgut caecum ( f i g u r e 9) i s a s p h e r i c a l chamber (40-45um) that l i e s above and a n t e r i o r to the esophagus. I t extends from the j u n c t i o n of the esophagus and the a n t e r i o r midgut towards the a n t e r i o r p o r t i o n of the copepod. The e p i t h e l i a l c e l l s a r e columnar with many c e l l s having a p i c a l v e s i c l e s and m i c r o v i l l i . Some of the j u n c t i o n s between the c e l l s -25-of t h i s c a v i t y are furrowed and have l a r g e v a c u o l e - l i k e s t r u c t u r e s . The c e l l s v a r y i n h e i g h t form 3-17 um and have some bas a l i n v a g i n a t i o n s which are ringed by the bas a l l a m i n a . In many r e g i o n s l o n g i t u d i n a l muscle can be seen i n c l o s e a s s o c i a t i o n with the bas a l lamina. There appears to be three or p o s s i b l y four c e l l types found i n the midgut caecum. F i g u r e s 9 and 10 are c r o s s s e c t i o n s through the midgut caecum i l l u s t r a t i n g these c e l l , types. C e l l t ype '1' i s u s u a l l y seen i n c l u s t e r s of three or f o u r c e l l s t h a t l i e a t the b a s a l p o r t i o n of the gut e p i t h e l i u m . These c e l l s u s u a l l y have a c e n t r a l nucleus with a prominent n u c l e o l u s . The a p i c a l p o r t i o n s of t h e s e c e l l s do not reach the lumen. There appears to be a random d i s t r i b u t i o n of G o l g i bodies, mitochondria and rough endoplasmic r e t i c u l u m . The c y t o p l a s m c o n t a i n s l o o s e r i b o s o m e s and some small s p h e r i c a l v e s i c l e s (0.2 um). C e l l type '2' has basal i n v a g i n a t i o n s and a c e n t r a l l y l o c a t e d nucleus. I t has a very dense cytoplasm which i n c l u d e s random m i t o c h o n d r i a and rough endoplasmic r e t i c u l u m . The G o l g i bodies tend to be very d i l a t e d . Numerous e x o c y t o t i c v e s i c l e s were found i n the cytoplasm. This i s suggestive of a mainly s e c r e t o r y f u n c t i o n . In a d d i t i o n to the e x o c y t o t i c f u n c t i o n of opaque v e s i c l e s , some c e l l s i n the midgut caecum c o n t a i n e d l a r g e c y t o p l a s m i c masses w i t h e l e c t r o n dense p a r t i c u l a t e m a t t e r . They are found mainly i n the a p i c a l -26-one-half of the c e l l and tend to i n c r e a s e i n s i z e towards the l u m i n a l s u r f a c e . The c y t o p l a s m i c d e n s i t y i s s i m i l a r to that d e s c r i b e d f o r c e l l type '2' but the number of e l e c t r o n opaque v e s i c l e s has g r e a t l y d i m i n i s h e d . T h i s c e l l type was found mainly i n the midgut caecum and i n the t r a n s i t i o n between the midgut caecum and the a n t e r i o r midgut. In f i g u r e 8, e v i d e n c e can be seen f o r s u b s t a n t i a l c e l l u l a r s e c r e t i o n s i n t o the lumen. I t appears that t h i s s e c r e t i o n i s of the macro-apocr i n e or h o l o c r i n e t y p e . In many s e c t i o n s , the dense c e l l s o f the midgut caecum appear to be ex t r u d i n g t h e i r c e l l u l a r c o n t e n t s . C e l l type '3' i s the most abundant c e l l type i n the midgut caecum. The basement membrane i s i n v a g i n a t e d , sometimes q u i t e deeply, and there i s a round nucleus most o f t e n b a s a l l y to c e n t r a l l y l o c a t e d . Lamellate rough endoplasmic r e t i c u l u m i s found c l o s e l y a s s o c i a t e d with the nucleus. Mitochondria a r e d i s t r i b u t e d randomly and the G o l g i b o d i e s a r e l e s s d i l a t e d and q u i t e numerous. Both e l e c t r o n opaque and e l e c t r o n t r a n s l u c e n t s m a l l v e s i c l e s a re found i n the c y t o p l a s m . In some s e c t i o n s t h e s e s m a l l opaque v e s i c l e s can be seen f u s i n g to the plasma membrane. A n t e r i o r Midgut The a n t e r i o r midgut b e g i n s a t the j u n c t i o n of the midgut caecum and ends at the p o s i t i o n o f the t h i r d p a i r of pereiopods. The e p i t h e l i a i s made up of p s e u d o s t r a t i f i e d -27-c u b o i d a l c e l l s ( f i g u r e 11) t h a t range i n h e i g h t from 5 to 25 um w i t h b a s a l i n v a g i n a t i o n s i n most c e l l s up to 1 um i n t o the c y t o p l a s m . The e p i t h e l i a l c e l l s a r e j o i n e d at t h e i r l u m i n a l s u r f a c e s by prominent gap j u n c t i o n s and tend to be h i g h l y c o n v o l u t e d i n the a n t e r i o r p o r t i o n o f the a n t e r i o r m i d g ut. In most s e c t i o n s , the e p i t h e l i a l c e l l s had round n u c l e i with a prominent n u c l e o l u s and s c a t t e r e d c h r o m a t i n . As i n the midgut caecum, the three c e l l types are present, but t h e r e i s a g r e a t e r c o n t i n u i t y i n t h e i r cytoplasmic d e n s i t i e s ( f i g u r e 12). A f o u r t h c e l l t y p e , type '4', can be seen i n t h i s r e g i o n of the g u t . I t has l u m i n a l m i c r o v i l l i , abundant bas a l m i t o c h o n d r ia , some rough e n d o p l a s m i c r e t i c u l u m and an abundance of small dense v e s i c l e s ( f i g u r e 13). F i g u r e s 14, 15 and 16 are c r o s s s e c t i o n s t h r o u g h the a n t e r i o r p o r t i o n of the a n t e r i o r midgut. C e l l type '1' i s u l t r a s t r u c t u r a l l y i d e n t i c a l to those seen i n the midgut caecum. C e l l type '2' has an abundance of rough endoplasmic r e t i c u l u m and has v e s i c l e s c o n t a i n i n g p a r t i c l e s of h i g h e l e c t r o n d e n s i t y . They a l s o c o n t a i n other l i p i d - l i k e e l e c t r o n opaque d r o p l e t s . C e l l type '3' has a reduced amount of rough endoplasmic r e t i c u l u m and s t i l l has some s m a l l d i l a t e d G o l g i b o d i e s . The p r e s e n c e of a p i c a l l i p i d d r o p l e t s could s t i l l be seen i n the a n t e r i o r p o r t i o n , but were reduced i n number or - 2 8 -a b s e n t i n t h e p o s t e r i o r p o r t i o n o f t h e a n t e r i o r m i d g u t . Towards t h e p o s t e r i o r segment o f t h e a n t e r i o r m i d g u t , t h e c e l l s t e n d t o be l e s s c o n v o l u t e d and s h o r t e r ( 5 t o 14 um) . F i g u r e 17 i s a c r o s s s e c t i o n o f t h e p o s t e r i o r p o r t i o n o f t h e a n t e r i o r m i d g u t . In f i g u r e 1 9 , t h e h i g h l y c o n v o l u t e d b a s a l l a m i n a i s s e e n w i t h i t s a s s o c i a t e d band o f l o n g i t u d i n a l m u s c l e . Numerous v e s i c l e s c a n be s e e n i n t h e a p i c e s o f many c e l l s . In t h e p o s t e r i o r segment o f t h e a n t e r i o r m i d g u t ( f i g u r e 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 form a v a l v e - l i k e s t r u c t u r e a t t h e e n t r a n c e t o t h e p o s 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 The p o s t e r i o r m i d g u t l i e s i n t h e c e n t r e o f t h e body c a v i t y and i s s u r r o u n d e d by body t i s s u e s . A t t h e j u n c t i o n b e t w e e n t h e a n t e r i o r and p o s t e r i o r m i d g u t , t h e c e l l s a r e t h e same h e i g h t as t h o s e o f t h e p o s t e r i o r s e g m e n t o f t h e a n t e r i o r m i d g u t . They a r e l e s s c o n v o l u t e d and more c u b o i d a l i n s h a p e . The m i c r o v i l l i a r e s l i g h t l y s h o r t e r and l e s s d e n s e l y p a c k e d . The b a s a l l a m i n a i s h i g h l y c o n v o l u t e d and c l o s e l y a s s o c i a t e d w i t h l o n g i t u d i n a l m u s c l e ( f i g u r e 2 1 ) . The c e l l s a r e rimmed a t t h e i r a p i c e s by gap j u n c t i o n s . In f i g u r e 2 0 , c e l l t y p e ' 2 ' can be s e e n as a b r i d g e s t r u c t u r e between n e i g h b o u r i n g c e l l s and has p r o m i n e n t e l e c t r o n t r a n s -p a r e n t and e l e c t r o n opaque l u m i n a l v e s i c l e s . In some s e c t i o n s , t h e v e s i c l e s c a n be s e e n i n c l o s e a s s o c i a t i o n w i t h t h e -29-plasma membrane and o c c a s i o n a l l y open to the lumen a t the base of the m i c r o v i l l i . F i g u r e 22, i s a c r o s s s e c t i o n t h r o u g h the 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 . The p o s t e r i o r midgut i n the m i d d l e p o r t i o n ( f i g u r e 23) shows the c u b o i d a l shaped e p i t h e l i u m with n u c l e i and prominent n u c l e o l i . The c e l l s a v erage about 5.0 um i n height and have an al m o s t u n i f o r m c y t o p l a s m i c d e n s i t y . F i g u r e 24 shows these low c u b o i d a l e p i t h e l i a l c e l l s . The m i c r o v i l l i are reduced i n height and d e n s i t y . There are s e v e r a l basal lamina i n v a g i n a t i o n s and a t h i c k l o n g i t u d i n a l muscle l a y e r j u s t beneath these i n v a g i n a t i o n s . The p r o p o r t i o n of a p i c a l s e c r e t o r y v e s i c l e s i s m a r k e d l y reduced. C e l l types ' l 1 , '2', and '3' are p r e s e n t but c e l l type '1' i s much more common i n the a n t e r i o r p o r t i o n s of the midgut. Mitochondria and some rough endoplasmic r e t i c u l u m a re p r e s e n t as w e l l as a few G o l g i b o d i e s . O c c a s i o n a l l y v e s i c l e s a r e seen with e l e c t r o n dense c e n t r a l c o r e s . C e l l type '2' i s more evident i n t h i s midgut r e g i o n . I t i s o f t e n seen forming a 'bridge' a d j a c e n t to c e l l s of type '3'. M i t o c h o n d r i a and rough e n d o p l a s m i c r e t i c u l u m were present but the c e l l i s m a i n l y c h a r a c t e r i z e d by i t s extreme e l e c t r o n d e n s i t y . E l e c t r o n dense a p i c a l v e s i c l e s are a l s o found i n the c e l l s of the p o s t e r i o r midgut. C e l l type '3' i n t h i s region has bas a l lamina i n v a g i n -a t i o n s with i n t e r d i g i t a t i n g f o l d s i n the plasma membrane. The rough endoplasmic r e t i c u l u m i s found mainly i n a s s o c i a t i o n -30-with the plasma membrane. M i t o c h o n d r i a and G o l g i b o d i e s are p r e s e n t . V e s i c l e s are o f t e n seen budding o f f the G o l g i c i t e r n a e . The m i c r o v i l l i are s h o r t e r (0.5 - 0.7 um) and are l o o s e l y packed ( f i g u r e 27). Hindgut Two c r o s s s e c t i o n s a r e shown i n f i g u r e 25 and 26. In the a n t e r i o r hindgut ( f i g u r e 26) the c e l l s are elongated, compressed and somewhat wedge-shaped. There are numerous c u t i c l e l i n e d furrows. The e p i t h e l i a l c e l l s a r e squamous and have c i r c u l a r n u c l e i . Very few, i f any, rough endoplasmic r e t i c u l u m or G o l g i bodies were seen. The p o s t e r i o r p o r t i o n of the a n t e r i o r hindgut ( f i g u r e 25) i s c u t i c l e l i n e d and g r e a t l y f u r r o w e d . The c y t o p l a s m i s l e s s dense than the midgut e p i t h e l i a l c e l l s and devoid of most membrane bound o r g a n e l l e s . F i g u r e 28 shows the c u t i c l e l i n e d , f u r r o w e d r e g i o n of the p o s t e r i o r hindgut. Near the anus, the c u t i c l e i s s t r i a t e d ( f i g u r e 29) which i s t y p i c a l of the outer c u t i c l e l i n i n g which p r o t e c t s the copepod. B. Calanus Plumchrus A l o n g i t u d i n a l s e c t i o n t h r o u g h the d i g e s t i v e t r a c t of an a d u l t Calanus Plumchrus i s shown i n f i g u r e 30. Morph-o l o g i c a l l y , the gut i s d i v i d e d i n t o four r e g i o n s ; the esophagus, midgut d i v e r t i c u l u m , midgut, and the hindgut. -31-Esophagus The esophagus of Calanus plumchrus ( f i g u r e s 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 tube that bends a n t e r i o d o r s a l l y from i t s v e n t r a l o p e n i n g . I t becomes ovoid or round i n appearance 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 segments. There i s an e p i t h e l i a l l a y e r bounded by a b a s a l l a m i n a and surrounded by an inner l o n g i t u d i n a l m uscle l a y e r and bands of c i r c u l a r m u s c l e s . I t i s c u t i c l e l i n e d ( f i g u r e 32) with 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 region (5 um) narrowing to about 1 um i n i t s most d o r s a l segments. In i t s d o r s a l r e g i o n , i t opens to the midgut at the j u n c t i o n of the midgut and midgut d i v e r t i c u l u m . As i t enters the midgut, a c u t i c l e l i n e d r i d g e prevents the esophageal contents from s p i l l i n g d i r e c t l y i n t o the midgut d i v e r t i c u l u m . The c u t i c l e t h i n s r a p i d l y at t h i s o p e n i n g and the c e l l s l i n i n g the midgut and midgut d i v e r t i c u l u m develop m i c r o v i l l i on t h e i r luminal s u r f a c e s . Midgut The midgut forms the l a r g e s t part of the ali m e n t a r y c a n a l . On the d o r s a l s i d e of i t s a n t e r i o r end, a c o n i c a l midgut d i v e r t i c u l u m i s formed. At i t s beginning, the midgut i s wide and s a c - l i k e . I t narrows and becomes d i s p l a c e d v e n t r a l l y as i t proceeds p o s t e r i o r l y , then i t bends d o r s a l l y . In the middle region of the copepod, the midgut has a d o r s a l p o s i t i o n and p r o c e e d s backwards to the j u n c t i o n of the -32-h i n d g u t . The w i d e s t p a r t o f t h e m i d g u t h a s a t r i a n g u l a r l u m i n a l s h a p e ( f i g u r e 33) a n d as i t b e n d s d o r s a l l y , t h e lumen becomes 'V shaped ( f i g u r e 34). F o u r r e g i o n s o f t h e m i d g u t c a n be d e f i n e d on t h e b a s i s o f t h e i r e p i t h e l i a l c e l l l i n i n g s . I n t h e a n t e r i o r m i d g u t d i v e r t i c u l u m p o r t i o n w h i c h s t a r t s a t t h e f r o n t o f t h e e s o p h a g e a l o p e n i n g , t h e r e i s a u n i f o r m l a y e r on n o n -v a c u o l a t e d e p i t h e l i a l c e l l s . Upon e n t e r i n g t h e wide s a c -l i k e p o r t i o n o f t h e m i d g u t , t h e e p i t h e l i a l l i n i n g t a k e s on a s l i g h t l y d i f f e r e n t a p p e a r a n c e , b ut i s n o n - v a c u o l a t e d . In t h e c e n t r a l w i d e s t p o r t i o n o f t h e m i d g u t t h e r e i s a g l a n d u l a r r e g i o n o f e p i t h e l i a l c e l l s w h ich a r e h i g h l y v a c u o -l a t e d . 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 , t h e e p i t h e l i a l c e l l s l o s e t h e i r v a c u o l a t e d a p p e a r a n c e and l o o k v e r y s i m i l a r t o t h e e p i t h e l i a l l i n i n g f o u n d a t t h e j u n c t i o n o f t h e m i d g u t and m i d g u t d i v e r t i c u l u m . A t t h e j u n c t i o n o f t h e m i d g u t and t h e h i n d g u t , t h e e p i t h e l i a l l i n i n g t a k e s on a h i g h l y c o n v o l u t e d a p p e a r a n c e and c o n s t r i c t s t o form a m i d g u t v a l v e . T h e e p i t h e l i a l l i n i n g o f t h e m i d g u t d i v e r t i c u l u m i s made up o f compressed c o l u m n a r c e l l s o f a s i n g l e c e l l t y p e . T h e s e c e l l s a r e t h e 'R * c e l l s ( n o m e n c l a t u r e from A r n a u d e t a l . , 1 9 7 8 ) . The 'R' c e l l s h a v e s a c e n t r a l l y l o c a t e d n u c l e i w i t h h e t e r o c h r o m a t i n l y i n g a d j a c e n t t o t h e n u c l e a r e n v e l o p e or c o n t i n u o u s l y d i s p e r s e d t h r o u g h o u t t h e n u c l e i . They have an i n v a g i n a t e d and u n d u l a t e d b a s a l l a m i n a . -33-Some muscle i s seen at the bas a l lamina r e g i o n . The c e l l s vary i n height from 40 to 70 um. In the a p i c a l regions of these c e l l s , there are t i g h t l y compressed m i c r o v i l l i (7 - 12 um t a l l ) . Beneath the m i c r o v i l l i i s an e l e c t r o n dense l a y e r . In t h i s r e g i o n , the m i c r o v i l l i have c o n s t r i c t e d bases. The a p i c a l c e l l regions c o n t a i n e d numerous m i t o c h o n d r i a w i t h t u b u l a r c r i s t a e and abundant e l e c t r o n t r a n s l u c e n t v e s i c l e s . A few medium e l e c t r o n dense v e s i c l e s a r e found and o f t e n v e s i c l e s c o n t a i n i n g e l e c t r o n dense p a r t i c u l a t e matter are a l s o seen ( f i g u r e 36). The b a s a l l a m i n a i s h i g h l y d e v e l o p e d and a d j a c e n t to the h i g h l y convoluted plasma membrane. There a r e some i n t e r m i t t e n t b r e a k s i n the b a s a l lamina where the plasma membrane i s i n c l o s e contact with the m y o e p i t h e l i a l c e l l s . The h i g h l y c o n v o l u t e d b a s a l plasma membrane i s packed i n i t s i n t e r d i g i t a t i n g f o l d s with m i t o c h o n d r i a . In many s e c t i o n s f r e q u e n t G o l g i bodies and some rough endoplasmic r e t i c u l u m were seen. F i g u r e 35 i l l u s t r a t e s a s e c t i o n t h r o u g h the 'R' c e l l s o f the midgut d i v e r t i c u l u m and f i g u r e 37 shows a high power view of the a p i c a l s u r f a c e . Note the v e s i c l e s open to the luminal s u r f a c e . At the j u n c t i o n o f the midgut d i v e r t i c u l u m and the midgut, the e p i t h e l i a l c e l l s take on a l e s s densely packed more columnar shape. The boundary muscle l a y e r i s more h i g h l y developed and the mitochondria appear as l a r g e d i l a t e d s t r u c t u r e s . The n u c l e i are prominent and c e n t r a l l y l o c a t e d . -34-The b a s a l plasma membrane i n v a g i n a t i o n s are s l i g h t l y reduced. The a p i c a l s u r f a c e has numerous v e s i c l e s present ( f i g u r e 39) . In the m i d d l e r e g i o n o f the midgut, p o s t e r i o r to the midgut d i v e r t i c u l u m and a n t e r i o r to the d o r s a l segment, g l a n d u l a r . e p i t h e l i a l c e l l s are found. There appears to be three d i f f e r e n t c e l l types found i n t h i s s e c t i o n of the midgut. In the t r a n s i t i o n to t h i s h i g h l y vacuolated r e g i o n , c e l l s s i m i l a r to 'R' type c e l l s a re s t i l l f o u n d . In t h i s r e g i o n , l a r g e v a c u o l a t e d 'B' c e l l s are found with b a s a l n u c l e i and prominent n u c l e o l i . The l u m i n a l s u r f a c e has short m i c r o v i l l i ( 1 - 3 um) . The cytoplasm i s of medium e l e c t r o n d e n s i t y w i t h some b a s a l plasma membrane c o n v o l u t i o n s , some mitochondria and numerous v e s i c l e s . T h e r e i s a l a r g e v a c u o l e (50 - 90 um) which g r e a t l y d i s t e n d s the c e l l . Numerous p i n o c y t o t i c i n c l u s i o n s can be seen i n the a p i c a l s u r f a c e . There are s e v e r a l pro-g r e s s i v e stages i n 'B' c e l l development. The l a r g e vacuolar apparatus i s c o n s i s t a n t w i t h the o l d e s t 'B' c e l l t y p e s . I t s f o r m a t i o n o c c u r s through the p r o g r e s s i v e uptake along i t s l u m i n a l s u r f a c e . Rough e n d o p l a s m i c r e t i c u l u m t ends to be a s s o c i a t e d w i t h the plasma membrane i n the b a s a l regions and between a d j a c e n t c e l l s . A few G o l g i b o d i e s are found i n a s s o c i a t i o n with the rough endoplasmic r e t i c u l u m . Whole c e l l e x t usions were sometimes seen i n 'B' c e l l s with an extremely l a r g e vacuolar apparatus. -35-A dense s t a i n i n g 'D' c e l l i s seen a d j a c e n t to and between many l a r g e vacuolated 'B1 c e l l s . I t has abundant v e s i c l e s and long m i c r o v i l l i (7 - 12 um) . I t has a dense nucleoplasm, numerous a p i c a l mitochondria, some endoplasmic r e t i c u l u m , G o l g i bodies and a convoluted b a s a l plasma membrane. It i s very s i m i l a r i n many resp e c t s to the ' R" c e l l type found i n the midgut d i v e r t i c u l u m . L i g h t l y s t a i n e d 'E' c e l l s are present i n many s e c t i o n s and a r e l e s s abundant than 'D* or 'B* c e l l s . They have a b a s a l n u c u l e u s w i t h l i g h t n u c l e o p l a s m and c o n t r a s t e d c h r o m a t i n and n u c l e o l u s . They have m i t o c h o n d r i a with a l i g h t m a t r i x i n t h e i r a p i c e s . Some rough e n d o p l a s m i c r e t i c u l u m was observed at the c e l l borders. G o l g i bodies were rare or not observed ( f i g u r e s 41 to 44). In the p o s t e r i o r p o r t i o n of the middle midgut adjacent and i n t r a n s i t i o n with the vacuolated r e g i o n , the 'F' c e l l t y pe i s pr o m i n e n t and i n most a r e a s e x c l u s i v e of o t h e r c e l l types. As the lumen c o n s t r i c t s and bends poster i o d o r s a l l y , i t t a k e s on a more invaginated and convoluted appearance. The c e l l s a re columnar to p s e u d o s t r a t i f i e d . They have c l u b shaped a p i c a l m i c r o v i l l i and l o n g e l o n g a t e d n u c l e i with a l i g h t nucleoplasm, a prominent n u c l e o l u s , and patchy h e t e r o c h r o m a t i n . They have g r a n u l a r and t u b u l a r rough endoplasmic r e t i c u l u m , many a p i c a l v e s i c l e s , d i l a t e d G o l g i b o d i e s , and numerous s c a t t e r e d m i t o c h o n d r i a . An e l e c t r o n dense l a y e r i s seen a t the base of the m i c r o v i l l i . The -36-m i c r o v i l l i b a s e s a r e h i g h l y c o n s t r i c t e d ( f i g u r e s 45, 47 and 48) . F i g u r e 38 shows 'R' t y p e c e l l s t h a t a r e f o u n d i n t h e p o s t e r i o r segment o f t h e m i d g u t . They a r e a l m o s t i d e n t i c a l t o t h e '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 e x c e p t t h a t t h e b a s a l r e g i o n s a r e much l e s s c o n v o l u t e d and t h e y a r e a s s o c i a t e d w i t h a dense m u s c u l a r band. The number o f a p i c a l m i t o c h o n d r i a a r e r e d u c e d and t h e b a s a l l a m i n a e v a g i n a t i o n s a r e l e s s compact. N e a r t h e j u n c t i o n o f t h e m i d g u t and h i n d g u t , t h e e p i t h e l i a l c e l l s become c u b o i d a l shaped and g r e a t l y r e d u c e d i n s i z e (30 um) . T h e r e a r e f e w e r c y t o p l a s m i c o r g a n e l l e s and d e n s e l y p a c k e d a p i c a l c l u b s h a p e d m i c r o v i l l i . T h e b a s a l l a m i n a i s t h i c k and c o n t i n u o u s w i t h the p l a s m a membrane i n v a g i n a t i o n s . D i s t i n c t m u s c u l a r b a n d s c a n be s e e n a t t h e base o f t h e s e c e l l s ( f i g u r e s 49 to 5 1 ) . A m i d g u t v a l v e forms a t t h e j u n c t i o n o f t h e m i d g u t and h i n d g u t ( f i g u r e s 40 a n d 4 6 ) . The c e l l s a r e t i g h t l y c o m p r e s sed i n t o e l o n g a t e s t r u c t u r e s . The lumen i s s e v e r e l y c o n t r i c t e d . H i n d g u t A t t h e m i d g u t / h i n d g u t j u n c t i o n , t h e c e l l s u r f a c e s i n t h e p r o m i n e n t t r i a n g u l a r r i d g e s a r e v i r t u a l l y f r e e o f a b r u s h b o r d e r or c u t i c l e l i n i n g . In t h e a n t e r i o r h i n d g u t r e g i o n s , t h e e p i t h e l i a l l i n i n g i s c u b o i d a l s h a p e d w i t h -37-l a r g e c e n t r a l n u c l e i and s c a t t e r e d or p a t c h y c h r o m a t i n . Deep i n v a g i n a t i o n s of the c u t i c l e l i n e d l u m i n a l s u r f a c e p r o j e c t between the c e l l s , sometimes to t w o - t h i r d s of the c e l l h e i g h t ( f i g u r e 5 3 ) . The h i n d g u t a b r u p t l y narrows as i t progresses p o s t e r i o r l y . M u s c u l a r bands c i r c l e the e n t i r e h i n d g u t . F i g u r e s 52, 54 and 56 show p r o g r e s s i o n s to the p o s t e r i o r p o r t i o n s of the copepod. Note t h a t the gut l i e s i n the d o r s a l segment and i s b r a c e d l a t e r a l l y by connective t i s s u e . The i n v a g i n a t i o n s of the p o s t e r i o r p o r t i o n a r e l i n e d by a t h i c k c u t i c l e ( f i g u r e 55) and the f l a t t e n e d e p i t h e l i a l c e l l s a r e reduced i n number. They have an e l a b o r a t e s t a r shaped appearance. Numerous l a r g e m i t o c h o n d r i a a r e c l o s e l y a s s o c i a t e d w i t h the c u t i c u l a r s u r f a c e and the b a n d i n g muscle l a y e r ( f i g u r e 57). Near the anus, the c e l l s become s m a l l e r and f l a t t e r , and have a d i s t i n c t l a y e r e d c u t i c l e s i m i l a r i n appearance to the esophagus and the outer c u t i c l e p r o t e c t i n g the copepod. Receptor Mediated Endocytosis ( T i g r i o p u s c a l i f o r n i c u s ) C o n t r o l m i c r o g r a p h s , f i g u r e s 59 to 62, show a r e a s of the copepod d i g e s t i v e t r a c t . In a l l s e c t i o n s viewed, no c o l l o i d a l gold could be found i n the c e l l s of the c o n t r o l g r o u p s . A l l a r e a s of the d i g e s t i v e t r a c t were examined i n s e v e r a l copepods. A f t e r 5 m i n u t e s i n c u b a t i o n f o l l o w e d by f i x a t i o n , c o l l o i d a l g o l d p a r t i c l e s c o u l d be found i n t h e lumen. - 3 8 -Luminal c o l l o i d a l gold was a l s o found i n the c o n t r o l groups but was never found bound to the plasma membrane or i n t e r n a l i z e d ( f i g u r e s 63 and 66) . These are high and low power micrographs i l l u s t r a t i n g the s i z e (15 - 20 um) of the c o l l o i d a l gold marker used. In f i g u r e s 64 and 65, a f t e r 15 minutes i n c u b a t i o n , the g o l d p a r t i c l e s c o u l d be seen adhered to the plasma membrane. Note the prominant gap j u n c t i o n between the e p i t h e l i a l c e l l s . Both s e c t i o n s are high power micrographs of c e l l type ' 3 ' i n the j u n c t i o n region of the midgut caecum and the a n t e r i o r midgut. C o l l o i d a l g o l d was found i n the cytoplasm of c e l l type '2' i n the middle to p o s t e r i o r p o r t i o n of the a n t e r i o r midgut ( f i g u r e 67). A high power micrograph of that region ( f i g u r e 68) shows the cytoplasmic c o l l o i d a l gold p a r t i c l e s . F l u i d Phase Endocytosis ( T i g r i o p u s c a l i f o r n i c u s ) C o n t r o l experiments f o r the f l u i d phase e n d o c y t o s i s experiment showed that the uptake of h o r s e r a d i s h peroxidase was a s p e c i f i c response. No endogenous peroxidase a c t i v i t y c o u l d be seen a f t e r v i e w i n g the s e c t i o n s . Those c o n t r o l s i n c l u d e d f o r the s p e c i f i c i t y of the process (those l a c k i n g 3 , 3 ' D i aminobenz idene) d i d not have p o s i t i v e i n c l u s i o n s . C o n t r o l m i c r o g r a p h s ( f i g u r e s 69 and 70) show r e g i o n s of the a n t e r i o r midgut and midgut caecum r e s p e c t i v e l y . Peroxidase p o s i t i v e a c t i v i t y was found to occur o n l y -39-i n c e l l t ype '3' i n the midgut caecum and the a n t e r i o r m i d g u t . F i g u r e 71 i l l u s t r a t e s a h i g h power view of the membrane bound peroxidase p o s i t i v e granules i n the cytoplasm of c e l l type '3' i n the anter i o r midgut. Horseradish peroxidase p o s i t i v e uptake products are sequestered i n membrane bound e n d o c y t o t i c v e s i c l e s and can be d i s t i n g u i s h e d by the dark gran u l a r or p a r t i c u l a t e appearance. W i t h i n 1 to 10 minute i n c u b a t i o n s , peroxidase could be seen i n e n d o c y t o t i c v e s i c l e s . I n i t i a l l y , i t i s found i n the p e r i p h e r a l p o r t i o n s of these v e s i c l e s adjacent to the membrane l i n i n g ( f i g u r e 74). As i n c u b a t i o n time i n c r e a s e s , these e n d o c y t o t i c v e s i c l e s become more concentrated with peroxidase p o s i t i v e p a r t i c u l a t e m a t t e r . W i t h i n 15 minutes, the v e s i c l e s are seen to have a c e n t a l dense area ( f i g u r e 75). Longer i n c u b a t i o n times (20-40 minutes) produced v e s i c l e s which appeared dark and almost homogeneously dense ( f i g u r e s 72 and 73) . No peroxidase p o s i t i v e a c t i v i t y was seen i n the c e l l s of type '2'. -40-D i s c u s s i o n : E x a m i n a t i o n of the d i g e s t i v e t r a c t s of T i g r i o p u s  c a l i f o r n i c u s and Calanus plumchrus r e v e a l s that t h e i r shape i s c o n s i s t e n t w i t h many copepods p r e v i o u s l y d e s c r i b e d . The r e s u l t s presented here are c o n s i s t e n t with those found by S u l l i v a n (1975), and S u l l i v a n and B i s a l p u t r a (1980) f o r T i g r i opus c a l i f o r n i c u s . No p r e v i o u s m o r p h o l o g i c a l s t u d i e s c o u l d be found on the d i g e s t i v e t r a c t of Calanus  p l u m c h r u s . However, a g r e a t s i m i l a r i t y e x i s t s between t h i s d e s c r i p t i o n w i t h t h o s e of o t h e r a u t h o r s p e r t a i n i n g to r e l a t e d copepods. T i g r i o p u s c a l i f o r n i c u s has a c u t i c u l i z e d esophagus, a midgut caecum, a n t e r i o r and p o s t e r i o r midgut r e g i o n s , an a n t e r i o r and a p o s t e r i o r hindgut ending in a c u t i c u l i z e d anus. C a l a n u s plumchrus has a c u t i c u l i z e d esophagus, a midgut d i v e r t i c u l u m , a d i f f e r e n t i a t e d midgut, and a long narrowing hindgut ending i n a c u t i c u l i z e d anus. Areas of r e g i o n a l 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 are c o r r e l a t e d to the p a r t i c u l a r f u n c t i o n s of the gut r e g i o n s . Morphological s i m i l a r i t i e s and d i f f e r e n c e s i n the u l t r a s t r u c t u r e w i l l be d i s c u s s e d with r e f e r e n c e to c e l l type and f u n c t i o n a l s i g n i f i c a n c e . Foregut The c h a r a c t e r i s t i c s of the foregut i n both copepods -41-are remarkably s i m i l a r . These foregut s t r u c t u r e s are a l s o c o n s i s t e n t w i t h t h o s e found i n o t h e r a r t h r o p o d s . Yonge (1924), d e s c r i b e d s i m i l a r f e a t u r e s f o r the c r a y f i s h w h i l e d e s c r i p t i o n s of comparable f o r e g u t f e a t u r e s i n c a l a n o i d copepods were d e s c r i b e d by many authors (Dakin, 1908; Lowe, 1935; M a r s h a l l and O r r , 1955; Park, 1966). Fahrenbach (1962) d e s c r i b e d a f o r e g u t s i m i l a r to those o b s e r v e d i n the h a r p a c t i c o i d , D i a r t h r o d e s cystoecus, while Lewis (1961) d e s c r i b e d the f o r e g u t of a c a l i g o i d copepod. Fahrenbach (1962), and Lang (1948), d e s c r i b e d s i m i l a r musculature i n the region of the esophagus i n other harpact-i c o i d s . The m u s c u l a t u r e i s i n v o l v e d i n the shape of the esophagus, i t s c o n t r a c t i o n s and movement of f o o d , and i s i m p o r t a n t i n the i n i t i a l c r u s h i n g of the i n g e s t e d food p a r t i c l e s . Nunez and Crawford (1976) d e s c r i b e d i n i t i a l d i g e s t i o n i n the esophagus of a m i l l i p e d e . L i t t l e or no s y n t h e s i s of d i g e s t i v e enzymes o c c u r s i n the esophagus, so i t i s suggested that the d i g e s t i v e enzymes pass i n t o the esophagus from the midgut. The fo r e g u t i n both T i g r iopus c a l i f o r n i c u s and Calanus  plumchrus appears to be a simple esophagus. This i s c o n s i s t a n t with the copepod genera ( E p i l a b i d o c e r a , (Park, 1966), Centro-pages, (Arnaud et a l , 1978), and T i g r i o p u s , ( S u l l i v a n and B i s a l p u t r a , 1 9 8 0 ) ) . In both o r g a n i s m s , a t h i c k c u t i c l e surrounds the l i n i n g of the esophagus. I t s t h i c k n e s s decreases -42-as t h e esophagus e x t e n d s d o r s a l l y . In a r t h r o p o d s , e s p e c i a l l y c r u s t a c e a n s , no e v i d e n c e c a n be f o u n d f o r a b s o r p t i o n or s e c r e t i o n on t h e v e n t r a l e s o p h a g u s . E v i d e n c e t h a t t h e d o r s a l c u t i c l e i s n o t e n t i r e l y i m p e r m e a b l e c a n be s e e n i n some i n s e c t s w h e r e c h o l e s t e r o l a b s o r p t i o n o c c u r s i n t h e f o r e g u t ( J o s h i and A g a r w a l , 1977). E i s n e r (1955) fo u n d e v i d e n c e f o r f a t a b s o r p t i o n i n t h e c o c k r o a c h f o r e g u t . The f o r e g u t o f many a t h r o p o d s i s more complex t h a n t h a t o b s e r v e d i n T i g r i o p u s or C a l a n u s . D i e t a r y d i f f e r e n c e s p r o b a b l y a c c o u n t f o r many o f t h e o b s e r v a t i o n s and i n f l u e n c e t h e d e v e l o p m e n t o f t h e f o r e g u t ( M c L a u g h l i n , 1983). S e v e r a l a r t h r o p o d s w e r e o b s e r v e d h a v i n g o n l y a s h o r t e s o p h a g u s and one or two chambers i n t h e f o r e g u t . In some, s e t a e a r e p r e s e n t w h i c h p r e v e n t t h e back r e g u r g i t a t i o n o f f o o d m a t e r i a l . In I d o t e a , an i s o p o d , f o o d i s c r u s h e d by l a t e r a l a m p u l l a e i n t h e f o r e g u t ( N a y l o r , 1955). E x t r e m e f o r e g u t c o m p l e x i t y has been o b s e r v e d i n t h e E u c a r i d s a n d t h e d e c a p o d s . ( M a u c h l i n e and F i s h e r , 1969; P i k e , 1947 ; M a y n a r d a n d Dando , 1974 ; H i n t o n and C o r e y , 1979). In i t s s i m p l e s t f o r m , t h e f o r e g u t f u n c t i o n s t o b r e a k up t h e f o o d p a r t i c l e s and a c t as a passageway to t h e m i d g u t s t r u c t u r e s where t h e c e l l s a c t i v e l y s e c r e t e d i g e s t i v e enzymes and c a r r y o u t l i p i d and n u t r i e n t a b s o r p t i o n . I t w o u l d a p p e a r t h a t i n 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 p l u m c h r u s the f o r e g u t c a r r i e s o ut a s i m i l a r f u n c t i o n and d o e s n o t - 4 3 -a c t i v e l y d i g e s t i n t o or absorb m a t e r i a l from the lumen. In the f o r e g u t of T i g r i opus C a l i f o r n i c u s there are b a s a l i n v a g i n a t i o n s i n the area opposite the muscle c e l l s t h a t have r a d i c a l i n t e r d i g i t a t i n g f o l d s . T h i s suggests e l a s t i c i t y i n c o n n e c t i o n w i t h the m u s c u l a r a c t i v i t i e s . The lumen of the foregut can be g r e a t l y distended to accommodate l a r g e good p a r t i c l e s . The c e l l s l i n i n g the esophagus are s t r e t c h e d d u r i n g the f e e d i n g p r o c e s s and the surrounding muscle l a y e r s c o n t r a c t to crush the good p a r t i c l e s i n the c u t i c l e l i n e d esophagus. T h i s r h y t h m i c s t r e t c h i n g and c o n t r a c t i o n serves to break up the ingested food p a r t i c l e s and to move them through the length of the esophagus. Midgut In T i g r i o p u s c a l i f o r n i c u s the midgut can be subdivided i n t o three d i s t i n c t r e g i o n s ; the midgut caecum, the a n t e r i o r midgut, and the p o s t e r i o r m i d g u t . Four e p i t h e l i a l c e l l types were found and i n the three regions t h e i r u l t r a s t r u c t u r e and frequency v a r i e d according to the f u n c t i o n . In C a l a n u s plumchrus the midgut can be d i v i d e d i n t o two main r e g i o n s , the midgut d i v e r t i c u l u m and the midgut, with f u r t h e r s u b d i v i s i o n s based on the u l t r a s t r u c t u r e of the f i v e c e l l types present. A. C e l l Types In T i g r i opus c a l i f o r n i c u s c e l l t ype ' 1 ' was found -44-occupying a b a s a l p o s i t i o n i n the midgut and the midgut caecum. I t s m o r p h o l o g i c a l c h a r a c t e r i s t i c s s u g g e s t that i t i s not a c t i v e i n s e c r e t i o n or a b s o r p t i o n , but p r o b a b l y f u n c t i o n s as a p r e c u r s o r or r e p l a c e m e n t c e l l f o r t h o s e that have been shed i n the e x t r u s i v e p r o c e s s e s o b s e r v e d i n many c e l l s . They are t y p i c a l of u n d i f f e r e n t i a t e d c e l l s . The b a s a l lamina of t h i s c e l l type i s much l e s s u n d u l a t e and probably not a c t i v e i n n u t r i e n t exchange. De P r i e s t e r (1971), d e s c r i b e d r e g e n e r a t i v e c e l l s c l u s t e r e d i n ' c e l l n e s t s ' i n which s e q u e n t i a l stages of d i f f e r e n t i a t i o n were found near the b a s a l p o r t i o n of the e p i t h e l i u m . L i t t l e e n d o p l a s m i c r e t i c u l u m , many r i b o s o m e s , and o n l y a few small mitochondria were u s u a l l y observed. Basal i n v a g i n a t i o n s are probably a s i g n of d i f f e r e n t i a t i o n . C e l l type '1' i n T i g r i o p u s c a l i f o r n i c u s i s s i m i l a r to those c e l l s d e s c r i b e d by De P r i e s t e r i n the f l y . In c e l l type '1' abundant ribosomes with l i t t l e endop-lasmic r e t i c u l u m or G o l g i and no l u m i n a l s u r f a c e c o n t a c t i s suggestive of some s y n t h e t i c a c t i v i t y but without export from the c e l l . In C a l a n u s p l u m c h r u s , the 'E' c e l l s a re p r o b a b l y s i m i l a r i n f u n c t i o n to c e l l type '1'. In decapods, isopods, and amphipods, the 'E' c e l l s have been d e s c r i b e d as embryonic and found i n the hepatopancreatic e p i t h e l i u m ( L o i z z i , 1971; S c h u l t z , 1976) . In Centropages t y p i c u s , Arnaud et a l . , (1978), d e s c r i b e d -45-th e 'E' c e l l as s l e n d e r w i t h a l i g h t c y t o p l a s m and few o r g a n e l l e s . They do however, r e a c h the l u m i n a l s u r f a c e and have slender m i c r o v i l l i . Some rough endoplasmic r e t i c u l u m i s present but G o l g i bodies and mitochondria i n many c e l l s were rare or unseen. H a l l b e r g and H i r c h e , (1980), d e s c r i b e d s i m i l a r c e l l s i n two genera of Calanus and termed them as u n d i f f e r e n t i a t e d c e l l s . In both c a l a n o i d s , mitochondria were observed. C e l l type '2' was found i n the midgut of T i g r iopus  c a l i f o r n i c u s . I t i s u s u a l l y more d a r k l y s t a i n e d than the surrounding c e l l s , with abundant rough endoplasmic r e t i c u l u m , ribosomes, G o l g i b o d i e s , m i t o c h o n d r i a and v a r i o u s s i z e d s e c r e t o r y v e s i c l e s , and i t i s l i k e l y to be h i g h l y s y n t h e t i c . D i l a t e d G o l g i bodies are i n d i c a t i v e of a c t i v e o r g a n e l l e s (Fawcett, 1969) . E n d o c y t o t i c a c t i v i t y was seen i n a l l c e l l s of type '2'. From the m i c r o s c o p y o b s e r v e d , and t h r o u g h s e r i a l s e c t i o n i n g , c e l l t ype '2' was found to undergo e x t r u s i o n of i t s c e l l u l a r contents i n t o the lumen. i n the a n t e r i o r p o r t i o n s of the gut i t ' s shape i s compressed by the neighbouring c e l l s and i s a f t e n seen i n the shape of a wedge w i t h a narrow l u m i n a l s u r f a c e . Many d i f f e r e n t shapes of c e l l type '2' were o b s e r v e d . Those c e l l s i d e n t i f i e d as type '2', v a r i e d mostly i n the number and s i z e of t h e i r v e s i c l e s . A p r o g r e s s i o n of shape from a l a r g e b a s a l wedge to a broad l u m i n a l f a c e was o b s e r v e d . T h i s shape p r o g r e s s i o n was -46-accompanied by e x t r u s i o n s i n t o the lumen. The more d i f f e r -e n t i a t e d the c e l l became, the l a r g e r was i t s luminal face and g r e a t e r was the p r o p o r t i o n and s i z e of the v e s i c l e s p r e s e n t . Many l i p i d d r o p l e t s were observed i n t h i s c e l l type. Together with the endocytosis observed, t h i s i s s u g g e s t i v e of an a b s o r p t i v e f u n c t i o n . A g a i n , from the o b s e r v a t i o n of the c e l l p r o g r e s s i o n and d i f f e r e n t i a t i o n , s e c r e t i o n i s l i k e l y to be i t u l t i m a t e f u n c t i o n . I t s o r g a n e l l e s a r e c h a r a c t e r i s t i c of p r o t e i n s y n t h e s i s . The b a s a l l a m i n a i n v a g i n a t i o n s of c e l l type '2' adds support to i t s r o l e i n c e l l u l a r metabolism. Lewis (1961), d e s c r i b e d the dark c e l l s of C a l i g o i d copepods as s e c r e t o r y c e l l s f o r e x t r a c e l l u l a r d i g e s t i o n . In C a l a n u s p l u m c h r u s , the 'F' c e l l s c a r r y out the s e c r e t o r y f u n c t i o n s . The 'F' c e l l s are columnar or c u b o i d a l i n shape and are c h a r a c t e r i z e d by s h o r t , stocky, c l u b shaped m i c r o v i l l i , abundant rough endoplasmic r e t i c u l u m , d i l a t e d G o l g i bodies, numerous s e c r e t o r y granules, and many mitocho-n d r i a . These 'F' c e l l s are s i m i l a r i n s t r u c t u r e to the F c e l l s d e s c r i b e d i n decapods which are b e l i e v e d to be d i g e s t i v e enzyme s e c r e t i n g . The morphology of the 'F' c e l l i s s i m i l a r to the s e c r e t o r y c e l l s d e s c r i b e d i n the i s o p o d (Jones e t a l , 1969) Eurydice p l u c h r a , and i n the d ecapods, Procambarus c l a r k i i ( L o i z z i , 1971). Arnaud et a l . , (1978) d e s c r i b e d the 'F' c e l l i n Centropages t y p i c u s . -47-They found i t to be s e c r e t o r y by a m a c r o a p o c r i n e p r o c e s s as w e l l as by a m i c r o a p o c r i n e p r o c e s s from the t i p s of the m i c r o v i l l i ( f i r s t d e s c r i b e d by Kurosumi i n 1 9 6 1 ) . This dual s e c r e t o r y process may be the r e s u l t of a p a r t i c u l a r l y i n t e n s e n u t r i t i o n a l phase. In T i g r iopus c a l i f o r n i c u s , c e l l type '3' i s commonly found t h r o u g h o u t the midgut. I t r e a c h e s the lumen and has l u m i n a l m i c r o v i l l i . The cytoplasm c o n t a i n s l a m e l l a t e rough endoplasmic r e t i c u l u m , m i t o c h o n d r i a, numerous G o l g i , r i b o s o m e s , and many v e s i c l e s . The i n t e r m e d i a t e amount of the s y n t h e t i c o r g a n e l l e s suggest that s e c r e t i o n i s not a major f u n c t i o n i n t h i s c e l l t y p e . The l i p i d m a t e r i a l i n the a p i c a l p o r t i o n s of t h i s c e l l and the p r e s e n c e of smooth e n d o p l a s m i c r e t i c u l u m s u g g e s t t h a t a b s o r p t i o n i s the major f u n c t i o n of c e l l type '3'. The s y n t h e t i c apparatus i s l i m i t e d and f u n c t i o n s to provide an i n t r a c e l l u l a r mechanism for the metabolism of absorbed m a t e r i a l . The h i g h d egree of b a s a l i n v a g i n a t i o n suggests a t r a n s p o r t f u n c t i o n between the products ingested and metabolized, and the haemocoel. C e l l type '4' i n T i g r i o p u s c a l i f o r n i c u s i s only found i n the a n t e r i o r m i d g ut. The e p i t h e l i a l c e l l s e xtend to the lumen and have a p i c a l m i c r o v i l l i . 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 rough endoplasmic r e t i c u l u m , and some u n d i l a t e d G o l g i b o d i e s . There a r e c y t o p l a s m i c m i t o c h o n d r i a and abundant e l e c t r o n dense v e s i c l e s . The u l t r a s t r u c t u r e i s s u g g e s t i v e o f l i p i d a b s o r p t i o n . The -48-p o l a r i t y of the o r g a n e l l e s with G o l g i bodies, rough endoplasmic r e t i c u l u m , and v e s i c l e s i n the a p i c a l p o r t i o n s of the c e l l suggests that c e l l type '4' does not p a r t i c i p a t e i n endocrine s e c r e t i o n . In C a l a n u s p l u m c h r u s , t h r e e a b s o r p t i v e c e l l t y p e s were observed. 'R' c e l l s have s e v e r a l c h a r a c t e r i s t i c morphological f e a t u r e s . They have long luminal m i c r o v i l l i , deep i n v a g i n a t i o n s of the plasma membrane, mitochondria, and a w e l l developed endoplasmic r e t i c u l u m . T h i s u l t r a s t r u c t u r e i s s u g g e s t i v e of an a b s o r p t i v e r o l e . They a r e a n a l o g o u s to c e l l s i n the i n t e s t i n a l d i v e r t i c u l u m of Calanus. h e l g o l a n d i o u s (Ong and Lake, 1970) and are s i m i l a r to midgut c e l l s i n M y t i l i c o l a  i n t e s t i n a l i s ( D u r f o r t , 1971) . They lack c e r t a i n c h a r a c t e r i s t i c s e x h i b i t e d i n other crustaceans a s s o c i a t e d with a b s o r p t i o n . L o i z z i (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 with a b s o r p t i v e c e l l s i n the c r a y f i s h , but no g l y c o c o l y x was observed i n c e l l type 'R'. The b a s a l plasma membrane i n v a g i n a t i o n s w i t h l a r g e numbers of a s s o c i a t e d mitochondria c o r r o b o r a t e s the evidence that 'R' c e l l s are i n v o l v e d i n the t r a n s f e r of m e t a b o l i t e s to the haemolymph. 'R' c e l l s a l s o have s i m i l a r i t i e s to the a b s o r p t i v e c e l l s o b s e r v e d i n the d i g e s t i v e t r a c t of Malacostracans. They i n c l u d e the S c e l l s i n O n i s c o u s a s e l l u s ( C l i f f o r d and W i t k u s , 1971), L c e l l s i n A t y a (Van Weel, 1955), R c e l l s i n C a r c i n u s maenas ( S t a n i e r e t a l . , 1968), and i n -49-the c r a y f i s h ( L o i z z i , 1971). Another a b s o r p t i v e c e l l type i s the 'D* c e l l . I t i s remarkably s i m i l a r i n many r e s p e c t s to the 'R' c e l l . They have a dense cytoplasm with abundant smooth v e s i c u l a r endoplasmic r e t i c u l u m . G o l g i b o d i e s a re p r e s e n t and the mitochondria are l a r g e l y s i m i l a r to those of the 'R' c e l l s . The b a s a l plasma membrane i s h i g h l y i n v a g i n a t e d . The 'D' c e l l s have l u m i n a l m i c r o v i l l i and a r e found adjacent to or near the vacuolar 'B' c e l l s which compress or p a r t i a l l y surround them. The f i n a l a b s o r p t i v e c e l l type found i n the midgut of Calanus plumchrus i s the XB' c e l l . I t has short m i c r o v i l l i , i n v a g i n a t i o n s o f the a p i c a l plasma membrane, and numerous v e s i c l e s . There i s the appearance of i n t e n s e p i n o c y t o t i c a c t i v i t y . 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 any d i s t i n c t p a r t i c u l a t e matter and are most l i k e l y i n v o l v e d i n the a b s o r p t i o n of p a r t i a l l y d i g e s t e d m a t e r i a l from the lumen. In many s e c t i o n s , a development of the l a r g e vacuolar a p p a r a t u s was seen. The development of 'B' c e l l s i s pro-g r e s s i v e . The vacuoles a r i s e from a f u s i o n of the p i n o c y t o t i c v e s i c l e s and the elements of the vacuolar complex. Arnaud et a l . , ( 1 9 7 8 ) , d e s c r i b e d the c o a l e s c e n s e of c y t o p l a s m i c dense bodies w i t h i n the vacuole i n Centrophages t y p i c u s . They suggested that autophagic a c t i v i t y would allow the vacuolar apparatus i n i t i a t i o n . In some s e c t i o n s , the c o n t e n t s o f l a r g e v a c u o l a r -50-'B' c e l l s were seen ex t r u d i n g i n t o the lumen. The p i n o c y t o t i c i n g e s t e d m a t e r i a l f u s e s and i s d i g e s t e d i n the vacuole. In the l a t t e r s t a g e s o f 'B' c e l l development, the c e l l s e x t r u d e t h i s d i g e s t e d m a t e r i a l i n t o the lumen where i t could then be reabsorbed elsewhere i n the gut. In Centropages  t y p i c u s , the d i g e s t e d and l i b e r a t e d m a t e r i a l i s probably r e a b s o r b e d by the 'R' 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 midgut (Arnaud et a l . , 1978). V a c u o l a r c e l l s l i k e t h o s e o f the midgut of Calanus  plumchrus, have been observed i n many crustaceans. Raymont et al.,(1974) d e s c r i b e d them i n copepods, Schmitz and S c h u l t z , (1969) found them i n isopods, Nath and P i l l a i (1973), i n Mysids, and many examples were found i n the decapods (Hirsh and Jacobs, 1930; D a l l , 1967; S t a n i e r et a l . , 1968; L o i z z i , 1971) . In newborn r a t s (Morris, 1968) some i n t e s t i n a l c e l l s have a c t i v e p r o t e i n p i n o c y t o s i s and the production of dense b o d i e s t h a t a r e lysosomal. Phagosomes are formed and the p i n o c y t o t i c m a t e r i a l i s d i g e s t e d i n the phagosomes and r e l e a s e d t o the lymphatic system. This i s s i m i l a r to 'B' c e l l a c t i o n i n C a l a n u s except for the d e s t i n a t i o n of the di g e s t e d and extruded m a t e r i a l . B. Midgut Caecum and Midgut D i v e r t i c u l u m The l a r g e s t c e l l s i n the d i g e s t i v e t r a c t of T i g r i o p u s  c a l i f o r n i c u s are found i n the midgut caecum. Many of the -51-e p i t h e l i a l c e l l s have b a s a l e v a g i n a t i o n s a s s o c i a t e d w i t h b a s a l l a m i n a i n v a g i n a t i o n s . Of the c e l l t y p e s present, type '3' i s the most abundant. C e l l type '2* i s p r e s e n t as w e l l as some c e l l type '1'. The l o n g m i c r o v i l l i i n the r e g i o n of the midgut adds evidence to the r o l e of the midgut caecum i n a b s o r p t i o n . C e l l types '2' and '3' o f t e n contained l i p i d d r o p l e t s . S c h u l t z and Kennedy (1976) suggested that the c e l l s c o n t a i n i n g l i p i d d r o p l e t s i n Dapnia are the major s i t e s of n u t r i e n t uptake. C e l l type '2' found i n the midgut caecum has both a b s o r p t i v e and s e c r e t o r y f u n c t i o n s . vonk (1960) found t h a t s e c r e t i o n was not r e s t r i c t e d to any one r e g i o n of the e n d o d e r m a l l y d e r i v e d e l e m e n t s . S c h u l t z and Kennedy (1976) found t h a t s e c r e t i o n i n Dapnia pulex was p o l a r i z e d to the a n t e r i o r midgut and d i g e s t i v e caeca. The p r e s e n c e of both type '2' and '3' c e l l s i n the midgut caecum of T i g r i o p u s c a l i f o r n i c u s i n d i c a t e s t h a t i t serves both a s e c r e t o r y and a b s o r p t i v e f u n c t i o n . Since c e l l t ype '3' i s found i n gr e a t e r numbers, the a b s o r p t i v e f u n c t i o n of the midgut caecum i s the major a c t i v i t y . In C a l a n u s p l u m c h r u s , the midgut d i v e r t i c u l u m i s l i n e d by o n l y one c e l l t y p e ; the 'R' c e l l . I t forms a u n i f o r m e p i t h e l i a l l a y e r t h a t i s no n - v a c u o 1 a t e d . Lowe (1935), d e s c r i b e d a s i m i l a r midgut d i v e r t i c u l u m s t r u c t u r e i n C a l a n u s f i n m a r c h i c u s . In c a l i g o i d s , the s e c r e t o r y 'B' -52-c e l l i s found p r i m a r i l y i n the a n t e r i o r midgut and foregut regions (Lewis, 1961) . The midgut d i v e r i t c u l u m of Calanus plumchrus i s s p e c i -a l i z e d f o r a b s o r p t i o n of n u t r i e n t s . I t s u l t r a s t r u c t u r e i s c o n s i s t a n t with a b s o r p t i v e c e l l s and i t s b a s a l e v a g i n a t i o n s , i n f o l d i n g s of the plasma membrane and numerous ba s a l mitoch-ondria i n d i c a t e i t s t r a n s p o r t r o l e of absorbed n u t r i e n t s . C. Midgut Regional S p e c i a l i z a t i o n s In T i g r i opus c a l i f o r n i c u s , the a n t e r i o r midgut i s l i n e d by e p i t h e l i a l c e l l s w i t h l u m i n a l i n v a g i n a t i o n s and e v a g i n a t i o n s of the b a s a l r e g i o n s . The c e l l types, e s p e c i a l l y type '3', tend to be more dense i n t h i s region of the midgut. C e l l type '1' i s more abundant here than i n the midgut caecum. This would suggest a g r e a t e r replacement requirement f o r the c e l l s l i n i n g the a n t e r i o r midgut. In c e l l type '2' and '3' more l i p i d d r o p l e t s were observed i n the cytoplasm. T h i s c o u p l e d w i t h the d e n s i t y of the c e l l s i n d i c a t e s a greater degree of n u t r i e n t a b s o r p t i o n . A f o u r t h c e l l type, type ' 4 1 , i s found e x c l u s i v e l y i n the a n t e r i o r m i d g ut. I t p r o j e c t s i n t o the lumen and has dense v e s i c l e s i n i t s cytoplasm. I t s major f u n c t i o n appears to be the a b s o r p t i o n of l i p i d s . In th brown shrimp, Pomaeus a z t e c u s , the c e l l s of the midgut are thought to f u n c t i o n i n the a b s o r p t i o n of l i p i d s , n u t r i e n t a b s o r p t i o n , osmoregulation, p e r i o t r o p h i c -53-membrane formation and s e c r e t i o n (Talbot et al.,1972). In A r t e m i a , a b r a c h i o p o d , Hootman and Conte (1974), c o r r e l a t e d the a p i c a l and b a s a l c e l l a m p l i f i c a t i o n s with a s s o c i a t e d m i t o c h o n d r i a to a b s o r p t i v e and o s m o r e g u l a t o r y f u n c t i o n s . In C a l a n u s p l u m c h r u s , the a n t e r i o r p o r t i o n o f the midgut, not i n c l u d i n g the d i v e r t i c u l u m , i s l i n e d by e p i t h e l i a l c e l l s i n the a n t e r i o r p o r t i o n which are n o n - v a c u o l a t e d , but p r o g r e s s i v e l y lead to a g l a n d u l a r region which i s h i g h l y vacuolated. The c e l l s adjacent to the midgut d i v e r t i c u l u m have an appearance s i m i l a r the the 'R' c e l l s . A f t e r a s h o r t t r a n s i t i o n from the a b s o r p t i v e 'R' c e l l s of the d i v e r t i c u l u m , the c e n t r a l midgut v a c u o l a r a p p a r a t u s i s f o u n d . I t c o n t a i n s a s m a l l number of 'E' c e l l s which are u n d i f f e r e n t i a t e d stem c e l l s . The m a j o r i t y of t h i s r e gion i s l i n e d by 'D' and 'B' c e l l t y p e s . The major f u n c t i o n o f the a n t e r i o r r e g i o n o f the midgut i s a b s o r p t i o n w i t h abundant p i n o c y t o t i c a c t i v i t y o c c u r r i n g i n the 'B' c e l l s . The 'D' c e l l s have c h a r a c t e r i s t i c s of ab s o r p t i v e c e l l s s i m i l a r to 1R 1 c e l l s but they are d i f f i c u l t to c h a r a c t e r i ze . In Carcinus maenus , Georgi (1969), i n t e r p r e t e d s i m i l a r c e l l s as being young c e l l s . In the m i d d l e r e g i o n s of the midgut, the e p i t h e l i a l l i n i n g i s a g a i n n o n - v a c u o l a t e d and made up of ' F' type c e l l s . These c e l l s have 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 with s e c r e t o r y c e l l s . The middle p o r t i o n of the midgut i n Calanus -54-plumchrus i s the probable s i t e of d i g e s t i v e enzyme s e c r e t i o n . In T i g r iopus c a l i f o r n i c u s the p o s t e r i o r midgut region has an e p i t h e l i a l c e l l l i n i n g of s m a l l e r c u b o i d a l shaped c e l l s . C e l l t y p e '1' i s s l i g h t l y more prominent i n t h i s region of the midgut and both c e l l types '2' and '3' a r e s t i l l p r e s e n t . P r e s e n c e of c e l l type '3' i n d i c a t e s that l i p i d a b s o r p t i o n i s o c c u r r i n g . Many c e l l s of the d e n s e l y s t a i n i n g type '2' a r e p r e s e n t i n the p o s t e r i o r midgut. Often they are seen forming ' b r i d g e - l i k e ' s t r u c t u r e s w i t h n e i g h b o u r i n g c e l l s . In many s e c t i o n s , s e c r e t i o n s could be observed from c e l l type '2'. The p o s t e r i o r midgut o f T i g r i o p u s c a l i f o r n i c u s p l a y s a d u a l r o l e i n a b s o r p t i o n and s e c r e t i o n . T o g e t h e r w i t h the g r e a t e r p r o p o r t i o n of c e l l type '1' and the s e c r e t i o n s o b s e r v e d i n c e l l type '2', the s e c r e t o r y f u n c t i o n of t h i s region appears to be of gr e a t e r i m p o r t a n c e than the a n t e r i o r midgut r e g i o n s . The p o s t e r i o r midgut f u n c t i o n s i n d i g e s t i v e enzyme s e c r e t i o n and n u t r i e n t a b s o r p t i o n . The p o s t e r i o r regions of the midgut of Calanus plumchrus has one c h a r a c t e r i s t i c e p i t h e l i a l c e l l t y p e ; type 'R'. The 'R' c e l l s i n t h i s region are s i m i l a r to those d e s c r i b e d i n the midgut d i v e r t i c u l u m , but t h e i r o r g a n e l l e s t r u c t u r e and b a s a l i n v a g i n a t i o n s are not as e l a b o r a t e . They r e t a i n the c h a r a c t e r i s t i c s of a b s o r p t i v e c e l l t y p e s , but have red u c e d b a s a l plasma membrane i n v a g i n a t i o n s , reduced b a s a l e v a g i n a t i o n s , l e s s mitochondria, and are bounded by a more -55-d i s t i n c t m u scle l a y e r . The b a s a l t r a n s f e r of d i g e s t e d m e t a b o l i t e s a p p e a r s , from the u l t r a s t r u c t u r e , to be l e s s important i n t h i s r e g i o n of the midgut. The e p i t h e l i a l c e l l s of the p o s t e r i o r r e g i o n near the j u n c t i o n o f the hindgut are much smaller and c u b o i d a l shaped. The luminal m i c r o v i l l i are shorter and l e s s numerous. The bas a l plasma membrane i n v a g i n a t i o n s are g r e a t l y reduced and l i n e d by t h i c k b a s a l l a m i n a . The e n d o p l a s m i c r e t i c u l u m i s much l e s s developed and the number of G o l g i bodies and mitochondria a r e r e d u c e d . A l t h o u g h s t i l l a b s o r p t i v e , i t s a b s o r p t i v e c a p a c i t y i s d i m i n i s h e d . The a b s o r p t i v e c e l l s i n the p o s t e r i o r r e g i o n s o f the midgut are analogous to c e l l s that occur i n the hepatop-ancreas of many arthropods. Neither T i g r i o p u s c a l i f o r n i c u s or Calanus plumchrus have a hepatopancreas. Dorman (1928), d e s c r i b e d the exchange between the c e l l s and the haemocoel i n the hepatopancreas of c r a y f i s h . Davis and Burnett (1964) d e s c r i b e d a b s o r p t i v e , s e c r e t o r y and embryonic c e l l s i n the c r a y f i s h h e p a t o p a n c r e a s . L o i z z i (1971) termed the a b s o r p t i v e c e l l s i n the c r a y f i s h 'R' c e l l s . Iron and f a t ab s o r p t i o n was s u g g e s t e d to oc c u r i n 'R' c e l l s ( S t a n i e r e t a l ., 1968) whereas a c c o r d i n g to S c h u l t z (1976), 'R' c e l l s absorb copper and 'F' c e l l s absorb i r o n . In c a l i g o i d s , the major f u n c t i o n o f the p o s t e r i o r midgut i s a b s o r p t i o n (Lewis, 1961). In T i g r i o p u s c a l i f o r n i c u s , the p o s t e r i o r midgut, -56-l i k e the h e p a t o p a n c r e a s , has e p i t h e l i a l c e l l t y p e s t h a t s e c r e t e d i g e s t i v e enzymes and a b s o r b s n u t r i e n t s . The p o s t e r i o r r e g i o n s of the midgut of Calanus plumchrus a l s o s e c r e t e d i g e s t i v e enzymes and absorb n u t r i e n t s , but the c e l l t y p e s c a r r y i n g out these f u n c t i o n s are r e g i o n a l i z e d . There are d i s c r e e t segments of c e l l t y p e s each h a v i n g a p a r t i c u l a r f u n c t i o n . In T i g r i o p u s c a l i f o r n i c u s many examples of gap j u n c t i o n s can be seen between the a p i c a l regions of adjacent e p i t h e l i a l c e l l s . They can be found between most of the e p i t h e l i a l c e l l s throughout the n o n - c u t i c u l i z e d regions of the d i g e s t i v e t r a c t . Such j u n c t i o n s a r e r e f e r r e d to a communicating j u n c t i o n s because the pore s i z e between the c o u p l e d c e l l s a l l o w s a v a r i e t y of s m a l l m o lecules (sugars, i o n s , amino a c i d s , n u c l e o t i d e s and vitamins) to be shared while e x c l u d i n g t h e i r macromolecules ( p r o t e i n s , n u c l e i c a c i d s and p o l y s a c c h -aride) . (Aberts et a l . / 1983). No e v i d e n c e of s e p t a t e j u n c t i o n s c o u l d be seen in e i t h e r T i g r i o p u s c a l i f o r n i c u s or Calanus plumchrus a f t e r the type of f i x a t i o n used. D. Hindgut The a n t e r i o r hindgut regions of T i g r iopus c a l i f o r n i c u s and C a l a n u s piumchrus a r e separated from t h e i r midgut by a midgut v a l v e . The e p i t h e l i a l l i n i n g a t t h i s j u n c t i o n i s h i g h l y c o n v o l u t e d . The lumen i s c o n s t r i c t e d and i n -57-some s e c t i o n s , i s n o t a p p a r e n t . The c e l l s o f t h e a n t e r i o r h i n d g u t o f T i g r i o p u s c a l i - f o r n i c u s a r e l i n e d by a t h i n c u t i c l e . The lumen i s c o n v o l u t e d and a t t i m e s a l m o s t s e p a r a t e s t h e e p i t h e l i a l c e l l s . The u l t r a s t r u c t u r e s u g g e s t s t h a t s y n t h e t i c a c t i v i t y d o e s n o t o c c u r , b u t t h e number o f m i t o c h o n d r i a s u g g e s t a h i g h e n e r g y r e q u i r e m e n t . V e r n o n e t a l . , ( 1 9 7 4 ) d e s c r i b e d a s i m i l a r h i n d g u t i n a t e r r e s t r i a l i s o p o d . F e c a l m a t e r i a l i n t h e f o r m o f p e l l e t s w e r e o f t e n o b s e r v e d i n t h i s r e g i o n o f t h e d i g e s t i v e t r a c t . Some a b s o r p t i o n may o c c u r i n t h e a n t e r i o r h i n d g u t . A b s o r p t i o n o f some n u t r i e n t s was o b s e r v e d i n t h e wasp (Edson and V i n s o n , 1977) . O s m o r e g u l a t i o n may be a f u n c t i o n o f t h i s r e g i o n ( S c h u l t z and Kennedy, 1976) due t o t h e a n a l i n t a k e o f water by a n t i -p e r i s t a t i c c o n t r a c t i o n s . D a l l (1967) f o u n d e v i d e n c e o f water u p t a k e and s a l t e x c r e t i o n i n t h e s h r i m p h i n d g u t . T o l m a n and S t e e l e (1976) fo u n d t h a t w ater r e g u l a t i o n i n t h e g u t was l i n k e d t o one or more i o n s . T h e e n e r g y f r o m t h e m i t o c h o n d r i a , i n t h e p r e s e n c e o f A T P a s e c o u l d be used t o a c t i v e l y pump th e i o n s i n t o t h e c e l l c a v i t i e s o f t h e c o c k r o a c h h i n d g u t . L i t t l e o r no m u s c l e was f o u n d a s s o c i a t e d w i t h t h e a n t e r i o r p o r t i o n s o f t h e h i n d g u t o f T i g r i o p u s c a l i f o r n i c u s . I o n t r a n s p o r t and o s m o r e g u l a t i o n w o u l d be f a s c i l i t a t e d by t h i s l a c k o f m u s c u l a t u r e . The p o s t e r i o r h i n d g u t has a c o n v o l u t e d lumen and ends i n a t h i c k c u t i c l e l i n e d anus. -58-In Calanus plumchrus, the hindgut i s a long and s t e a d i l y n a r r o w i n g t u b e . P o s t e r i o r to the midgut v a l v e , i t i s a s p h e r i c a l chamber of c u t i c l e l i n e d e p i t h e l i a l c e l l s w i t h a furrowed lumen e x t e n d i n g between a d j a c e n t c e l l s . The gut narrows a b r u p t l y and l i e s i n the d o r s a l segment of the copepod. I t ends at a t h i c k c u t i c l e l i n e d anus. Developed f e c a l p e l l e t s were o f t e n o b s e r v e d i n the h i n d g u t . The ba s a l c e l l s u r f a c e s are bounded by a prominent muscle l a y e r throughout most of the hindgut, but i s reduced p o s t e r i o r l y and i s not a p p a r e n t i n those c e l l s near the anus. These muscles c i r c l i n g the hindgut would serve to move the f e c a l p e l l e t down i t s c o n s i d e r a b l e l e n g t h . The presence of la r g e mitochondria i n the e p i t h e l i a l c e l l s suggest that osmoregulation and ion 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 r e g i o n s . P e r i t r o p h i c Membrane In some s e c t i o n s o f both T i g r iopus c a l i f o r n i c u s and Calanus plumchrus a p e r i t r o p h i c membrane was observed s u r r o -u n d i n g the f e c a l p e l l e t . In both copepods, the membrane i s seen i n the midgut and i s most obvious surrounding the f e c a l m a t e r i a l i n the p o s t e r i o r p o r t i o n s o f the midgut. Gauld (1975) reported that the p e r i t r o p h i c membrane surrounds the f e c a l p e l l e t s i n Calanoids and i s secreted by the p o s t e r i o r m i d g u t. In I s o p o d s , H o l d i c h (1975) s u g g e s t e d t h a t the membrane was s y n t h e s i z e d by the midgut caecum c e l l s . In -59-i n s e c t s , components of the p e r i t r o p h i c membrane may be produced i n pa r t by c e l l s i n the a n t e r i o r p o r t i o n of the midgut, and a second part by c e l l s i n the foregut (Smith, 1968) . In Daphnia, the foregut forms the p e r i t r o p h i c membrane. Burgos and G u t i e r r e z (1976) reported that the p e r i t r o p h i c membrane i s the g l y c o c a l y x a s s o c i a t e d w i t h , and a d j a c e n t t o , the m i c r o v i l l i . I t may act as a s u b s t r a t e f o r h y d r o l y t i c enzymes. Hootman and Conte (1974) propose t h a t i n the shrimp, g l y c o c a l y x and/or v e s i c l e s between the m i c r o v i l l i p roduce the p e r i t r o p h i c membrane. The membrane i s the endodermal e q u i v a l e n t of c u t i c l e and c o n s i s t s of c h i t i n o u s m i c r o f i b r i l s i n a p r o t e i n and p o l y s a c c h a r i d e matrix (Clarke et a l . , 1977; Schultz and Kennedy, 1976). In both T i g r i o p u s c a l i f o r n i c u s and Calanus plumchrus the flow of m a t e r i a l throughout the gut i s very i m p o r t a n t to the d i g e s t i v e p r o c e s s . In the esophagus m a s t i c a t i o n i s accomplished by the c o n t i n u a l c o n t r a c t i o n and expansion of the e p i t h e l i a l c e l l s i n c o n j u n c t i o n with the muscular a c t i v i t y . B asal i n v a g i n a t i o n s and i n t e r d i g i t a t i n g f o l d s of the plasma membrane expand to accommodate the food p a r t i c l e s and m uscle c o n t r a c t i o n s s e r v e to g r i n d the p a r t i c u l a t e matter and f o r c e i t through the esophagus. In the midgut and the a n t e r i o r r e g i o n s (caecum and d i v e r t i c u l u m ) t h i s p r o c e s s o f c o n t i n u a l c o n t r a c t i o n and expansion i s extremely important to d i g e s t i o n . P e r i s t a l t i c and a n t i p e r i s t a l t i c c o n t r a c t i o n s along the length of the - 6 0 -d i g e s t i v e t r a c t allow the food p a r t i c l e s to mix w i t h the d i g e s t i v e enzymes and to be presented to the s p e c i f i c regions of the gut where n u t r i e n t a b s o r p t i o n occurs. In T i g r i o p u s C a l i f o r n i c u s the caecum acts as a s i t e f o r d i g e s t i o n and s e c r e t i o n of enzymes. I t s s u r r o u n d i n g m u scle l a y e r a c t s as a pump to f o r c e the food m a t e r i a l back and f o r t h w i t h i n the caecum. By t h i s a c t i o n , the ingested food comes i n t o c o n t a c t with the d i g e s t i v e enzymes secr e t e d by the s p e c i a l i z e d c e l l s and a l s o comes i n t o contact w i t h the e p i t h e l i a l s u r f a c e of the a b s o r p t i v e c e l l s . The midgut has the a b i l i t y to expand and c o n t r a c t i n a rhythmic c y c l e that i s i n c o n j u n c t i o n with the caecum. In Calanus plumchrus t h i s process of rhythmic d i g e s t i v e t r a c t c o n t r a c t i o n s i s v e r y n e c e s s a r y s i n c e the s e c r e t o r y and a b s o r p t i v e c e l l s are 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 of d i g e s t i v e enzymes occurs mainly i n the p o s t e r i o r midgut. The r h y t h m i c c o n t r a c t i o n s o f the gut s e r v e to move the enzymes w i t h i n the d i g e s t i v e t r a c t and to move the food m a t e r i a l to the d i v e r t i c u l u m where some ab s o r p t i o n o c c u r s . The d i v e r t i c u l u m can a l s o c o n t r a c t and a c t l i k e a pump to f u r t h e r mix the food and enzymes and move them throughout the gut. In both copepods t h i s c o n t i n u a l churning a c t i o n provides a mechanism to ens u r e adequate d e g e s t i o n and a b s o r p t i o n of the r e l e a s e d n u t r i e n t s . -61-Endocytosis E n d o c y t o s i s i s the p r o c e s s o f i n t e r n a l i z a t i o n of e x t r a c e l l u l a r m a t e r i a l w i t h i n an i n v a g i n a t i o n of the plasma membrane. U l t i m a t e l y , the i n v a g i n a t i o n i s th o u g h t to giv e r i s e to an independent cytoplasmic v e s i c l e . The type of v e s i c l e formed i s named according to the c e l l type and the mode of e n d o c y t o s i s i n v o l v e d (Besterman and Low, 1983) . In t h i s study, two types of p i n o c y t o s i s (endocytosis) were s t u d i e d using e s t a b l i s h e d b i o l o g i c a l marker methodology. P i n o c y t o s i s o f s u b s t a n c e e n t e r i n g with the f l u i d content i s c a l l e d f l u i d phase p i n o c y t o s i s . P i n o c y t o s i s of substances bound to the 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 p i n o c y t o s i s . A d s o r p t i v e p i n o c y t o s i s i s f u r t h e r subdivided i n t o the pi n o c -t o s i s of markers bound n o n - s p e c i f i c a l l y , and p i n o c y t o s i s of l i g a n d s mediated v i a b i n d i n g to s p e c i f i c r e c e p t o r s on the c e l l s u r f a c e membrane (receptor mediated p i n o c y t o s i s ) (Besterman and Low, 1983; S i l v e r s t e i n et a l . , 1977). T i g r i o p u s c a l i f o r n i c u s e n d o c y t o t i c a c t i v i t y was s t u d i e d using two b i o l o g i c a l markers. S o l u b l e h o r s e r a d i s h peroxidase (HRP) was used to study f l u i d phase endocytosis and a c o l l o i d a l gold/wheat germ a g g l u t i n i n (WGA) c o n j u g a t e was used to study receptor mediated e n d o c y t o s i s . F l u i d Phase 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 -62-a p p e a r s t o be a b s o r b e d o n l y i n c e l l t y p e '3'. I t was fo u n d i n c y t o p l a s m i c v e s i c l e s w i t h i n t y p e '3' c e l l s o f t h e m i d g u t caecum and t h e a n t e r i o r m i d g u t . The i n t r a c e l l u l a r a c c u m u l a t i o n o f a f l u i d p hase marker i s p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f the m a r k e r i n t h e medium ( A l b e r t s e t a l . , 1983). H o r s e r a d i s h p e r o x i d a s e i s a p l a n t g l y c o p r o t e i n . Graham and K a r n o v s k y (1966) d e v e l o p e d a c y t o c h e m i c a l t e c h n i q u e t o o b s e r v e p e r o x i d a s e a c t i v i t y . They f o u n d t h a t 3,3' d i a m i -n o b e n z i d i n e (DAB) c o u l d l o c a l i z e t h e enzyme a c t i v i t y o f p e r o x i d a s e by f o r m i n g a dense r e a c t i o n p r o d u c t . DAB s e r v e d as a h y d r o g e n - d o n o r s u b s t r a t e i n t h e p r e s e n c e o f low c o n c e n -t r a t i o n s o f h y d r o g e n p e r o x i d e . The r e a c t i o n p r o d u c t s c an be made e l e c t r o n d ense w i t h osmium t e t r o x i d e ( H e r z o g and F a h i m i , 1973; Rennke e t a l . , 1979). A f t e r f i v e m i n u t e s i n c u b a t i o n , t h e p e r o x i d a s e p o s i t i v e v e s i c l e s f o u n d i n c e l l t y p e '3' show d e n s e p a r t i c u l a t e m a t t e r m a i n l y i n t h e p e r i p h e r y o f t h e v e s i c l e s . L o n g e r i n c u b a t i o n s r e s u l t e d i n s c a t t e r e d dense m a t t e r t h r o u g h o u t t h e v e s i c l e s . T he l o n g e s t i n c u b a t i o n t i m e s r e s u l t e d i n p e r o x i d a s e p o s i t i v e h o m o g e n o u s l y d e n s e v e s i c l e s . T h e y were fo u n d t h r o u g h o u t t h e c y t o p l a s m i n c l u d i n g t h e b a s a l p o r t i o n s o f t h e e p i t h e l i a l c e l l s . Due t o t h e e x t r e m e i m p e r m e a b i l i t y o f t h e c u t i c l e and t h e r a p i d p ostmortem d e g e n e r a t i o n o f t h e c e l l s l i n i n g t h e d i g e s t i v e t r a c t , l i t t l e work h a s b e e n c o m p l e t e d t o -63-d a t e on t h e e l e c t r o n m i c r o s c o p i c a n a l y s i s o f t h e g u t o f T i g r i o p u s c a l i f o r n i c u s . No l i t e r a t u r e c o u l d be f o u n d d e a l i n g w i t h t h e a b s o r p t i v e m e c h a n i s m s o f t h e d i g e s t i v e t r a c t a t t h e e l e c t r o n m i c r o s c o p e l e v e l . I n T i g r i o p u s c a l i f o r n i c u s , t h e u p t a k e o f s o l u b l e h o r s e r a d i s h p e r o x i d a s e f o u n d o n l y i n c e l l t y p e '3' i s c o n s i s t e n t w i t h th e u l t r a s t r u c t u r e d e s c r i b e d . The f a c t o r s c o n t r o l l i n g f l u i d p h a s e p i n o c y t o s i s a r e v e r y p o o r l y u n d e r s t o o d . In mammalian c e l l s , t h e m a g n i t u d e and d i r e c t i o n o f t h e o b s e r v e d e f f e c t s a r e v a r i a b l e and c e l l - t y p e d e p e n d e n t . The c h a r g e on t h e m a r k e r ( D a v i e s e t a l . , 1980) and t h e s t a g e o f t h e c e l l c y c l e ( B e r l i n e t a l . , 1980) h a v e been d e s c r i b e d as f a c t o r s t h a t r e g u l a t e f l u i d p h ase p i n o c y -t o s i s . A r o l e f o r c a l c i u m has been d e s c r i b e d i n t h e amoeba. The i n d u c t i o n p r o c e s s i n c l u d e s an i n c r e a s e d plasma membrane i o n i c p e r m e a b i l i t y , and a c a l c i u m i n f l u x . I t h a s b e e n s u g g e s t e d t h a t t h e i n c r e a s e d c a l c i u m i n t h e c y t o s o l p r o v i d e s a s i g n a l f o r t h e a r r a n g e m e n t and c o n t r a c t i o n o f a membrane a s s o c i a t e d c o n t r a c t i l e f i l a m e n t n e t w o r k . T h i s r e s u l t s i n i n v a g i n a t i o n and v e s i c u l a t i o n ( P r u s c h , 1980). The m a t e r i a l i n t e r n a l i z e d i n Amoeba p r o t e u s may be s e l e c t i v e l y e x t r u d e d from the c e l l i f i t i s p e r c e i v e d b y t h e c e l l as p o t e n t i a l l y h a r m f u l . D e g r e d a t i o n o f t h e s o l u t e m a c r o m o l e c u l e s a p p e a r s t o be t h e most common form o f i n t r a c e l l u l a r m o d i f i c a t i o n -64-and l i k e l y i n v o l v e s t h e l y s o s o m e ( P o o l e e t a l . , 1980). P i n o c y t o s i s may a l s o s e r v e as a pathway f o r t h e t u r n o v e r o f t h e plasma membrane, l i p i d s and/or p r o t e i n s . H o w e v e r , t h i s i s p r o b a b l y o n l y a segment o f t h e f u n c t i o n s o f p i n o c y t o s i s . In T i g r i o p u s c a l i f o r n i c u s , t h e use o f a h o r s e r a d i s h p e r o x i d a s e marker p r o v i d e d e v i d e n c e i n s u p p o r t o f t h e a b s o r p t i v e f u n c t i o n o f c e l l t y p e '3'. A d s o r p t i v e E n d o c y t o s i s ( T i g r i o p u s c a l i f o r n i c u s ) In T i g r i o p u s c a l i f o r n i c u s , g o l d c o l l o i d s (17 - 20um) were o b s e r v e d a d h e r e d t o t h e p l a s m a membrane a t t h e b a s e o f t h e m i c r o v i l l i and w i t h i n t h e c y t o p l a s m o f c e l l t y p e s '2' and '3'. The m a j o r i t y o f the g o l d p a r t i c l e s o b s e r v e d were found i n c e l l t y p e '3' o f t h e m i d g u t caecum and a n t e r i o r m i d g u t . In t h e a n t e r i o r m i d g u t r e g i o n s , n u m e r o u s g o l d p a r t i c l e s w e r e a l s o 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' . R e c e p t o r m e d i a t e d e n d o y c t o s i s i n v o l v e s t h e l i g a n d b i n d i n g t o membrane r e c e p t o r s f o l l o w e d by c l u s t e r i n g o f he 1 i g a n d - r e c e p t e r c o m p l e x e s i n t o c o a t e d p i t s (Besterman and Low, 1983) . R e s e a r c h e s u s i n g mammalian c e l l s have p r o v i d e d e v i d e n c e t h a t t h e r e g i o n s o f t h e plasma membrane a r e c o a t e d on t h e i r c y t o p l a s m i c s u r f a c e s w i t h a p r o t e i n , c l a t h r i n ( P e a r s e , 1978; L i n d o n and R o t h , 1983; B r e t s c h e r and P e a r s e , 1 9 8 4 ) . I t has been g e n e r a l l y assumed t h a t t h e c o a t e d v e s i c l e s -65-p i n c h o f f and d e l i v e r the l i g a n d to lysosomes or o t h e r i n t r a c e l l u l a r compartments and then r e c y c l e the c l a t h e r i n coated v e s i c l e s and re c e p t o r s back to the plasma membrane (Pearse, 1980) . Willingham and Pastan (1980) suggested that i n f i b r o b -l a s t s , these coated v e s i c l e s remain attached to the plasma membrane and the l i g a n d - r e c e p t o r s are t r a n s f e r r e d to uncoated v e s i c l e s from an opening i n the c l a t h e r i n coat or adjacent plasma membrane. These uncoated v e s i c l e s are termed 'recepto-somes'. Monsigny e t a l (1980) d e s c r i b e d the s p e c i f i c i t y of wheat germ A g g l u t i n i n (WGA) f o r g l y c o p r o t e i n r e c e p t o r s on the c e l l s u r f a c e . C o l l o i d a l gold i s one of the most r e a d i l y v i s u a l i z e d markers used i n e l e c t r o n m i c r o s c o p y . H o r i s b e r g e r (1979) d e s c r i b e d a wide range of p r o t e i n s t h a t can be adsorbed very t i g h t l y to gold under a p p r o p r i a t e c o n d i t i o n s . A wheat germ a g g l u t i n i n / c o l o i d a l gold conjugate was used as a b i o l o g i c a l marker f o r e n d o c y t o s i s . I n T i g r i o p u s c a l i f o r n i c u s , no coated p i t s or receptosomes could be seen. The r e s u l t s o b s e r v e d u s i n g the gold/WGA probes were c o n s i s t e n t w i t h the u l t r a s t r u c t u r e o f c e l l types '2' and '3'. Secti o n s of the e n t i r e d i g e s t i v e t r a c t showed t h a t the a b s o r p t i o n of gold/WGA was s p e c i f i c for c e l l types 12' and 13'. The use of t h i s b i o l o g i c a l marker r e i n f o r c e s the -66-c o n c l u s i o n s made from the observed u l t r a s t r u c t u r e and shows that a b s o r p t i o n occurs i n the midgut caecum and the a n t e r i o r midgut. The exact nature of the i n t e r n a l i z a t i o n p r o c e s s remains to be e l u c i d a t e d . S t u d i e s of the a b s o r p t i v e and e n d o c y t o t i c mechanisms in the d i g e s t i v e t r a c t a r e a e x c i t i n g a r e a of r e s e a r c h . However, i n T i gr i opus c a l i f o r n i c u s t h i s p r o v e d to be a c h a l l e n g i n g t e c h n i c a l problem. The r e l a t i v e impermeability of the c u t i c l e l i n i n g and the r a p i d post mortem degeneration are d i f f i c u l t problems to overcome. For the b e n e f i t of f u t u r e r e s e a r c h e r s , i t might be p o s s i b l e to improve on the r e s u l t s that have been presented. To s t u d y f l u i d phase p i n o c y t o s i s o t h e r s might t r y to i n c r e a s e the c o n c e n t r a t i o n of h o r s e r a d i s h p e r o x i d a s e and t h e r e b y i n c r e a s e the a b s o r p t i v e t r a n s i t time or they might employ the use of s o l u b l e dextrans or f e r r i t i n . The s t u d y of 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 might a l s o be improved by i n c r e a s i n g the c o n c e n t r a t i o n of the b i o l o g i c a l marker and i t s t r a n s i t time i n the d i g e s t i v e t r a c t or by s e l e c t i n g a s e r i e s of d i f f e r e n t l e c t i n s conjugated w i t h c o l l o i d a l g o l d . A l s o , the use of an i n t e r m e d i a t e monoclonal a n t i b o d y r a i s e d a g a i n s t the l e c t i n of c h o i c e and conjugated to c o l l o i d a l gold may amplify the s e n s t i v i t y of the receptor mediated event. The problems of r a p i d p o s t mortem degeneration and the c u t i c l e i m p e r m e a b i l i t y may be d e c r e a s e d i f , a t the -67-i n i t a l time of f i x a t i o n , 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 or p o s t e r i o r p o r t i o n s of the copepod a r e c a r r i e d out to allow r a p i d f i x a t i o n p e n e t r a t i o n . A l s o , o t h e r f i x a t i v e s many be employed t h a t have a f a s t e r p e n e t r a t i o n t i m e . Enzymes t h a t s o f t e n the i n v e r t e b r a t e c u t i c l e might a l s o be employed to enhance the p e n e t r a t i o n of the f i x a t i v e . In summary, the d i g e s t i v e t r a c t of T i g r i o p u s c a l i f o r n i c u s i s 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 midgut, and an a n t e r i o r and p o s t e r i o r hindgut. The d i g e s t i v e t r a c t o f C a l a n u s plumchrus c o n s i s t s of a c u t i c u l a r i z e d esophagus, a midgut d i v e r t i c u l u m , a d i v e r s i f i e d midgut, and a hindgut. In both copepods, the esophagus and i t s a s s o c i a t e d m u s c u l a t u r e f u n c t i o n s to draw the food i n t o the gut. The l i m i t e d d i g e s t i o n observed i n the d o r s a l esophagus r e s u l t s from the d i g e s t i v e enzymes secreted i n the midgut and moved forward by a n t i p e r i s t a l t i c c o n t r a c t i o n s . The midgut caecum and a n t e r i o r midgut of T i g r i o p u s  c a l i f o r n i c u s has c e l l s s p e c i a l i z e d f o r abs o r p t i o n and s e c r e -t i o n . The a n t e r i o r regions have a gre a t e r a b s o r p t i v e c a p a c i t y . In C a l a n u s p l u m c h r u s , the midgut d i v e r i t i u l u m i s s p e c i a l i z e d f o r a b s o r p t i o n . The a n t e r i o r midgut regions are high v a c u o l i z e d and mainly a b s o r p t i v e . S e c r e t o r y f u n c t i o n s occur i n the p o r t i o n of the midgut p o s t e r i o r to the vacuolated -68-r e g i o n and the p o s t e r i o r end of the midgut has c e l l s s p e c i a l i z e d for a b s o r p t i o n . The hindgut of both copepods i s a c u t i c u l i z e d chamber s p e c i a l i z e d f o r the movement of the f e c a l p e l l e t and p o s s i b l y osmoregulation. The o v e r a l l shape of the d i g e s t i v e t r a c t o f both 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 piumchrus i s s i m i l a r to that observed i n other arthropods. P e r i s t a l t i c a c t i v i t y i n the gut s e r v e s to move the i n g e s t e d food p o s t e r i o r l y through the d i g e s t i v e t r a c t . A n t i p e r i s t a l t i c c o n t r a c t i o n s s e r v e t o move the ingested m a t e r i a l to the regions of the gut s p e c i a l i z e d f o r ab s o r p t i o n and s e c r e t i o n . R e g i o n a l s p e c i a l i t i e s and d i f f e r e n c e s i n c e l l type and d i s t r i b u t i o n probably r e l a t e to the abundance and type of food i n g e s t e d as w e l l as to the h a b i t a t i n which the copepods are f o u n d . Sex and s e a s o n a l v a r i a t i o n s would a l s o a f f e c t the m o r p h o l o g i c a l s t r u c t u r e of the d i g e s t i v e t r a c t . -69-Key f o r f i g u r e s A anus AH a n t e r i o r hindgut AM a n t e r i o r midgut B l b a s a l lamina c c u t i c l e E esophagus fm f e c a l m a t e r i a l g G o l g i bodies H hindgut L lumen M muscle m mitochondria MC midgut caecum Md midgut MD midgut d i v e r t i c u l u m MdV - midgut valve mvb m u l t i - v e s i c u l a r bodies MV m i c r o v i l l i N nucleus No n u c l e o l u s PH p o s t e r i o r hindgut PM p o s t e r i o r midgut pm p e r i o t r o p h i c membrane RER - rough endoplasmic r e t i c u l u m SER " - smooth endoplasmic r e t i c u l u m Gj gap j u n c t i o n V v e s i c l e Vac - vacuole 1 c e l l type 1 2 c e l l type 2 3 c e l l type 3 4 c e l l type 4 B 'B' c e l l type D 1D' c e l l type E 'E' c e l l type F 'F" c e l l type R 'R1 c e l l type -70-L 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 regions of the 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 ( s a g i t t a l s e c t i o n ) . L i g h t micrograph showing the o r a l and mandibular r e g i o n s . The esophagus surrounded by muscle. Esophagus and midgut caecum of T i g r i o p u s c a l i f o r n i c u s . A h i g h e r power l i g h t m i c r o g r a p h showing the c e l l s of the midgut caecum and t h e 'H' shaped esophagus. 70 a -71-An e l e c t r o n micrograph of the esophagus i n 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 plasma membrane i n v a g i n a t i o n s , the muscle l a t e r , and the t y p i c a l c u t i c l e l i n e d 'H' shape. 71a -72-F i g u r e 7. A higher power view of the c u t i c l e l i n i n g the esophagus. F i g u r e 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 through the midgut caecum and a n t e r i o r m i d g u t . Note the e x t r u s t i o n o f c e l l contents i n the midgut caecum. F i g u r e 9. A low power e l e c t r o n m i c r o g r a p h of a cro s s s e c t i o n through the midgut caecum i n T i g r iopus c a l i f o r n i c u s . 72 a -73-F i g u r e s 10 and 11. Two low power e l e c t r o n micrographs o f t h e 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 l i p i d v e s i c l e s found i n many c e l l s . 73a -74-F i g u r e 12. A high power e l e c t r o n micrograph of e p i t h e l i a l c e l l s i n the a n t e r i o r midgut. Note t h e d e n s e l y packed l u m i n a l m i c r o v i l l i and the prominant gap j u n c t i o n . F i g u r e 13. C e l l 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 m i d g u t . Note t h e p r e s e n c e of e l e c t r o n dense v e s i c l e s . 74a -75-F i g u r e s 14 and 15. L i g h t m i c r o g r a p h s of the c e l l s of the a n t e r i o r midgut. Note the presence of d a r k l y s t a i n i n g c e l l type *2'. F i g u r e 16. An 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 m i d g u t . The c e l l s a r e more c o m p r e s s e d than those seen i n the midgut caecum. 75a -76-F i g u r e s 17 and 18. L i g h t micrographs of the a n t e r i o r m i d g u t . The lumen v a r i e s c o n s i d e r a b l y i n shape d e p e n d i n g upon c o n t r a c t i o n or the presence of food p a r t i c l e s . F i g u r e 19. An 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 i n T i g r i opus c a l i f o r n i c u s . L i p i d v e s i c l e s are a b u n d a n t . Note t h e b a s a l m u s c l e bands. In many c e l l s , the G o l g i bodies are q u i t e d i l a t e d . 76a - 7 7 -F i g u r e 2 0 . An 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 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 ' b r i d g e ' shaped c e l l type ' 2 ' . F i g u r e 2 1 . 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 midgut. The ba s a l lamina appears continuous with the plasma membrane i n v a g i n a t i o n s . - 7 8 -F i g u r e s 22 and 23. L i g h t micrographs of the p o s t e r i o r m i d g u t o f T i g r i o p u s c a l i f o r n i c u s . 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 and more c u b o i d a l i n shape. T h i s area of the gut l i e s c e n t r a l l y i n the body c a v i t y . F i g u r e 24. An e l e c t r o n m i c r o g r a p h of some c e l l s i n the p o s t e r i o r m i d g ut. The m u s c l e bands a s s o c i a t e d w i t h the b a s a l lamina are w e l l developed. Note the decrease i n the d e n s i t y of the m i c r o v i l l i and the c u b o i d a l shape of the c e l l s . 78a - 7 9 -and 2 6 . Two l i g h t m i crographic views (cross s e c t i o n s ) t h r o u g h t h e a n t e r i o r h i n d g u t i n T i g r i o p u s c a l i f o r n i c u s . The lumen i s c u t i c u l i z e d and f u r r o w e d . In f i g u r e 2 6 i t appears as t i g h t l y c o n s t r i c t e d . T h i s e l e c t r o n m i c r o g r a p h shows t h a t 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 p o s t e r i o r midgut a r e low and c u b o i d a l shaped. The b a s a l c e l l s u r f a c e s a re i n v a g i n a t e d and t h e a p i c a l m i t o c h o n d r i a are re d u c e d i n s i z e and number. 79 a -80-F i g u r e 28. The c u t i c l e l i n e d f u r r o w e d lumen of the p o s t e r i o r hindgut i s shown in t h i s e l e c t r o n micrograph. F i g u r e 29. The c u t i c l e i n t h e r e g i o n o f the anus. It takes on the t y p i c a l banded appearance of the copepod p r o t e c t i v e c u t i c l e . 80 a -81-F i g u r e 30. 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 d i g e s t i v e t r a c t of Calanus plumchrus. F i g u r e 31. L i g h t micrograph of the c u t i c l e l i n e d esoph-agus. Fig u r e 32, F i g u r e 33 F i g u r e 34 An e l e c t r o n the r e g i o n prominant micrograph of the of the e s o p h a g u s muscle. c u t i c l e from Note t h e The a n t e r i o r p o r t i o n of the midgut. Note t h e p r e s e n c e o f l a r g e v a c u o l a t e d c e l l s surrounded by darker s t a i n i n g e p i t h e l i a . In the lume n In C a l a n u s c e n t r e of the vacuolar r e g i o n , the i s o f t e n s e e n t o be 1U' s h a p e d . plumchrus, the e p i t h e l i a l c e l l s l i n i n g the gut are compressed. There are l a r g e numbers of vacuolated c e l l s . 81a -82-F i g u r e s 35 to 37. 'R' c e l l s of the midgut d i v e r t i c u l u m . Note the v e s i c l e s present near the m i c r o v i 1 1 i . In ( f i g u r e 35) these c e l l s , the b a s a l plasma membrane i s h i g h l y i n v a g i n a t e and the b a s a l c e l l p o r t i o n s e v a g i n a t e . In f i g u r e 37, v e s i c l e s can be seen open to the cytoplasm (arrow head). F i g u r e 38. 'R' type c e l l found i n the p o s t e r i o r region of the midgut. They are remarkably s i m i l a r i n appearance to the 'R' c e l l s of the midgut d i v e r t i c u l u m . The b a s a l p o r t i o n s are l e s s i n v a g i n a t e d and the mitochondria l e s s densely packed. F i g u r e 39. An e l e c t r o n micrograph of an 'R' type c e l l i n the t r a n s i t i o n between midgut d i v e r t i c u l u m and midgut. F i g u r e 40. A l i g h t m i c r o g r a p h o f the midgut v a l v e between the midgut and h i n d g u t . compressed arrangement of the c e l l s . Note the ep i t h e l i a1 82 a -83-F i g u r e 41. The g l a n d u l a r region of the midgut of Calanus  p l u m c h r u s i s shown. Note the p r e s e n c e 'D' and 1E" type of 'B' c e l l s F i g u r e 42. The l a r g e v a c u o l e of the 'B' c e l l 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 . N o t e t h e 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 s u r f a c e . F i g u r e 43. Basal 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 region of the midgut. Note the ba s a l nucleus of the vacuolated "B1 c e l l s . F i g u r e 44 •E 1 and r e g i o n . and o n l y 'D' c e l l s i n the g l a n d u l a r midgut The 'E' c e l l s are much l e s s dense l i g h t l y s t a i n e d . 83a - 8 4 -F i g u r e 45. 'F' c e l l t y p e i n the midgut of C a l a n u s  p1umch 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 m i c r o v i l l i are t y p i c a l l y club-shaped. F i g u r e 46. A high power l i g h t micrograph of the midgut valve seen i n f i g u r e 40. F i g u r e s 47 and 48. S e c t i o n s of the s e c r e t o r y 'F' type c e l l s i n the midgut. Note the d i l a t e d G o l g i bodies and the granular rough endoplasmic r e t i c u l u m . F i g u r e s 49 to 51. In the p o s t e r i o r p o r t i o n of the midgut near the midgut v a l v e . The e p i t h e l i a l c e l l s are s h o r t e r and have a w e l l developed ba s a l lamina continuous with plasma membrane i n v a g i n a t i o n s . O r g a n e l l e s 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 'club' shape. 84 a - 8 5 -F i g u r e 52. A l i g h t m i c r o g r a p h o f t h e m i d d l e p o r t i o n o f t h e h i n d g u t . The l u m e n t e n d s t o h a v e a s t a r s h a p e d a p p e a r a n c e and i s c u t i c l e 1 i n e d . F i g u r e 53. A s e c t i o n t h r o u g h t h e a n t e r i o r p o r t i o n o f t h e h i n d g u t i s s e e n . N o t e t h e d e e p l u m i n a l i n v a g i n a t i o n s a n d t h e p r o m i n e n t n u c l e i . F i g u r e 54. A l o w power m i c r o g r a p h o f t h e p o s t e r i o r p o r t i o n o f t h e h i n d g u t i n C a l a n u s p l u m c h r u s . Note i t s d o r s a l p o s i t i o n . I t i s c o n s i d e r a b l y r e d u c e d i n s i z e as c o m p a r e d w i t h f i g u r e 53. F i g u r e 55. T h i s e l e c t r o n m i c r o g r a p h shows t h e c u t i c l e l i n i n g t h e l u m i n a l i n v a g i n a t i o n o f t h e c e l l s i n t h e p o s t e r i o r p o r t i o n o f t h e h i n d g u t . F i g u r e 56. A t r a n s v e r s e s e c t i o n showing t h e p o s t e r i o r h i n d g u t and some m u s c u l a t u r e . F i g u r e s 57 and 58. Two e l e c t r o n m i c r o g r a p h s o f t h e c u t i c l e l i n i n g o f t h e p o s t e r i o r h i n d g u t c e l l s n e a r t h e r e g i o n o f t h e 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 -86-F i g u r e 59. 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 a n t e r i o r midgut of T i g r i o p u s c a l i f o r n - i c u s . F i g u r e 60. C o n t r o l micrograph of c o l l o i d a l gold absorption i n the region of the midgut caecum. Note densely s t a i n i n g c e l l type '2'. F i g u r e 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 midgut i n T i g r i o p u s c a l i f o r n i c u s . C e l l t y p e s '2' and '3' are seen. 86 a -87-F i g u r e 62. A r e g i o n of the p o s t e r i o r midgut. Note the presence of a m u l t i v e s i c u l a r body (mvb) i n t h e ' b r i d g e ' s h a p e d c e l l t y p e '2'. C o l l o i d a l gold / WGA c o n t r o l . F i g u r e 63. Luminal gold c o l l o i d s are seen a f t e r 5 minutes i n c u b a t i o n . The c e l l s u r f a c e viewed belongs to c e l l type '3'. 87a -88-F i g u r e s 64 and 65. A f t e r 15 minute i n c u b a t i o n s , the gold c o l l o i d s are seen adhered to the plasma membrane of c e l l type '3'. Note the presence of the gap j u n c t i o n . F i g u r e 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 the lumen of the d i g e s t i v e t r a c t i n the r e g i o n of the p o s t e r i o r m i d g u t . A r r o w s i n d i c a t e the 17 to 20 um c o l l o i d s . 88a -89-F i g u r e 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 of the a n t e r i o r midgut i n T i g r i o p u s  c a l i f o r n i c u s . Gold p a r t i c l e s were found i n the cytoplasm of c e l l type '2'. F i g u r e 68. 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 the region shown by the a s t e r i x i n f i g u r e 67. Numerous gold p a r t i c l e s can be observed. 89 a -90-F i g u r e 69. C o n t r o l m i c r o g r a p h f o r h o r s e r a d i s h p e r o x i d a s e 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  c a l i f o r n i c u s . F i g u r e 70. C o n t r o l m i c r o g r a p h o f a s e c t i o n t h r o u g h the midgut 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 seen. 90a -91-F 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 o f c e l l type '3' i n the a n t e r i o r midgut as see 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 can be seen (arrows). F i g u r e 72. A s e c t i o n o f c e l l t y p e '3' i n the midgut caecum. At the arrows, v e s i c l e s c o n t a i n i n g p o s i t i v e h o r s e r a d i s h 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 can be s e e n . T h e y a r e r e a d i l y d i s t i n g u i s h a b l e from o t h e r dense v e s i c l e s i n the cytoplasm. F i g u r e 73. Membrane bound v e s i c l e s wi th posi t i v e peroxidase r e a c t i o n product can be seen i n the cytoplasm of c e l l type '3' i n the midgut caecum. 91a -92-F i g u r e 74. A f t e r a 1 to 10 minute i n c u b a t i o n , v e s i c l e s c o n t a i n i n g h o r s e r a d i s h p e r o x i d a s e can be seen i n the c y t o p l a s m o f c e l l t ype '3'. With s h o r t i n c u b a t i o n s , t h 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 i s found m a i n l y i n the p e r i p h e r y of the v e s i c l e s (see a r r o w s ) . This i s a s e c t i o n of the a n t e r i o r midgut. F i g u r e 75. The arrows i n d i c a t e p e r o x i d a s e c o n t a i n i n g v e s i c l e s a f t e r a 15 m i n u t e i n c u b a t i o n . The c e n t r a l p o r t i o n s of the v e s i c l e s appear denser than that observed with short incuba-t i o n s . T h i s s e c t i o n represents the t r a n s i t i o n of the e p i t h e l i a l c e l l s between the midgut caecum and the a n t e r i o r midgut. 92a -93-References: A l b e r t s , B., Bray, D., Lewis, J . , Raff, M., Roberts, F. and Watson, J.D. (1983). M o l e c u l a r B i o l o g y o f t h e C e l l . Garland P u b l i s h i n g Inc., New York. Arnaud, J . B r u n e t , M. and Mazza, J . (1978). C e l l T i s s u e Res. 187, 333-353. B a c c a r i , S. and Renaud-Morant, J . (1974). Arch. Z o o l . Exp. Gen. 115, 607-620. Barker, P.L. and Gibson, R. (1977). J . Exp. Mar. B i o l . E c o l . 26, 297-394. Barker, P.L. and Gibson, R. (1978). J . Exp. Mar. B i o l . E c o l . 32, 177-196. B e r l i n , R.D. and O l i v e r , J.M. (1980). J . C e l l B i o l . 85, 660-671. Besterman, J.M. and Lowe, R.B. (1983). Biochem. J . 210, 1-13. B r e s c i a n i , J . and Lutzen, J . (1961). Crustaceana 3, 9-23. B r e t s c h e r , M.S. and Pearse, B.M.F. (1984). 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