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The digestive tract of a harpacticoid copepod, Tiqriopus californicu: a light and electron microscope… Sullivan, Druscilla Shirley 1978

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THE DIGESTIVE TRACT OF A HARPACTICOID COPEPOD, T i g r i o p u s c a l i f o r n i c u s . A LIGHT AND ELECTRON MICROSCOPE STUDY. By DRUSCILLA SHIRLEY SULLIVAN B.Sc. (Hons.) , U n i v e r s i t y o f B.C., 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER, 1977 (~c) DRUSCILLA SHIRLEY SULLIVAN i n the Department of Botany In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements f o r an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or p u b l i c a t i o n of th i s thesis fo r f inanc ia l gain sha l l not be allowed without my written permission. Department of BOTANY The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date DECEMBER 5. 1977. i Abstract A study on the digestive tract of a harpacticoid copepod, 2i3lio£Ss c a l i f o r n i c u s , was c a r r i e d out using techniques of l i g h t and electron microscopy. I t was found that a curved, c u t i c u l i z e d esophagus extends from the ventral mouth to the midgut. I t s musculature and shape allows f a i r l y large food p a r t i c l e s to enter the gut. The noncuticulized portion of the digestive tract consists of; 1. A single, anterior, spherical midgut caecum, 2. An anterior midgut extending from the midgut caecum to the j o i n t at the beginning of the urosome, 3. A posterior midgut extending almost the length of the urosome. The c u t i c u l i z e d hindgut can be divided, s t r u c t u r a l l y , into anterior and posterior regions. I t i s suggested that the anterior hindgut functions in ion and water regulation as well as begins the formation of a f a e c a l p e l l e t . The posterior hindgut compacts the faecal p e l l e t and retains i t u n t i l defaecation. At the l i g h t and electron microscope l e v e l s four c e l l types could be distinguished. By studying the c e l l ' s position i n the gut, electron density, amount of l i p i d , amount and type of vesiculation and the abundance and p o s i t i o n of the c e l l * s organelles, functions f o r these c e l l s were determined: 1. c e l l type one i s an embryonic c e l l which w i l l replace c e l l s worn away or l o s t in secretion. 2. C e l l type two functions mainly i n the synthesis and secretion of proteins and also plays a r o l e i n l i p i d absorption. 3. C e l l type three appears to function mainly in l i p i d absorption. 4. C e l l type four also functions in l i p i d absorption but this c e l l i s only found i n the anterior midgut i i and the type of v e s i c l e s found i n t h i s c e l l suggest a d i f f e r e n t type of a b s o r p t i o n i s o c c u r r i n g than i n c e l l type t h r e e . From the abundance of each c e l l type, the l e n g t h o f the m i c r o v i l l i , the development of the b a s a l lamina and l u m i n a l p r o j e c t i o n s , the f o l l o w i n g c o n c l u s i o n s were made: 1. The midgut caecum f u n c t i o n s mainly f o r 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 . 2. The a n t e r i o r midgut a l s o f u n c t i o n s f o r n u t r i e n t a b s o r p t i o n but p l a y s a more important r o l e i n merocrine and e x o c r i n e s e c r e t i o n . The presence of c o n c r e t i o n s i n c e l l types two and three o f the a n t e r i o r midgut suggest a r o l e i n e x c r e t i o n , water or i o n r e g u l a t i o n . 3. The p o s t e r i o r midgut f u n c t i o n s mainly i n a b s o r p t i o n , though some h o l o c r i n e s e c r e t i o n i s e v i d e n t . i i i TABLE OF CONTENTS Abs t r a c t ................................................. i TABLE OF CONTENTS . . i i i LIST OF FIGURES ............................. v. .... V , , V ACKNOWLEDGEMENTS v i i INTRODUCTION .... . . . . 1 MATERIALS AND METHODS , 9 RESULTS . . ......... ... . ............ ........ ............... 12 Esophagus 12 Midgut Caecum 14 A n t e r i o r Midgut ......................................... 19 P o s t e r i o r Midgut 22 Hindgut ......................... ....................... 25 A n t e r i o r Hindgut ....................................... 26 P o s t e r i o r Hindgut ...................................... 26 DISCUSSION . ... . .... .................. ........... 28 Esophagus 28 Musculature .... ............... 30 B a s a l Lamina 33 C e l l Type One . ......................................... 34 C e l l Type Two .......................................... 36 C e l l Type Three ........................................ 38 Midgut Caecum ............ ..... . . ....................... 41 A n t e r i o r Midgut 43 P o s t e r i o r Midgut ....................................... 49 P e r i t r o p h i c Membrane ................................... 52 S e c r e t i o n .............................. ...... ............. 54 Hindgut KEY FOE FIGURES FIGURES TABLE ONE LITERATURE CITED V LIST OF FIGURES Figure 1a. Scanning electron micrograph of an adult male la. c a l i f o r n i c u s . ..................... . . ....... 6 5b Figure 1b. Scanning electron micrograph of an adult female T. c a l i f o r n i c u s and her egg sac. .............. , 66b Figure 1c. Whole mount of male and female Tigriopus c a l i f o r n i c u s . ................................. 67b Figure 2. S a g i t t a l section of T. californicus.. 67b Figure 3. Transverse section through the esophagus. ...... 68b Figure 4. Transverse section through the midgut caecum. .. , 68b Figure 5. Esophagus projecting into anterior midgut. ..... 68b Figure 6. Esophagus entering midgut and becoming continuous with i t 68b Figure 7. Light micrograph of the posterior midgut. ...... 69b Figure 8. Anterior hindgut lacking c u t i c l e . ............., 69b Figure 9. S a g i t t a l section showing the t r a n s i t i o n from anterior hindgut to posterior hindgut. ........ 69b Figure 10. Dilated posterior hindgut. 69b Figure 11. S a g i t t a l section showing the entrance of the esophagus i n t o the midgut. .................... 70b Figure 12. Light micrograph of c e l l type one. ............ 70b Figure 13. Light micrograph of c e l l types one, two and three. 70b Figure 14. C e l l type two being extruded into the lumen. ., 70b Figure 15. Micrograph of a s a g i t t a l section of the posterior midgut. . ................... .... ............... 70b Figure 16. Low powered electron micrograph of esophagus. . 71b vi Figure 17. Esophagus i n midgut lumen. .................... 71b Figure 18. Microtubules of esophagus 71b Figure 19. Electron micrograph of a portion of the midgut caecum. .......................... 72b Figure 20. Exocytosis i n c e l l type three. ................ 72b Figure 21. Basal lamina in midgut caecum 72b Figure 22. Extrusion of c e l l in midgut caecum. 72b Figure 23. Electron micrograph of c e l l type one. ......... 72b Figure 24. Electron micrograph of c e l l type two and three. 72b Figure 25. Secretion of c e l l type two i n anterior midgut. 73b Figure 26. Electron micrograph of anterior midgut. ....... 74b Figure 27. Multivesicular bodies of anterior midgut. ..... 75b Figure 28. Evaginations of anterior midgut. .............. 76b Figure 29. C e l l type four of anterior midgut. ............ 76b Figure 30.,Electron micrograph of the posterior midgut. 76b Figure 31. C e l l type two of posterior midgut. 76b Figure 32. P a r t i a l l y extruded c e l l of posterior midgut. .. 77b Figure 33. Gap l e f t by extruded c e l l i n posterior midgut. 77b Figure 34. Electron micrograph of anterior hindgut. ...... 78b Figure 35. Anterior region of posterior hindgut. 78b Figure 36. Posterior region of posterior hindgut. ........ 78b Figure 37. Electron micrograph of the anus region. ....... 78b Figure 38. High magnification micrograph of c u t i c l e at anus. ........................................,/ 78b v i i ACKNOWLEDGEMENTS I wish t o express g r a t i t u d e t o my s u p e r v i s o r Dr. T. B i s a l p u t r a f o r i n t r o d u c i n g me t o the e l e c t r o n microscope and f o r h i s v a l u a b l e s uggestions on technigues and i n t e r p r e t a t i o n s . Thanks are a l s o extended to Dr. A. G. Lewis f o r h i s h e l p f u l c r i t i c i s m and f o r s u p p l y i n g the copepod under study and t o Dr. I. I l l g f o r v a l i d a t i n g the i d e n t i f i c a t i o n of 2i2£iP£JJ§ c a l i f o r n i c u s . For i d e a s on my p r o j e c t and sugg e s t i o n s f o r follow-up p r o j e c t s I wish t o thank Dr. F. J . R. T a y l o r . To c l o s e f r i e n d s D. Walker, Dr. L. O l i v e i r a and J . K a l l e y I am indebted f o r t h e i r i d e a s on my p r o j e c t and su g g e s t i o n s concerning microscope technigues. With the encouragement, and a s s i s t a n c e of Tom S u l l i v a n my work on t h i s p r o j e c t was always enjoyable and r e l a x e d . , Summer support was s u p p l i e d by an NRC grant awarded t o Dr. T. B i s a l p u t r a . 1 INTRODUCTION Few studies, at both l i g h t and electron microscope l e v e l s , have examined the complete digestive t r a c t of any crustacean. This study, on the digestive tract of a harpacticoid copepod Tigriopus c a l i f o r n i e u s , i s meant to f i l l t h i s gap for marine i n t e r t i d a l copepods. , Comparisons of the digestive t r a c t s and t h e i r c e l l s in other arthropods are made in order to show their differences and s i m i l a r i t i e s . , By such comparisons, the functions suggested for the morphology of the gut regions and t h e i r c e l l s i n other arthropods may be related to T. c a l i f o r n i c u s ^ In studies of the arthropod digestive t r a c t confusion often arises over the differences between the terms diverticulum, caecum, digestive gland and hepatopancreas. k diverticulum i s generally defined as a blind-ending tubular or sac-like out-pushing from a cavity and thus i s a very general term. In t h i s study a caecum was defined as a diverticulum s p e c i f i c a l l y of the digestive tract and i s not extensively branched. Digestive d i v e r t i c u l a that are extensively branched are call e d digestive glands or hepatopancreas {Meglitsch 1972). Studies on other copepods, such as Calanus finmarchicus (Dakin 1908; Marshall arid Orr 1955) and Diarthrodes cysteocus (Fahrenbach 1961) suggested f i v e main regions of the gat: a muscular foregut {esophagus) l i n e d by c u t i c l e , three d i f f e r e n t i a t e d but not i s o l a t e d midgut regions including an anterior midgut diverticulum, and a c u t i c u l a r i z e d hindgut occupying the l a s t abdominal segment(s). Caligoid copepods have 2 a foregut, two anterior d i v e r t i c u l a and a midgut which ends at the junction between the cephalothorax and the fourth thoracic leg. Posterior to the midgut i s the hindgut (Lewis 1961). In branchiopods, Artemia sali n a was found to have a foregut, a midgut with two caeca and a hindgut (Hootman and Conte 1974). Schultz and Kennedy (1976) described the o v e r a l l shape and c e l l s of a Cladoceran (Paphnia p u l e x ) d i g e s t i v e t r a c t . Studies of various malacostracan orders show s i m i l a r i t i e s i n that they a l l have foregut and hindgut regions. However, the form of the midgut and the number and form of the midgut caecum varies. The isopod, Armadillidium vulgare, has a midgut i n the form of a hepatopancreas (Vernon, Herold and Hitkus 1974) while amphipods have a true midgut with an excretory caecum extending from the posterior region of the midgut (Shyamasundari and Rao 1976). Caridina l a e v i s x a shrimp studied by P i l l a i (1960), has three midgut regions with three blind anterior midgut caeca as well as a hepatopancreas. Few studies have been done on the arthropod foregut. Hurthy (1975) has studied the c u t i c l e of the cockroach (Periplaneta americana) foregut and Thomson and Holling (1976) have proposed a model to explain the 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 a c t i v i t i e s of the blowfly (Phormia r e t i n a l foregut. Knowledge of the phenomenon of a n t i p e r i s t a l s i s i n the arthropod gut i s extremely important to the understanding of where digestion, secretion and absorption of nutrients occurs and how they are related to each other. P e r i s t a l s i s occurs i n the esophagus, anterior midgut and hindgut while a n t i p e r i s t a l t i c movements occur i n the foregut and posterior midgut. , This 3 a l l o w s a b s o r p t i o n to occur i n the a n t e r i o r midgut and d i g e s t i o n i n the p o s t e r i o r midgut {Vonk 1960). The p e r m e a b i l i t y of the f o r e g u t c u t i c l e i s under q u e s t i o n . Yonge (1924) s t a t e d t h a t no a b s o r p t i o n occurs i n the esophagus. On the other hand, E i s n e r (1955) found f a t was absorbed i n the cockroach f o r e g u t and J o s h i and Agarwal (1977) found c h o l e s t e r o l was absorbed by the f o r e g u t of some omnivorous and c a r n i v o r o u s i n s e c t s . , Mucus and p o s s i b l y amylase are s e c r e t e d i n t h i s r e g i o n of the gut (Vonk 1960). E l e c t r o n microscope s t u d i e s on the midgut caecum of v a r i o u s Crustacea have suggested f u n c t i o n s f o r t h i s gut r e g i o n . Ong and Lake (1970), i n s t u d y i n g a c a l a n o i d copepod d e s c r i b e d the midgut caecum c e l l s and concluded t h a t they d i d not produce enzymes but churn and produce mucopolysaccharides as w e l l as absorb amino a c i d s . In v a r i o u s P e r a c a r i d a i t was found a b s o r p t i o n o c c u r s i n the midgut caecum (Donadey 1969) and M o r i t z , Storch and Buchheim (1973) suggested: 1. r e s o r p t i o n ^ 2. storage of l i p i d , 3. s t o r a g e of glycogen, and 4. s e c r e t i o n occur here. In a e u c a r i d , C a l l i n e c t e s sapidus, glycogen i s s t o r e d i n the "Midgut Glands" (Hinget, Rouse and Maurer 1977). The malaeostracan midgut s t r u c t u r e and c e l l components have been s t u d i e d and may be used to understand the f u n c t i o n s of the T. c a l i f o r n i c u s midgut. The midgut by d e f i n i t i o n i s of endodermal o r i g i n but Holdich (1973) suggested the isopod midgut i s ectodermal i n o r i g i n and t h e r e f o r e may not be a t r u e midgut. McMurrich (1897) a l s o s t u d i e d the midgut of t e r r e s t r i a l i s o p o d s . T r a n s p o r t of g l y c i n e and sodium a c r o s s the marine shrimp (Penaeus marginatus) midgut was s t u d i e d by Ahearn (1976) and van 4 Weel (1955) studied secretion, r e s t i t u t i o n and resorption i n the midgut glands of another decapod (fttya spinipes).- In the brown shrimp jPenacus aztecus) two types of midgut e p i t h e l i a were observed. There were dark c e l l s with abundant rough endoplasmic reticulum (E. R. ), ribosomes and mitochondria with a l i g h t matrix and there were l i g h t c e l l s (Talbot, Clark and Lawrence 1972). In malacostracans the hepatopancreas c a r r i e s on s i m i l a r functions as the midgut of T. c a l i f o r n i c u s . The hepatopancreas, functioning for food absorption, purine metabolism (Vonk 1960), secretion of digestive enzymes and storage of l i p i d s , glycogen and minerals has been studied i n malacostracans (Dorman 1928; Davis and Burnett 1964; Hartenstein 1964; Bunt 1968; L o i z z i 1968; Stanier, Boodhouse and G r i f f i n 1968; L o i z z i and Peterson 1969; Steves 1969; and Lawrence 1976). Schultz (1976), in a study of an amphipod (Gammarus minus) described: 1. l i g h t staining R-cells which had large numbers of l i p i d droplets and few golgi bodies; 2. F - c e l l s with many dilat e d golgi bodies and 3. large vacuolated B-cells with apocrine secretion. These c e l l s were also described by Reddy (1938) i n the crab (Paratelphusa hydrpdrpmus) and L o i z z i (1971) i n the c r a y f i s h . Hany studies have been c a r r i e d out on insect midguts and by looking at the c e l l types described, comparisons can be made between these c e l l s and those i n T.. californicus.. Differences between c e l l s and the kinds of c e l l s i n d i f f e r e n t regions of the gut suggest functions for these regions. Sud (1968) described the p r i n c i p a l and goblet c e l l s of insects i n general. In Calliphora erythrocephala (a f l y ) , de Priester (1971) described 5 columnar (absorptive) c e l l s , r e g e n e r a t i v e c e l l s and a t h i r d c e l l type which he suggested may be e n d o c r i n a l i n f u n c t i o n . The c e c r o p i a midgut has "Goblet c e l l s " , which are suggested t o f u n c t i o n f o r potassium t r a n s p o r t from haemolymph to the midgut lumen, and columnar c e l l s but l a c k r e g e n e r a t i v e c e l l s (Anderson and Harvey 1966). Flower and F i l s h i e (1976) s t u d i e d the membranes and j u n c t i o n s of "Goblet c e l l s " i n Lepidopteran l a r v a e and S c h u l t z and J u n g r e i s (1977), using scanning e l e c t r o n m i c r o s c o p i c techniques, t r i e d t o r e l a t e the c e l l ' s s t r u c t u r e to the f u n c t i o n of i o n t r a n s p o r t . , L i p o p h i l i c and c u p r o p h i l i c c e l l s were found i n the b l o w f l y l a r v a e midgut (Waterhouse and Wright 1960). T h i s copper-accumulating r e g i o n was s t u d i e d by F i l s h i e , Poulson and Waterhouse (1971) i n Drosophjla l a r v a e and by S o h a l , P e t e r s and H a l l (1977) i n the a d u l t h o u s e f l y . The s t r u c t u r e of a mosquito XCulex t a r s a l i s ) midgut has been d e s c r i b e d by Houk (1977) and d i f f e r e n c e s i n c e l l s between the 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 of male and female mosquitoes (JRudin and Hecker 1976) and sugar cane b e e t l e s {P r o t a e t i a acumina ta) (Cheung and Low 1975) were s t u d i e d . In c a l i g o i d copepods two c e l l t y p e s were found. A non-:vacuolated, a b s o r p t i v e c e l l of a squamous s o r t i s found i n the p o s t e r i o r midgut while a columnar c e l l i s found i n the a n t e r i o r midgut (Lewis 1961). The vacuolated, s e c r e t o r y type c e l l s are squamous a f t e r undergoing merocrine s e c r e t i o n (Lewis 1961) . A d r a g o n f l y JAeshna cyanea) has s p e c i a l i z e d smooth E. R. i n i t s midgut c e l l a p i c e s and they are suggested t o f u n c t i o n i n t h e r e s y n t h e s i s of t r i g l y c e r i d e s (Andries 1977) . Midgut musculature i n JDrosofihiia was s t u d i e d by Gartner 6 ( 1 9 7 6 ) . M o r i ( 1 9 6 9 , 1976) d e s c r i b e d " t h e f o r m a t i o n o f t h e v i s c e r a l m u s c u l a t u r e and o r i g i n o f t h e m i d g u t e p i t h e l i u m i n a H e m i p t e r a ( G e r r i s P a l l i d u m i n s u l a r i s ) . A d e v e l o p m e n t a l a n d c y t o l o g i c a l s t u d y o f t h e f l e s h f l y ( S a r c o p h a g a b u l l a t a ) m i d g u t was u n d e r t a k e n by N o p a n i t a y a and M i s c h ( 1 9 7 4 ) . The t y p e s o f s e c r e t o r y p r o c e s s e s a r e e x p l i c i t l y d e f i n e d by R h o d i n (1974) and K u r o s u m i ( 1 9 6 1 ) . R h o d i n d e f i n e s a p o c r i n e s e c r e t i o n a s s e c r e t i o n where a " d r o p l e t l i f t s t h e c e l l s u r f a c e t o g e t h e r w i t h a r i m o f s u r r o u n d i n g c y t o p l a s m " . The r u p t u r e d membrane i s mended and t h e c e l l i s n o t l o s t . M e r o c r i n e s e c r e t i o n (a synonym f o r e x o c y t o s i s ) , - a c c o r d i n g t o R h o d i n ( 1 9 7 4 ) , i s where t h e " b o u n d a r y membrane o f t h e s e c r e t o r y g r a n u l e f u s e s w i t h t h e c e l l membrane and t h e c o n t e n t s o f t h e g r a n u l e a r e d i s c h a r g e d i n t o t h e l u m e n " . I n h o l o c r i n e s e c r e t i o n c e l l o r g a n e l l e s , i n c l u d i n g t h e n u c l e u s , d i s i n t e g r a t e c h a n g i n g t h e e n t i r e c e l l i n t o a h u g e s e c r e t o r y s u b s t a n c e w h i c h i s e x t r u d e d i n t o t o ( A l i k h a n 1 9 6 9 ) . K u r o s u m i ' s (1961) d e f i n i t i o n s o f t h e s e p r o c e s s e s a r e s i m i l a r b u t i n c l u d e s u b c a t e g o r i e s . A p o c r i n e a n d •'ec'crine' ( e x o c y t o s i s ) s e c r e t i o n a r e c o n s i d e r e d t o be u n d e r a s i n g l e l i g h t m i c r o s c o p i c c a t e g o r y o f m e r o c r i n e s e c r e t i o n . A p o c r i n e s e c r e t i o n i s d i v i d e d i n t o macro- and m i c r o - a p o c r i n e s e c r e t i o n d e p e n d i n g o n t h e q u a n t i t y o f m a t e r i a l ' d e c a p i t a t e d ' f r o m t h e c e l l . E c c r i n e s e c r e t i o n i n c l u d e s p r o c e s s e s b o t h v i s i b l e and n o n - v i s i b l e 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 . The l a t t e r i s t h o u g h t t o be a m o l e c u l a r f o r m o f s e c r e t i o n t h r o u g h t h e i n t a c t p l a s m a membrane. I n v a r i o u s a r t h r o p o d s , d e s c r i p t i o n s a n d f u n c t i o n s f o r t h e c u t i c u l a r i z e d h i n d g u t have b e e n g i v e n . . L i p i d , t r i o l e i n and 7 disaccharides are absorbed by the cut i c u l a r i z e d anal v e s i c l e of a braconid wasp ( H i c r o p l i t i s croceipes) (Edson and Vinson 1977). The blowfly j C a l l i p h o r a erythrocephala) rectum was described by Gupta and Berridge (1966) ..„• They described three c e l l types: r e c t a l , junctional and c o r t i c a l c e l l s . The c o r t i c a l c e l l i s suggested to function i n ion and water transport. The r e c t a l pads of the cockroach (Periplaneta americana) have also been described and a s i m i l a r system of solute and non-solute-coupled water transport suggested (Oschman and Wall 1969). In Ouabain-sensitive enzyme (Tolman and Steele 1976) and c e l l u l a r junctions (Hoirot and Noirot-Timothee 1976) have been implicated i n t h i s process. In crustacea p e r i s t a l t i c movements continually draw water into the gut v i a the mouth and a n t i - p e r i s t a l t i c movements continually draw water into the gut via the anus. These processes, their function and means of eliminating the excess water accumulated, have been described by Fox (1952). Fox (1952) suggested that the functions for such water intake are: 1. acts as an enema for defaecation and 2. stretches gut walls to i n i t i a t e a n t i p e r i s t a l t i c muscle contractions. Excess water i s passed through the gut wall into the blood and out of the body. Using radioisotopes. B a l l (1967) experimented with t h i s system and concluded that water uptake and s a l t excretion take place in the gut of hypo-osmoregulating crustacea. , The hindgut of a t e r r e s t r i a l isopod <&rmadillidium vulgare) was described (Vernon, Herold and Witkus 1974) and confirms an e a r l i e r study on another t e r r e s t r i a l isopod JOniscus ascellus) in which microtubule bundles were reported. It was suggested t h e i r role 8 was in orientating osmoregulatory c e l l s of the hindgut (Witkus, G r i l l o and Smith 1969). Studies on the enzymes of Crustacea have shown the presence of lipases {Eisner 1955; G i l b e r t and O'Connor 1970), esterases, amylase, maltase {Alikhan 1969) and saccharase as well as protein digesting enzymes such as proteinase, carboxypeptidase, aminopeptidase and peptidase {Bond 1934; Vonk 1960). Microorganisms i n the hindgut have been - suggested to supply digestive enzymes fo r c e l l u l o s e and hemicellulose digestion i n the american cockroach (Bignell 1977). In Crustacea pH differences are known to occur i n d i f f e r e n t regions of the digestive tract and t h i s a f f e c t s the a c t i v i t y of various enzymes (e.g. Sinha 1975-invertase a c t i v i t y in a f l y ) . The purpose of t h i s study was to describe, at the l i g h t and electron microscope l e v e l s , the digestive t r a c t of a harpacticoid copepod, Tiqriopus c a l i f o r n i c u s . From such a description, and comparisons with s i m i l a r r e s u l t s i n other arthropods, i t i s hoped that functional r e l a t i o n s h i p s between the c e l l s and regions of the gut w i l l become evident. By studying the processes of secretion, digestion and absorption i n T.. c a l i f o r n i c u s . s i m i l a r processes i n other crustacea w i l l be better understood. By using T. c a l i f o r n i c u s . an i n t e r t i d a l marine copepod, i t may be possible to understand and predict the e f f e c t s disturbances in the marine environment would have on marine food webs. 9 MATERIALS AND METHODS Tigriojgus 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. The s p e c i e s i d e n t i t y was confirmed by Dr. P. L. I l l g <0. of Washington). The copepods were maintained i n sea water at room temperature i n the l a b o r a t o r y under f l o r e s c e n t l i g h t c o n d i t i o n s . G o l d f i s h food and mixed a l g a l c u l t u r e s were s u p p l i e d as food and the sea water was changed approximately every t h r e e months. Once a good b a c t e r i a l c u l t u r e was developed i n the c u l t u r e d i s h e s , continued f e e d i n g was no longer necessary.. The m a t e r i a l was prepared f o r l i g h t microscope study i n the f o l l o w i n g manner: A 2-hour primary f i x a t i o n was c a r r i e d out e i t h e r i n 5% g l u t a r a l d e h y d e o r 555 g l u t a r a l d e h y d e and 5% paraformaldehyde i n 0.2H phosphate b u f f e r (pH 7.3). A f t e r washing i n the b u f f e r , t h e copepods were p o s t - f i x e d i n 2% osmium t e t r o x i d e i n phosphate b u f f e r f o r a minimum of f i v e hours, f o l l o w e d by r i n s i n g i n b u f f e r and d i s t i l l e d water. The specimens were then s t a i n e d i n 555 u r a n y l a c e t a t e i n d i s t i l l e d wafer. A f t e r washing, and a p p r o p r i a t e dehydration they were embedded i n e i t h e r Spurr*s or J.B. ,4 embedding media (P o l y s c i e n c e , Warrington, P e n n s y l v a n i a ) . Specimen b l o c k s were trimmed and 1- t o 2-micron s e c t i o n s were cut using a B e i c h e r t OM03 Oltramicrotome or a Du Pont-S o r v a l JB4 microtome. S e c t i o n s were f l o a t e d onto an e t h a n o l -d i s t i l l e d water s o l u t i o n on a cleaned microscope s l i d e . The s l i d e was then heat d r i e d and the s e c t i o n s s t a i n e d with T o l u i d i n e Blue or A n i l i n e Blue Black. S e c t i o n s were observed 10 under the Zeiss Photomicroscope. The d i f f i c u l t y i n preparing organisms such as small copepods for electron microscopic study i s perhaps responsible for the r e l a t i v e lack of research i n t h i s area. As experienced by Rigdon and Mensik (1976) i n the brown shrimp, Penaeus aztecus, post-mortem degeneration occurs r a p i d l y along the digestive t r a c t . The basic problem i n f i x i n g and subsequent processing steps of such tissues i s the r e l a t i v e impermeability of the c u t i c l e . In preparations for electron microscope study, paraformaldehyde was found to reduce post-mortem changes i n the tissue, owing, perhaps, to i t s comparatively fast penetration through the c u t i c l e . In t h i s work 3% paraformaldehyde was used in conjunction with buffered glutaraldehyde (3%) as the primary f i x a t i v e . Secondary f i x a t i o n , as in the preparation for l i g h t microscope sections, was i n 2% osmium tetroxide i n phosphate buffer. To ensure dehydration of the tissue a prolonged dehydration i n alcohol was used (30-60 minutes exposure f o r each step of graded alcohol solution) followed by a prolonged bath i n propylene oxide. To compensate for the slow penetration of embedding resin through t h i s formidable c u t i c l e b a r r i e r , a 7 to 10 day i n f i l t r a t i o n period was allowed. The polymerized blocks were trimmed and sectioned on a Reichert 0MD3 Oltramicrotome with a Du-Pont diamond knife. The sections were mounted on C o l l o i d i o n or Formvar coated copper grids and stained, f i r s t i n a saturated solution of Oranyl Acetate for 30 minutes followed by 10 minutes in lead c i t r a t e (Reynolds 1963). , The specimens were observed on a Zeiss EM9S 11 e l e c t r o n microscope. Pr e p a r a t i o n of m a t e r i a l f o r the scanning e l e c t r o n microscope i n v o l v e d f i x i n g the t i s s u e f o r one hour i n 2% g l u t a r a l d e h y d e i n c a c d d y l a t e b u f f e r . A f t e r washing, the copepods were f r e e z e d r i e d and mounted on aluminium stubs by Pe l c o C o l l o i d a l m e t a l i c p a i n t . 12 'RESULTS The scanning electron micrographs of adult male and female Tigriopus c a l i f o r n j c u s copepods (Figures 1a and 1b) show the copepod's body shape. A mating pair of T. c a l i f o r n i c u s i s shown in the l i g h t micrograph (Figure 1c). A longitudinal section of the digestive tract of an adult female T. c a l i f o r n i c u s i s i l l u s t r a t e d in i t s entirety i n Figure 2. On a morphological basis the digestive t r a c t s h a l l be subdivided into the esophagus, midgut caecum, anterior midgut, posterior midgut and two regions of the hindgut. The following descriptions are r e f e r r i n g to the general adult T^ c a l i f o r n i c u s copepod. Esophagus The c u t i c u l a r esophagus l i e s c e n t r a l l y i n the anterior quarter of the cephalothorax, approximately 100um from the rostrum. From the mouth at i t s anterior ventral end, the tubular esophagus curves s l i q h t l y i n an anterior d i r e c t i o n . ,• Dahl (1956) also observed t h i s form of esophaqus in other copepods. In T. c a l i f o r n i c u s the ventral esophaqus has two large setae projecting into dips i n the esophaqus c u t i c l e . The esophaqus opens into the midqut at the junction of the anterior midqut and the midqut caecum (Fiqures 6 and 11). In cross section the lumen of the esophaqus frequently takes the form of an nH", dependinq upon the s i z e and quantity of food that may pass to the midqut via the esophagus (Fiqure 3). Food i n the ventral esophaqus lumen i s coarcer r e l a t i v e to that in the dorsal esophagus lumen* I t i s apparent that t h i s »*HM form r e f l e c t s the a b i l i t y of the esophaqus to enlarqe and c o n s t r i c t 13 fr e e l y when the need ar i s e s . The lumen of the esophagus i s enclosed in three layers. A d i s t i n c t c u t i c l e separates the esophagus lumen from the nucleated, homogeneously dense, e p i t h e l i a l c e l l s 5-15um t a l l . These c e l l s are themselves bound by a c i r c u l a r muscle layer of approximately 10um i n thickness. Longitudinal musculature l i e s in l a t e r a l association with the esophagus. , The c u t i c l e i s made up of three layers. The e p i c u t i c l e i s a very thin (0.02um) layer of electron dense material forming a smooth, unbroken margin between the lumen and the inner c u t i c l e layers. The middle layer i s less dense and of uniform thickness <0.2um) and the innermost c u t i c l e layer, which varies greatly i n width, i s of low electron density. The e p i t h e l i a l c e l l s are bound by a plasma membrane which makes deep invaginations, both at the basal regions of the c e l l s as well as at the c u t i c u l a r regions of the c e l l s . Each c e l l contains an often i r r e g u l a r l y shaped nucleus <0.7um diameter) with an electron dense c i r c u l a r and central n u c l e o l i (0.2um diameter)., There i s l i t t l e dispersed heterochromatin. Mitochondria are f a i r l y abundant, especially i n the region where the esophagus enters the midgut. The electron dense mitochondria are mainly c i r c u l a r or s l i g h t l y oblong i n p r o f i l e with a diameter of about O.&ura. The number of c r i s t a e , per se c t i o n a l view, varies but i s f a i r l y low. L i t t l e endoplasmic reticulum (E. B.,) i s found but ribosomes and metabolites are abundant throughout the cytoplasm and give i t a dense appearance (Figure 16). Frequently, multivesiculate electron dense bodies are evident, especially where the esophaqus occurs i n the qut 14 lumen. M i c r o t u b u l e - l i k e s t r u c t u r e s (0.02um diameter) extend i n many d i r e c t i o n s p a r a l l e l to the esophagus c u t i c l e l i n i n g the lumen (Figure 18). D i r e c t l y apposed t o the e p i t h e l i u m i s a l a y e r of c i r c u l a r muscle wi t h l o n g i t u d i n a l muscle extending between the d o r s a l and v e n t r a l arms of the esophagus. The k, H, I and Z bands of the c i r c u l a r muscle sarcomers (averaging 2.5um long) resemble those of v e r t e b r a t e s k e l e t a l muscles. Once the esophagus has reached the j u n c t i o n between the a n t e r i o r midgut and the midgut caecum, i t protrudes i n t o the gut lumen. At t h i s p o i n t the esophagus i s no l o n g e r ensheathed by c i r c u l a r muscle and t h e e p i t h e l i a l t i s s u e i s drawn away from the c u t i c l e (Figure 17). The o r g a n e l l e s of the e p i t h e l i a l t i s s u e are e s s e n t i a l l y the same as before they e n t e r the midgut lumen, except f o r an i n c r e a s e i n u n i d e n t i f i e d , e l e c t r o n dense, m u l t i -v e s i c u l a r b odies. At the p o i n t where t h e esophagus has penetrated the midgut lumen, the c u t i c l e l i n i n g the esophagus lumen i s continuous with the c u t i c l e l i n i n g the outer perimeter of the esophagus. Where the esophagus i s continuous with the midgut the c u t i c l e i s r e p l a c e d by m i c r o v i l l i . Although there were no s i g n s o f s e c r e t i o n , some d i g e s t i o n had occurred i n the lumen of t h e esophagus as c o l l o i d a l m a t e r i a l was f r e g u e n t l y seen. Midgut Caecum The midgut caecum i s a s p h e r i c a l chamber averaging 40um i n diameter ( F i g u r e s 4 and 5). I t l i e s a n t e r i o r t o , and above the esophagus a t approximately 15um from the a n t e r i o r t i p of the copepod. The caecum i s 20-25um beneath the d o r s a l body s u r f a c e and i s l o c a t e d at a c o n s t r i c t i o n forming the j u n c t i o n between 15 the esophagus and the anterior midgut. The c o n s t r i c t i o n i s approximately 60-70um below the dorsal surface of the animal. The columnar c e l l s of the constricted region are among the largest c e l l s l i n i n g the digestive tract and, as throughout the whole midgut, plasma membrane t i g h t junctions (Zonula occludens) occured at c e l l apices. The caecum e p i t h e l i a l c e l l s appear to be pseudostratified columnar c e l l s and contain many ve s i c l e s at thei r apical ends (Figure 4). The nuclei are located i n the middle or basal regions of the c e l l s . . Even at the l i g h t microscope l e v e l m i c r o v i l l i form a d i s t i n c t brush border over the entire region (Figures 4 to 6). Deep furrows and spherical c a v i t i e s suggest secretory a c t i v i t i e s of large vacuoles along these surfaces. The e p i t h e l i a l c e l l s of the caecum are columnar with an average height of 7.6um (Figure 19). At the base of the c e l l s there are numerous i n t e r d i g i t a t i o n s of the plasma membrane (Figure 21). Although these together with the basal lamina may extend into the cytoplasm up to one third of the height of the c e l l they more commonly penetrate only 0.6uinto the cytoplasm of the c e l l . These basal lamina invaginations can, occasionally, be seen to pinch off the basal region of a c e l l . In these regions the basal lamina forms a concentric ring around the basal region of the c e l l almost i s o l a t i n g i t from the rest of the c e l l and midgut caecum. I t i s i n these i r r e g u l a r invaginations that longitudinal muscle i s most obvious. No c i r c u l a r muscle was seen to surround t h i s region of the midgut although the basal lamina may appear, i n some regions, to be a form of muscle. I t i s most evident (averaging 0.2um wide) i n 1 6 r e g i o n s where only minor i n v a g i n a t i o n s occur., The mitochondria are f a i r l y s c a t t e r e d throughout the c e l l s with a p o s s i b l e i n c r e a s e i n numbers a t the apex and base. They are r a t h e r s m a l l <0.6um diameter) and c i r c u l a r i n p r o f i l e and possess a v a r i a b l e number of l a m e l l a t e c r i s t a e (two t o many with a predominace of 8 c r i s t a e per m i t o c h o n d r i a l s e c t i o n ) which extend the l e n g t h of the o r g a n e l l e . The m i t o c h o n d r i a l matrix i s homogeneously dense so t h a t c r i s t a l membranes may become obscured. Ribosoraes are o f t e n a s s o c i a t e d w i t h E. R. The l a t t e r i s e x t e n s i v e l y developed i n most c e l l s and, along with f r e e polysomes, tends to aggregate b a s a l l y and along n u c l e a r and plasma membranes. The l a r g e c i r c u l a r or s l i g h t l y convoluted n u c l e i l i e c e n t r a l l y or b a s a l l y and average 3um i n diameter. They c o n t a i n s c a t t e r e d areas of heterochromatin and/or d i s t i n c t n u c l e o l i (1ura diameter). The pars amorpha and nucleonema o f the n u c l e o l u s are f r e q u e n t l y e v i d e n t at higher m a g n i f i c a t i o n s . The m i c r o v i l l i i n t h i s r e g i o n of the midgut are 2um long (Figure 19). They form a comparatively compact row l a c k i n g a d e f i n i t e p e r i t r o p h i c membrane while having a 1 membrane* resembling a g l y c o c a l y x l y i n g p a r a l l e l and between the m i c r o v i l l i . O c c a s i o n a l l y there are r e g i o n s where the m i c r o v i l l i have been pushed a s i d e and s m a l l gaps occur. The m i c r o v i l l i , i n c r o s s s e c t i o n , appear to c o n t a i n a minimum of 6 m i c r o f i b r i l s which can a l s o be seen extending the l e n g t h of the m i c r o v i l l i when cut l o n q i t u d i n a l l y . A l o n q i t u d i n a l s e c t i o n a l s o shows an e l e c t r o n dense r e q i o n a t the t i p of the m i c r o v i l l i although no t e r m i n a l web at the apex of the c e l l c o u l d be observed. , 17 At the u l t r a s t r u c t u r a l l e v e l d i f f e r e n c e s between c e l l types are even more s t r i k i n g than a t the l i g h t microscope l e v e l . C e l l type one i s seen to l i e a t the base of the gut e p i t h e l i u m i n c l u s t e r s of t h r e e o r more c e l l s ( F i g u r e 12 and 23). In a c r o s s s e c t i o n of the midgut caecum an average of three of these c l u s t e r s i s e v i d e n t . , B a s a l l a m i n a l i n d e n t a t i o n s o f t e n i n v o l v e d the c e l l s adjacent to t h i s c e l l type but seldom a c t u a l l y penetrated the cytoplasm of c e l l type one. The c e n t r a l n u c l e u s , with i t s s i n g l e or o c c a s i o n a l l y double n u c l e o l u s , i s o n l y s l i g h t l y l a r g e r than the average nucleus of t h i s r e g i o n . Some mitochondria , rough E. R. .,, and g o l g i are randomly d i s t r i b u t e d i n the cytoplasm of these c e l l s . The c y t o p l s m i c d e n s i t y of the c e l l s seems to be mainly a r e s u l t of l o o s e ribosomes. Some v e s i c l e s , averaging 0.Sum diameter, are present i n these c e l l s . Host of these v e s i c l e s , though membrane bound, were e l e c t r o n t r a n s p a r a n t . O c c a s i o n a l l y a c e l l c o n t a i n e d v e s i c l e s with an e l e c t r o n dense c o r e . These v e s i c l e s were 0.2u® i n diameter. L i p i d i n c l u s i o n s were not observed i n these c e l l s and t h e c e l l s were never observed t o reach the lumen. In the midgut caecum, approximately one c e l l i n seven i s c e l l type two (F i g u r e s 19 and 24 ) . T h i s c e l l type has r e g u l a r i n v a g i n a t i o n s of the b a s a l lamina and has a n u c l e o l a t e d c e n t r a l nucleus. The randomly d i s p e r s e d mitochondria are s l i g h t l y more abundant than i n c e l l type one. Rough E. R. and l o o s e ribosomes make the cytoplasm appear extremely dense. The g o l g i bodies are u s u a l l y so d i l a t e d t h a t i n d i v i d u a l c i s t e r n a e can seldom be d i s c e r n e d . These g o l g i are abundant throughout the cytoplasm and are i n t i m a t e l y a s s o c i a t e d with v e s i c l e s (averaging 18 0.07 um diameter) which contain a core of dense material and can be seen to fuse with each other. Shen the vesicles become larger (0.3um diameter) the density of their contents becomes greatly reduced u n t i l eventually they appear as large (1.0um) electron opague vesicles. These vesicles have been seen to undergo exocytosis (Figure 20). From the sections taken, i t appears t h i s c e l l type i s pyramidal in i t s three dimensional shape. The c e l l s are usually wider at the base with a pointed apex or wider at the apex with a smaller base. In a l l c e l l s of this type there was a portion of the c e l l reaching the lumen and having m i c r o v i l l i . Small l i p i d vesicles were occasionally seen at the very apex of the c e l l . In addition to exocytosis, mentioned above, holocrine or merocrine secretion was observed at the l i g h t microscope and electron microscope l e v e l (Figures 14 and 22). Cytoplasmic masses of various s i z e s containing i d e n t i f i a b l e membrane bound organelles were frequently seen i n the lumen of the digestive t r a c t . Through s e r i a l sections these masses could be traced to c e l l type two. After t h i s secretion process, the density of these c e l l s was greatly reduced. C e l l type three, the most prevalent c e l l type, has the ultra-structure of a t y p i c a l exocrine c e l l (Figures 19 and 24). The basement membrane i s invaginated, the nucleus i s generally basal and i s surrounded by varyinq amounts of lamellate rough E. R. Electron micrographs of t h i s c e l l type show more portions of randomly di s t r i b u t e d mitochondria than i n the other two c e l l types. Golgi bodies are t y p i c a l l y located i n association with the nucleus and v e s i c l e s of a l l sizes (0.2-2.Oum) are seen i n 19 the a p i c a l h a l f of the c e l l . Some of the l a r g e r v e s i c l e s may c o n t a i n evenly d i s t r i b u t e d e l e c t r o n t r a n s l u c e n t or opague p a r t i c u l a t e m a t e r i a l . Fusion between v e s i c l e s i s a common occurrence. Hany f a i r l y s m a l l v e s i c l e s at the apex can be seen f u s i n g t o the plasma membrane i n the r e g i o n s between m i c r o v i l l i (Figure 20). L i p i d d r o p l e t s were a l s o seen a t the apex of these c e l l s . , Interior Midcjut The a n t e r i o r midgut extends approximately 350um beyond the j u n c t i o n of the esophagus with the midgut caecum and ends a t the second c o n s t r i c t i o n o f the d i g e s t i v e t r a c t at the p o s i t i o n of the t h i r d p a i r of swimming l e g s (pereiopods). T h i s r e g i o n of the gut l i e s c o n s i s t e n t l y 50-70um below the d o r s a l s u r f a c e and 40-50um from the v e n t r a l s u r f a c e of the copepod body. I t s lumen averages 65um i n h e i g h t but i n c r e a s e s i n c r o s s s e c t i o n a l width from 60 to 150um towards the p o s t e r i o r end ( F i g u r e 5). The p s e u d o s t r a t i f l e d c u b o i d a l c e l l s of the extremely convoluted a n t e r i o r midgut average 8um i n height and have a b a s a l lamina, as d e s c r i b e d f o r the caecum, which may extend 0.6um i n t o the cytoplasm o f the e p i t h e l i a l c e l l bases (Figure 25). In the a n t e r i o r midgut the number of s e m i - c i r c u l a r e v a g i n a t i o n s from the b a s a l r e g i o n s of the c e l l s has g r e a t l y i n c r e a s e d when compared to the midgut caecum. As i n the caecum each e v a g i n a t i o n has l o n g i t u d i n a l muscle a s s o c i a t e d with i t . C i r c u l a r muscle i s more p r e v a l e n t i n the a n t e r i o r midgut but i s s t i l l not continuous. Each c e l l has a r e l a t i v e l y c e n t r a l oblong nucleus (2.Sum by 1.5um) c o n t a i n i n g a c e n t r a l n u c l e o l u s and/or s c a t t e r e d heterochromatin. , Mitochondria i n these c e l l s appear to be evenly distributed throughout the c e l l s . They are f a i r l y small (0.2um diameter) and c i r c u l a r or s l i g h t l y elongated i n shape and contain well developed p a r a l l e l c r i s t a e . The three c e l l types of the midgut caecum are present, though less extreme i n cytoplasmic density differences, and there i s possible evidence for a fourth c e l l type (Figures 25, 26 and 29). C e l l type one i s just as prevalent i n the anterior midgut as i n the midgut caecum and maintains the same ul t r a s t r u c t u r e (Figure 28). C e l l type two appears to have even more rough E. R. in t h i s region of the midgut than the caecum (Figure 26). Most of t h i s rough E. R. i s i n the basal two-thirds of the c e l l , being so compact in the basal half of the c e l l as to v i r t u a l l y exclude a l l other organelles. At the c e l l apex the c i s t e r n a l rough E. R. / r t y p i c a l of the base, has become less continuous and l e s s well defined. The golgi bodies of t h i s c e l l type, as i n the caecum, are abundantly d i s t r i b u t e d throughout the c e l l and are not necessarily associated with the nucleus. The large golgi bodies are composed of many dil a t e d cisternae completely surrounded by small (O.Oftum diameter) v e s i c l e s . Most of the golgi-associated vesicles are s l i g h t l y electron opague or transparent. These vesicles become larger (O.ftum diameter) and are often seen to have membrane-like structures as i n c l u s i o n s . The occasional golgi body i s associated with v e s i c l e s which are more electron dense and have a very electron dense central core. These vesicles were described i n the midgut caecum and, as i n the caecum, they become larger and the core becomes less electron dense as they progress to the c e l l apex. Another type 21 of v e s i c l e not observed i n the caecum i s f a i r l y electron dense and contains a c l u s t e r of very electron dense bodies. These bodies sometimes appear to be composed of, or surrounded by, membranes. Holocrine or merocrine secretion of the a p i c a l third of these c e l l s occurs i n t h i s region of the gut as well as i n the caecum (Figure 25). These c e l l s have numerous l i p i d - l i k e , homogeneously electron opaque, droplets 0.1-0.3um i n diameter. C e l l type three has a much smaller supply of rough E. R. which i s more evenly distributed throughout the c e i l (Figures 26 and 28). I t s broken and dispersed nature, as well as the presence of c i r c u l a r cross sections of rough E. R. , suggest that at least some of the rough E. R. i s tubular. Though s t i l l d i l a t e d , the g o l g i bodies are noticeably smaller and fewer i n number than i n c e l l type two of t h i s region of the midgut. The electron transparent v e s i c l e s of these c e l l s are of comparable size and number as in c e l l type two and also contain loose membrane structures as inclusions. Vesicles with electron dense cores, c h a r a c t e r i s t i c of c e l l type two, were e s s e n t i a l l y absent from these c e l l s . L i p i d droplets were observed at the c e l l apices. The cytoplasm of the fourth c e l l type, with i t s basal nucleus, i s s i m i l a r i n electron density to c e l l type three but d i f f e r s from t h i s c e l l type i n a variety of ways (Figures 25, 26 and 29). Rough E. R. i n these c e l l s i s even more broken up and rare. The golgi bodies of these c e l l s are smaller and s l i g h t l y less dilated than i n c e l l type three. Electron transparent vesic l e s of s i m i l a r size and number as i n c e l l type three occur and are evenly di s t r i b u t e d however, c e l l type four i s characterized by another type of vesi c l e . This c e l l type has an extremely abundant supply of small vesicles {0.1-0.4um diameter) containing material of various degrees of electron density. These vesicles dominate the a p i c a l t h i r d to half of the c e l l . C e l l type four extends from the basal lamina to the lumen but appear to lack m i c r o v i l l i . The m i c r o v i l l i i n t h i s region are shorter than in the midgut caecum (1um) (Figure 28) and occasionally l i e f l a t i n areas where food or adjacent c e l l s are apposed. Neigher a glycocalyx nor a peritrophic membrane were evident i n t h i s region of the midgut although small, apparently hollow v e s i c l e s (0.07um diameter) were randomly dispersed at the apices of the m i c r o v i l l i . When food material was seen i n t h i s region of the gut i t was already p a r t i a l l y digested. This was indicated by the lack of recognizable organelles inside the food c e l l s . Instead, one finds diatom fr u s t u l e s that are s t i l l together but only contain material s l i g h t l y greater i n electron density than that distributed throughout the gut lumen. Occasionally bacteria c e l l s were seen, either as independent c e l l s or i n c l u s t e r s of three to eight c e l l s . 7  Posterior Midqut The posterior midgut extends approximately 175um from the anterior midgut t o the hindgut. The lumen height i s f a i r l y constant at 45-60ua with a width of 100-110um (Figure 7). This region of the midgut l i e s in the center of the body cavity surrounded by approximately 20um of body tissue. The unfurrowed low cuboidal or pseudostratifled squamous 23 c e l l s of t h i s r e g i o n average 3-4um i n hei g h t (Figure 15). They appear, under the l i g h t microscope, t o be much more uniform i n s t r u c t u r e and s t a i n i n g p r o p e r t i e s than the c e l l s of the a n t e r i o r midgut and, i n c o n t r a s t to t h a t r e g i o n , the c e l l s of the p o s t e r i o r midgut c o n t a i n no or few s e c r e t o r y v e s i c l e s ( F i g u r e s 30 and 31).. The brush border o f these c e l l s appears t h i n n e r (0.if-0.6um) than those of the a n t e r i o r 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 midgut have a l e s s c onvoluted b a s a l lamina than more a n t e r i o r r e g i o n s of the midgut. The b a s a l lamina extends 0.5um i n t o the cytoplasm. C i r c u l a r muscle (O.ftum wide) and l o n g i t u d i n a l muscle i s much more common, though s t i l l not c o n s i s t e n t l y present, i n t h i s r e g i o n o f the midgut. Extremely f l a t t e n e d n u c l e i (40um l o n g by 0.7um t a l l ) a re l o c a t e d i n the widest p o r t i o n o f - t h e c e l l s and so, owing t o the p s e u d o s t r a t i f i e d nature of the t i s s u e , t h e r e may be a second nucleus l o c a t e d between a nucleus and the midgut lumen. The n u c l e i e i t h e r c o n t a i n one dense c i r c u l a r o r s l i g h t l y oblong n u c l e o l u s (1um by 0.6um) but more commonly c o n t a i n a f a i r l y s c a r c e supply o f d i f f u s e heterochromatin. The three c e l l types o r i g i n a l l y found i n the midgut caecum are present i n the p o s t e r i o r midgut. C e l l type one i s more common than i n t h e more a n t e r i o r r e g i o n s of the midgut and i s present next t o the b a s a l lamina (F i g u r e s 32 and 33). These c e l l s are e s p e c i a l l y e l ongate and f l a t t e n e d . They have mitochondria (0.7um l o n g by 0.3um diameter) with l a m e l l a t e c r i s t a e extending the len g t h of the mitochondria. The matrix o f the mitochondria i s e l e c t r o n dense. Rough E. R. i s s c a r c e though the c e l l s are f i l l e d with e l e c t r o n dense p a r t i c u l a t e 24 m a t e r i a l . G o l g i bodies were not observed. ; C e l l type two i s even more e v i d e n t i n t h i s r e g i o n of the midgut than i n the a n t e r i o r midgut (Figure 31). I t s pyramidal shape, with a narrow base and wider apex, i s g r e a t l y exaggerated. I n some re g i o n s t h i s c e l l type i s separated by only 3-4 c e l l s o f type t h r e e and the arms of the apex o f t h i s c e l l type may extend t o touch the next arm of i t s neighbouring c e l l type two ( F i g u r e s 30 and 31). & c e n t r a l o r a p i c a l nucleus i s found i n these c e l l s and has a s i m i l a r e l e c t r o n d e n s i t y as the r e s t of the c e l l matrix. A c e n t r a l n u c l e o l u s i s common. Some e l e c t r o n dense mitochondria and rough E. B. c o u l d be observed but the main c h a r a c t e r i s t i c s o f the c e l l : are i t s extreme e l e c t r o n d e n s i t y and i t s v e s i c l e s . The v e s i c l e s (0.2um diameter) appear as l e s s e l e c t r o n dense r e g i o n s i n the c e l l . In the c e n t e r o f these v e s i c l e s t h e r e i s u s u a l l y a more e l e c t r o n dense c i r c u l a r r e g i o n . These v e s i c l e s accumulate a t the apex of the c e l l s and th e r e i s evidence t h a t they may play a p a r t i n merocrine s e c r e t i o n . The h o l o c r i n e - t y p e of s e c r e t i o n i s l e s s common i n the p o s t e r i o r midgut than i n the more a n t e r i o r r e g i o n s of the midgut. H o l o c r i n e s e c r e t i o n , however, was observed ( F i g u r e s 32 and 33). O c c a s i o n a l l y a l a r g e r e l e c t r o n opague v e s i c l e (1.8um by 0.7um) occurs i n the a p i c a l r e g i o n o f the c e l l . The m i c r o v i l l i o f t h i s c e l l type (averaging 0.Sum long) are not as compactly arranged as i n oth e r r e g i o n s of the midgut. They are f r e q u e n t l y a s s o c i a t e d with l a r g e masses of medium e l e c t r o n d e n s i t y , p a r t i c u l a t e m a t e r i a l . These masses of p a r t i c u l a t e m a t e r i a l were about 17um deep. They f o l l o w e d the gut e p i t h e l i u m the l e n g t h o f t h e a p i c a l arms of c e l l type two. The v a r i a t i o n i n e l e c t r o n d e n s i t y of t h i s m a t e r i a l was not as grea t as i n the a n t e r i o r midgut and a membrane, p o s s i b l y the p e r i t r o p h i c membrane, was more c o n s i s t e n t l y present around t h i s mass of di g e s t e d m a t e r i a l . Food m a t e r i a l , i n the form of whole b a c t e r i a l c e l l s and empty diatom f r u s t u l e s was immersed i n t h i s m a t e r i a l . The t h i r d c e l l type i s l e s s e l e c t r o n dense than c e l l type two and has a r e g u l a r b a s a l lamina (Figures 30 and 31). I t s nucleus tends t o be s l i g h t l y more i r r e g u l a r i n i t s o u t l i n e and co n t a i n s d i s p e r s e d heterochromatin and/or a s p h e r i c a l n u c l e o l u s . The mitochondria (1um long and 0.3um diameter) have a s l i g h t l y l e s s e l e c t r o n dense matrix than c e l l type two mitochondria but the number and shape of the c r i s t a e appear the same. The plasma membrane of t h i s c e l l type f r e q u e n t l y forms i n t e r d i g i t a t i n g f o l d s with rough E. H. f o l l o w i n g the plasma membrane o f both c e l l s i n v o l v e d . The rough E. S. i s f a i r l y s c a r c e and t a k e s the form-. of s h o r t s e c t i o n s l o o s e l y f o l l o w i n g the plasma membrane. G o l g i bodies are abundant and are made up o f 5-7 u n d i l a t e d c i s t e r n a e . , E l e c t r o n t r a n s p a r e n t , membrane bound v e s i c l e s bud o f f from these c i s t e r n a e . These c e l l s extend from the b a s a l lamina to the gut lumen. Hindgut The t o t a l l e n g t h o f t h e hindgut i s 80-90um but i t appears to be subd i v i d e d i n t o two r e g i o n s (Figure 9). C l o s e s t to the p o s t e r i o r midgut and c e n t r a l l y l o c a t e d i s a r e g i o n 30-40um i n diameter and l e n g t h . T h i s p o r t i o n of the hindgut i s l i n e d by elongated wedge-shaped e p i t h e l i a l c e l l s with the lumen 26 characterized by numerous narrow c l e f t s and l i n e d by a layer of l i g h t l y stained c u t i c l e (Figures 8 and 35). From t h i s region to the dorsal anal opening, located between the uropods, i s the second portion of the hindgut. This region i s frequently d i l a t e d and i s heavily c u t i c u l a r i z e d . This posterior region of the hindqut lacks or has a minimal e p i t h l i a l l i n i n g (Figure 36) and the lumen i s often f i l l e d with faecal p e l l e t s . Anterior Hindgut The simple squamous e p i t h e l i a l c e l l s of the hindqut average 3um in height (Figure 8). They have a basal lamina s i m i l a r to that seen throuqhout the digestive tract except that the invaginations are much reduced and less complicated. The nuclei of these c e l l s are f a i r l y c i r c u l a r (1.5um diameter) and-contain the regular scatterings of heterochromatin. Any E. B. and the few golgi that are found i n these c e l l s occur around the nuclei. Mitochondria in these c e l l s are predominately elongate i n cross section (about 1um long) and contain well developed c r i s t a e in a dense matrix. M i c r o v i l l i , so c h a r a c t e r i s t i c of the midqut, are gradually replaced by c u t i c l e s i m i l a r to that described for the esophagus (Figures 34 and 35). This i s the region of the digestive t r a c t where defined faecal p e l l e t s may be found, surrounded by a peritrophic membrane. Posterior Hindqut Between the anterior hindgut and the anus i s the l a s t region of the cope pod digestive tract (Figure 14). . Here the n u c l e i , l y i n g i n the center of the simple squamous c e l l s , are elonqate (2.5 um long) and l i e with t h e i r main axis p a r a l l e l to the c u t i c l e (Figure 36). The mitochondria are evenly 27 d i s t r i b u t e d and are s l i g h t l y elongate (0.Sum long) with few c r i s t a e i n a matrix of low e l e c t r o n d e n s i t y . E. R. , g o l g i and v e s i c l e s of any d e s c r i p t i o n are e s s e n t i a l l y absent. The p o s t e r i o r hindgut i s l i n e d by c u t i c l e s i m i l a r to t h a t found i n the esophagus and a n t e r i o r hindgut except t h a t the middle r e g i o n of the c u t i c l e {the e n d o c u t i c l e ) i s t h i c k e r (0.7um th i c k ) and takes on the s t r i a t e d appearance t y p i c a l o f the c u t i c l e p r o t e c t i n g the o u t s i d e of the copepod {Figure 37). Within or j u s t i n s i d e o f the e p i c u t i c l e (outermost dense r e g i o n of the c u t i c l e ) e l e c t r o n t r ansparent c i r c l e s a re e v i d e n t (0.03urn diameter) {Figure 38) . 28 DISCUSSION Examination of the shape of the digestive t r a c t of Tigriopus c a l i f o r n i c u s reveals that i t i s a t y p i c a l copepod digestive t r a c t . As i n calanoid copepods {Lowe 1935) and harpacticoid copepods (Fahrenbach 1961), the digestive t r a c t of T, c a l i f o r n i c u s i s made up of a c u t i c u l a r i z e d esophagus, a midgut divided into two regions with a single conical anterior extension, and a c u t i c u l a r i z e d hindgut. There i s a f a i r degree of regional s t r u c t u r a l s p e c i a l i z a t i o n which can, i n general, be correlated to p a r t i c u l a r functions of the gut regions. The noncuticulized regions of the digestive t r a c t , however, also showed c o n t i n u i t i e s i n basic structure and c e l l c h a r a c t e r i s t i c s . Esophagus The c h a r a c t e r i s t i c s of the 3V californicvis esophagus are sim i l a r to those described f o r the cr a y f i s h (Yonge 1924), calanoid copepods (Dakin 1908; Lowe 1935; Marshall and Orr 1972), c a l i g o i d copepods (Lewis 1961), and another harpacticoid copepod (Fahrenbach 1961) and even a cladoceran (Schultz and Kennedy 1976). The muscle system seen in t h i s copepod esophagus and extensively described for other harpacticoid copeods (Lang 1948; Fahrenbach 1961), creates a s t r u c t u r a l base for the shape of the esophagus and undoubtedly functions i n c o n t r o l l i n g the extent of d i l a t i o n and relaxation of the esophagus lumen. These d i l a t i o n s and relaxations may serve to control the speed of the movement of food to the midgut. The *H* shape of the esophagus lumen indicates a high degree of c o n t r a c t i l i t y and would allow great v a r i a b i l i t y i n the amount of material that can be ingested. 29 In the ventral portion of the esophagus f a i r l y large setae were observed passing through the esophagus lumen. These setae, l i k e those of the cockroach (Hurthy 1975), do not appear to act as a f i l t e r i n g mechanism. However they may function i n mastication. The setae ex i s t i n association with a dip i n the c u t i c l e l i n i n g and thus, along with the arms of the ventral esophagus, form a type of mortar and pestle. The f a c t that the particulate material i n the ventral arm of the esophagus i s much coarser than that found i n the more anterior and dorsal portions of the esophagus suggests a cert a i n degree of physical breakdown. There i s a question as to whether the microtubules seen throughout the esophagus epithelium and es p e c i a l l y i n d i r e c t association with the c u t i c l e may be involved i n enzyme and/or mucus export by the esophagus c e l l s . I t i s most l i k e l y that these microtubules take on the more s t r u c t u r a l function of maintaining the esophagus shape. The microtubules are probably not involved i n the export of enzymes and/or mucus from the e p i t h e l i a l c e l l s because there i s l i t t l e sign that the e p i t h e l i a l c e l l s are secretory. Although the mitochondria of these c e l l s are small, they are r e l a t i v e l y abundant to supply the energy reguired for the esophagus and i t s c e l l s to undergo conformational changes. Although l i t t l e synthesis of enzymes occurs in the esophagus there are indications that digestion has taken place. This same si t u a t i o n was seen in a millipede and so i t was suggested that digestive enzymes were passed i n t o the esophagus from the midgut (Nunez and Crawford 1976). The dense multivesicular bodies found i n the e p i t h e l i a l 30 c e l l s i n c r e a s e i n number as the esophagus approaches the midgut. T h i s may suggest, along with the apparent decrease i n c u t i c l e t h i c k n e s s , t h a t some m a t e r i a l i s being absorbed and p o s s i b l y accumulated i n the esophagus. T h i s may a l s o be a r e g i o n where minerals and i o n s are r e g u l a t e d . Evidence that the c u t i c l e i s not an impenetrable b a r r i e r and c o u l d f u n c t i o n i n such processes i s seen i n the f a c t t h a t i n omnivorous and c a r n i v o r o u s i n s e c t s , c h o l e s t e r o l i s absorbed i n the f o r e g u t (Joshi and Agarwal 1977). Yonge (1924) s t a t e d t h a t t h e r e was no a b s o r p t i o n i n the esophagus although the c u t i c l e was semipermeable. E i s n e r (1955) found t h a t i n c o m p l e t e l y d i g e s t e d f a t i s absorbed i n the cockroach f o r e g u t . In the midgut of the h o u s e f l y , Musca domestical c o n c r e t i o n s c o n t a i n i n g phosphorous, c h l o r i n e , c a l c i u m , i r o n and copper occur and are s i m i l a r i n appearance t o the e l e c t r o n dense s t r u c t u r e s found i n the T.: c a l i f o r n i c u s esophagus (Sohal, P e t e r s and H a l l 1977). I n the h o u s e f l y , these c o n c r e t i o n s were thought to play a r o l e i n e x c r e t i o n . These c o n c r e t i o n s were a l s o r e p o r t e d i n another h a r p a c t i c o i d s t u d i e d by Fahrenbach (1961). In the shrimp, s a l t (NaCI) e x c r e t i o n occurs i n the a n t e r i o r d i v e r t i c u l u m ( D a l l 1967)., Musculature The musculature of the r e g i o n from the caecum to the p o s t e r i o r midgut shows a t r e n d . There i s a f a i r l y i r r e g u l a r arrangement of s t r i a t e d c i r c u l a r muscle and l o n g i t u d i n a l muscle i n the caecum. The muscles of the a n t e r i o r midgut becomes a f a i r l y continuous arrangement of c i r c u l a r muscle by the p o s t e r i o r midgut. The most e v i d e n t l o n g i t u d i n a l muscle, as i n 31 Calanus (Lowe 1935), occur i n a s s o c i a t i o n with t h e midgut e v a g i n a t i o n s . From s e r i a l s e c t i o n s o f Ti3£io£3S c a l i f o r n i c u s , i t appears t h a t these e v a g i n a t i o n s may i n f a c t be s p h e r i c a l bodies attached t o the main p o r t i o n of the midgut by a narrower neck p o r t i o n . T h i s o b s e r v a t i o n makes i t l e s s c o n c l u s i v e t h a t t h i s s e t of muscles i s , i n f a c t , l o n g i t u d i n a l . Instead, they could be c i r c u l a r , s urrounding or p i n c h i n g the neck p o r t i o n of these e v a g i n a t i o n s . The f u n c t i o n f o r such a muscle system i s not c l e a r but i t i s obvious t h a t a l o n g i t u d i n a l muscle system i n c o n j u n c t i o n with the c i r c u l a r muscles would be u s e f u l i n p e r i s t a l t i c type c o n t r a c t i o n s . I t i s known t h a t 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 o f the gut occur i n the b l o w f l y (Thomson and H o l l i n g 1976), Daphnia (Schultz and Kennedy 1976) and c a l a n o i d copepods (Mar s h a l l and Orr 1972). In Daghnia p e r i s t a l s i s moves food through the esophagus t o the a n t e r i o r midgut and a n t i p e r i s t a l s i s moves d i g e s t i v e j u i c e s from the p o s t e r i o r midgut to t h e a n t e r i o r midgut. T h i s system i s suggested t o be a mechanism f o r d i g e s t i o n i n the a n t e r i o r midgut, s e c r e t i o n i n the p o s t e r i o r midgut and movement of n o n d i g e s t i b l e m a t e r i a l out of the d i g e s t i v e t r a c t as faece s (Vonk 1960; S c h u l t z and Kennedy 1976). In the cockroach, l i p a s e i s known to be s e c r e t e d by the midgut and the midgut caecum, yet i t i s found i n the f o r e g u t . E i s n e r (1955) suggested 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 i n the foregut may f a c i l i t a t e t h i s movement of enzymes. T h i s system of food flow i s r e m i n i s c e n t o f the d i g e s t i v e and f i l t e r i n g system of malacostracans (Megl i t s h 1972). In decapods, amphipods and c a l a n o i d copeods the non-s t r i a t e d longitudinal myofilaments l i e between the c i r c u l a r myofilaments and the basal lamina (Dakin 1908; Lowe 1935; L o i z z i 1971; Schultz 1976), while i n isopods the longitudinal filaments are external to the c i r c u l a r (Donadey 1971). Schultz and Kennedy (1976) suggested the muscle system of Daphnia i s h e l i c a l and lacks the true c i r c u l a r or long i t u d i n a l muscle c h a r a c t e r i s t i c s . From the appearance of the caecum and anterior midgut 'longitudinal* muscle that i s not associated with the evaginations, the muscle system of T. c a l i f o r n i c u s appears to be simi l a r to that of Daphnia. In these regions the 'longitudinal» muscle appears more as i f i t were c i r c u l a r muscle cut i n a diagonal fashion. The muscle of the posterior midgut appears to be much les s longitudinal or c i r u c l a r and may be h e l i c a l . I f i n fact the muscle system of t h i s copepod i s a continuum, i t would be concluded that at the posterior midgut the helix has become so tight that i t i s lined e n t i r e l y by muscle which, when sectioned, always reveals a diagonal or obligue view. In f a c t , in the region of the posterior midgut the muscle i s no longer s t r i a t e d . &s i n the cray f i s h (Yonge 1924), the hindgut lacks c i r c u l a r muscle. This i s probably associated with a lack of regular p e r i s t a l s i s . . Fahrenbach (1961) found the hindgut undergoes a series of twitches during defecation rather than smooth p e r i s t a l s i s . L o i z z i (1971) described the myo-epithelial network of the cr a y f i s h hepatopancreas as being composed of longitudinal muscle f i b e r s formed from branches of c i r c u l a r f i b e r s such that the res u l t i n g perpendicular myofibrils shared the same sarcolemma. Yonge (1924) proposed that the function of the hepatopancreatic 33 muscle system was to make p o s s i b l e the movement o f d i g e s t i v e j u i c e s out of the h e p a t o p a n c r e a t i c t u b u l e and of p a r t i a l l y d i g e s t e d n u t r i e n t s i n t o t h i s t u b u l e . ; He concluded that c o n t r a c t i o n of the c i r c u l a r muscle produced the outward flow while c o n t r a c t i o n of the l o n g i t u d i n a l f i b e r s caused t h e inward flow. I f the muscle system i s as d e s c r i b e d by L o i z z i (1971), then c o n t r a c t i o n would i n v o l v e both l o n g i t u d i n a l and c i r c u l a r muscles simultaneously so t h a t i n e f f i c i e n t b a l l o o n i n g would not occur. I f the midgut caecum and the a n t e r i o r midgut systems are s i m i l a r f u n c t i o n a l l y t o the hepatopancreas, then i t seems reasonable to expect the same type o f musculature as i n the hepatopancreas. The muscle system i n T^ . c a l i f o r n i c u s , as d e s c r i b e d i n t h i s study, would allow s i m i l a r p e r i s t a l t i c , a n t i p e r i s t a l t i c and pumping a c t i o n s of the gut as d i s c u s s e d i n other arthropods above. With a d d i t i o n a l i n f o r m a t i o n concerning the amount of d i g e s t i o n t h a t has occured i n the v a r i o u s r e g i o n s of the T. c a l i f o r n i c u s gut and o b s e r v a t i o n s of where a b s o r p t i o n has occured, i t seems most probable t h a t the same s i t u a t i o n o c c u r s as i n Daphnia. ; That i s , 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 move food and d i g e s t i v e enzymes from the p o s t e r i o r midgut to the a n t e r i o r midgut and midgut caecum where a b s o r p t i o n o c c u r s . B a s a l Lamina The b a s a l lamina of the midgut appears to f o l l o w an o p p o s i t e t r e n d t o t h a t o f the musculature of the midgut. In the midgut caecum and a n t e r i o r midgut the b a s a l lamina i s most prominent and shows i t s g r e a t e s t i n v a g i n a t i o n s and c o m p l e x i t i e s . In the p o s t e r i o r midgut the b a s a l lamina tends t o form d e l i c a t e dips at random i n t e r v a l s . The function of the basal lamina has been suggested to be water {Pease 1956) and ion regulation ( F i l s h i e , Poulson and Waterhouse 1971; Hootman and Conte 1974; Burgos and Gutierrez 1976). The c l a s s i c function of any type of surface area expansion i s to increase surface area for exchange. The extensiveness of the basal lamina suggests that i t functions in exchange of material of some sort between the gut epithelium and the underlying haemocoel (Beams and Anderson 1957). I t would follow, therefore, that the greatest amount of exchange between the underlying tissues and the gut epithelium occurs i n the caecum and the anterior midgut. In the r a t , Na, K-ATPase a c t i v i t y has been shown to be high i n the basal l a t e r a l i n t e s t i n a l epithelium (Crane 1975; Mircheff and Wright 1976). The structure of the basal lamina i s that of a gr i d i n association with an amorphous material and t h i s association i s suggested to serve as a mechanical support and an avenue for f i l t r a t i o n (Terzakis 1967)* The fact that mitochondria of the e p i t h e l i a are often found i n close association with these invaginations suggests an active metabolic role (Waterhouse and Wright 1960; Nopanitaya and Misch 1974). The mitochondria would supply the energy reguired f o r transport of materials against a concentration gradient. C e l l types one, two and three were evident to some degree along the whole extent of the midgut and so i t seems most appropriate to discuss each c e l l type as an i n d i v i d u a l e n t i t y and then discuss any variances i n number, c h a r a c t e r i s t i c s or form that may occur i n the various regions of the gut. 35 C e l l Type One The b a s a l p o s i t i o n o f c e l l type one i n T i g r i o p u s c a l i f o r n i c u s and the c h a r a c t e r i s t i c s of i t s o r g a n e l l e s suggest i t t o be an u n d i f f e r e n t i a t e d c e l l . These c e l l s would develop and r e p l a c e c e l l s t h a t had worn away or were l o s t i n h o l o c r i n e s e c r e t i o n . The d i s t r i b u t i o n of the c e l l s , o f t e n i n the e v a g i n a t i o n s almost excluded from the gut e p i t h e l i u m , suggest they are , at t h i s stage o f development, independent from the a b s o r p t i v e - s e c r e t i v e f u n c t i o n o f the gut. I f the f u n c t i o n of the b a s a l lamina i s as d i s c u s s e d above, i t would be l o g i c a l t o f i n d t h a t a c e l l not a c t i v e l y f u n c t i o n i n g i n exchange would have a comparatively smooth b a s a l lamina. The b a s a l lamina subtending c e l l t ype one i s r e l a t i v e l y smooth. In i n s e c t s r e g e n e r a t i v e c e l l s occur i n c l u s t e r s c a l l e d n i d i and i t has been suggested t h a t once b a s a l i n f o l d i n g s occur, the c e l l s have a l r e a d y begun d i f f e r e n t i a t i o n <Sud 1968; de P r i e s t e r 1971). although these r e g e n e r a t i v e c e l l s u s u a l l y o c c u r r e d i n c l u s t e r s t h e r e was l i t t l e s i g n o f c e l l d i v i s i o n . In the a d u l t isopod s t u d i e d by H a r t e n s t e i n (1964) , no m i t o t i c a c t i v i t y was found i n the d i g e s t i v e t r a c t . In T i g r i o p u s c a l i f o r n i c u s the abundance of ribosomes with l i t t l e E. 5. or g o l g i bodies i n c e l l type one suggests t h a t , while p r o t e i n s y n t h e s i s i s o c c u r r i n g , p r o t e i n products are not n e c e s s a r i l y being exported o u t s i d e o f the c e l l . Mitochondria are f a i r l y abundant t o provide the energy r e q u i r e d f o r the processes of c e i l d i v i s i o n and d i f f e r e n t i a t i o n . The simple o r g a n i z a t i o n o f the cytoplasm with few membranes or r e s i d u a l vacuoles but l a r g e numbers of f r e e ribosomes i s t y p i c a l o f 36 u n d i f f e r e n t i a t e d c e l l s (Smith 196 8) . This f i r s t c e l l type of Tigrioj>u,s c a l i f o p j c u s appears t o be comparable t o the E - c e l l s of decapods, amphipods and isopods ( l o i z z i 1971; C l i f f o r d and witkus 1971; S c h u l t z 1976). These E-c e l l s are embryonic c e l l s forming the d i s t a l m i t o t i c r e g i o n of the hepatopancreatic e p i t h e l i u m . T h i s embryonic r e g i o n o f the hepatopancreas undergoes c e l l d i v i s i o n t o produce an i n t e r m e d i a t e c e l l type ( T - c e l l ) which then d i f f e r e n t i a t e s to a l i g h t c e l l type ( L - c e l l ) . In the L - c e l l , s e c r e t o r y products begin t o form (van S e e l 1955) and accumulate u n t i l the L - c e l l becomes an e x t r u s i o n c e l l at which time merocrine s e c r e t i o n occurs ( P i l l a i 1960). The empty c e l l i s now the dark c e l l (D-c e l l ) which i s a b l e t o r e s t o r e i t s e l f t o the l i g h t c e l l again and continue through t h i s c y c l e o f s e c r e t i o n . , In t h i s s i t u a t i o n the embryonic c e l l s o f the hepatopancreas r e p l a c e c e l l s t h a t are worn away., although T. c a l i f o r n i c u s does not have a hepatopancreas, the E - c e l l of t h i s organ i n other Crustacea can b e compared to the u n d i f f e r e n t i a t e d embryonic c e l l o f T. c a l i f o r n i c u s . C e l l Ty.De Two The second c e l l type found i n the midgut of T i q r i o p u s c a l i f o r n i c u s i s seen to be very d a r k l y s t a i n e d when viewed under the l i g h t and the e l e c t r o n microscope. I t has abundant rough E. R. , ribosomes, mitochondria and d i l a t e d g o l g i bodies with s e c r e t o r y v e s i c l e s i n a l l stages of maturation, s i z e and p o s i t i o n . a l l of these o r g a n e l l e s and t h e i r abundance makes i t l i k e l y t h a t such c e l l s are s y n t h e t i c c e l l s . D i l a t e d g o l g i bodies with loose arrangements of lacunae are s u g g e s t i v e of very 37 a c t i v e g o l g i bodies (Fawcett 1969) . E x o c y t o s i s was f r e q u e n t l y seen i n these c e l l s i r r e s p e c t i v e of t h e i r s t a t e of maturation. E x o c y t o s i s and a process s i m i l a r t o merocrine or h o l o c r i n e s e c r e t i o n o c c u r r e d i n the mature c e l l s . a f t e r t h i s l a t t e r process o f s e c r e t i o n the c e l l i t s e l f and the s e c r e t e d products i n the gut lumen appear much lower i n e l e c t r o n d e n s i t y . The c e l l l o s e s i t s m i c r o v i l l i and i t s o r g a n e l l e s become l e s s i d e n t i f i a b l e but the m a t e r i a l being extruded remains i n t a c t , f o r a time. The shape of t h i s second c e l l type i s that of a wedge, with i t s base e i t h e r at t h e b a s a l or a p i c a l end. T h i s shape suggests a process of maturation and d i f f e r e n t i a t i o n . The c e l l c o n d i t i o n with the broad base probably r e p r e s e n t s a younger c e l l , while those possessing the wider apex c o u l d be the mature s t a t e b e f o r e merocrine or h o l o c r i n e s e c r e t i o n . The next s t e p of t h i s process i s f o r the apex of the c e l l t o protrude i n t o the gut lumen, and f o r the plasma membrane to break down to extrude the c e l l products and o r g a n e l l e s . another f a i r l y common c h a r a c t e r i s t i c of t h i s second c e l l type i s the accumulation of l i p i d d r o p l e t s i n the a p i c a l t h i r d of the c e l l . These d r o p l e t s are homogeneously dense and have a f a i r l y c o n s i s t e n t diameter. T h i s , along with the occurrence of en d o c y t o s i s , suggests that a b s o r p t i o n of d i g e s t e d products was o c c u r r i n g i n these c e l l s . The s e c r e t o r y stage o f t h i s c e l l type may be c o n s i d e r e d i t s u l t i m a t e f u n c t i o n . From the o r g a n e l l e s and t h e i r abundance i t i s obvious that t h i s c e l l f u n c t i o n s i n p r o t e i n s y n t h e s i s . While the c e l l i s s y n t h e s i z i n g i t appears to a l s o f u n c t i o n i n l i p i d 38 absorption. The basal lamina invaginations of these c e l l s adds credence to the idea that these c e l l s are d i r e c t l y active i n the metabolism of the gut. This i s i n contrast to crabs (Reddy 1938) and c a l i g o i d copepods (Lewis 1961) where the dark c e l l s are apparently only involved in secretion. This c e l l type most approaches the F - c e l l ( F i b r i l l a r c e l l ) of decapods and amphipods (Loi z z i 1971; Schultz 1976). The F-c e l l i s described as being f i b r i l l a r with a b a s i o p h i l i c cytoplasm, developing vacuoles and functioning i n enzyme synthesis. In crabs these c e l l s are c a l l e d 'dark c e l l s * (van Weel 1955; P l i l a i 1960). These c e l l s are described as having a f a i n t l y frothy cytoplasm with minute f i b r i l s extending from the base of the c e l l to i t s apex ( P i l l a i 1960). In the isopod hepatopancreas the basophilic c e l l s are B-cells ( C l i f f o r d and Witkus 1971) . In copepods, randomly spaced columnar c e l l s with a very densely staining cytoplasm have been described. These c e l l s are not as frequent as other c e l l s i n the gut. In c a l i g o i d copepods they have been c a l l e d type B c e l l s and th e i r granules have been suggested to function i n e x t r a c e l l u l a r digestion (Lewis 1961). In calanoid copepods t h i s •dark* or 'compact* c e l l type i s also present (Lowe 1935; Marshall and Orr 1972) and they are described by Dakin f1908) as being smaller, more regular, hexagonal c e l l s that are very wide for their depth. Fahrenbach (196 1) did not describe a c e l l comparable to c e l l type two. C e l l type two of the T. c a l i f o r n i c u s digestive t r a c t , therefore, i s s i m i l a r to the enzyme synthesizing c e l l s of other crustacea i n both structure and function. 39 C e i l 22RS. Three C e l l type three i s common throughout the noncuticularized gut of X*. c a l i f o r n i c u s . I t i s much l i g h t e r i n staining c h a r a c t e r i s t i c s than c e l l type two and tends to have larg e r , less electron dense secretory vesicles. The intermediate amounts of rough E. R. , go l g i , mitochondria and loose ribosomes suggest secretion i s not a major function of t h i s c e l l . The function of this c e l l may be indicated by the small v e s i c l e s of l i p i d material and smooth E. R. that accumulate i n the a p i c a l portions of the c e l l . Thus, i t appears that absorption i s their major function and synthesis i s limited to i n t r a c e l l u l a r metabolism of the c e l l products absorbed. These c e l l s show a greater and more consistent development of basal i n f o l d i n g than i n c e l l type two and i t could be suggested that t h i s t h i r d c e l l type i s active i n transport between the gut lumen and the haemocoel. In insects there does not appear to be a d i f f e r e n t i a t i o n between the c e l l types two and three so the c e l l s that are not regenerative or goblet c e l l s are ca l l e d ' p r i n c i p a l c e l l s * by Sud (196 8) and 'columnar' c e l l s by Smith (1968) . These c e l l s are said to function for both absorption and secretion. Smith (1868) suggested that although some pinocytosis may occur, most nutrients are taken up d i r e c t l y through the a p i c a l plasma membrane as small molecules not detectable in the electron microscope. Decapods and amphipods have two c e l l types that appear to f i t the function and description of the t h i r d c e l l type of T. c a l i f o r n i c u s . The f i r s t i s a storage c e l l (R-cell) which 40 a l s o f u n c t i o n s i n a b s o r p t i o n and c o n t a i n s n u t r i e n t s t o r e s ( L o i z z i 1971; S c h u l t z 1976). The second i s a b l i s t e r - l i k e c e l l ( B - c e l l ) which s e c r e t e s d i g e s t i v e enzymes and c o n t a i n s a s i n g l e l a r g e vacuole and a scanty b a s i o p h i l i c cytoplasm ( L o i z z i 1971; S c h u l t z 1976). The isopod hepatopancreas has a c i d o p h i l i c c e l l s ( S - c e l l s ) which may be comparable t o the t h i r d c e l l type under d i s c u s s i o n f o r T. c a l i f o r n i c u s ( C l i f f o r d and Kitkus 1971) . The ' l i g h t c e l l s * of the c r a b d i g e s t i v e t r a c t are columnar, narrow c e l l s t h a t tend t o v a c u o l i z e ( P i l l a i 1960). a c c o r d i n g t o van Seel (1955), these c e l l s are most common i n the midgut gland of shrimp (Atyidae) and are a stage of a c e l l c y c l e i n which s e c r e t o r y products are formed and s t o r e d b e f o r e being extruded. In c a l a n o i d copepods these ' l i g h t * c e l l s are l a r g e and t h e i r heavy v a c u o l a t i o n f o r c e s the cytoplasm and nucleus t o the base of the c e l l s . They a l s o have p r o t r u s i o n s extending i n t o the lumen of the gut (Dakin 1908; Lowe 1935; M a r s h a l l and Orr 1972). The f u n c t i o n of c e l l t ype a i n the midgut o f c a l i g o i d copepods, a c e l l comparable u l t r a s t r u c t u r a l l y t o c e l l type three i - n J. •. . c a l i f o r n i c u s , i s a b s o r p t i o n o f d i g e s t e d food m a t e r i a l s (Lewis 1961). The l a r g e v a c u o l a t e d c e l l s o f c a l a n o i d copepods were a l s o found i n a h a r p a c t i c o i d cop e pod, D i a r t jh odes cy s to ecus, s t u d i e d by Pahrenbach (1961). The shape o f these c e l l s i s a p p a r e n t l y dependent upon p h y s i c a l f a c t o r s such as the presence or absence of food i n the gut. C e l l type three of T._ c a l i f o r n i c u s i s comparable i n s t r u c t u r e t o c e l l s i n decapods, amphipods and other copepods. The predominating f u n c t i o n o f t h e s e c e l l s v a r i e s from order to order. In T. c a l i f o r n i c u s , as i n c a l i g o i d copepods, c e l l type 41 three f u n c t i o n s mainly f o r a b s o r p t i o n but undergoes some s e c r e t i o n . In decapods and amphipods, separate c e l l types c a r r y out each of these f u n c t i o n s r a t h e r e x c l u s i v e l y . Each r e g i o n of the n o n c u t i c u l a r i z e d Tr c a l i f o r n i c a s gat had each of these three c e l l types and a f o u r t h c e l l type was found i n the a n t e r i o r midgut. Each r e g i o n o f the gut w i l l be d i s c u s s e d independently, p o i n t i n g out v a r i a n c e s and other unigue f e a t u r e s and t h e i r f u n c t i o n s . Midgut Caecum The midgut caecum of T j g r i o p u s c a l i f o r n i c u s c o n t a i n s the l a r g e s t c e l l s of t h e whole midgut and there are a moderate number of e v a g i n a t i o n s at the base of the c e l l s . The b a s a l lamina i s deeply i n v a g i n a t e d . The t h r e e c e l l types d e s c r i b e d above are present with c e l l type three being the most abundant, c e l l type two next f o l l o w e d by c e l l type one. T h i s f a c t , along with the r e l a t i v e s c a r c i t y of lu m i n a l e v a g i n a t i o n s , suggests merocrine or h o l o c r i n e s e c r e t i o n i s f a i r l y uncommon. A g l y c o c a l y x around the m i c r o v i l l i may be present although i t s appearance i s c l o s e r t o t h a t o f the p a r t i c u l a t e m a t e r i a l i n the lumen of the caecum or the a n t e r i o r midgut., The f a c t t h a t the m i c r o v i l l i i n t h i s r e g i o n a re the l o n g e s t of the midgut suggests a g r e a t a b s o r p t i v e f u n c t i o n f o r t h i s r e g i o n . Crane (1975) has proposed a membrane model f o r the a c t i v e passage of glucose and p a s s i v e passage of f r u c t o s e from the gut lumen through the d i g e s t i v e - a b s o r p t i v e m i c r o v i l l i border i n t o the e p i t h e l i a l c e l l s . In the f l y a n t e r i o r midgut the m i c r o v i l l i were the l o n g e s t of the midgut and were d e f i n i t e l y surrounded by a g l y c o c a l y x {de P r i e s t e r 1971). I t has r e c e n t l y 42 been suggested that the microfilaments found inside the m i c r o v i l l i may give i t c o n t r a c t i l e a b i l i t i e s {Bodewald, Newman and Karnovsky 1976). This process would then act to 'pump* material, which has moved inside the m i c r o v i l l i , into the cytoplasm below {Boyd and Parsons 1969). In T. c a l i f o r n i c u s , l i p i d droplets were very common i n the anterior midgut c e l l types two and three and so i t i s suggested, as for Daphnia (Schultz and Kennedy 1976) and isopods (Donadey 1969), that t h i s i s one of the major s i t e s of nutrient uptake. Ong and Lake (1970) believe the midgut diverticulum of Calanus helgoiandicus does not produce enzymes but churns and produces mucopolysaccharides and absorbs amino acids to be transported by the basal mitochondria pump to the haemocoel., In malacostracans the midgut caecum stores glycogen (Singet, Souse and Maurer 1977) and produces most of the digestive enzymes. In Daphnia the anterior midgut, as well as the midgut caecum, produces digestive enzymes (Schultz and Kennedy 1976). P i l l a i (1960) suggests there i s very l i t t l e difference between the c e l l s of the midgut caecum and the c e l l s of the midgut in the shrimp (Caridina l a e y i s ) . Amphipods have storage ( R - c e l l s ) , f i b r i l l a r (F-cells) and vacuolated (B-cells) c e l l s in the i r midgut diverticulum. The function of these c e l l s i s resorption, storage of l i p i d and glycogen as well as secretion (Horitz, Storch and Buchheim 1973). Thus, as i n T. californicus,. both secretion and absorption take place i n th i s region of the midgut. In calanoid copepods the whole anterior midgut, including the diverticulum, i s li n e d by a uniform layer of nonvacuolated cubical c e l l s {Lowe 1935). Marshall and Orr (1972) suggest that in calanoid copepods food i s constantly pushed backwards and forwards in the wide midgut caecum and anterior midgut. In c a l i g o i d copepods, secretory c e l l type B i s primarily i n the foregut and anterior midgut regions suggesting t h i s i s the region where secretion occurs (Lewis 1961). In the harpacticoid copepod studied by Fahrenbach {1961) , the midgut diverticulum i s made up of large vacuolated c e l l s projecting into the lumen. In most of the Crustacea described above, as i n T. c a l i f o r n i c u s . the midgut caecum or diverticulum functioned mainly i n absorption of nutrients. In some crustaceans, the nutrients absorbed are stored and i n others the caecum also produces enzymes. Anterior Midgut In Tigriopus c a l i f o r n i c u s the anterior midgut has a basal lamina with the greatest number of evaginations and invaginations. The c e l l s and m i c r o v i l l i are smaller than those of the caecum and the m i c r o v i l l i lack a surface coat. In Daphnia (Schultz and Kennedy 1976), the glycocalyx of these m i c r o v i l l i i s also not evident. A m i c r o v i l l i glycocalyx i s present in the midgut c e l l s of decapods (Talbot, Clark and Lawrence 1972) and the H-cells of the c r a y f i s h hepatopancreas (Loizzi 1971). I t i s lacking i n the bilobed hepatopancreas of isopods and amphipods ( C l i f f o r d and Witkus 1971; Schultz 1976) as well as in the caecum and midgut of Cala mis (Ong and Lake 1970). This region of the midgut has the greatest number of luminal extrusions and along with deep invaginations from the lumen into the c e l l s , the surface area f o r absorption and aa t r a n s p o r t i s g r e a t l y i n c r e a s e d . Of the t h r e e c e l l t y pes, c e l l type one appears to be s l i g h t l y more abundant here than i n the midgut caecum and merocrine or h o l o c r i n e s e c r e t i o n o f c e l l type two i s obvious. C e l l type t h r e e i s n o t i c e a b l y more e l e c t r o n dense than i n the caecum. The i n c r e a s e i n abundance of c e l l type one suggests a g r e a t e r l o s s of gut c e l l s i n t h i s r e g i o n e i t h e r through wear or through s e c r e t i o n . The d e n s i t y o f c e l l type t h r e e may r e f l e c t a g r e a t e r amount of n u t r i e n t a b s o r p t i o n i n t h i s r e g i o n o f the gut when compared to the caecum and, indeed, t h e r e appears to be a s i g n i f i c a n t i n c r e a s e i n the number of l i p i d d r o p l e t s i n both c e l l types two and t h r e e . In t h i s r e g i o n of the T i g r i o p u s c a l i f o r n i c u s midgut t h e r e are two c h a r a c t e r i s t i c s not found i n any other r e g i o n o f the midgut. These a r e : 1. the presence o f c e l l type f o u r , and 2. c e l l u l a r c o n c r e t i o n s i n c e l l types two and t h r e e . The v e s i c l e s of c e l l type f o u r , from t h e i r shape, s i z e and e l e c t r o n d e n s i t y , are s u g g e s t i v e of e i t h e r l i p i d a b s o r p t i o n , endocrine s e c r e t i o n or zymogenic s e c r e t i o n . The s c a r c i t y of rough E. R. v i r t u a l l y e l i m i n a t e s the l a s t p o s s i b i l i t y . The p o s s i b i l i t y o f endocrine s e c r e t i o n by these c e l l s i s open due to the f a c t t h a t i n i n s e c t s there i s some evidence t h a t the s e c r e t i o n of d i g e s t i v e enzymes i s under hormonal c o n t r o l (Higglesworth 1965). However, f o r protease p r o d u c t i o n i n the i n s e c t i n t e s t i n e a c e p h a l i c endocrine system r a t h e r than an i n t e s t i n a l endocrine system i s i n c o n t r o l ( G a r c i a and Garcia 1977). The p o l a r i t y i n t h i s c e l l type f o u r of E. R.. and g o l g i bodies b a s a l and v e s i c l e s a p i c a l makes i t u n l i k e l y that the s m a l l v e s i c l e s i n these c e l l s are endocrine i n T. c a l i f o r n i c u s . Thus, i t appears the most l i k e l y p o s s i b i l i t y f o r the f u n c t i o n of the f o u r t h c e l l type i s l i p i d a b s o r p t i o n . I t i s known t h a t l i p i d a b s o r p t i o n may occur by at l e a s t two processes. One process would be molecular i n c o r p o r a t i o n by the c e l l through the plasma membrane such as appears to occur i n the c e l l types two and three., Such a system would occur i f d i g e s t i o n were e x t r a c e l l u l a r , as Bond (1934) suggested. I n r a t s sucrase i s co n c e n t r a t e d at the brush border plasma membrane ( H i r c h e f f and Wright 1976) and t h i s enzyme i s suggested t o r e l e a s e glucose from i n t r a l u m i n a l sucrose. T h e r e f o r e the more e a s i l y t r a n s p o r t a b l e hexose i s a v a i l a b l e f o r t r a n s p o r t through the plasma membrane. In the corn borer, D i a t r a e a g r a n d i o s e l l a , the enzyme f o r the s u b s t r a t e i s l o c a t e d i n the membrane of the c e l l t h a t w i l l absorb the products of the r e a c t i o n and i s not s e c r e t e d i n t o the gut lumen (Turunen and Chippendale 1977). Thus, d i g e s t i o n of the l i p i d i s not t r u l y e x t r a c e l l u l a r , i n the c r a y f i s h hepatopancreas, l i p a s e d i s t r i b u t i o n on the s t r i a t e d border c o u l d be c o r r e l a t e d with the presence or absence of a fil a m e n t o u s (glycocalyx) l a y e r a s s o c i a t e d with the m i c r o v i l l i ( L o i z z i and Peterson 1969). G i l b e r t and 0*Connor (1970) suggested l i p a s e s occur e x t r a c e l l u l a r l y i n arthropods, l i p o l y s i s g r e a t l y i n c r e a s e d a b s o r p t i o n but the f a c t t h a t i t i s not a p r e r e q u i s i t e f o r a b s o r p t i o n (Weintraub and T i e t z 1973) suggests a second means o f l i p i d uptake by c e l l s . , The other process o f l i p i d a b s o r p t i o n i s p i n o c y t o t i c or e n d o c y t o t i c i n c o r p o r a t i o n i n t o c y t o p l a s m i c v e s i c l e s . The v e s i c l e s are then passed d i r e c t l y t o the E. R. and g o l g i system for metabolism (Lentz 1971). Phagocytosis of f a t droplets was never observed i n the crab and so i t was suggested that g l y c e r o l and f a t t y acid products of e x t r a c e l l u l a r digestion were resynthesized into f a t s i n the l i g h t c e l l s (van Seel 1955). Although i t was not determined which method of l i p i d absorption occurs i n TigrioBus c a l i f o r n i c u s , c e l l types two and three as well as c e l l type four contain l i p i d . The electron dense multivesiculate bodies found i n t h i s region of the Tigriopus c a l i f o r n i c u s m i d g u t may be concretions formed by an accumulation or excretory process. These concretions may also be r e s i d u a l lysosome bodies as are found i n the f l y midgut (de P r i e s t e r 1971). P i l l a i (1960) found concretions of the shrimp, Caridina l a e v i s , to be formed by the midgut c e l l s and believes that they represent secretory products. The gut, with i t s close association to the external environment and the haemocoel, would be an obvious place for secretory and/or osmoregulatory processes to occur. Fahrenbach (1962) presumed the anterior midgut concretions in the harpacticoid copepod, Diarthrgdes cystoecus. to be excretory as i n the housefly. The housefly concretions, containing high concentrations of phosphorus, s u l f u r , chlorine, calcium, iron and copper are i n i t i a l l y deposited within golgi ve s i c l e s , lamellar bodies and r e s i d u a l bodies (Sohal, Peters and Hall 1977). The fact that the multivesiculate bodies of 2i3£iopus c a l i u f o r n i c u s appear to be lamellar or residual bodies suggests they may be concretions of a s i m i l a r nature to those of the housefly and D. cystoecus. In many insects midgut goblet c e l l s , o r i g i n a l l y thought to 47 function as a reservoir of secretion (Sud 1968), function i n ion regulation. I t was found in the Cecropja midgut that large amounts of calcium and magnesium and lesser amounts of potassium and sodium are taken up by i s o l a t e d mitochondria., This process was s t o i c h i o m e t r i c a l l y linked to phosphate uptake and electron transport. In contrast to plasma membrane transport systems which are ouabain-sensitive, neither mitochondrial ion accumulation nor potassium transport across the midgut i s i n h i b i t e d by ouabain {Anderson and Harvey 1966). The o v e r a l l transport i s that of potassium from the haemolymph i n t o the gut lumen to be excreted {Flower and F i l s h i e 1976). The mitochondria, intimately involved with the c e l l c avity projections of the goblet c e l l s , are thought to be the s i t e of ion exchange. Specialized c e l l junctions occur to join these goblet c e l l s to the columnar c e l l s . These junctions may control the opening and closing of the goblet c e l l apex and thus the extrusion of the cavity matrices (Schultz and Jungreis 1977). C e l l s s i m i l a r to these were not seen i n Tigriopus c a l i f o r n i c u s but c e l l types two and three appear to possess a s i m i l a r function as suggested by the presence of concretions, invaginated basal lamina and mitochondria.. The functions of the insect midgut include; secretion of digestive enzymes, absorption of the products of digestion, absorption and regulation of water (Berridge and Oschman 1972; Cheung and Low 1975), and possibly a storage depot for food reserves (Smith 1968). In the dragonfly i t i s suggested that resynthesis of t r i g l y c e r i d e s occurs i n specialized midgut E. R. (Andries 1977). In the brown shrimp, Penaeus aztecus. the 48 a n t e r i o r midgut c e l l s a r e hypothesized to f u n c t i o n i n l i p i d s t o r a g e , s e c r e t i o n , p e r i t r o p h i c membrane fo r m a t i o n , a b s o r p t i o n and p o s s i b l y osmoregulation ( T a l b o t , C l a r k and Lawrence 1972). In the mosguito (Hudin and Hecker 1976) and i n Daphnia (Schu l t z and Kennedy 1976); the a n t e r i o r midgut i s the important organ i n a b s o r p t i o n s i n c e the caecum i s r e l a t i v e l y s m a l l compared to the organisms with l a r g e hepatopancreatic organs. In the brachiopod, ftrtemia. 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 and a s s o c i a t e d mitochondria are c o r r e l a t e d with a b s o r p t i o n and osmoregulation (Hootman and Conte 1974). In an e a r l y d e s c r i p t i o n of the t e r r e s t r i a l i sopod midgut, HcHurrich (1897) s t a t e d t h a t the a n t e r i o r midgut was not a t a l l g l a n d u l a r or a b s o r p t i v e and t h a t i t was l i n e d by impermeable c u t i c l e . He suggested a l l d i g e s t i o n and a b s o r p t i o n occurred i n the hepatopancreas. In c a l a n o i d copepods s e c r e t i o n i s s a i d t o occur i n the a n t e r i o r midgut r e g i o n s . Often enzyme a c t i v i t y i s a f f e c t e d by pH o f the medium i n which i t i s present. The f a c t t h a t t h e r e i s a d e f i n i t e change i n pH i n the middle v e r s u s the a n t e r i o r and p o s t e r i o r midgut i n D r o s o p h i l a ( F i l s h i e , Poulson and waterhouse 1971) suggests t h a t the midgut f u n c t i o n s i n some s p e c i f i c way to s u i t the gut matrix t o the enzymes that i t i s s e c r e t i n g . The most s u i t a b l e pH f o r i n v e r t a s e a c t i v i t y i n D i p t e r a occurred only i n t h e most a n t e r i o r p o r t i o n o f the midgut (Sinha 1975)., Thus, as f o r i n s e c t s , shrimp, h a r p a c t i c o i d and c a l a n o i d copepods, the a n t e r i o r midgut of T. c a l i f o r n i c u s f u n c t i o n s f o r both a b s o r p t i o n of n u t r i e n t s and s e c r e t i o n of enzymes. D e f i n i t e c o n c l u s i o n s c o n cerning whether osmoregulation, e x c r e t i o n or storage of n u t r i e n t s occured i n the T. c a l i f o r n i c u s a n t e r i o r midgut c o u l d not be made although the p o s s i b i l i t e s e x i s t . In the shrimp C a r j d i n a l a e y i s ^ , the p e r i t r o p h i c membrane i s suggested t o be formed i n t h i s r e g i o n o f the midgut but i t c e r t a i n l y was not obvious i n T. c a l i f o r n i c u s . P o s t e r i o r fiidgut The p o s t e r i o r midgut c e i l s o f T i a r i o p u s c a l i f o r n i c u s are o n e - h a l f the s i z e of the a n t e r i o r midgut c e l l s and one t h i r d the s i z e of the midgut caecum c e l l s . T h i s , however, may be due t o the pressure of food aggregates t h a t are r e t a i n e d i n t h i s r e g i o n of the gut. In the cockroach, the r a t e of food passage i n the gut decreases towards the anus and so i t i s concluded t h a t d i g e s t i o n occurs i n the p o s t e r i o r r e g i o n s as w e l l as more a n t e r i o r l y ( B i g n e l l 1977). In the c o r n borer l i p o l y t i c enzymes were only s e c r e t e d by the p o s t e r i o r midgut (Turunen and Chippendale 1977). Proteases accumulate i n the p o s t e r i o r midgut of a b e e t l e , Attenoqenus megatoma, although i t i s not known whether s e c r e t i o n of the enzyme i s a c t u a l l y g r e a t e r or t h a t the enzymes simply accumulate here {Baker 1976). , That an enzyme accumulates i n the p o s t e r i o r midgut a l s o suggests that some d i g e s t i o n occurs here. L i p i d a b s o r p t i o n occurs i n t h e T i g r i o D j i s c a l i f o r n i c u s p o s t e r i o r midgut and c e l l type two, which i s suggested t o produce enzymes and be i n v o l v e d i n s e c r e t i o n , a l s o occurs here. The 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 t h i s p o s t e r i o r r e g i o n of the midgut are minimal and thus suggest there i s l e s s exchange between these c e l l s and the haemocoel. Although the m i c r o v i l l i are one-half the l e n g t h of the r e s t of the midgut and are q u i t e 50 i r r e g u l a r , absorption s t i l l appears to occur to a considerable degree as indicated by the number and size of l i p i d droplets i n the c e l l s . In the f l y , poor f i x a t i o n of the posterior midgut i s blamed on the high l i p i d content of these c e l l s (de Prie s t e r 1971). , Only c e l l types one, two and three are present i n the posterior midgut of Tigriopus c a l i f o r n i c u s and there are s l i g h t l y more of c e l l type one than i n the anterior midgut. This suggests that there i s a greater need for replacement c e l l s which follows with the finding that secretion of c e l l type two i s more d e f i n i t e l y of a holocrine than a merocrine nature. This secretion was also observed i n Daphnia where gaps i n the digestive t r a c t resulted from the t o t a l extrusion of groups of three to four c e l l s (Schultz and Kennedy 1976)., In calanoid copepods the c e l l s of the posterior midgut either revert back to being s i m i l a r to those of the midgut caecum (Lowe 1935) or the whole midgut i s made up of s i m i l a r c e l l s (Marshall and Orr 1972). In c a l i g o i d copepods the fact that c e l l type A predominates in the posterior portion of the midgut suggests that t h i s region i s the main s i t e of absorption (Lewis 1961) . In many arthropods the processes of digestion (via proteases, lipases and carbohydrate s p l i t t i n g enzymes (Barns and Goodfellow 1968)), purine metabolism, absorption and storage of glycogen, f a t and copper, occur i n the hepatopancreas (Vonk 1960). T. c a l i f o r n i c u s does not have such an organ but s i m i l a r c e l l s and the same processes that occur in the hepatopancreas also appear to occur i n the T_. c a l i f o r n i c u s midgut. This 51 s i m i l a r i t y i n the c e l l s and function of the T^ . c a l i f o r n i c u s midgut and the hepatopancreas makes i t appropriate to b r i e f l y discuss the hepatopancreas. The hepatopancreas of the crayfish i s the most thoroughly studied and consists of two i d e n t i c a l lobes lying along the ga s t r o - i n t e s t i n a l tract i n the dorsal part of the cephalothorax. As i n the midgut, exchange between the c e l l s and the haemocoel occurs through the basement membrane of the c e l l s (Dorman 1928). The lobes of the hepatopancreas consist of tubules with embryonic c e l l s d i s t a l l y next to absorptive c e l l s . Secretory and f i b r i l l a r c e l l s are more proximally located. From l a b e l l i n g studies i t was found that a l l the c e l l s i n the tubules arise from embryonic c e l l s at the apex (Davis and Burnett 1964). Thus, the embryonic c e l l s are at f i r s t absorptive, then secretory, then ' f i b r i l l a r * (containing abundant E. R. ) and f i n a l l y they die. L o i z z i (1968) suggests that the absorptive and b a s i o p h i l i c c e l l s originate separately i n the d i s t a l t i p of the hepatopancreas and only the l a t t e r give r i s e to the secretory c e l l s . In Crustacea a major portion of the hepatopancreas was found to be l i p i d (Steves 1969; Lawrence 1976). The absorptive c e l l s of the hepatopancreas are termed R-c e l l s and they are comparable to c e l l type three of T„ californicus.. The R-cells have l i p i d droplets, short cisternae of rough E. R. , smooth E. R. and golgi bodies with flattened cisternae (L o i z z i 1971). These c e l l s are occasionally seen to have electron dense inclusions in t h e i r mitochondria suggestive of heavy metals (Bunt 1968). In crabs the R-cells 52 absorb i r o n and f a t ( S t a n i e r , Woodhouse and G r i f f i n 1968). according t o S c h u l t z (1976) the R - c e l l s absorb copper while the F - c e l l s absorb i r o n . C e l l type two of T. c a l i f o r n i c u s i s comparable to the f i b r i l l a r c e l l s o f the c r a y f i s h hepatopancreas which are suggested t o f u n c t i o n i n the s e c r e t i o n of a non-zymogenic p r o t e i n (Bunt 1968). These F - c e l l s a l s o absorb m a t e r i a l from the lumen by means of bulk t r a n s f e r as opposed t o con t a c t d i g e s t i o n and molecular t r a n s p o r t suggested f o r B - c e l l s ( L o i z z i 1971), In isopods the hep a t o p a n c r e a t i c e p i t h e l i u m i s formed from i n t e r s p e r s e d s m a l l , a c i d o p h i l i c a b s o r p t i v e S - c e l l s and l a r g e r , b a s i o p h i l i c a b s o r p t i v e and s e c r e t i v e B - c e l l s with a d i s t a l embryonic r e g i o n which g i v e s r i s e to both of the l a t t e r c e l l types (Schultz 1976). The f u n c t i o n of the T. c a l i f o r n i c u s p o s t e r i o r midgut, l i k e the hepatopancreas, i s s e c r e t i o n o f d i g e s t i v e enzymes and a b s o r p t i o n of n u t r i e n t s . The major o f these two f u n c t i o n s i n 3k c a l i f o r n i c u s i s s e c r e t i o n while the major f u n c t i o n o f the p o s t e r i o r midgut of c a l i g o i d copepods i s a b s o r p t i o n . P e r i t r o p h i c Membrane The p e r i t r o p h i c membrane, t y p i c a l of c r u s t a c e a n s , was not c o n s i s t e n t l y evident i n T. c a l i f o r n i c u s u n t i l f a e c a l p e l l e t s were formed. O c c a s i o n a l l y m a t e r i a l was seen i n a p p o s i t i o n to the m i c r o v i l l i but t h i s was more l i k e l y the membrane g l y c o c a l y x , m a t e r i a l t h a t had been extruded from the c e l l s , or d i g e s t e d m a t e r i a l i n the lumen. In isopods t h i s membrane i s hypothesized t o be formed by the endodermal midgut caecum c e l l s (Holdich 1973). In i n s e c t s the p e r i t r o p h i c membrane i s found around the food i n t h e midgut 53 and i s reported to be formed either along the length of the midgut or at the narrow c l e f t between the midgut and the foregut (Smith 1968; Nunez and Crawford 1976)., Specialized protein producing c e l l s i n the most anterior portion of the midgut produce the f i r s t component of the peritrophic membrane (Smith 1968; F i l s h i e , Poulson and Waterhouse 1971; Nopanitaya and Misch 1974). & second layer i s supplied by the foregut and a t h i r d layer i s a thi n sheet making a 20um dense layer containing fibrous material (Smith 1968). In calanoid copepods the peritrophic membrane, which surrounds the faecal p e l l e t s , i s secreted by the posterior midgut (Gauld 1975)., This membrane i s the endodermal equivalent to the ectodermal c u t i c l e and i s made up of chitinous (Clarke, Temple and Vincent 1977) m i c r o f i b r i l s embedded i n a protein and polysaccharide group substance (Schultz and Kennedy 1976). In the shrimp the peritrophic membrane i s an extremely thin and transparent chitinous membrane found i n the midgut surrounding the faecal p e l l e t s (Forster 1953; P i l l a i 1960). This peritrophic membrane has been suggested t o protect the midgut c e l l s from damage and to act as a fine f i l t r a t i o n device (Forster 1953; Smith 1968). Gauld (1975) suggested that the calanoid peritrophic membrane acts to keep the faeces i n a compact p e l l e t which w i l l sink r a p i d l y out of the water column in which the copepods are feeding.. In isopods an acid mucopolysaccharide coat replaces the peritrophic membrane and i s suggested to function i n protecting the gut c e l l s (Hartenstein 1964). Burgos and Gutierrez (1976) concluded that the peritrophic membrane i s the glycocalyx associated with the 54 m i c r o v i l l i and i s a f i l a m e n t o u s m a t e r i a l with compartments adjacent t o the m i c r o v i l l i . . They suggested that t h i s s t r u c t u r e c o u l d f u n c t i o n 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. Beams and Anderson (1957) argued t h a t the porous nature o f the p e r i t r o p h i c membrane r e s u l t s from t h e p e n e t r a t i n g m i c r o v i l l i . i n the brown shrimp the p e r i t r o p h i c membrane resembles the m i c r o v i l l a r s u r f a c e coat and i s p r e s e n t , though v a r i a b l e , through out the midgut ( T a l b o t , C l a r k and Lawrence 1972). S t u d i e s on the b r i n e shrimp, Artemia s a l i n a , show that f u s i o n of the g l y c o c a l y x and/or v e s i c l e s between m i c r o v i l l i produce the p e r i t r o p h i c membrane of the midgut and hindgut (Hootman and Conte 1974). The place where the p e r i t r o p h i c membrane i s formed appears to be v a r i a b l e . In c a l a n o i d copepods, and p o s s i b l y i n T. c a l i f o r n i c u s . i t appears t o be formed i n the p o s t e r i o r midgut. In C i r r i p e d i a and c a r i d e a the membrane l i n e s the whole midgut. The p e r i t r o p h i c membrane of Daphnia i s formed by the fo r e g u t . The f u n c t i o n s of t h i s membrane ( f i l t r a t i o n b a r r i e r , p r o t e c t i o n f o r mid.gut c e l l s , compacter of f a e c a l p e l l e t s ) are f a i r l y obvious and a c c e p t a b l e . , Food was not c o n s i s t e n t l y present i n the T. c a l i f o r n i c u s gut nor was the p e r i t r o p h i c membrane., This f a c t made i t d i f f i c u l t t o make c o n c l u s i o n s on the formation or form of the membrane i n T. - c a 1 i f o r n i c u s t S e c r e t i o n The types o f s e c r e t o r y processes t h a t appear to occur i n the arthropod d i g e s t i v e t r a c t cover a wide range. E x o c y t o s i s of s e c r e t o r y products i s ev i d e n t i n a l l of the n o n c u t i c u l i z e d p o r t i o n s of the T«_ c a l i f o r n i c u s d i g e s t i v e t r a c t and most probably merocrine or p o s s i b l y h o l o c r i n e s e c r e t i o n occurs i n the 55 anterior midgut and midgut caecum. In the anterior midgut the c e l l s that are extruding into the lumen s t i l l have many organelles and a nucleus., This s i t u a t i o n , by d e f i n i t i o n , i s merocrine secretion. On the other hand, c e l l s are frequently found in the posterior midqut that have been completely expelled from the gut epithelium. In t h i s case holocrine secretion appears to have occurred. In some decapods secretion i n the hepatopancreas has been stated to be holocrine while i n the crab, secretion appears to be merocrine (van Weel 1955; Stanier, Woodhouse and G r i f f i n 1968). This conclusion was drawn from the fact that there was not an increase i n mitotic figures in the embryonic zone with increased secretory a c t i v i t y . Secretion i n the shrimp (Caridina la e y i s j digestive d i v e r t i c u l a i s merocrine ( P i l l a i 1960). Diqestive enzyme secretion i n D^ aghjEiia i s holocrine (Schultz and Kennedy 1976) i n contrast to the apocrine or merocrine method suggested by L o i z z i (1971) to occur i n other decapods, isopods ( C l i f f o r d and Witkus 1971) and amphipods (Schultz 1976). In c a l i g o i d copepods secretion of c e l l type B i s merocrine f o r t h i s c e l l i s seen to undergo r e s t i t u t i o n (Lewis 1961). When t h i s c e l l ruptures, almost the whole surface i s destroyed and the edges of the membrane are l e f t projecting into the lumen., Such loose membranes were not seen i n the lumen of T. c a l i f o r n i c u s where secretion i s thought to be occurring. Instead, the c e l l seems to have extruded into the lumen and then undergoes decomposition u n t i l the plasma membrane i s disintegrated and the c e l l remnants and products are free i n the lumen. Schultz (1976) believes that extrusion by the b l i s t e r - l i k e c e l l (B-cell) 56 of the amphipod h e p a t o p a n c r e a t i c caeca, as i n the c r a y f i s h hepatopancreas ( L o i z z i 1971) , i s an a c t i v e process of a p o c r i n e p i n c h i n g o f f of the a p i c a l complex. Another c o n t r o v e r s y , other than whether merocrine o r h o l o c r i n e s e c r e t i o n i s o c c u r r i n g , concerns whether the e x t r u s i o n s i n t o the lumen are a r e s u l t o f degeneration of the c e l l s or are a t r u e form of s e c r e t i o n . In i n s e c t s i t i s suggested t h a t t h i s i s a degenerative process t h a t occurs d u r i n g s t a r v a t i o n (Smith 1968). A l i k h a n (1969) found the number o f e x t r u s i o n s d i d not i n c r e a s e with i n c r e a s i n g maltase a c t i v i t y and so A l i k h a n d i d not f e e l the e x t r u s i o n s were a form of s e c r e t i o n . I t was suggested t h a t d u r i n g s t a r v a t i o n the decrease i n gut s i z e sgueezes the c e l l s out, De P r i e s t e r (1971) f o l l o w s t h i s l i n e of re a s o n i n g , s u g g e s t i n g t h a t the e x t r u s i o n s are a means of e l i m i n a t i n g degenerated c e l l components t h a t have undergone autophagy. Bunt (1968) found t h a t the degeneration and r u p t u r i n g of mature type H - c e l l s {storage c e l l s ) of the c r a y f i s h hepatopancreas has been m i s i n t e r p r e t e d as s e c r e t i o n , and t h a t e x t r u s i o n i s a c t u a l l y c a r r i e d out by F - c e l l s ( f i b r i l l a r c e l l s ) . The merocrine and/or h o l o c r i n e s e c r e t i o n seen i n the midgut of T f t c a l i f o r n i c u s may be a degenerative process as d i s c u s s e d above. P o s s i b l e evidence f o r t h i s i s the a b s o r p t i o n o f l i p i d by the c e l l s t h a t are l a t e r s e c r e t e d . , S t u d i e s on animals (Takahashi, P h i l p o t t and Miguel 1970; Howse and H e l f o r d 1972) and p l a n t s (flcLean 1968; Schuster, Hershenov and Aaronson 1968) have i n d i c a t e d t h a t one of the c h a r a c t e r i s t i c s of senescence i s storage of l i p i d s . In f a c t , such a degenerative process may be the means by which the f i n a l f u n c t i o n o f the c e l l types i n v o l v e d 57 i s enacted. The l i p i d s absorbed previous t o t h i s stage are p o s s i b l y metabolized and passed on to the haemocoel j u s t as i n any other absorbing c e l l . Hindgut The c e l l s of the a n t e r i o r hindgut of T i g r i o p u s c a l i f o r n i c u s ^ as i n the t e r r e s t r i a l i s o p o d (Vernon, H e r o l d and Witkus 1974), suggest l i t t l e s y n t h e s i s i s o c c u r r i n g yet the w e l l developed mitochondria suggest an energy r e g u i r i n g f u n c t i o n f o r these c e l l s . The deep i n d e n t a t i o n s of the lumen i n t o the c e l l s and the t h i n n e s s of the c u t i c l e l i n i n g t h i s r e g i o n of the hindgut a l s o suggest a b s o r p t i o n and t r a n s p o r t of m a t e r i a l s from the gut lumen i n t o the e p i t h e l i a l c e l l s . F a e c a l p e l l e t s are f r e q u e n t l y seen i n t h i s r e g i o n of the gut. They may be s t o r e d here to take advantage of t h e l a s t chance to absorb n u t r i e n t s from the d i g e s t e d remains. Many n u t r i e n t s a r e absorbed through the anal v e s i c l e of the wasp (Edson and Vinson 1977). In an ant the f a e c a l f l u i d c o n t a i n s high l e v e l s o f enzymes a c t i v e i n the degradation of p r o t e i n , c h i t i n and s t a r c h (Martin 1975). The occurrence o f a n t i p e r i s t a l t i c a c t i v i t e s of the arthropod hindgut, t o draw water i n t o the hindgut (Marshall and Orr 1972; S c h u l t z and Kennedy 1976), suggests a f u n c t i o n of osmoregulation f o r t h i s r e g i o n . In s m a l l e r c r u s t a c e a anal d r i n k i n g i s continuous while i n the a d u l t s of l a r g e r s p e c i e s i t occurs i n i n t e r m i t t e n t b u r s t s (Fox 1952). In the b r i n e shrimp osmoregulatory a b i l i t y of the gut e p i t h e l i u m i s i m p l i e d by the f a c t t h a t i t i s c o n t i n u o u s l y t a k i n g up hyper-, i s o - , or hypotonic medium ( r e l a t i v e t o the haemolymph) making i t hypo-osmotic and t a k i n g up water (Croghan 1958). Water uptake and 58 s a l t excretion were also found to occur i n the Metapenaeus shrimp gut (Dall 1967). Insect osmoregulation by the hindgut has been studied to a great extent (Berridge and Oschman 1972). The blowfly has c o r t i c a l c e l l s arranged in the form of four cones projecting into the r e c t a l lumen. These c e l l s function for ion and water transport (Gupta and Berridge 1966). This system has extensive i n t e r c e l l u l a r spaces surrounded by e p i t h e l i a l c e l l s but is o l a t e d from the haemocoel. Material i n these i n t e r c e l l u l a r sinuses must come either from/ or through, the hindgut e p i t h e l i a l c e l l s . The same sort of arrangement of c e l l s , sinuses and junctions occurs i n the much studied r e c t a l papillae of the cockroach.. Water regulation of the hindgut involves regulation of one or more ions (possibly potassium). With the aid of energy producing mitochondria which are abundant and an ATPase (Tclman and Steele 1976), the ion i s act i v e l y pumped int o c a v i t i e s i n the c e l l s . In the locust high levels of a sodium-potassiua-activated ATPase were present i n microsomal fract i o n s of the rectum only (Peacock 1976). Water then enters these c a v i t i e s from the cytoplasm of the c e l l s thus creating a chain of events with the ultimate source of water being the gut lumen and the ultimate destination of the water being the haemocoel (Smith 1968). The c u t i c l e of the hindgut plays a part in t h i s process by not allowing the passage of large molecules from the lumen into the c e l l c a v i t i e s , such molecules are excreted (Oschman and Wall 1969). In Tiqriopus c a l i f o r n i c u s the thin c u t i c l e with i t s many invaginations penetrating in t o the e p i t h e l i a l c e l l s and the 59 presence of mitochondria suggest the p o s s i b i l i t y of an osmoregulatory process s i m i l a r , though simpler, to that described above. The r e l a t i v e l y f l a t basal lamina of the T. c a l i f o r n i c u s anterior hindgut, as in the cockroach, allows ions that are resorbed by the e p i t h e l i a l c e l l s to diffuse away before e q u i l i b r a t i o n i s complete (Oschman and Wall 196 9). This means that there would not be a proportional flow of water into the r e c t a l pads.; The fact that no muscular system was noted i n the Tigriopus c a l i f o r n i c u s hindgut adds credence to the theory that osmoregulation through ion transport may occur here. This lack of musculature may allow the necessary recycling of the ions lost from the hindgut e p i t h e l i a i n the osmoregulation process (Oschman and Wall 1969; Uoirot and Noirot-Timothee 1976). Studies have shown that a muscle layer between the r e c t a l pads and the haemolymph can act, to some extent, as a physiological barrier to the movement of water and ions (Oschman and Wall 1969). In a t e r r e s t r i a l isopod the hindgut i s also suggested to be involved in water and ion movement and microtubule bundles are believed to function for support of the large c e l l s , movement of water and ions through the c e l l s and aligning the mitochondria (Witkus, G r i l l o and Smith 1969). In the sugar cane beetle, ion secretion occurs i n the posterior midgut (Cheung and Low 1975). In summary, the T. c a l i f o r n i c u s digestive tract i s composed of a c u t i c u l a r i z e d esophagus, a midgut caecum, an anterior and posterior midgut, and an anterior and posterior hindgut. Table one provides a summary of the c e l l types, t h e i r location, 60 s t r u c t u r e and f u n c t i o n . The esophagus has a w e l l developed muscle system which may f u n c t i o n t o draw food i n t o the gut.„ The musculature and shape of the esophagus allows f o r d i l a t i o n of the esophagus lumen to accommodate l a r g e diatoms and other such food. The c e l l s of the esophagus do not possess the o r g a n i z a t i o n normally a s s o c i a t e d with enzyme s y n t h e s i s and the c u t i c l e would probably i n h i b i t s e c r e t i o n i n t o the lumen of such d i g e s t i v e enzymes. However, some d i g e s t i o n does occur here, probably as a r e s u l t of enzymes s e c r e t e d by 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 v e n t r a l esophagus appears to f u n c t i o n i n the t r i t u r i t i o n of food. The d o r s a l esophagus, with i t s t h i n n e r c u t i c l e , appears to be i n v o l v e d i n accumulation of e l e c t r o n dense m a t e r i a l , p o s s i b l y s t o r a g e m a t e r i a l . In the midgut caecum the abundance o f c e l l t y p e t h r e e , presence of abundant l i p i d d r o p l e t s , great number of i n v a g i n a t i o n s of the e p i t h e l i a l a p i c e s , the l e n g t h of the m i c r o v i l l i and development of the b a s a l lamina suggest t h i s r e g i o n of the gut f u n c t i o n s mainly i n 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 . The a n t e r i o r midgut a l s o f u n c t i o n s f o r n u t r i e n t a b s o r p t i o n but another major r o l e appears t o be merocrine s e c r e t i o n . T h i s r e g i o n of the midgut i s unique i n having e l e c t r o n dense c o n c r e t i o n s found i n the c e l l types two and t h r e e . I t i s a l s o unique i n having a f o u r t h c e l l type. T h i s c e l l type appears t o f u n c t i o n i n l i p i d a b s o r p t i o n . The p o s t e r i o r midgut f u n c t i o n s mainly i n s e c r e t i o n , though some a b s o r p t i o n i s e v i d e n t . The a n t e r i o r hindgut probably 61 f u n c t i o n s mainly f o r osmoregulation and as a l a s t chance at absorbing n u t r i e n t s from the now forming f a e c a l p e l l e t s . J u s t before e x c r e t i o n , f a e c a l p e l l e t s are kept i n the h e a v i l y c u t i c u l i z e d p o s t e r i o r hindgut. T h i s r e g i o n o f the hindgut may f u n c t i o n i n compacting the f a e c a l p e l l e t s . I t can be seen, t h e r e f o r e , t h a t 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 of T. c a l i f o r n i c u s i s s i m i l a r t o other copepods. Other arthropods, t y p i c a l l y malacostracans, have a hepatopancreas branching o f f o f the midgut. T. c a l i f o r n i c u s does not have a hepatopancreas but the f u n c t i o n s of t h i s organ are f u l f i l l e d by the midgut. The c e l l types d e s c r i b e d f o r a v a r i e t y of other arthropods from c r u s t a c e a n s to i n s e c t s occur i n T . , c a l i f o r n i c u s . Food m a t e r i a l , i n the form of b a c t e r i a l c e l l s and diatom f r u s t u l e s , when present i n the T^ c a l i f o r n i c u s gut, o c c u r r e d i n aggregates. One aggregate i s found i n the lumen of the a n t e r i o r midgut, another i n the lumen of the p o s t e r i o r midgut and a f a e c a l p e l l e t aggregate i n the p o s t e r i o r hindgut. Each aggregation of food took the form of an oblong bolus. No matter where i n the midgut food m a t e r i a l was seen, i t was e q u a l l y d i g e s t e d . I t was not u n t i l the hindgut t h a t maximum d i g e s t i o n appears to have been completed. These o b s e r v a t i o n s along with s t u d i e s of the gut musculature suggest 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 have moved food m a t e r i a l back and f o r t h i n the gut lumen from r e g i o n s where a b s o r p t i o n occurs (midgut caecum and a n t e r i o r midgut) t o r e g i o n s where d i g e s t i o n occurs ( a n t e r i o r midgut and p o s t e r i o r midgut). In most cases the gut lumen was v o i d o f food m a t e r i a l of 62 any form. I t seems probable t h a t the shock of f i x a t i o n o r h a n d l i n g causes the copepod t o e j e c t the c o n t e n t s of i t s gut. Continuous u n i c u l t u r e f e e d i n g s t u d i e s would give an i n d i c a t i o n of the food p r e f e r e n c e s and growth p a t t e r n s of t h i s copepod. R a d i o a c t i v e l y l a b e l l i n g the l i p i d s and/or p r o t e i n s of food sources o f the copepod would be an i d e a l way to f i n d , f a i r l y c o n c l u s i v e l y , the a s s i m i l a t i o n a b i l i t y and r a t e s . Pulse l a b e l l i n g with such r a d i o a c t i v e food would allow one to f o l l o w the passage of food through the gut. Such a pulse l a b e l l i n g method would a l s o give an i n d i c a t i o n of a b s o r p t i o n r a t e s and where i n the gut each of these n u t r i e n t s was being absorbed. KEY FOR FIGURES A-anus a i - f i r s t antenna A2-second antenna A h - a n t e r i o r hindgut e p i t h e l i a l c e l l s AM-anterior midgut e p i t h e l i a l c e l l s Ap-a ppendage B-basal lamina c - c u t i c l e Ct-cephalothorax D-diatom f r u s t u l e E-esophagus e p i t h e l i a l t i s s u e E C - e x c r e t i n g (ed) c e l l ER—endoplasmic r e t i c u l u m ES-egg sac E x - e x t e r n a l environment f - f a e c a l m a t e r i a l g - g o l g i H-haemocoel L-lumen 1 - l i p i d H-muscle m-mitochondria MC-midgut caecum e p i t h e l i a l c e l l s M c - c i r c u l a r muscle M l - l o n g i t u d i n a l muscle !3t-microtubules 64 M v - m i c r o v i l l i M V-multivesicular bodies N-nucleus No-nucleolus HT-nervous t i s s u e 0- ovary Od-oviduct P - p e r i t r o p h i c membrane P1, 2, 3, 4-pereiopods 1, 2, 3, and 4 P h - p o s t e r i o r hindgut e p i t h e l i a P H - p o s t e r i o r midgut e p i t h e l i a R-rostrum r-ribosomes RER-rough endoplasmic r e t i c u l u m S-secreted m a t e r i a l SER-smooth endoplasmic r e t i c u l u m T j - t i g h t j u n c t i o n V - v e s i c l e Z-Z band of muscle sarcomeres 1- c e l l type one 2- c e l l type two 3- c e l l type three 4- c e l l type four • - r e g i o n where h o l o c r i n e s e c r e t i o n may have o c c u r r e d . LEAF 65 OMITTED IN PAGE NUMBERING. 65a Figure 1a. Scanning e l e c t r o n micrograph of an a d u l t male T. c a l f f o r n i c n s . Note the enlarged t i p o f the f i r s t antennae. LEAF 66 OMITTED IN PAGE NUMBERING. 66 a F i g u r e 1b. , Scanning e l e c t r o n micrograph of an a d u l t female T. c a l j f r o n j c u s . Note the egg sac c r a d l e d by the p o s t e r i o r pereiopods. LEAF 67 OMITTED IN PAGE NUMBERING. 67 a F i g u r e 1c., Low m a g n i f i c a t i o n l i g h t micrograph o f a whole mount of a mating p a i r of a d u l t T i q r i o p u s c a l i f o r n i c u s . F i g u r e 2. S a g i t t a l s e c t i o n o f T. c a l i f o r n i c u s A Arrows i n d i c a t e the p o s i t i o n o f t r a n s v e r s e s e c t i o n s i n the p l a t e s i n d i c a t e d . 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 i s evident as i s the c u t i c l e l i n i n g the esophagus and hindgut. LEAF 68 OMITTED IN PAGE NUMBERING. 68 a Figure 3. The esophagus surrounded by muscle proximally and haemocoel d i s t a l l y . Figure 4. Hidgut caecum regions showing abundant v e s i c l e s undergoing p o s s i b l e s e c r e t i o n . Figure 5. This micrograph shows the esophagus e n t e r i n g the a n t e r i o r midgut i n the re g i o n where the midgut caecum opens i n t o the a n t e r i o r midgut. Figure 6. The midgut caecum and esophagus i s seen i n t h i s micrograph to have fused with the a n t e r i o r midgut. Note the c u t i c l e of the esophagus ends abruptly t o give way to m i c r o v i l l i of the midgut c e l l s . LEAF 69 OMITTED IN PAGE NUMBERING. 69a Fi g u r e 7. The p o s t e r i o r midgut, c e l l s i n t h i s r e g i o n have fewer v e s i c l e s t h a t are d i s t i n c t l y s m a l l e r than those of the a n t e r i o r midgut. Note the d i f f e r e n t c e l l s i z e s , the l a r g e r c e l l s l i n e the v e n t r a l s u r f a c e o f the gut. F i g u r e 8. The a n t e r i o r hindgut e p i t h e l i a , note the r a t h e r t h i c k and dense c e l l s l a c k i n g c u t i c l e . T h i s r e g i o n o f the hindgut has l i t t l e muscle t i s s u e around the d i g e s t i v e t r a c t but i t i s surrounded by the b a s a l lamina and the haemocoel. F i g u r e 9. A p o r t i o n of a s a g i t t a l s e c t i o n of the t r a n s i t i o n area between the a n t e r i o r and the p o s t e r i o r hindgut. Note the t h i c k e n i n g of the c u t i c l e and absence of e p i t h e l i a l t i s s u e . F i g u r e 1 0 . The p o s t e r i o r hindgut as i t appeared when d i l a t e d . The r e g i o n appears t o l a c k an e p i t h e l i a l l i n i n g but i s l i n e d by r e l a t i v e l y t h i c k c u t i c l e . 69b LEAF 70 OMITTED IN PAGE NUMBERING. 70a F i g u r e 11,, The entrance of the esophagus i n t o the a n t e r i o r midgut of the d i g e s t i v e t r a c t i s shown i n t h i s s a g i t t a l s e c t i o n . Note c u t i c l e of esophagus ends a b r u p t l y where m i c r o v i l l i of midgut begins. F i g u r e 12, A c l u s t e r of c e l l type one not extending to the lumen are seen i n t h i s micrograph., Note the l a r g e n u c l e i i n these c e l l s . F i g u r e 13. T h i s l i g h t micrograph shows c e l l types one, two and three. C e l l type two and t h r e e a r e seen t o extend from the b a s a l lamina to the lumen. Fi g u r e 14. C e l l type two, being extruded i n t o the a n t e r i o r midgut lumen. C e l l types one and three can a l s o be seen i n t h i s f i g u r e . F i g u r e 13 and 14 may be taken as evidence f o r a process o f s e c r e t i o n o f c e i l t y p e two. F i g u r e 15, 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 midgut and the haemocoel d i r e c t l y below the e p i t h e l i a l b a s a l lamina are seen i n t h i s micrograph. The c e l l s are s m a l l e r and appear p s e u d o - s t r a t i f i e d . C e l l types one, two and three are s t i l l present., LEAF 71 OMITTED IN PAGE NUMBERING. 71a F i g u r e 16. Esophagus lumen and e p i t h e l i a l t i s s u e surrounded by c i r c u l a r muscle. Esophagus lumen takes on an •H * appearance. F i g u r e 17. Esophagus as i t e n t e r s a n t e r i o r midgut. Note l a c k of c i r c u l a r muscle and u n i d e n t i f i e d m u l t i v e s i c u l a t e bodies i n e p i t h e l i a l c e l l s . F i g u r e 18. Higher m a g n i f i c a t i o n of r e g i o n c u t i c l e showing l o n g i t u d i n a l and a s s o c i a t e d microtubules. next to esophagus c r o s s s e c t i o n s of LEAF 72 OMITTED IN PAGE NUMBERING. V 72a Figure 19. Midgut caecum e p i t h e l i a l c e l l s . The abundant vesicles of c e l l type three are evident as are the well developed golgi bodies of c e l l type two., Figure 20. Exocytosis of c e l l type three in midgut caecum. M i c r o v i l l i i n both longitudinal and cross section are shown. Figure 21. Higher magnification showing i n t e r d i g i t a t e d basal lamina of midgut caecum. Figure 22. M i c r o v i l l i of merocrine or holocrine micrograph. Figure 23. Three type one c e l l s are shown l y i n g at the basal lamina. Note that they do not reach the lumen. Figure 24. C e l l type two (on right) and c e l l type three (on l e f t ) are shown. Note large vesicles of c e l l type three and smaller ones of c e l l type two. the midgut caecum and evidence for secretion are shown in th i s LEAF 73 OMITTED IN PAGE NUMBERING. 73a F i g u r e 25. In 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, deep furrows extend from the lumen and almost reach the b a s a l lamina of the c e l l s . Bote s e c r e t i o n i n the upper r i g h t c o r n e r of the micrograph, a l s o note the c e l l type f o u r i n the lower mid p o r t i o n o f the micrograph. 73b LEAF 74 OMITTED IN PAGE NUMBERING. 7ua F i g u r e 26. Although c e l l types two and t h r e e can be d i s t i n g u i s h e d i n t h i s region of the gut, t h e i r d i f f e r e n c e s i n o v e r a l l e l e c t r o n d e n s i t y are l e s s obvious. The f o u r t h c e l l type of the a n t e r i o r midgut i s enclosed i n the c i r c l e o u t l i n e d by a dark l i n e . L o n g i t u d i n a l muscle i s seen i n a s s o c i a t i o n with the i n v a g i n a t i o n s of the b a s a l lamina. In t h i s micrograph m u l t i v e s i c u l a r bodies can be seen LEAF 75 OMITTED IN PAGE NUMBERING. 75 a F i g u r e 27. T h i s h i g h e r m a g n i f i c a t i o n micrograph shows the m u l t i v e s i c u l a r bodies o f the a n t e r i o r midgut. Note t h a t many v e s i c l e s , o f t e n with l a m e l l a t e s t r u c t u r e s , make up one body. L E A F 76 OMITTED IN PAGE NUMBERING . 76 a F i g u r e 28. The a n t e r i o r midgut i s seen, i n t h i s micrograph, t o possess e v a g i n a t i o n s a s s o c i a t e d with c e l l s of type one. C e l l types two and th r e e a re l e s s d i f f e r e n t i a t e d from eachother. C e l l type two i s l e s s e l e c t r o n dense and c e l l type t h r e e i s more e l e c t r o n dense than i n the midgut caecum. Note the l o n g i t u d i n a l muscle a s s o c i a t e d with the i n v a g i n a t i o n of the b a s a l region of the c e l l s . F i g u r e 29. T h i s i s a high e r m a g n i f i c a t i o n of the c e l l type four of F i g u r e 25. Note the great number o f 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 s e c r e t o r y products. V e s i c l e s a t a l l stages of maturation a r e e v i d e n t . The v e s i c l e s may be: 1. absorbed l i p i d , 2. zymogenic, or 3. e n d o c r i n a l . These v e s i c l e s are very s i m i l a r , i n s i z e (300-400mu) and appearance, t o the g a s t r i n - p r o d u c i n g c e l l s of the stomach i n higher organisms. F i g u r e 30. In the p o s t e r i o r midgut the b a s a l lamina shows fewer 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 and the m i c r o v i l l i , o f t e n only a s s o c i a t e d with c e l l type two, are s h o r t e r and l e s s r e g u l a r l y arranged. Note the c i r c u l a r muscle a t the base of t h e c e l l s . F i g u r e 31. In t h i s micrograph c e l l type two, i n the p o s t e r i o r midgut, are seen t o send 'arms* t o eachother. 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 m a t e r i a l s are common i n these c e l l s . LEAF 77 OMITTED IN PAGE NUMBERING. 77 a F i g u r e 32 and 33. These two micrographs are evidence of h o l o c r i n e s e c r e t i o n i n the p o s t e r i o r midgut. The i n c r e a s e d number o f c e l l type one a l s o adds credence t o the theory that h o l o c r i n e s e c r e t i o n i s o c c u r r i n g . Bote the d i s r u p t i o n of the m i c r o v i l l i where t h i s form of s e c r e t i o n i s o c c u r r i n g and the c l o s e p o s i t i o n of c e l l type one i n both micrographs. L E A F 7 8 O M I T T E D I N P A G E N U M B E R I N G . 78 a Fi g u r e 34., In the a n t e r i o r hindgut the m i c r o v i l l i become s h o r t e r and more s c a r c e . Rough E. R. i s abundant as are some v e s i c l e s . The b a s a l lamina i s l e s s i n v a g i n a t e d and the c e l l s are s i g n i f i c a n t l y s m a l l e r . F i g u r e 35. In the p o s t e r i o r hindgut the m i c r o v i l l i have been t o t a l l y r e p l a c e d by a t h i n c u t i c l e and f a e c a l m a t e r i a l has re p l a c e d t h e more r e c o g n i z a b l e food m a t e r i a l found more a n t e r i o r l y i n the d i g e s t i v e t r a c t lumen. F i g u r e 36. T h i s e l e c t r o n micrograph shows the t r a n s i t i o n from the f i n e r c u t i c l e to the denser c u t i c l e i n the most p o s t e r i o r r e g i o n s of the p o s t e r i o r hindgut. The e p i t h e l i a l t i s s u e i n these r e g i o n s i s minimal. F i g u r e 37. In the r e g i o n of the anus the c u t i c l e i s a p p a r e n t l y i d e n t i c a l i n s t r u c t u r e to the c u t i c l e surrounding the outer r e g i o n s of the body. F i g u r e 38. T h i s i s a higher m a g n i f i c a t i o n o f the r e g i o n o f the c u t i c l e i n d i c a t e d by the dashed box. Note the e l e c t r o n t r a n s p a r a n t r e g i o n s l y i n g next to the e p i c u t i c l e . TABLE 1. CELL TYPE LOCATION* STRUCTURE FUNCTION REFERENCES TO SIMILAR CELLS IN OTHER ARTHROPODS ONE PMDG -docs not reach lumen AMDG -found i n c l u s t e r s of 2 -4 MDGC c e l l s -basal lamina i n f o l d i n g s i s o l a t e t h i s c e l l type but do not penetrate cytoplasm -some mitochondria -minimal E.R. or g o l g i -varying abundance of ribosomes -replace c e l l s worn away or l o s t during secretion -vanWeel - P i l l a i -Hartenstein -Smith -Sud - C l i f f o r d and Witkus -de P r i e s t e r - L o i z z i -Schultz 955 960 964 ]968 ]968 197] ] 971 197] 1976 -Dakin )908" -Lowe ]9 35 -Reddy ]938 -van. Weel j 955 - P i l l a i I960 -Fahrenbach ]96J -Lewis ]96] - C l i f f o r d and Witkus ]97] - L o i z z i ' )97] -Marshall and Orr J972 -Schultz ]976 TWO PMDG AMDG MDGC -reaches lumen and has m i c r o v i l l i -has regular invaginations of basal lamina -randomly dispersed mitochondria -rough E.R. and loose ribosomes produce a very dense cytoplasm - d i l a t e d g o l g i bodies -exocytotic v e s i c l e s with e l e c t r o n opaque centers -some l i p i d droplets a p i c a l l y - i n ant.midgut i t cont-ains e l e c t r o n dense, -synthesis of enzymes -exocytotic and holocrine or merocrine secre-t i o n -some l i p i d absorption THREE MDGC -reaches lumen and has -mainly absorption -Dakin ]908 AMDG m i c r o v i l l i -some secretion of -Lowe ]935 PMDG -invaginated basal lamina enzymes -van Weel ] 955 -randomly d i s t r i b u t e d mito. - P i l l a i I960 -basal lamellate rough E.R. -Fahrenbach 196] -numerous golgi bodies -Lewis 196] -small and large v e s i c l e s -Smith 1968 seen throughout the c e l l -Sud 1968 - l i p i d v e s i c l e s a p i c a l l y i n - C l i f f o r d and Witkus J97] a n t e r i o r midgut contain - L o i z z i 197] electron dense m u l t i v e s i c u l - -Marshall and Orr 1972 ate bodies -Schultz 1976 FOUR AMDG -reaches lumen and has m i c r o v i l l i -minor invaginations of basal lamina -mitochondria s l i g h t l y more abundant b a s a l l y -minimal rough E.R. . -small and undilated g o l g i bodies -abundant supply of small e l e c t r o n dense v e s i c l e s a p i c a l l y -appears to be l i p i d absorption * - i n order of decreasing frequency -AHDG=antcrior midgut -MDGC=midgut ctocum -PMDG=postorior midgut LITERATURE CITED Ahearn, G.A. 1976. 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