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UBC Theses and Dissertations

Parasitism of Trichoptera by Bunodera mediovitellata (Digenea:Allocreadiidae) and the encapsulation responses Caira, Janine Nicole 1981

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PARASITISM OF TRICIIOrTERA BY 3UN0DERA MEDIOVITELLATA (DIGENEA: ALLOCREADIIDAE) AND THE ENCAPSULATION RESPONSES by JANINE NICOLE CAIRA B.Sc., U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f Zoology We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h Columbia J u l y , 1981 (c) J a n i n e N i c o l e C a i r a , 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f <^-OQ I The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 Date ABSTRACT The i n c i d e n c e o f p a r a s i t i s m by Bunodera m e d i o v i t e l l a t a i n T r i c h o p t e r a i n T i n Can Creek was i n v e s t i g a t e d . L a r v a e o f Lepidostoma r o a f i had a low i n c i d e n c e o f i n f e c t i o n , w h i l e P s y c h o g l y p h a a l a s c e n s i s l a r v a e had a h i g h i n c i d e n c e o f i n f e c t i o n . The e n c a p s u l a t i o n r e a c t i o n s o f t h e s e T r i c h o p t e r a l a r v a e t o i n t e r n a l Epon i m p l a n t s , and m e t a c e r c a r i a e o f t h e p a r a s i t e e n c y s t e d w i t h i n t h e s i l k g l a n d s , were examined i n v i v o t o d e t e r m i n e t h e d e t a i l s o f t h e c e l l u l a r e n c a p s u l a t i o n r e a c t i o n s o f T r i c h o p t e r a t o f o r e i g n o b j e c t s i n b o t h o f t h e s e s i t e s , and t o de t e r m i n e w h i c h hemocytes a r e i n v o l v e d i n t h e r e a c t i o n . The hemocytes o f l a r v a e o f P. a l a s c e n s i s were c a p a b l e o f e n c a p s u l a t i n g Epon i m p l a n t s w i t h i n t h e hemocoele; t h e r e a c t i o n t o o k a p p r o x i m a t e l y twenty days l o n g e r t h a n t h o s e o f o t h e r i n s e c t s t h a t have been m a i n t a i n e d a t 20°C. The hemocytes o f l a r v a e o f P. a l a s c e n s i s a r e a l s o c a p a b l e o f e n c a p s u l a t i n g t h e m e t a c e r c a r i a e o f t h e i r n a t u r a l p a r a s i t e B. m e d i o v i t e l l a t a i n t h e s i l k g l a n d s d e s p i t e t h e normal absence o f hemocytes from t h e s i l k g l a n d lumen. The hemocyte c a p s u l e s around b o t h t h e n o n - l i v i n g and l i v i n g o b j e c t s was t y p i c a l o f t h e ' o r d i n a r y e n c a p s u l a t i o n r e a c t i o n ' d e s c r i b e d by S a l t (1970). The c a p s u l e c o n s i s t e d o f an i n n e r l a y e r o f f l a t t e n e d c e l l s w hich were d e n s e l y packed and an o u t e r l a y e r o f c e l l s w h i c h were n o t f l a t t e n e d t o t h e same e x t e n t as t h o s e o f the i n n e r l a y e r . i i The hemocytes wh i c h took p a r t i n b o t h c a p s u l e s around i m p l a n t s and c a p s u l e s around p a r a s i t e s were f l a t t e n e d e l o n g a t e d hemocytes wh i c h p r o b a b l y c o r r e s p o n d t o p l a s m a t o c y t e s o r g r a n u l o c y t e s . However, t h e p r o d u c t i o n o f a Summary F a t e Map o f f r e e c i r c u l a t i n g hemocytes demonstrated t h a t hemocyte t y p e s a r e s t a g e s i n t h e development o f a number of l i n e s o f hemocyte t y p e s . I t appears t h a t t h e hemocytes t a k i n g p a r t i n t h e e n c a p s u l a t i o n r e a c t i o n s b e l o n g to two o f t h e s e l i n e s o f hemocyte t y p e s . i i i TABLE OF CONTENTS Page : I . ' INTRODUCTION 1 I I . MATERIALS AND METHODS.. 5 1. F i e l d S t u d i e s 5 2. E x p e r i m e n t a l and L a b o r a t o r y S t u d i e s 5 A. E x p e r i m e n t a l A n i m a l s 5 ( i ) T r i c h o p t e r a 5 ( i i ) Clams 6 B. R e a r i n g T r i c h o p t e r a 6 C. S t a t i s t i c a l A n a l y s i s 9 D. E x p e r i m e n t a l I n f e c t i o n 9 E. S i l k Gland D i s s e c t i o n P r o c e d u r e 10 F. M e t a c e r c a r i a l D e s c r i p t i o n 10 G. H i s t o l o g y 10 H. M e l a n i n T e s t 11 I . E l e c t r o n M i c r o s c o p y 11 J . I m p l a n t a t i o n P r o c e d u r e 12 K. Hemocyte E x a m i n a t i o n 13 I I I . RESULTS 16 A. T r i c h o p t e r a L a r v a e I n v o l v e d and L i f e C y c l e . 16 B. N a t u r a l I n f e c t i o n s 22 C. E x p e r i m e n t a l I n f e c t i o n s 25 D. M e t a c e r c a r i a l C y s t s from the Two S p e c i e s o f T r i c h o p t e r a 27 E. M e t a c e r c a r i a l D e s c r i p t i o n 28 F. The S i l k G l a n d s : Normal S t r u c t u r e 32 i v G. The Hemocyte Complex 42 H. R e a c t i o n t o I m p l a n t s . . 49 ( i ) Seven Day I m p l a n t s 52 ( i i ) T h i r t y - F i v e Day I m p l a n t s 55 I . Host Response t o t h e P a r a s i t e 58 ( i ) New I n f e c t i o n s 58 ( i i ) W e l l E s t a b l i s h e d I n f e c t i o n s 66 ( i i i ) O l d I n f e c t i o n s . . . 77 J . Hemocytes I n v o l v e d i n t h e E n c a p s u l a t i o n Response 80 IV. DISCUSSION 86 A. P a r a s i t e s o f T r i c h o p t e r a and t h e I n c i d e n c e o f the P a r a s i t e Bunodera m e d i o v i t e l l a t a i n i t s T r i c h o p t e r a n Host 86 B. Encystment o f A l l o c r e a d i i d s 91 C. E n c a p s u l a t i o n R e a c t i o n s 94 ( i ) Time o f R e a c t i o n 97 ( i i ) L a y e r s o f t h e C a p s u l e s 99 (a) N o n - L i v i n g I m p l a n t s . . . . . . . . . . . . . . . . . . . 99 (b) L i v i n g P a r a s i t e s 101 (c) Comparison o f t h e R e a c t i o n s t o L i v i n g and N o n - L i v i n g F o r e i g n O b j e c t s 106 ( i i i ) Hemocytes I n v o l v e d i n t h e R e a c t i o n 107 (a) N o n - L i v i n g I m p l a n t s 107 (b) Hemocytes I n v o l v e d i n t h e R e a c t i o n t o P a r a s i t e s 110 (c) Summary F a t e Map as a S o l u t i o n I l l D. Hemocyte A c t i v a t i o n 119 E. Wound H e a l i n g 121 V. BIBLIOGRAPHY 125 V I . APPENDIX 132 V LIST OF TABLES T a b l e Page . 1. A n a l y s i s o f t h e e f f e c t o f dat e o f c o l l e c t i o n , s p e c i e s o f T r i c h o p t e r a and s i z e o f case on the v a r i a n c e o f t h e number o f m e t a c e r c a r i a l c y s t s p e r l a r v a 26 2. C o m p a r i s i o n o f encystment and e n c a p s u l a t i o n o f m e t a c e r c a r i a e o f Bunodera m e d i o v i t e l l a t a between two s p e c i e s o f T r i c h o p t e r a 29 3. Data m a t r i x f o r c e l l s 1-24 o f P s y c h o g l y p h a  a l a s c e n s i s ( F i g u r e 11).....: 45 4. F a m i l i e s o f Digen e a , e x c l u d i n g t h e A l l o c r e a d i i d a e , whose members p a r a s i t i z e T r i c h o p t e r a 87 5. A l l o c r e a d i i d s whose second i n t e r m e d i a t e h o s t s a r e i n v e r t e b r a t e s o t h e r t h a n i n s e c t s 92 6. A l l o c r e a d i i d s whose second i n t e r m e d i a t e h o s t s are i n s e c t s 93 v i LIST OF FIGURES Figure Page 1. Schematic diagrams of the set-up for rearing Trichoptera larvae 8 2. Mean case lengths of the two species of Trichoptera over the twenty-four month c o l l e c t i n g period. 18 3. The l i f e cycle of Bunodera mediovitellata 21 4. Incidence of parasitism by Bunodera  mediovitellata i n the two species of Trichoptera 24 ,5. Longitudinal sections of metacercariae encysted within s i l k glands of Trichoptera 31 6. Excysted metacerGaria of Bunodera  mediovitellata 34 7. Longitudinal section of an uninfected s i l k gland of Psychoqlypha alascensis 37 8. Surface view of a s i l k gland of Psychoqlypha  alascensis demonstrating hexagonal e p i t h e l i a l c e l l s with polymorphic n u c l e i 37 9. Magnified view of a longitudinal section through a s i l k gland of Psychoglypha  alascensis demonstrating d i f f u s e nature of the e p i t h e l i a l c e l l nucleus 39 10. Schematic diagram of the construction of a s i l k gland 41 11. Morphologically d i s t i n c t hemocytes seen i n hemolymph smears of Psychoglypha alascensis... 4 4 12. Cladogram of hemocytes of Psychoglypha  alascensis 48 13. Summary Fate Map of hemocyte lineages 51 14. Cross sections of the hemocyte response of Psychoglypha alascensis to an Epon s l i v e r implanted in the abdominal hemocoele and removed a f t e r seven days 54 v i i 15. C r o s s s e c t i o n o f t h e response o f P s y c h o g l y p h a  a l a s c e n s i s t o an Epon s l i v e r i m p l a n t e d i n t h e abdominal hemocoele and removed a f t e r 35 days. 57 16. M a g n i f i e d v i e w o f response o f P s y c h o g l y p h a  a l a s c e n s i s t o a 35 day o l d Epon i m p l a n t 60 17. L o n g i t u d i n a l s e c t i o n o f a young m e t a c e r c a r i a o f Bunodera m e d i o v i t e l l a t a w i t h i n t h e e p i t h e l i a l w a l l o f t h e s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s 63 18. M a g n i f i e d v i e w o f a l o n g i t u d i n a l s e c t i o n o f : t h e s i l k g l a n d e p i t h e l i u m a d j a c e n t t o t h e p a r a s i t e . 6 3 19. M a g n i f i e d v i e w o f a l o n g i t u d i n a l s e c t i o n o f t h e s i l k g l a n d e p i t h e l i u m a c r o s s from t h e p a r a s i t e 65 20. L o n g i t u d i n a l s e c t i o n o f a m e t a c e r c a r i a o f Bunodera m e d i o v i t e l l a t a e s t a b l i s h e d w i t h i n t h e s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s . 6 8 21. M a g n i f i e d v i e w s o f two a r e a s o f a , l o n g i t u d i n a l s e c t i o n t h r o u g h a m e t a c e r c a r i a o f Bunodera m e d i o v i t e l l a t a e n c y s t e d w i t h i n t h e s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s 70 22. E l e c t r o n m i c r o g r a p h s o f c a p s u l e s around m e t a c e r c a r i a e o f Bunodera m e d i o v i t e l l a t a e n c y s t e d w i t h i n t h e lumen o f a s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s 73 23. T r a n s v e r s e s e c t i o n o f a m e l a n i z e d m e t a c e r c a r i a o f Bunodera m e d i o v i t e l l a t a i n w h i c h hemocytes have e n t e r e d t h e s i l k g l a n d lumen and a t t a c h e d t o t h e p a r a s i t e 76 24. C r o s s s e c t i o n o f a s i l k g l a n d o f P s y c h o g l y p h a  a l a s c e n s i s c o n t a i n i n g two m e l a n i z e d m e t a c e r c a r i a e o f Bunodera m e d i o v i t e l l a t a 79 25. L o n g i t u d i n a l s e c t i o n t h r o u g h a m e l a n i z e d m e t a c e r c a r i a l c y s t o f Bunodera m e d i o v i t e l l a t a . 79 26. Drawings o f sample hemocytes from hemolymph smears and i n v o l v e d i n o u t e r l a y e r o f c a p s u l e s 83 27. Summary F a t e Map 115 ACKNOWLEDGMENTS I w i s h t o e x p r e s s my s i n c e r e g r a t i t u d e t o my s u p e r v i s o r , Dr. G.G.E. Scudder f o r h i s s u p p o r t and e n c o u r a g i n g g u i d a n c e t h r o u g h o u t t h i s s t u d y . I n a d d i t i o n I w i s h t o thank Dr. D.R. Brooks f o r b r i n g i n g c l a d i s t i c s t o U.B.C, and f o r a s s i s t i n g me w i t h t h e c l a d i s t i c a n a l y s i s o f t h e i n s e c t hemocytes. I a l s o thank Mr. L. Veto who p a t i e n t l y g u i d e d me t h r o u g h t h e p r e p a r a t i o n o f my m a t e r i a l f o r e l e c t r o n m i c r o s c o p y . Dr. J . Maze a s s i s t e d w i t h t h e s t a t i s t i c a l a n a l y s i s , and I thank him. Many th a n k s t o Dr. M.J. Kennedy f o r showing me h i s s a m p l i n g s i t e s i n T i n Can Creek and f o r a l l o w i n g me t o see h i s u n p u b l i s h e d m a n u s c r i p t on t h e l i f e c y c l e o f Bunodera m e d i o v i t e l l a t a . I am a l s o g r a t e f u l t o Dr. G.B. Wiggi n s f o r i d e n t i f y i n g t h e specimens o f T r i c h o p t e r a . I am e s p e c i a l l y i n d e b t e d t o Kate Shaw, Lynn V a s i n g t o n and C a r o l Mc N i c h o l f o r t h e i r h e l p b o t h i n s a m p l i n g T i n Can Creek and i n the p r e p a r a t i o n o f t h i s m a n u s c r i p t . 1 I . INTRODUCTION D e s p i t e t h e p r o t e c t i v e n a t u r e o f t h e i r tough e x o s k e l e t o n , i n s e c t s a r e o f t e n s u b j e c t t o i n s u l t by v a r i o u s pathogens. A c c o r d i n g t o S a l t (1970), depending on t h e s i z e and n a t u r e o f t h e pathogen, th e i n s e c t w i l l r e s p ond i n one o f t h r e e f a s h i o n s . F i r s t l y , s m a l l i n d i v i d u a l o r g a n i s m s , such as b a c t e r i a and v i r u s e s may be d e a l t w i t h t h r o u g h p h a g o c y t o s i s ; 'phagocytes' e n g u l f t h e p a r t i c l e s and t h u s e l i m i n a t e them from t h e hemolymph. S e c o n d l y , p a r t i c l e s t o o l a r g e t o be p h a g o c y t i s e d , such as metazoan p a r a s i t e s , a r e surrounded by a number of b l o o d c e l l s w hich adhere t o t h e s u r f a c e o f t h e p a r a s i t e : t h e e n t i r e s t r u c t u r e i s termed a c a p s u l e , t h e p r o c e s s i s termed e n c a p s u l a t i o n . T h i r d l y , clumps of b a c t e r i a o r o t h e r f o r e i g n p a r t i c l e s may be i s o l a t e d i n a n o d u l e : t h i s p r o c e s s i s a c o m b i n a t i o n o f p h a g o c y t o s i s and e n c a p s u l a t i o n . In h i s e x a m i n a t i o n o f t h e metazoan p a r a s i t e s o f i n s e c t s , Maupus (1899) was one o f t h e f i r s t t o d i s t i n g u i s h between th e terms ' e n c a p s u l a t i o n ' and 'encystment'; he r e s t r i c t e d t h e f i r s t term t o t h e r e a c t i o n o f t h e h o s t , and t h e second t o t h e a c t i v i t y o f t h e p a r a s i t e . S i n c e h i s t i m e , t h e i n v e s t i g a t i o n o f t h e c e l l u l a r e n c a p s u l a t i o n r e a c t i o n s o f i n s e c t s t o t h e i r metazoan p a r a s i t e s has become o f i n t e r e s t t o two groups o f r e s e a r c h e r s : (1) t h o s e w i s h i n g t o p r o t e c t e c o n o m i c a l l y i m p o r t a n t i n s e c t s from t h e i r p a r a s i t e s ( F r e e , 1970) , f o r example th e s i l k moth Bombyx sp. 2 (Sato e t a l . 1976), and (2) t h o s e w i s h i n g t o e l i m i n a t e c e r t a i n i m p o r t a n t i n s e c t p e s t s such as mosquitoes ( A n d r e a d i s and H a l l , 1976), w e e v i l s (Bosch and D i e t r i c k , 1959), s p r u c e s a w f l i e s (Hawboldt, 1947) , and f l e a s (Chen, 1934) . I n o r d e r t o o b t a i n a b r o a d and t h o r o u g h u n d e r s t a n d i n g o f t h e c e l l u l a r d e f e n s e r e a c t i o n s o f i n s e c t s , i t i s n e c e s s a r y t o examine t h e r e a c t i o n s i n as many o r d e r s o f i n s e c t s as p o s s i b l e . U n f o r t u n a t e l y , t h e d e t a i l e d e x a m i n a t i o n s o f t h e e n c a p s u l a t i o n r e a c t i o n s o f i n s e c t s have c e n t e r e d around the r e a c t i o n s o f a l i m i t e d number o f i n s e c t o r d e r s . The r e a c t i o n s o f a number of o t h e r i n s e c t groups i n c l u d i n g T r i c h o p t e r a have never been c l o s e l y examined. The m e t a c e r c a r i a e o f t h e a l l o c r e a d i i d d igenean Bunodera m e d i o v i t e l l a t a Z i m b a l i k and Roytman n a t u r a l l y e n c y s t w i t h i n the s i l k g l a n d s o f T r i c h o p t e r a l a r v a e (Kennedy, i n l i t . ) . T h i s s i t e p r o v i d e s an i n t e r e s t i n g system i n w h i c h t o examine t h e e n c a p s u l a t i o n r e a c t i o n o f an a q u a t i c i n s e c t t o a n a t u r a l i n t e r n a l p a r a s i t e . A l t h o u g h , t h e e n c a p s u l a t i o n r e a c t i o n s o f T r i c h o p t e r a t o f o r e i g n o b j e c t s , u s u a l l y l o c a t e d i n t h e hemocoele, have been examined p r e v i o u s l y ( L i n s t o w , 1897; Thompson, 1915; Neuhaus, 1940; B u r n s , 1961), t h e s e a c c o u n t s a r e s u p e r f i c i a l and, as t h e a u t h o r s were more i n t e r e s t e d i n t h e p a r a s i t e s than t h e h o s t r e a c t i o n , t h e d e t a i l s o f t h e c a p s u l e a r e poor. I t i s now g e n e r a l l y a c c e p t e d t h a t hemocytes a r e t h e main agents o f e n c a p s u l a t i o n o f f o r e i g n o b j e c t s i n 3 i n s e c t s ( S a l t , 1963). A l t h o u g h i t has been suggested t h a t o t h e r components i n c l u d i n g c e l l s o f t h e f a t body (Thompson and Thompson, 1921), and c o n n e c t i v e t i s s u e ( S c h e l l , 1952) may be i n v o l v e d i n t h e r e a c t i o n , S a l t (1963) d i s m i s s e s t h e s e c i t i n g s as s t r u c t u r e s t h a t have a c c i d e n t l y been e n c a p s u l a t e d by t h e hemocytes, o r c e l l s t h a t have been m i s t a k e n f o r c e l l s o t h e r t h a n hemoctyes. The s t u d y o f t h e e n c a p s u l a t i o n r e s ponse o f T r i c h o p t e r a l a r v a e t o m e t a c e r c a r i a e o f B. m e d i o v i t e l l a t a i s worthy o f a t t e n t i o n f o r s e v e r a l r e a s o n s : (1) t h e e n c a p s u l a t i o n r e s p o n s e o f T r i c h o p t e r a o r o t h e r a q u a t i c i n s e c t s t o metazoan p a r a s i t e s has not been examined i n d e t a i l ; (2) t h e hemocyte complex o f T r i c h o p t e r a and t h e hemocytes i n v o l v e d i n t h e e n c a p s u l a t i o n r e a c t i o n have not been examined i n d e t a i l ; (3) t h e s i l k g l a n d s a r e an u n u s u a l s i t e f o r t h e encystment o f a d i g e n e a n , and t h e r e f o r e an u n u s u a l s i t e i n which t o examine the hemocyte response e s p e c i a l l y s i n c e hemocytes a r e n o t n o r m a l l y p r e s e n t w i t h i n t h e lumen o f t h e g l a n d . Thus, t h e purposes o f t h i s s t u d y were: (1) t o c o n f i r m t h e d e t a i l s o f t h e l i f e c y c l e o f B. m e d i o v i t e l l a t a and i n v e s t i g a t e t h e i n f e c t i o n o f t h e T r i c h o p t e r a l a r v a e i n v o l v e d ; (2) t o examine t h e e n c a p s u l a t i o n r e a c t i o n o f t h e s e T r i c h o p t e r a l a r v a e t o m e t a c e r c a r i a e o f B. m e d i o v i t e l l a t a e n c y s t e d w i t h i n t h e hemocyte-free lumen o f the s i l k g l a n d s ; and (3) t o de t e r m i n e which hemocytes t a k e p a r t i n t h e c a p s u l e f o r m a t i o n . In a d d i t i o n , t h e r e a c t i o n o f the T r i c h o p t e r a l a r v a e t o n o n - l i v i n g i m p l a n t s l o c a t e d 4 w i t h i n t h e hemocoele, was a l s o s t u d i e d , i n o r d e r t o compare t h i s w i t h t h e e n c a p s u l a t i o n r e sponse t o t h e p a r a s i t e i n t h e s i l k g l a n d s . 5 I I . MATERIALS AND METHODS 1. F i e l d S t u d i e s The l i f e c y c l e and n a t u r a l i n f e c t i o n s o f T r i c h o p t e r a were s t u d i e d by p e r i o d i c s a m p l i n g o f t h e fauna i n T i n Can Creek, a s m a l l stream t h a t f l o w s from t h e U n i v e r s i t y Endowment Lands t h r o u g h Musqueum P a r k , and e n t e r s t h e S t r a i t o f G e o r g i a near t h e mouth o f t h e n o r t h arm o f t h e F r a s e r R i v e r . Samples were t a k e n w i t h a d i p n e t , a t a p p r o x i m a t e l y t h r e e month i n t e r v a l s b e g i n n i n g i n A p r i l 1979 and e n d i n g May 1981. 2. E x p e r i m e n t a l and L a b o r a t o r y S t u d i e s A. E x p e r i m e n t a l A n i m a l s ( i ) T r i c h o p t e r a L a r v a l T r i c h o p t e r a were c o l l e c t e d from t h e . b o t t o m d e t r i t u s o f t h e edges o f T i n Can Creek w i t h a s m a l l d i p n e t . The c o l l e c t i o n s were t a k e n from t h e stream as i t p a s s e s t h r o u g h Musqueum P a r k , on t h e s o u t h s i d e o f South West Marine D r i v e as i t meets 4 1 s t Avenue, a p p r o x i m a t e l y 100 meters downstream from t h e t u n n e l i n t o w h i c h i t f l o w s from underneath a h o u s i n g development. A l l samples were t r a n s p o r t e d from t h e f i e l d t o t h e l a b o r a t o r y i n f o u r - l i t e r p l a s t i c b u c k e t s f i l l e d w i t h s t r e a m w a t e r . I n t h e l a b o r a t o r y t h e b u c k e t s were p l a c e d i n a c o n s t a n t t e m p e r a t u r e chamber s e t a t 10°C and l a t e r s o r t e d i n t o s p e c i e s . The l a r v a e were f e d w i t h d e c a y i n g l e a v e s a l s o c o l l e c t e d from t h e s i t e i n T i n Can Creek. ( i i ) Claras Clams ( P i s i d u m sp.) were c o l l e c t e d i n n e t f u l s o f sand from th e bottom o f T i n Can Creek. The clams were c a r r i e d t o t h e l a b o r a t o r y i n t h e same b u c k e t s as t h e l a r v a l T r i c h o p t e r a . I f t h e samples were t o be k e p t f o r a p e r i o d o f t i m e t h e clams were removed from t h e s e b u c k e t s and k e p t s e p a r a t e l y i n a e r a t e d b u c k e t s w i t h a l a y e r o f sand on t h e bottom. B. R e a r i n g T r i c h o p t e r a The a p p a r a t u s used t o r e a r t h e T r i c h o p t e r a l a r v a e t o a d u l t s i s i l l u s t r a t e d i n F i g u r e 1. T h i s i s a d e s i g n s i m i l a r t o t h a t o f Neander (1928); however, i t has been s l i g h t l y m o d i f i e d by t h e a d d i t i o n o f a g l a s s p e t r i - p l a t e v i e w i n g l i d . I n a d d i t i o n , t o f a c i l i t a t e t h e r e a r i n g o f c a d d i s f l y l a r v a e r e q u i r i n g moving w a t e r , the whole c o n t a i n e r was p l a c e d i n a p l a s t i c d i s h pan. The pan was then p l a c e d i n t o a s i n k and t h e hose from t h e c o l d water t a p was clamped t o t h e pan and a l l o w e d t o r u n c o n s t a n t l y a t a medium speed. C a d d i s f l y l a r v a e were examined and s e p a r a t e d i n t o d i f f e r e n t m o r p h o l o g i c a l t y p e s (those p o s s e s s i n g a d o r s a l c e n t e r i n g hump on t h e f i r s t abdominal segment and t h o s e p o s s e s s i n g no hump); twenty l a r v a e o f each t y p e were p l a c e d i n each r e a r i n g cage a t one t i m e . The l a r v a e were f e d ad l i b i t u m w i t h d e c a y i n g l e a v e s c o l l e c t e d from T i n Can Creek. 7 FIGURE 1 Schematic diagrams o f t h e s e t - u p f o r r e a r i n g T r i c h o p t e r a l a r v a e , (a) r e a r i n g chamber, (b) r e a r i n g chamber s e t up i n moving w a t e r system. 9 The r e a r i n g c o n t a i n e r s were v i s u a l l y i n s p e c t e d a t l e a s t b i w e e k l y . Imagos seen c l i n g i n g t o t h e c h e e s e c l o t h a t t h e t o p o f t h e c o n t a i n e r s were p l a c e d i n a s o l u t i o n o f 70% e t h a n o l and s e n t t o Dr. G.B. Wiggins a t t h e R o y a l O n t a r i o Museum i n Tor o n t o f o r i d e n t i f i c a t i o n . C. S t a t i s t i c a l 7Analysis I n o r d e r t o d e t e r m i n e i f t h e number o f c y s t s p e r i n d i v i d u a l was a f f e c t e d by t h e s i z e o f t h e c a s e , s p e c i e s o f T r i c h o p t e r a , o r d a t e o f c o l l e c t i o n , an a n a l y s i s o f v a r i a n c e u s i n g t h e U.B.C. TAnovar program (Greg and O s t e r l i n , 1978) was a p p l i e d . D. E x p e r i m e n t a l I n f e c t i o n E i g h t l a r v a e o f each o f t h e s p e c i e s o f c a d d i s f l y , m easuring between 10 and 15 mm. i n l e n g t h ( i n c l u d i n g t h e case) , were removed from t h e i r c a s e s and p l a c e d i n d e c h l o r i n a t e d w a t e r i n w e l l s o f w e l l - p l a t e s s e t up under a s t e r e o d i s s e c t i n g m i c r o s c o p e . Clams i n s m a l l p e t r i -d i s h e s were opened w i t h m i c r o f o r c e p s and c e r c a r i a e were removed from t h e clams by b r e a k i n g the r e d i a l membranes. Ten t o 20 c e r c a r i a e were p i p e t t e d i n t o each o f t h e w e l l s c o n t a i n i n g T r i c h o p t e r a l a r v a e . These l a r v a e were v i s u a l l y examined f o r p e n e t r a t i o n o f c e r c a r i a e o v e r a p e r i o d o f a p p r o x i m a t e l y one hour. A f t e r t h r e e hours the l a r v a e were d i s s e c t e d and examined f o r c e r c a r i a e b o t h i n t h e body c a v i t y , and i n p a r t i c u l a r w i t h i n t h e s i l k g l a n d s . 10 As t h e a n i m a l s b e i n g i n f e c t e d were from a n a t u r a l p o p u l a t i o n , and t h e r e f o r e n ot n e c e s s a r i l y u n i n f e c t e d , e i g h t non-e x p e r i m e n t a l f i e l d caught l a r v a e o f each s p e c i e s were i s o l a t e d , d i s s e c t e d and a l s o examined f o r c e r c a r i a e w i t h i n t h e body c a v i t y and s i l k g l a n d s . E. S i l k G l a n d D i s s e c t i o n P r o c e d u r e T r i c h o p t e r a l a r v a e i n d e c h l o r i n a t e d water were d i s s e c t e d under a s t e r e o d i s s e c t i n g m i c r o s c o p e u s i n g two p a i r s o f m i c r o f o r c e p s . One p a i r was used t o f i r m l y h o l d t h e p r o t h o r a x , w h i l e t h e o t h e r p a i r was used t o g r a s p t h e p o s t e r i o r p o r t i o n o f t h e head and p u l l i t s h a r p l y f o r w a r d and o f f . Because t h e s i l k g l a n d s a r e a t t a c h e d i n t h i s r e g i o n o f t h e head, t h e y were u s u a l l y p u l l e d o ut i n t a c t , a l o n g w i t h t h e head. F. M e t a c e r c a r i a l D e s c r i p t i o n F i v e m e t a c e r c a r i a e were t e a s e d from t h e i r c a p s u l e s i n P. a l a s c e n s i s and removed from t h e i r c y s t s u s i n g two f i n e d i s s e c t i n g n e e d l e s . The e x c y s t e d m e t a c e r c a r i a e were p r e p a r e d a c c o r d i n g t o t h e p r o t o c o l o f Chubb (196 2) and s t a i n e d w i t h E r l i c h ' s h e m a t o x y l i n . Drawings were done w i t h t h e a i d o f a d r a w i n g t u b e . Measurements were done u s i n g an o c u l a r m i c r o m e t e r . G. H i s t o l o g y The s i l k g l a n d s o f T r i c h o p t e r a c o n t a i n i n g m e t a c e r c a r i a e were embedded i n wax and s t a i n e d w i t h E r l i c h ' s h e m a t o x y l i n 11 and e o s i n Y a c c o r d i n g t o t h e method d e s c r i b e d i n C u l l i n g (1963). S e c t i o n s o f m e t a c e r c a r i a e w i t h i n t h e s i l k g l a n d s o f t h e two s p e c i e s o f T r i c h o p t e r a were compared and t h e f o l l o w i n g c h a r a c t e r i s t i c s n o t e d : (1) g e n e r a l appearance o f t h e c e r c a r i a (2) p o s i t i o n o f c e r c a r i a w i t h i n t h e g l a n d (3) damage t o t h e g l a n d (4) h o s t r e a c t i o n t o t h e c e r c a r i a . H. M e l a n i n T e s t Ten p a r a f f i n s e c t i o n s were p r e p a r e d from each o f two pigmented m e t a c e r c a r i a e c o l l e c t e d on J u l y 28, 1979. Ten o f t h e s e s e c t i o n s ( f i v e from each m e t a c e r c a r i a ) were t r e a t e d w i t h t h e F e r r o u s I r o n Technique o f Pearse (1972) , a t e c h n i q u e s p e c i f i c f o r s t a i n i n g m e l a n i n . T h i s t e s t s t a i n s m e l a n i n dark g r e e n , w h i l e t h e background remains f a i n t ' g r een o r c o l o u r l e s s . The r e m a i n i n g t e n s e c t i o n s were t r e a t e d w i t h t h e m e l a n i n - s p e c i f i c b l e a c h , p o t a s s i u m permanganate f o l l o w e d by o x a l i c a c i d ( P e a r s e , 1972). The b l e a c h e d s e c t i o n s were then t r e a t e d w i t h t h e F e r r o u s I r o n Technique f o r m e l a n i n . I . E l e c t r o n M i c r o s c o p y M e t a c e r c a r i a l c y s t s t o be examined under t h e e l e c t r o n m i c r o s c o p e were p r e p a r e d a c c o r d i n g t o the p r o t o c o l o f R. Dean ( p e r s o n a l c o m m u n i c a t i o n ) . Owing t o t h e dense n a t u r e o f t h e c y s t s however, th e p r o t o c o l was a l t e r e d t o i n c r e a s e p e n e t r a t i o n . One drop o f P h o t o f l o w d i l u t e d 1:100 was added t o t h e f i x a t i o n and wash s o l u t i o n s t o reduce t h e s u r f a c e t e n s i o n - a r o u n d the c y s t s . A l l f i x a t i o n , wash, d e h y d r a t i o n w i t h p r o p y l e n e o x i d e , and i n f i l t r a t i o n t i m e s were extended t o o v e r n i g h t . The c y s t s were embedded i n S p u r r s , a low v i s c o s i t y r e s i n . The f i x a t i o n , wash, d e h y d r a t i o n w i t h p r o p y l e n e o x i d e , i n f i l t r a t i o n and embedding were c a r r i e d o ut under vacuum. The e n t i r e p r o t o c o l used i s g i v e n i n Appendix 1. Both t h i c k (4u) s e c t i o n s and t h i n ( a p p r o x i m a t e l y 200A) s e c t i o n s were c u t u s i n g a S o r v a l MT-1 u l t r a m i c r o t o m e e q uipped w i t h a g l a s s k n i f e . T h i c k s e c t i o n s were s t a i n e d w i t h a s o l u t i o n o f methylene b l u e i n 1% bo r a x w h i c h had been s i t t i n g f o r some t i m e and t h e r e f o r e had a c q u i r e d polychrome s t a i n i n g q u a l i t i e s . T h i n s e c t i o n s were p l a c e d on g r i d s c o a t e d w i t h c a r b o n . These s e c t i o n s were s t a i n e d w i t h l e a d c i t r a t e and examined a t 60 kv. w i t h a Z e i s s EM-10. J . I m p l a n t a t i o n P r o c e d u r e Each o f s i x c a d d i s f l y l a r v a e was p l a c e d i n a s m a l l v i a l a t t a c h e d t o a CO2 tank f o r 4.5 minutes and t h u s a n e s t h e t i z e d . The l a r v a was t h e n removed from i t s c a s e and a t t a c h e d w i t h p l a s t i c i n e t o t h e bottom o f a s m a l l w e i g h i n g d i s h f i l l e d w i t h d e c h l o r i n a t e d w a t e r . A s m a l l l o n g i t u d i n a l i n c i s i o n was made on t h e r i g h t d o r s a l s i d e o f t h e f i r s t t o t h i r d abdominal segments o f t h e a n i m a l , and an Epon 812 s l i v e r m easuring a p p r o x i m a t e l y 0.2 x 1.00 mm. was i m p l a n t e d w i t h m i c r o f o r c e p s i n t o t h i s a r e a s u c h t h a t i t was l y i n g f r e e w i t h i n t h e h e m o c o e l e . The l a r v a was t h e n r e t u r n e d t o i t s c a s e and p l a c e d i n a 300 ml. p r e p a r a t i o n d i s h c o n t a i n i n g d e c a y i n g l e a v e s and d e c h l o r i n a t e d w a t e r . The l a r v a e were k e p t a e r a t e d i n a c o n s t a n t t e m p e r a t u r e chamber a t 10°C. A f t e r e i t h e r s e v e n o r 35 d a y s , e a c h o f t h r e e l a r v a e c o n t a i n i n g i m p l a n t s was removed f r o m i t s c a s e and a n e s t h e t i z e d f o r t e n m i n u t e s a s a b o v e . The a r e a o f t h e i n c i s i o n , ( r e c o g n i z a b l e by t h e p i g m e n t a t i o n o f t h e wound h e a l i n g r e a c t i o n a t t h e s i t e o f t h e i n c i s i o n o f t h e i n t e g u m e n t ) was t h e n s l i t w i t h a r a z o r t o o l and t h e h e m o c o e l e examined f o r t h e i m p l a n t . I m p l a n t s f o u n d l y i n g f r e e w i t h i n t h e h e m o c o e l e a t t h i s t i m e were i m m e d i a t e l y p l a c e d i n t o f i x a t i v e a n d embedded i n Epon 812 a c c o r d i n g t o t h e p r o c e d u r e o f R. Dean ( p e r s o n a l c o m m u n i c a t i o n ) . The Epon 812 b l o c k s were s e c t i o n e d a t 4u w i t h a S o r v a l MT-1 u l t r a m i c r o t o m e e q u i p p e d w i t h a g l a s s k n i f e . S e c t i o n s were s t a i n e d w i t h m e t h y l e n e b l u e ( p o s s e s s i n g p o l y c h r o m a t i c s t a i n i n g a b i l i t y ) i n 1% b o r a x . K. Hemocyte E x a m i n a t i o n Hemocytes were h e a t f i x e d w i t h i n t h e i n s e c t by p l a c i n g t h e l a r v a l T r i c h o p t e r a i n a b e a k e r o f 6 0°C w a t e r f o r f i v e m i n u t e s . I m m e d i a t e l y a f t e r f i x a t i o n , hemolymph was removed f r o m t h e abdomen o f t h e i n s e c t s u s i n g a f i n e l y drawn g l a s s p i p e t t e . The hemolymph was t h e n smeared on a g l a s s s l i d e and a l l o w e d t o a i r d r y . E a c h s l i d e was p r e p a r e d and s t a i n e d w i t h Giemsa a c c o r d i n g t o t h e p r o t o c o l o f A r n o l d and H i n k s (19 7 9 ) . The d r y s t a i n e d smears were c o v e r e d w i t h permount and #1 c o v e r s l i p s . Each s l i d e was scanned under emersion o i l f o r as many d i f f e r e n t hemocytes as p o s s i b l e . Photographs of t h e s e c e l l s were t a k e n u s i n g a camera adapted t o a m i c r o s c o p e and Ektachrome Tungsten 50 ASA f i l m . The Wagner method o f q u a n t i t a t i v e parsimony a n a l y s i s ( c l a d i s t i c ) ( F a r r i s , 1970) was used t o a n a l y z e t h e d a t a . T h i s method produces a b r a n c h i n g diagram (cladogram) o f t h e s h o r t e s t r o o t e d t r e e f o r t h e d a t a s e t . A l i s t o f the c h a r a c t e r s used t o c o n s t r u c t t h e d a t a m a t r i x i s g i v e n below: A- a r e a o f c e l l ( n u c l e u s a r e a + c y t o p l a s m area) B- n u c l e u s a r e a C- c y t o p l a s m a r e a / n u c l e u s a r e a D- p o s i t i o n o f n u c l e u s E- shape o f c e l l F- compactness of c y t o p l a s m G- compactness o f n u c l e u s H- c y t o p l a s m g r a n u l a t i o n I - c y t o p l a s m v a c u o l a t i o n . Only t h o s e c e l l s whose c y t o p l a s m and n u c l e u s were r e a d i l y d i s t i n g u i s h a b l e were used i n t h e a n a l y s i s . F o r t h o s e c h a r a c t e r s i n v o l v i n g a r e a measurments (A,B,C) photographs of each c e l l were t r a n s p o s e d t o 1 mm.2 graph paper and t h e number o f squares c o v e r e d by t h e c y t o p l a s m and n u c l e u s o f each c e l l was c o u n t e d . C e l l s were a r r a n g e d i n a s c e n d i n g o r d e r o f a r e a and s e q u e n t i a l p a i r w i s e comparisons o f p e r c e n t a g e d i f f e r e n c e was made. Only i f t h i s d i f f e r e n c e was 15.5% o r g r e a t e r was a new c h a r a c t e r s t a t e a s s i g n e d t o t h e c e l l . F o r example, t h e measurements f o r t h e hemocyte c y t o p l a s m a r e a were 12, 13, 16, 18, 18, e t c . (mm.2). The p e r c e n t a g e d i f f e r e n c e between 12 and 13 i s a p p r o x i m a t e l y 7% t h e r e f o r e t h e s e c e l l s a r e coded w i t h i n t h e same c h a r a c t e r s t a t e (eg. 0 ) . The d i f f e r e n c e between 13 and 16 however, i s 19% t h e r e f o r e 16 r e p r e s e n t s a new s t a t e o f t h e c y t o p l a s m a r e a c h a r a c t e r (eg. 1 ) . 16 I I I . RESULTS A. T r i c h o p t e r a L a r v a e I n v o l v e d and L i f e C y c l e Twelve p e r i o d i c samples o f T i n Can Creek c o n s i s t e n t l y demonstrated t h e pr e s e n c e and c o e x i s t e n c e o f two m o r p h o l o g i c a l l y d i s t i n c t t y p e s of c a d d i s f l y l a r v a e . The f i r s t t y p e o f l a r v a was d i s t i n g u i s h e d by t h e p r e s e n c e o f a median d o r s a l hump on t h e f i r s t abdominal segment i n a d d i t i o n t o a l a t e r a l hump a t each s i d e o f t h i s segment. The second t y p e o f l a r v a p o s s e s s e d o n l y t h e two l a t e r a l humps. Case measurements i n d i c a t e d a s i z e d i f f e r e n c e between t h e two t y p e s o f T r i c h o p t e r a l a r v a e . Measurements o f t h e c a s e s o f 248 l a r v a e p o s s e s s i n g t h e d o r s a l c e n t e r i n g hump had a mean l e n g t h o f 14.07 - 1.19mm. ;however, case measurements o f 492 l a r v a e p o s s e s s i n g no median d o r s a l hump had a mean l e n g t h o f 8.18 - .50 mm. A h i s t o g r a m d e m o n s t r a t i n g t h i s s i z e d i f f e r e n c e f o r t h e t w e l v e s a m p l i n g p e r i o d s i s g i v e n i n F i g u r e 2. L a r v a l d e t e r m i n a t i o n showed t h e s m a l l e r s p e c i e s t o b e l o n g t o the L e p i d o s t o m a t i d a e , and t h e l a r g e r t o t h e L i m n e p h i l i d a e . I n d i v i d u a l s o f t h e two t y p e s were p l a c e d s e p a r a t e l y i n t o r e a r i n g c o n t a i n e r s , i n o r d e r t o o b t a i n a d u l t s f o r i d e n t i f i c a t i o n ; f o u r a d u l t s o f t h e s m a l l e r s p e c i e s (1 f e m a l e , 3 males) and one male o f t h e l a r g e r s p e c i e s emerged. Dr. G.B. Wiggins i d e n t i f i e d t h e s m a l l e r s p e c i e s as Lepidostoma r o a f i ( Milne) 17 FIGURE 2 Mean case lengths of the two species of Trichoptera over the twenty-four month c o l l e c t i n g period. n=40 n=25 33 n= 6 n = 26 n = 4 4 n=9 n = 6 n = 5 n= 12 h = 25 n= 17 n= 15 n=l2 n=3 o CVI CD CO h = n=2l h=l5 n=l99 _n^216l CVI O CO CD —I— CM May 4,1981 March 17,1981 Feb.l, 1981 Sept. 11,1980 May 21, 1980 March 20 ,1980 A u g . 30 ,1979 July 9 , 1979 July 5 , 1979 June 22,1979 May 3 0 , 1 9 7 9 May 1,1979 May 4,1981 March 17, 1981 Feb.l,1981 Sept. II, 1980 May 21,1980 March 20,1980 A u g . 3 0 , 1 9 7 9 July 9,1979 July 5 ,1979 June 22 ,1979 May 3 0 , 1979 May 1,1979 CD O a c o o ~o o E E OCXJCO II QJ II XCO ~2L E E — tO CD 00 n UJ 1 1 xcoz c o_i E o £ •5 a) i CO o 19 ( L e p i d o s t o m a t i d a e ) and t h e l a r g e r s p e c i e s as P s y c h o g l y p h a  a l a s c e n s i s (Banks) ( L i m n e p h i l i d a e ) . I n T i n Can Creek t h e l a r v a e o f b o t h s p e c i e s o f T r i c h o p t e r a a c t as second i n t e r m e d i a t e h o s t s i n t h e l i f e c y c l e o f t h e trematode Bunodera m e d i o v i t e l l a t a ( A l l o c r e a d i i d a e ) . The a d u l t o f B. m e d i o v i t e l l a t a i n h a b i t s the i n t e s t i n e o f t h e t h r e e - s p i n e d s t i c k l e b a c k , G a s t e r o s t e u s  a c u l e a t u s L. Eggs o f t h e p a r a s i t e a r e r e l e a s e d f rom t h e a d u l t i n s i t u , and a r e passed o ut w i t h t h e f e c e s o f t h e f i s h . A m i r a c i d i u m emerges from each egg and e n t e r s a c l a m o f t h e genus P i s i d i u m . A s p o r o c y s t d e v e l o p s w i t h i n t h e g i l l l a m e l l a e o f t h e c l a m , and l a t e r as a r e d i a w i t h i n t h e d i g e s t i v e g l a n d . C e r c a r i a e a r e r e l e a s e d from t h e r e d i a . Each c e r c a r i a l e a v e s t h e clam and i s swept i n t o t h e case o f a T r i c h o p t e r a l a r v a v i a t h e c i r c u l a t i o n o f wa t e r g e n e r a t e d by abdominal v e n t i l l a t i o n movement. The c e r c a r i a then a t t a c h e s t o t h e abdomen o f t h e l a r v a , p e n e t r a t e s t h r o u g h t h e integument and e v e n t u a l l y r e a c h e s one o f t h e s i l k g l a n d s . The m e t a c e r c a r i a t h e n e n t e r s t h e s i l k g l a n d , e n c y s t s and d e v e l o p s w i t h i n t h e c y s t . When t h e T r i c h o p t e r a l a r v a i s i n g e s t e d by a s t i c k l e b a c k , c o n t a c t w i t h t h e d i g e s t i v e j u i c e s o f t h e stomach r e l e a s e s t h e m e t a c e r c a r i a f rom i t s c y s t . The m e t a c e r c a r i a t h e n moves t o the i n t e s t i n e o f t h e f i s h and d e v e l o p s i n t o t h e a d u l t . The l i f e c y c l e o f B. m e d i o v i t e l l a t a as d e s c r i b e d by Dr. M.J. Kennedy ( i n l i t . ) was t h u s c o n f i r m e d and i s diagrammed i n F i g u r e 3 . FIGURE 3 The l i f e c y c l e o f Bunodera m e d i o v i t e l l a t a . ADULT in intestine develop d then REDIA B. N a t u r a l I n f e c t i o n s D e t a i l e d d i s s e c t i o n s o f 25 i n d i v i d u a l s o f each s p e c i e s o f T r i c h o p t e r a c o l l e c t e d May 30, 1979 demonstrated t h e p r e s e n c e o f t h e m e t a c e r c a r i a e o f B. m e d i o v i t e l l a t a w i t h i n t h e s i l k g l a n d s o f b o t h s p e c i e s o f c a d d i s f l y l a r v a e ; m e t a c e r c a r i a e o f t h e trematode were found nowhere e l s e i n the body o f e i t h e r s p e c i e s o f c a d d i s f l y . I n a d d i t i o n , one u n e n c y s t e d p r o g e n e t i c m e t a c e r c a r i a was d i s c o v e r e d f l o a t i n g w i t h i n t h e abdominal hemocoele o f a specimen o f P. a l a s c e n s i s c o l l e c t e d September 11, 1980; i t was i d e n t i f i e d by Dr. D.R. Brooks as an u n d e s c r i b e d s p e c i e s b e l o n g i n g t o t h e f a m i l y W a r e t r e m a t i d a e , w i t h morphology s i m i l a r t o t h a t o f t h e members o f t h e genus Megasolena. E x a m i n a t i o n o f t h e s i l k g l a n d s o f a number o f i n d i v i d u a l s o f P. a l a s c e n s i s and L. r o a f i i n d i c a t e d t h a t t h e r e was a d i f f e r e n c e i n i n c i d e n c e o f i n f e c t i o n between t h e two s p e c i e s . The mean p e r c e n t a g e i n c i d e n c e o f i n f e c t i o n (at l e a s t one m e t a c e r c a r i a per i n d i v i d u a l ) o f 242 i n d i v i d u a l s o f P. a l a s c e n s i s was 33.7 - 14.90%; however, the i n c i d e n c e i n 497 i n d i v i d u a l s o f L. r o a f i was 3.34 - 4.4%. The i n c i d e n c e o f i n f e c t i o n i n b o t h s p e c i e s o f T r i c h o p t e r a o ver t h e two y e a r c o l l e c t i o n p e r i o d i s i l l u s t r a t e d i n F i g u r e 4. The i n c i d e n c e o f i n f e c t i o n i n L. r o a f i was a t i t s l o w e s t (0-2%) on May 30, 1979, J u l y 30, 1979, May 21, 1980, and March 17, 1981; i t was h i g h e s t (18%) on J u l y 9, 1979. The i n c i d e n c e o f i n f e c t i o n i n P. a l a s c e n s i s was a t a low o f 4% on March 17, 1981. The 23 FIGURE 4 I n c i d e n c e o f p a r a s i t i s m by Bunodera m e d i o v i t e l l a t a i n t h e two s p e c i e s o f T r i c h o p t e r a . o= no l a r v a e o f Lepidostoma r o a f i c o l l e c t e d on t h a t d a t e . n = 4 0 n = 33 n=44 ) n=25 n = 26 n=9 n=6 n = 6 n=5 n= 12 n=25 n= 17 -i i 1 r May4, 1981 March 17,1981 Feb. 1,1981 Sept. II, 1980 May 21,1980 March 20,1980 Aug. 30 ,1979 July 9 ,1979 July 5, 1979 June 22,1979 May 3 0 , 1979 May 1,1979 a a cnoo r o srV r o -CM n LU II n= 15 n=l2 o o n=3 o o n = 11 n = 2l n=l5 n=199 n=2!6 o o May 4,1981 March 17,1981 Feb. l , 1981 Sept. 11,1980 May 21,1980 March 20 ,1980 Aug. 30,1979 ' July 9, 1979 July 5 ,1979 June 2 2 , 1 9 7 9 May 30,1979 May 1,1979 c o o "5 o -5 ^ < £ > s f CM ^ <sJ" cn r6 II ^ II LU " x t o z 8 c c . 2 o •*- -—-^ ED 0 S h i g h e s t i n c i d e n c e s o f i n f e c t i o n i n P. a l a s c e n s i s (75%) were on J u l y 5, 1979, J u l y 9, 1979 and March 20, 1980. Samples were not t a k e n i n June o r J u l y o f 1980, t h e r e f o r e , i t i s d i f f i c u l t t o a s s e s s whether o r not t h i s h i g h p e r c e n t p a r a s i t i s m i n t h e summer i s a r e p e a t e d t r e n d . Many o f t h e T r i c h o p t e r a had m u l t i p l e i n f e c t i o n s o f more t h a n one m e t a c e r c a r i a p e r i n d i v i d u a l . The i n f e c t i o n s ranged from one t o t e n m e t a c e r c a r i a e p e r i n d i v i d u a l i n P. a l a s c e n s i s (max. 5 c y s t s p e r gland) and from one t o f o u r i n L. r o a f i (max. t h r e e c y s t s per g l a n d ) . The r e s u l t s o f t h e a n a l y s i s o f v a r i a n c e (Table 1) i n d i c a t e d t h a t d a t e o f c o l l e c t i o n and t h e s p e c i e s o f T r i c h o p t e r a , b o t h had a s i g n i f i c a n t e f f e c t on t h e v a r i a n c e i n t h e number o f c y s t s p e r i n d i v i d u a l (P<.05). S i z e o f t h e c a s e , however, d i d n o t appear t o have a s i g n i f i c a n t e f f e c t (P .05). C. E x p e r i m e n t a l I n f e c t i o n s I n o r d e r t o d e t e r m i n e i f t h e d i f f e r e n c e i n i n c i d e n c e o f i n f e c t i o n between the two s p e c i e s o f T r i c h o p t e r a was a r e s u l t o f d i f f e r e n t i a l p e n e t r a t i o n o f t h e c a d d i s f l y l a r v a e by t h e c e r c a r i a e , p e n e t r a t i o n was examined e x p e r i m e n t a l l y . Enough swimming c e r c a r i a e were o b t a i n e d t o examine p e n e t r a t i o n o f f i v e l a r v a e o f each s p e c i e s o f T r i c h o p t e r a . W i t h i n t h e one hour o b s e r v a t i o n p e r i o d , t h r e e c e r c a r i a e were seen t o drop t h e i r t a i l s and p e n e t r a t e one l a r v a and one c e r c a r i a t o drop i t s t a i l and p e n e t r a t e a n o t h e r l a r v a o f t h e f i v e i n d i v i d u a l s o f L. r o a f i exposed t o them. Upon d i s s e c t i o n , a l l o f t h e 26 T a b l e 1. A n a l y s i s o f t h e e f f e c t o f da t e o f c o l l e c t i o n , s p e c i e s o f T r i c h o p t e r a and s i z e o f case on the v a r i a n c e o f t h e number o f m e t a c e r c a r i a l c y s t s p e r l a r v a . Source D.f. Mean Square F v a l u e F P r o b a b i l i t y Date 11 6 .09 3.19 0.0037* S p e c i e s 1 13.55 7.09 0.0109* S i z e 18 2.33 1.22 0.2929 *= S i g n i f i c a n t a t .05 l e v e l . r e m a i n i n g t h r e e l a r v a e , i n a d d i t i o n t o t h e f i v e non-e x p e r i m e n t a l f i e l d caught l a r v a e examined were found t o c o n t a i n no m e t a c e r c a r i a e e i t h e r i n t h e body c a v i t y o r w i t h i n t h e s i l k g l a n d s . Two c e r c a r i a e were seen t o drop t h e i r t a i l s and p e n e t r a t e one l a r v a and one c e r c a r i a t o drop i t s t a i l and p e n e t r a t e a n o t h e r l a r v a o f t h e f i v e i n d i v i d u a l s o f P. a l a s c e n s i s exposed t o t h e c e r c a r i a e . D i s s e c t i o n showed t h e r e m a i n i n g t h r e e exposed l a r v a e t o c o n t a i n no c e r c a r i a e w i t h i n t h e body c a v i t y o r s i l k g l a n d s . Of the f i v e n o n - e x p e r i m e n t a l f i e l d caught l a r v a e o f P. a l a s c e n s i s d i s s e c t e d and examined f o r c e r c a r i a e , one c o n t a i n e d two m e t a c e r c a r i a e w i t h i n one s i l k g l a n d ; t h e r e m a i n i n g f o u r c o n t a i n e d no m e t a c e r c a r i a e . The numbers o f c e r c a r i a e e n t e r i n g each s p e c i e s o f T r i c h o p t e r a were t o o s m a l l t o a n a l y z e s t a t i s t i c a l l y . However, as c e r c a r i a e o f B. m e d i o v i t e l l a t a were seen t o p e n e t r a t e i n d i v i d u a l s o f b o t h s p e c i e s o f T r i c h o p t e r a , i t seems l i k e l y t h a t t h e d i f f e r e n c e i n t h e i n c i d e n c e o f i n f e c t i o n between t h e two s p e c i e s o f c a d d i s f l y l a r v a e i s not due t o d i f f e r e n t i a l p e n e t r a t i o n by t h e c e r c a r i a e . D. M e t a c e r c a r i a l C y s t s from t h e Two S p e c i e s o f T r i c h o p t e r a I t seems l i k e l y t h a t b o t h s p e c i e s o f T r i c h o p t e r a s e r v e as the normal i n t e r m e d i a t e h o s t s f o r t h e m e t a c e r c a r i a e o f B. m e d i o v i t e l l a t a a l t h o u g h t h e r e was a d i f f e r e n c e i n i n c i d e n c e o f i n f e c t i o n . To check t h a t b o t h h o s t s a r e a b l e t o s u p p o r t t h e p a r a s i t e l i f e c y c l e , m e t a c e r c a r i a e e n c y s t e d w i t h i n t h e s i l k g l a n d s were t a k e n from b o t h l a r v a e o f P. a l a s c e n s i s and L. r o a f i , s e c t i o n e d and examined. The r e s u l t s o f t h i s e x a m i n a t i o n a r e summarized i n T a b l e 2. Between the two s p e c i e s o f T r i c h o p t e r a t h e r e appeared t o be no d i f f e r e n c e i n s i z e o f t h e p a r a s i t e , i t s l o c a t i o n w i t h i n t h e s i l k g l a n d , t h e h o s t ' s r e a c t i o n t o t h e p a r a s i t e or t h e appearance o f t h e p a r a s i t e : m e t a c e r c a r i a e remained a l i v e i n b o t h h o s t s p e c i e s . A m e t a c e r c a r i a from a l a r v a o f L. r o a f i and one from P. a l a s c e n s i s are shown i n F i g u r e 5. T h i s p r e l i m i n a r y s t u d y demonstrated no d i f f e r e n c e between m e t a c e r c a r i a e e n c y s t e d w i t h i n t h e two s p e c i e s o f T r i c h o p t e r a . Because the l a r v a e o f P. a l a s c e n s i s were u s u a l l y more abundant, and had a much h i g h e r i n c i d e n c e o f i n f e c t i o n t h a n l a r v a e o f L. r o a f i , i n d i v i d u a l s o f P. a l a s c e n s i s were used t h r o u g h o u t t h e r e s t o f t h e s t u d y . E. M e t a c e r c a r i a l D e s c r i p t i o n S i n c e a knowledge o f t h e anatomy o f t h e m e t a c e r c a r i a i s o f some use f o r a g i n g t h e v a r i o u s m e t a c e r c a r i a l c y s t s , t h i s s tage was s t u d i e d and i s d e s c r i b e d f o r t h e f i r s t t i m e : a l l measurements a r e i n m i c r o m e t e r s . Body l e n g t h 311 (min. 275-max. 340) . Body w i d t h 184 (min. 137-max. 231) . C u t i c l e a s p i n o s e . A n t e r i o r s u c k e r 66 (min. 48-max. 75) by 71 (min. 55-max. 87) b o r d e r e d l a t e r a l l y by two m u s c u l a r p a p i l l a e and w i t h f o u r d o r s a l p a p i l l a e . No t r a c e s o f 29 T a b l e 2. C o m p a r i s i o n o f encystment and e n c a p s u l a t i o n o f m e t a c e r c a r i a e o f Bunodera m e d i o v i t e l l a t a between two s p e c i e s o f T r i c h o p t e r a . C h a r a c t e r i s t i c s P s y c h o g l y p h a Lepidostoma a l a s c e n s i s r o a f i S i z e o f m e t a c e r c a r i a e (diameter a c r o s s c y s t ) 120- 175 x 120- 170u 130- 195 x 119-150u G e n e r a l appearance of m e t a c e r c a r i a e H e a l t h y H e a l t h y P o s i t i o n o f m e t a c e r c a r i a e w i t h i n t h e s i l k g l a n d D i r e c t l y w i t h i n lumen of g l a n d D i r e c t l y w i t h i n lumen of g l a n d Damage t o t h e g l a n d E p i t h e l i a l l a y e r o f g l a n d w a l l d e g e n e r a t i n g E p i t h e l i a l l a y e r o f g l a n d w a l l d e g e n e r a t i n g Host r e a c t i o n t o m e t a c e r c a r i a e Hemocytes v i s i b l e around c y s t Hemocytes v i s i b l e around c y s t 3 0 FIGURE 5 L o n g i t u d i n a l s e c t i o n s o f m e t a c e r c a r i a e e n c y s t e d w i t h i n s i l k g l a n d s o f T r i c h o p t e r a . (a) C y s t w i t h i n s i l k g l a n d s of Lepidostoma r o a f i ( H ematoxylin and e o s i n Y, 6 75x), (b) C y s t w i t h i n s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s ( H ematoxylin and e o s i n Y, 6 75 x). • H= hemocyte, LG= lumen of t h e s i l k g l a n d , P= p a r a s i t e , VS= v e n t r a l s u c k e r o f m e t a c e r c a r i a . 31 p e n e t r a t i o n g l a n d s o b s e r v e d . Remains o f eyesp o t pigment c l e a r l y v i s i b l e on e i t h e r s i d e o f t h e esophagus between pharynx and b i f u r c a t i o n o f the g u t . S t y l e t seen i n one m e t a c e r c a r i a . Prepharynx 8 (min. 5-max. 11) l o n g . Pharynx 29 (min. 25-max. 31) l o n g by 32 (min. 24-max. 44) wide. Esophagus 31 b i f u r c a t i n g a t c e n t e r p o i n t o f acetabulum. I n t e s t i n a l caeca t h i c k , e x t e n d i n g i n t o p o s t e r i o r o n e - q u a r t e r o f body. Acetabulum i n a n t e r i o r o n e - h a l f o f body 69 (min. 63-max. 90) by 67 (min. 50-max. 88) . Two round t e s t e s tandem, a n t e r i o r t e s t i s 17 by 17, p o s t e r i o r t e s t i s 21 by 20. Ovary p o s t e r i o r t o . a c e t a b u l u m 10 by 11 Ootype r e g i o n v i s i b l e . U t e r u s n ot v i s i b l e . V i t e l l i n e f o l l i c l e s e x t r a c a e c a l r a n g i n g from o r a l s u c k e r t o t i p s o f caecae. E x c r e t o r y v e s i c l e n o t v i s i b l e . One o f t h e e x c y s t e d m e t a c e r c a r i a e i s i l l u s t r a t e d i n F i g u r e 6. F. The S i l k G l a n d s : Normal S t r u c t u r e S i n c e one o f t h e aims o f t h i s s t u d y was t o examine t h e c e l l u l a r r e s ponse o f t h e T r i c h o p t e r a l a r v a e t o t h e pr e s e n c e o f a p a r a s i t e e n c y s t e d w i t h i n t h e i r s i l k g l a n d s , i t was f i r s t n e c e s s a r y t o examine t h e normal c o n s t r u c t i o n and h i s t o l o g y o f u n i n f e c t e d g l a n d s . Changes due t o p a r a s i t i s m o f s i l k g l a n d s c o u l d t h e n be d e t e c t e d and d e s c r i b e d i n r e l a t i o n t o t h e normal morphology o f the g l a n d s . E x a m i n a t i o n s o f whole u n f i x e d s i l k g l a n d s i n d i c a t e d 33 FIGURE 6 E x c y s t e d m e t a c e r c a r i a o f Bunodera m e d i o v i t e l l a t a . Drawing done w i t h t h e a i d o f a drawing t u b e . c= i n t e s t i n a l c a e c a , e= eye s p o t p i g m e n t a t i o n , es= esophagus, mp= m u s c u l a r p a p i l l a , o= o v a r y , os= o r a l s u c k e r , p=pharynx, pp= p r e p h a r y n x , t= t e s t i s , v= v i t e l l a r i a , vs= v e n t r a l s u c k e r . 34 t h a t t h e s e g l a n d s were l o n g s a c s c o n s t r i c t e d a p p r o x i m a t e l y 1 mm. from th e a n t e r i o r end, and b l i n d a t t h e p o s t e r i o r end. When examined under s u r f a c e l i g h t i n g w i t h a s t e r e o d i s s e c t i n g m i c r o s c o p e , th e w a l l o f t h e g l a n d s appeared t o be composed o f l a r g e , t i g h t l y packed h e x a g o n a l c e l l s . L o n g i t u d i n a l s e c t i o n s o f u n i n f e c t e d s i l k g l a n d s f i x e d i n B o u i n ' s showed t h e g l a n d s t o be t u b u l a r w i t h a s i n g l e l a y e r e d e p i t h e l i u m and a lumen a p p r o x i m a t e l y 50-70u i n d i a m e t e r . The s i l k w i t h i n t h e lumen appeared as a homogeneous amorphous s u b s t a n c e . Of t h e 15 u n i n f e c t e d g l a n d s l o n g i t u d i n a l l y s e c t i o n e d , no hemocytes were seen i n t h e lumen o f any o f t h e g l a n d s . The e p i t h e l i a l c e l l s o f t h e g l a n d were 15-25u i n h e i g h t , and t h e r e appeared t o be a gap i n t h e e p i t h e l i a l l a y e r c o r r e s p o n d i n g t o an i n t e r c e l l u l a r space e v e r y 90-170u (arrows i n F i g u r e 7 ) . The gaps i n t h e e p i t h e l i a l l a y e r o f one s i d e o f t h e g l a n d c o r r e s p o n d e d t o t h e c e n t e r o f t h e u n i n t e r r u p t e d a r e a s o f t h e o u t e r s i d e o f t h e g l a n d , which s u g g e s t e d the c l o s e p a c k i n g o f h e x a g o n a l c e l l s . A l o n g i t u d i n a l s u r f a c e - s e c t i o n o f t h e w a l l o f t h e s i l k g l a n d a l s o i n d i c a t e d h e x a g o n a l c e l l s w i t h l a r g e p o l y m o r p h i c n u c l e i ( F i g u r e 8 ) . These n u c l e i , when seen i n s e c t i o n appeared v a r i a b l e i n shape and d i f f u s e ( F i g u r e 9 ) . A s c h e m a t i c d i a g r a m o f a p o r t i o n o f a s i l k g l a n d i s g i v e n i n F i g u r e 10. Note t h a t a c r o s s s e c t i o n o f t h i s g l a n d would show i n t e r c e l l u l a r gaps i n t h e e p i t h e l i u m o f t h e g l a n d a t t h e arrows i n F i g u r e 10. 36 FIGURE 7 L o n g i t u d i n a l s e c t i o n o f an u n i n f e c t e d s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s . Arrows i n d i c a t e i n t e r c e l l u l a r s p a c e s . E= e p i t h e l i a l c e l l s , LG= lumen o f t h e s i l k g l a n d . ( H e m a t o x y l i n and e o s i n Y, 200x). FIGURE 8 S u r f a c e v i e w o f a s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s d e m o n s t r a t i n g hexagonal e p i t h e l i a l c e l l s w i t h p o l y m o r p h i c n u c l e i . E= e p i t h e l i a l c e l l , NE= p o l y m o r p h i c n u c l e u s . ( H e m a t o x y l i n and e o s i n Y, 6 7 5 x ) . 37 38 FIGURE 9 M a g n i f i e d v i e w o f a l o n g i t u d i n a l s e c t i o n t h r o u g h a s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s d e m o n s t r a t i n g d i f f u s e n a t u r e o f t h e e p i t h e l i a l c e l l n u c l e u s . LG= lumen o f s i l k g l a n d , NE= e p i t h e l i a l c e l l n u c l e u s . ( H e m a t o x y l i n and e o s i n Y, 675x). 9 40 FIGURE 10 Schematic diagram o f t h e c o n s t r u c t i o n o f a s i l k g l a n d . Arrows i n d i c a t e j u n c t i o n s o f e p i t h e l i a l c e l l s , seen as gaps i n l o n g i t u d i n a l s e c t i o n ( F i g u r e 7 ) . N u c l e i have been marked i n o n l y i n t h e f o r e g r o u n d o f t h e diagram. 41 hexagonal epidermal ce l l polymorphic nucleus 42 G. The Hemocyte Complex B e f o r e e x a m i n i n g th e hemocyte response of P. a l a s c e n s i s t o a p a r a s i t e i t was n e c e s s a r y t o examine the complex o f f r e e - c i r c u l a t i n g hemocytes o f u n i n f e c t e d l a r v a e . Once t h e morphology o f t h e f r e e - c i r c u l a t i n g hemocytes i s e s t a b l i s h e d i t s h o u l d be p o s s i b l e t o compare them w i t h t h o s e t a k i n g p a r t i n t h e e n c a p s u l a t i o n r e s p o n s e and attempt t o d e t e r m i n e ( i ) how t h e hemocytes change d u r i n g t h e e n c a p s u l a t i o n r e s p o n s e , ( i i ) which o f t h e f r e e hemocyte t y p e s t a k e p a r t i n t h e r e a c t i o n , and ( i i i ) i f any hemocytes n o t seen i n t h e hemocyte smears t a k e p a r t i n t h e e n c a p s u l a t i o n r e s p o n s e . F i g u r e 11 d e p i c t s t h e 24 m o r p h o l o g i c a l l y d i s t i n c t hemocytes found by s c a n n i n g hemocyte smears. A c l a d i s t i c a n a l y s i s was done on t h e s e c e l l s t o d e t e c t any p a t t e r n o f r e l a t i o n s h i p p r e s e n t among them. The s t a t e s f o r each c h a r a c t e r , i n a d d i t i o n t o t h e d a t a m a t r i x c o n s t r u c t e d f o r t h e s e hemocytes a r e g i v e n i n T a b l e 3. The n u c l e i o f t h e hemocytes o b s e r v e d had a range i n s i z e o f 3-16u, w h i l e t h e c e l l l e n g t h ranged from 5-41u. I t w i l l be seen t h a t c e l l s #9 and 21 coded w i t h t h e same c h a r a c t e r s t a t e sequence, and t h e r e f o r e r e p r e s e n t t h e same ' t y p e 1 o f hemocyte. A l l o f t h e r e m a i n i n g 23 hemocytes p o s s e s s a unique c o d i n g sequence and t h e r e f o r e each r e p r e s e n t s a d i f f e r e n t hemocyte 't y p e ' . The c l a d o g r a m c o n s t r u c t e d from t h e d a t a m a t r i x o f d i f f e r e n t hemocytes from T a b l e 3 i s shown i n F i g u r e 12. 43 FIGURE 11 M o r p h o l o g i c a l l y d i s t i n c t hemocytes seen i n hemolymph smears o f P s y c h o g l y p h a a l a s c e n s i s . C e l l s have been numbered t o f a c i l i t a t e f u r t h e r r e f e r e n c e . (Giemsa, 2200x). 44 11 4 5 Table 3. Data matrix for c e l l s 1-24 of Psychoglypha alascensis (Figure 11). A l l measurements i n mm.2 from graph paper tracings of c e l l s i n Figure 11. Defi n i t i o n s of character states are given below. A- Area of c e l l (nuclear area + cytoplasmic area) 0= 10 1= 12.5 to 15.5 2= 21 to 27 3= 32 B- Area of nucleus 0= 6 to 11.5 1= 15 to 19 C- Cytoplasm/nucleus area 0= nucleus area greater than cytoplasm 1= 1.1:1 to 1.28:1 a r e a 2= 1.56:1 to 1.9:1 3= 3.3:1 to 3.9:1 4= 4.3:1 D- Nucleus po s i t i o n 0= central 1= eccentric E- Shape of c e l l 0= round 1= some v a r i a t i o n of spindled 2= presence of pseudopodia F- Compactness of nucleus 0= dense 1= loose G- Compactness of cytoplasm 0= dense 1= loose H- Cytoplasm granulation 0= no granulation 1= some small granules I- Cytoplasm vacuolation 0= no vacuolation 1= vacuolation C e l l C h a r a c t e r C e l l Number 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16 . 17. 18. 19. 20. 21. 22. 23. 24. A B 2 0 1 0 2 1 3 1 2 1 0 0 1 0 2 1 1 0 1 0 2 0 2 0 1 0 2 1 2 0 1 0 2 0 2 0 0 0 2 1 1 0 1 0 2 0 2 0 C D E F G H I 3 1 0 1 0 0 0 2 1 1 0 0 0 0 2 1 0 1 0 0 1 2 1 0 1 1 0 1 2 0 1 1 1 0 1 0 1 2 0 0 0 0 1 0 0 0 0 0 0 2 0 0 1 0 0 0 0 0 0 0 1 0 0 1 0 0 0 1 0 0 2 0 1 1 0 0 1 2 1 0 1 0 0 0 1 0 2 0 1 0 0 2 0 1 0 1 0 0 2 1 1 1 0 0 1 2 1 1 0 0 0 1 2 1 2 0 1 0 0 3 0 1 0 1 0 1 0 0 0 0 0 0 0 3 0 1 1 1 0 0 0 0 0 0 1 0 0 2 1 1 0 1 0 1 3 1 0 0 1 0 1 4 1 0 1 0 0 0 47 FIGURE 12 Cladogram o f hemocytes o f P s y c h o g l y p h a a l a s c e n s i s . C o n s t r u c t e d from d a t a m a t r i x i n T a b l e 3. Numbered hemocytes c o r r e s p o n d t o t h o s e i n F i g u r e 11. The number o f synapomorphies c h a r a c t e r i z i n g each b r a n c h i s i n d i c a t e d a t each mid-branch p o i n t . 48 The p a r t i c u l a r unique c h a r a c t e r s (synapomorphies) a r e marked i n a l o n g each b r a n c h . When t h e branches n ot c h a r a c t e r i z e d by any synapomorphies ( w i t h z e r o b r a n c h l e n g t h s ) a re c o l l a p s e d down t o t h e nodes, t h e t r e e becomes a•Summary Fate Map o f p o s s i b l e d e v e l o p m e n t a l pathways open t o d e v e l o p i n g hemocytes ( C a i r a , B r o o k s , Scudder, i n prep.) ( F i g u r e 1 3 ) . The Summary F a t e Map p o s t u l a t e s s i x hemocyte t y p e s o f P. a l a s c e n s i s w h i c h were n o t seen on t h e s l i d e s examined f o r hemocytes. These unseen t y p e s a r e r e p r e s e n t e d by b l a c k c i r c l e s on t h e f a t e map i n F i g u r e 13. • I n summary t h e n , e x a m i n a t i o n o f hemocytes r e v e a l e d a t l e a s t 24 m o r p h o l o g i c a l l y d i s t i n c t t y p e s o f hemocytes. C l a d i s t i c a n a l y s i s o f t h e s e c e l l s showed t h a t many o f t h e hemocytes seen were s t a g e s i n t h e development o f s e v e r a l l i n e s o f d i s t i n c t hemocyte t y p e s . H. R e a c t i o n t o I m p l a n t s The s i l k g l a n d s a r e an u n u s u a l s i t e i n which t o f i n d digenean encystment and i t i s not a s i t e i n whi c h one would e x p e c t t o f i n d a hemocyte r e a c t i o n t o a p a r a s i t e . The g l a n d l i e s w i t h i n t h e hemocoele, however, t h e lumen o f t h i s g l a n d i s not p a r t o f t h e hemocoele, and c o n s e q u e n t l y does n ot p o s s e s s a normal hemocyte p o p u l a t i o n . B e f o r e e x a m i n i n g the response o f P. a l a s c e n s i s t o a p a r a s i t e e n c y s t e d w i t h i n t h e s i l k g l a n d , i t seemed a d v i s a b l e t o examine t h e hemocyte response o f P. a l a s c e n s i s t o f o r e i g n 50 FIGURE 13 Summary F a t e Map o f hemocyte l i n e a g e s . P r o d u c e d by c o l l a p s i n g a l l c e l l s o f P s y c h o g l y p h a a l a s c e n s i s w i t h z e r o b r a n c h l e n g t h s i n t h e clad o g r a m i n F i g u r e 12 t o nodes. Synapomorphic c h a r a c t e r s ( c o r r e s p o n d i n g t o th o s e i n T a b l e 3) a r e marked by l e t t e r a l o n g each b r a n c h . Numbered hemocytes c o r r e s p o n d t o t h o s e i n F i g u r e 11. /= a s i n g l e f o r w a r d s h i f t i n a c h a r a c t e r s t a t e , / / = - t w o c o n s e c u t i v e f o r w a r d s h i f t s i n a c h a r a c t e r s t a t e , X= a r e v e r s a l o f one s t a t e o f t h e c h a r a c t e r , ©= p r e c u r s o r c e l l (node) p o s t u l a t e d by Wagner a n a l y s i s o f t h e d a t a m a t r i x (Table 3 ) , but not found among c e l l s examined i n hemolymph smears. o b j e c t s w i t h i n an a r e a a c c e s s i b l e t o hemocytes such as t h e hemocoele. Hence the competence of the c e l l u l a r immune system o f P. a l a s c e n s i s was t e s t e d by e x a m i n i n g t h e response o f t h e l a r v a e t o i n e r t p i e c e s o f Epon i m p l a n t e d w i t h i n t h e hemocoele. Two response p e r i o d s were c h o s e n , one s h o r t ( a p p r o x i m a t e l y 7 d a y s ) , and one l o n g ( a p p r o x i m a t e l y 35 d a y s ) . ( i ) Seven Day I m p l a n t s Of t h e s i x l a r v a e r e c e i v i n g i m p l a n t s , one d i e d b e f o r e t h e seven day p e r i o d was c o m p l e t e . I n a n o t h e r l a r v a , one o f t h e i m p l a n t s had moved i n t o t h e a r e a o f t h e i n c i s i o n . s o t h a t o n e - h a l f o f t h e Epon was w i t h i n t h e a n i m a l w h i l e t h e o t h e r h a l f was exposed t o t h e a i r . As p a r t o f t h e r e a c t i o n around t h i s i m p l a n t was t h e c l o s i n g o f t h e i n t e g u m e n t a l wound, i t was not examined f o r an e n c a p s u l a t i o n r e a c t i o n . Owing t o t h e s m a l l s i z e and t r a n s p a r e n t n a t u r e o f t h e i m p l a n t s , d i s s e c t i o n o f l a r v a e r e c e i v i n g i m p l a n t s l e d t o t h e r e t r i e v a l o f Epon s l i v e r s f r o m o n l y two o f t h e f o u r r e m a i n i n g i n d i v i d u a l s . S e c t i o n s o f t h e s e s l i v e r s showed a s p o t t y hemocyte r e s p o n s e . A s e c t i o n o f one o f t h e s e Epon s l i v e r s i s shown i n F i g u r e 14. Some a r e a s o f t h e i m p l a n t appear c o m p l e t e l y f r e e o f hemocytes (D i n F i g u r e s 14 a and b ) . In o t h e r a r e a s , however, s e v e r a l hemocytes have become g r e a t l y e x t e n d e d and have f l a t t e n e d a g a i n s t t h e i m p l a n t , f o l l o w i n g t h e c o n t o u r s o f i t s edge (Q i n F i g u r e s 14a and b ) . I n y e t o t h e r a r e a s o f t h e i m p l a n t groups o f hemocytes have f l a t t e n e d 53 FIGURE 14 C r o s s s e c t i o n s o f t h e hemocyte response o f P s y c h o g l y p h a  a l a s c e n s i s t o an Epon s l i v e r i m p l a n t e d i n t h e abdominal hemocoele and removed a f t e r seven d a y s , (a) (Methylene b l u e , 6 7 5 x ) , (b) (Methylene b l u e , 2200x). D= h e m o c y t e - f r e e a r e a o f i m p l a n t , EI= Epon i m p l a n t , F= f l a t t e n e d hemocyte, FN= f r e e hemocyte n u c l e u s , LH= a r e a o f i m p l a n t c o v e r e d by c a p s u l a r l a y e r s o f hemocytes, Q= a r e a o f i m p l a n t c o v e r e d by o n l y a few hemocytes. 55 a g a i n s t t h e edge o f t h e i m p l a n t f o r m i n g p a r t o f a c a p s u l e o f hemocytes a p p r o x i m a t e l y 15-20u i n d i a m e t e r (L i n F i g u r e s 14a and b ) . S e v e r a l hemocytes had n u c l e i w h i c h s t a i n e d d i s t i n c t l y w i t h l a r g e p e r i p h e r a l c h r o m a t i n g r a n u l e s , as w e l l as s e v e r a l c e n t r a l and e c c e n t r i c c h r o m a t i n g r a n u l e s . I n some a r e a s o n l y n u c l e i o f hemocytes a r e v i s i b l e d i r e c t l y a g a i n s t t h e s u r f a c e o f t h e i m p l a n t ( FN i n F i g u r e 14a and b ) . T h i s , however, does n ot appear t o be t h e case i n a l l a r e a s o f t h e i m p l a n t ; i n some a r e a s f l a t t e n e d b u t i n t a c t hemocytes ar e v i s i b l e ( F L i n F i g u r e 14b) d i r e c t l y a g a i n s t t h e s u r f a c e o f t h e i m p l a n t . ( i i ) T h i r t y - F i v e Day I m p l a n t s Four o f t h e s i x l a r v a e r e c e i v i n g Epon i m p l a n t s were s t i l l a l i v e 35 days l a t e r ; d i s s e c t i o n l e d t o t h e r e t r i e v a l o f Epon s l i v e r s from t h r e e o f t h e f o u r r e m a i n i n g i n d i v i d u a l s . A g a i n , however, one o f t h e s e i m p l a n t s was l o c a t e d d i r e c t l y w i t h i n t h e wound h e a l i n g r e a c t i o n o f t h e integument and t h u s n o t examined. S e c t i o n s o f t h e Epon s l i v e r s showed them t o be a l m o s t t o t a l l y s u r r o u n d e d by hemocytes ( F i g u r e 1 5 ) . I n c l u d e d i n t h e hemocyte c a p s u l e was some d a r k l y s t a i n i n g f a t body t i s s u e ( F B i n F i g u r e 1 5 ) . I t appeared t h a t a f t e r 35 days t h e hemocyte c a p s u l e around t h e i m p l a n t and f a t body t i s s u e was composed o f two d i s t i n c t p a r t s , an i n n e r a r e a o f d e n s e l y packed hemocytes (30-70u i n w i d t h ) , and an o u t e r a r e a o f l o o s e l y packed hemocytes (30-80u i n w i d t h ) . I n the i n n e r dense a r e a o f t h e c a p s u l e , b o t h t h e hemocytes and t h e i r 56 FIGURE 15 C r o s s s e c t i o n o f t h e r e s p o n s e o f P s y c h o g l y p h a a l a s c e n s i s t o an Epon s l i v e r i m p l a n t e d i n t h e abdominal hemocoele and removed a f t e r 35 days. EI= Epon i m p l a n t , FB= f a t body t i s s u e , H= hemocyte (Methylene b l u e , 200x). 57 1 5 n u c l e i were e x t r e m e l y f l a t t e n e d and the c e l l u l a r and n u c l e a r b o u n d a r i e s o f t h e hemocytes were v e r y d i f f i c u l t t o d i s c e r n (I i n F i g u r e 1 6 ) . I n most a r e a s the hemocytes adhered d i r e c t l y t o each o t h e r w i t h l i t t l e o r no i n t e r c e l l u l a r s p a c i n g . I n c o n t r a s t t o t h e c e l l s o f t h e i n n e r l a y e r , t h e hemocytes o f t h e o u t e r a r e a o f t h e c a p s u l e were o n l y s l i g h t l y f l a t t e n e d (0 i n F i g u r e 1 6 ) . In some a r e a s round n u c l e i w i t h p e r i p h e r a l and e c c e n t r i c c h r o m a t i n g r a n u l e s were c l e a r l y v i s i b l e . I n a d d i t i o n , t h e r e appeared t o be more i n t e r c e l l u l a r space t h a n i n t h e i n n e r l a y e r , and i n some a r e a s hemocyte b o u n d a r i e s were c l e a r l y v i s i b l e . I t appears t h e n , t h a t t h e hemocytes o f P. a l a s c e n s i s ar e c a p a b l e o f r e s p o n d i n g t o f o r e i g n o b j e c t s l o c a t e d i n t h e hemocoele a f t e r seven days and o f t o t a l l y e n c a p s u l a t i n g them a f t e r 3 5 d a y s . I . Host Response t o t h e P a r a s i t e P a r a s i t e i n f e c t i o n s were judged t o be "new", " w e l l e s t a b l i s h e d " o r " o l d " on c o m p a r a t i v e morphology and ar e d e s c r i b e d s e p a r a t e l y below. ( i ) New I n f e c t i o n s I n s i l k g l a n d s removed from f i e l d c o l l e c t e d c a d d i s f l y l a r v a e , s e v e r a l m e t a c e r c a r i a e d i d not appear t o be s i t u a t e d d i r e c t l y w i t h i n t h e lumen o f t h e g l a n d . When t h e s e g l a n d s were s e c t i o n e d , i t was d i s c o v e r e d t h a t the m e t a c e r c a r i a e were a c t u a l l y s i t u a t e d w i t h i n t h e e p i t h e l i a l l a y e r o f t h e s i l k 59 FIGURE 16 M a g n i f i e d v i e w o f r e s p o n s e o f P s y c h o g l y p h a a l a s c e n s i s t o a 35 day o l d Epon i m p l a n t . EI= Epon i m p l a n t , FB= f a t body t i s s u e , 1= i n n e r l a y e r o f c a p s u l e , N= hemocyte n u c l e u s , 0= o u t e r l a y e r o f c a p s u l e . (Methylene b l u e , 6 7 5 x ) . 1 6 61 g l a n d w a l l . These m e t a c e r c a r i a e - w e r e s m a l l (100-105u i n diameter) and appeared t o have a t h i n c y s t w a l l . I t h e r e f o r e , c o n s i d e r e d t h e s e m e t a c e r c a r i a e t o be immature, however, t h e i r e x a c t ages were n o t known. One o f t h e s e young m e t a c e r c a r i a e i s shown i n F i g u r e 17. The e p i t h e l i a l l a y e r o f t h e s i l k g l a n d b o t h a d j a c e n t t o t h e m e t a c e r c a r i a f o r about lOOu and i n t h e w a l l o f t h e g l a n d o p p o s i t e t h e m e t a c e r c a r i a f o r about 200u, appeared t o be d e g e n e r a t i n g (DE i n F i g u r e 1 7 ) . The e p i t h e l i u m was d i s c o n t i n u o u s and much t h i n n e r t h a n t h e normal e p i t h e l i u m o f t h e s i l k g l a n d (5-10u as opposed t o 10-20u). I n a d d i t i o n , hemocytes appeared t o have moved t o some a r e a s o f t h e d e g e n e r a t i n g e p i t h e l i a l l a y e r b o t h a d j a c e n t t o and a c r o s s from t h e p e n e t r a t i n g m e t a c e r c a r i a e . F i g u r e 18 i s a m a g n i f i e d v i e w o f t h e a r e a o f t h e g l a n d a d j a c e n t t o t h e m e t a c e r c a r i a . The hemocytes i n t h i s a r e a appeared f l a t t e n e d i n some s e c t i o n s , b u t r e t a i n e d t h e i r rounded o u t l i n e i n o t h e r s . F i g u r e 19 shows the a r e a o f t h e g l a n d o p p o s i t e the p e n e t r a t i n g m e t a c e r c a r i a . The f l a t t e n e d n u c l e i and c y t o p l a s m o f t h e hemocytes a r e r e a d i l y d i s t i n g u i s h a b l e . I n most a r e a s two t o t h r e e l a y e r s o f hemocytes a r e p r e s e n t . The hemocytes l y i n g d i r e c t l y a g a i n s t t h e e p i t h e l i u m o f t h e s i l k g l a n d a r e v e r y f l a t t e n e d , t h e n u c l e i are l - 2 u i n h e i g h t . Hemocytes composing t h e second and t h i r d l a y e r o f t h e c a p s u l e a r e not as f l a t t e n e d , t h e n u c l e i a r e 2-4u i n h e i g h t . 62 FIGURE 17 L o n g i t u d i n a l s e c t i o n o f a young m e t a c e r c a r i a o f Bunodera m e d i o v i t e l l a t a w i t h i n t h e e p i t h e l i a l w a l l o f t h e s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s . DE= de g e n e r a t e d e p i t h e l i u m , H= hemocyte, LG= lumen o f th e s i l k g l a n d , NE= e p i t h e l i a l c e l l n u c l e u s , P= p a r a s i t e . ( H e m a t o x y l i n and e o s i n Y, 200x). FIGURE 18 M a g n i f i e d v i e w o f a l o n g i t u d i n a l s e c t i o n o f t h e s i l k g l a n d e p i t h e l i u m a d j a c e n t t o t h e p a r a s i t e . DE= d e g e n e r a t e d e p i t h e l i u m , H= hemocyte, LG= lumen o f t h e s i l k g l a n d , P= p a r a s i t e . ( H e m a t o x y l i n and e o s i n Y, 675x) 64 . FIGURE 19 Magnified view of a longitudinal section of the s i l k gland epithelium across from the parasite. Demonstrating flattened hemocytes against the surface of the s i l k gland. DE= degenerated epithelium, H= hemocyte, LG= lumen of the s i l k gland. (Hematoxylin and eosin Y, 2200x). G5 19 T h e r e f o r e , i t seems t h a t b e f o r e t h e m e t a c e r c a r i a e a c t u a l l y c o n t a c t e d t h e o p p o s i t e w a l l o f t h e s i l k g l a n d , t h e e p i t h e l i u m o f t h e g l a n d had begun t o de g e n e r a t e and hemocytes had moved t o t h e w a l l s o f t h e s i l k g l a n d . ( i i ) W e l l E s t a b l i s h e d I n f e c t i o n s The m a j o r i t y o f m e t a c e r c a r i a e t a k e n from f i e l d c o l l e c t e d c a d d i s f l y l a r v a e were s i t u a t e d d i r e c t l y w i t h i n t h e lumen o f t h e p o s t e r i o r o n e - t h i r d o f t h e s i l k g l a n d s . S e c t i o n s showed t h e c y s t s t o t o t a l l y o c c l u d e t h e lumen o f the s i l k g l a n d , as t h e c y s t s were w i d e r than t h e g l a n d i t s e l f (130-190u) ( F i g u r e 2 0 ) . C o n s e q u e n t l y , s i l k p r oduced by t h e a r e a o f t h e g l a n d l y i n g between t h e m e t a c e r c a r i a and t h e b l i n d end o f t h e g l a n d c o u l d no l o n g e r move f o r w a r d . T h i s d i s t a l a r e a o f t h e g l a n d u s u a l l y showed s i g n s o f d e g e n e r a t i o n . L i g h t m i c r o s c o p e s e c t i o n s o f Epon embedded m e t a c e r c a r i a e s t a i n e d w i t h methylene b l u e showed t h e c y s t w a l l s e c r e t e d by t h e p a r a s i t e as an amorphous p u r p l e - p i n k l a y e r l y i n g d i r e c t l y a d j a c e n t t o t h e p a r a s i t e (C i n F i g u r e 2 1 ) . Next t o t h e c y s t w a l l i n some s e c t i o n s was a t h i n l a y e r o f s i l k w h i c h appeared t o be amorphous and s t a i n e d b l u e (S i n F i g u r e 2 1 ) . I n some c a s e s t h i s l a y e r o f s i l k was n o t p r e s e n t , p r o b a b l y owing t o t h e growth o f the p a r a s i t e p u s h i n g t h e s i l k away from t h i s a r e a . The a r e a n e x t t o t h e s i l k was a d e n s e l y l a y e r e d a r e a s h i c h s t a i n e d a v e r y dark b l u e (I i n F i g u r e 2 1 ) . Hemocyte n u c l e i were v i s i b l e i n some a r e a s o f t h i s 67 FIGURE 20 L o n g i t u d i n a l s e c t i o n o f a m e t a c e r c a r i a o f Bunodera  m e d i o v i t e l l a t a e s t a b l i s h e d w i t h i n t h e s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s . FH= a r e a o f p a r a s i t e f r e e o f hemocytes, H= hemocyte, LG= lumen o f t h e s i l k g l a n d , P= p a r a s i t e . ( H e m a t o x y l i n and e o s i n Y, 6 7 5 x ) . 69 FIGURE 21 Magnified views of two areas of a longitudinal section through a metacercaria of Bunodera mediovitellata encysted within the s i l k gland of Psychoglypha  alascensis, (a) (Methylene blue, 2200x), (b) (Methylene blue, 2200 x). A= amorphous pink substance, C= cyst wall, F= flattened hemocyte, 1= inner layer of the capsule, N= hemocyte nucleus, 0= outer layer of the capsule, R= rounded hemocyte, S= s i l k . 7 0 l a y e r (N i n F i g u r e 21). However, t h e m a j o r i t y o f t h e hemocytes were so d e n s e l y packed t h a t t h e c e l l o u t l i n e s were not c l e a r and t h e l a y e r appeared f i b r o u s . There was no e v i d e n c e o f t h e e p i t h e l i a l l a y e r o f t h e w a l l o f t h e s i l k g l a n d i n t h i s a r e a . I n a d d i t i o n , amorphous masses o f homogeneous m a t e r i a l w h i c h s t a i n e d p i n k w i t h methylene b l u e were v i s i b l e a t t h e i n n e r a r e a o f t h i s l a y e r (A i n F i g u r e 21). The outermost l a y e r around t h e m e t a c e r c a r i a was one of l o o s e l y packed hemocytes which s t a i n e d v e r y l i g h t b l u e . (0 i n F i g u r e 21). I n many a r e a s i n d i v i d u a l c e l l s were e a s i l y r e c o g n i z a b l e . The hemocytes i n t h i s o u t e r l a y e r were not as f l a t as were t h o s e i n t h e i n n e r l a y e r and t h e r e was much i n t e r c e l l u l a r space. I n s e v e r a l a r e a s u n f l a t t e n e d hemocytes were v i s i b l e towards t h e o u t e r a r e a o f t h e c a p s u l e (R i n F i g u r e 21). I n t h i s o u t e r a r e a t h e r e was no e v i d e n c e o f t h e e p i t h e l i u m o f t h e s i l k g l a n d . I t appeared t h a t t h e w a l l o f t h e s i l k g l a n d around t h e p a r a s i t e had been t o t a l l y r e p l a c e d by a c a p s u l e composed o f two l a y e r s o f hemocytes, one c o n t a i n i n g d e n s e l y packed c e l l s and one c o n t a i n i n g l o o s e l y packed c e l l s . E l e c t r o n m i c r o g r a p h s o f s e c t i o n s t h r o u g h m e t a c e r c a r i a e e n c y s t e d w i t h i n t h e lumen o f s i l k g l a n d s , s u p p o r t t h e above o b s e r v a t i o n s . V i s i b l e a g a i n s t t h e tegument o f t h e p a r a s i t e i s t h e amorphous c y s t w a l l a p p r o x i m a t e l y 5u i n t h i c k n e s s (C i n F i g u r e s 22a and b ) . Next t o t h a t t h e r e appeared t o be a f i b r o u s l a y e r composed o f compact c e l l s (I i n F i g u r e s 22a 72 FIGURE 22 E l e c t r o n m i c r o g r a p h s o f c a p s u l e s around m e t a c e r c a r i a e o f Bunodera m e d i o v i t e l l a t a e n c y s t e d w i t h i n t h e lumen o f a s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s t ( a ) demonstrates i n n e r l a y e r o f t h e c a p s u l e (2500x), (b) demonstrates o u t e r l a y e r o f t h e c a p s u l e (1875x). C= c y s t w a l l , I = i n n e r l a y e r o f c a p s u l e , 0= o u t e r l a y e r o f c a p s u l e , P= p a r a s i t e , T= tegument o f p a r a s i t e . b and b ) ; d e t a i l s o f t h e c e l l s were not v i s i b l e . The e l e c t r o n m i c r o g r a p h s do n o t show t h e d e t a i l s o f t h e o u t e r l a y e r o f t h e c a p s u l e , a l t h o u g h l o o s e l y 'packed' m a t e r i a l was v i s i b l e a t t h e o u t s i d e edge o f t h e c a p s u l e i n one s e c t i o n (0 i n F i g u r e 22b). I t appears t h a t hemocytes do not i m m e d i a t e l y e n t e r i n t o t h e lumen o f t h e s i l k g l a n d and t o t a l l y e n c a p s u l a t e t h e m e t a c e r c a r i a upon i t s p e n e t r a t i o n . S e c t i o n s o f some m e t a c e r c a r i a e c l e a r l y showed t h a t t h e hemocytes had t o t a l l y r e p l a c e d t h e e p i t h e l i a l w a l l o f t h e s i l k g l a n d , b o t h d i r e c t l y around t h e p a r a s i t e , and a p p r o x i m a t e l y 150u on e i t h e r s i d e o f t h e p a r a s i t e , so t h a t t h e c a p s u l e was s u r r o u n d i n g b o t h t h e p a r a s i t e and t h e s i l k o f t h e g l a n d . However, l o n g i t u d i n a l s e c t i o n s o f t h e s e c a p s u l e s showed t h a t t h e hemocytes had n o t pass e d t h r o u g h t h e s i l k t o a s s o c i a t e w i t h t h e a r e a s o f t h e p a r a s i t e remote from t h e g l a n d w a l l (FN i n F i g u r e 2 0 ) . T h e r e f o r e , a r e a s o f t h e p a r a s i t e remained f r e e o f hemocytes w i t h i n t h e lumen o f t h e g l a n d . In s e c t i o n s o f o t h e r c a p s u l e s , p r o b a b l y o f o l d e r i n f e c t i o n s , hemocytes had moved i n so t h a t a l l a r e a s o f t h e p a r a s i t e were e n c a p s u l a t e d . In t h e s e s e c t i o n s , hemocytes had b l o c k e d t h e lumen o f t h e g l a n d on e i t h e r s i d e o f t h e p a r a s i t e so t h a t t h e m e t a c e r c a r i a e were i s o l a t e d from t h e r e s t o f t h e g l a n d ( F i g u r e 2 3 ) . S e v e r a l o f t h e s i l k g l a n d s s e c t i o n e d c o n t a i n e d more tha n one m e t a c e r c a r i a w i t h i n t h e lumen o f t h e g l a n d . 75 FIGURE 23 T r a n s v e r s e s e c t i o n o f a m e l a n i z e d m e t a c e r c a r i a o f Bunodera m e d i o v i t e l l a t a i n w h i c h hemocytes have e n t e r e d t h e s i l k g l a n d lumen and a t t a c h e d t o t h e p a r a s i t e . A l l o f t h e s u r f a c e o f t h e p a r a s i t e i s c o v e r e d w i t h hemocytes. CA= a r e a i n which hemocytes have e n t e r e d t h e lumen o f t h e s i l k g l a n d and a t t a c h e d t o t h e p a r a s i t e , MM= m e l a n i z e d m e t a c e r c a r i a . ( H e m a t o x y l i n and e o s i n Y, 67 5 x ) . 76 2 3 I n m u l t i p l e i n f e c t i o n s such as t h i s , a l l m e t a c e r c a r i a e were su r r o u n d e d by f l a t t e n e d hemocytes ( F i g u r e 2 4 ) . I n a d d i t i o n , t h e e p i t h e l i u m o f t h e s i l k g l a n d l y i n g between each m e t a c e r c a r i a had o f t e n been r e p l a c e d by hemocytes (H i n F i g u r e 2 4 ) ; i n no case was e p i t h e l i a l r e g e n e r a t i o n d e t e c t e d . M e t a c e r c a r i a e i n s i d e many o f t h e s e c y s t s appeared t o be i n t a c t . When s e v e r a l o f t h e u n f i x e d c y s t s were broken open, t h e m e t a c e r c a r i a e r e l e a s e d were a l i v e and a b l e t o move. ( i i i ) O l d I n f e c t i o n s M e t a c e r c a r i a e removed from a t o t a l o f 15 l a r v a e o f P. a l a s c e n s i s c o l l e c t e d i n l a t e J u l y and August o f b o t h 1979 and 1980 were found t o be c o v e r e d w i t h a brown pigment In wax s e c t i o n s o f m e t a c e r c a r i a e s t a i n e d w i t h h e m a t o x y l i n and e o s i n Y t h i s m a t e r i a l appeared as a b r i t t l e amorphous -brown s u b s t a n c e l y i n g between t h e i n n e r l a y e r o f t h e hemocyte c a p s u l e and t h e c y s t w a l l o f t h e p a r a s i t e ( F i g u r e 2 5 ) . When t h e s e brown m e t a c e r c a r i a e were s e c t i o n e d and t r e a t e d w i t h t h e F e r r o u s I r o n Technique f o r m e l a n i n (Pearse 1972), t h e brown a r e a s s t a i n e d a dark g r e e n i s h brown, w h i l e t h e m e t a c e r c a r i a e and t h e r e s t o f t h e s i l k g l a n d s t a i n e d l i g h t g r e e n . When o t h e r s e c t i o n s o f t h e s e m e t a c e r c a r i a e were t r e a t e d w i t h p o t a s s i u m permanganate and o x a l i c a c i d , a m e l a n i n s p e c i f i c b l e a c h f o l l o w e d by t h e m e l a n i n s p e c i f i c s t a i n , a l l a r e a s o f t h e c a p s u l e ( i n c l u d i n g what was 78 FIGURE 24 C r o s s s e c t i o n o f a s i l k g l a n d o f P s y c h o g l y p h a a l a s c e n s i s c o n t a i n i n g two m e t a c e r c a r i a e o f Bunodera m e d i o v i t e l l a t a . Note t h a t t h e s i l k g l a n d e p i t h e l i u m between t h e two m e t a c e r c a r i a e has been r e p l a c e d by hemocytes. H= hemocyte, P= p a r a s i t e . ( H e m a t o x y l i n and e o s i n Y, 200x). FIGURE 25 L o n g i t u d i n a l s e c t i o n t h r o u g h a m e l a n i z e d m e t a c e r c a r i a l c y s t o f Bunodera m e d i o v i t e l l a t a . C= c y s t w a l l , H= hemocyte, M= m e l a n i n , P= p a r a s i t e . ( H e m a t o x y l i n and e o s i n Y, 67 5 x ) . 79 80 o r i g i n a l l y brown p i g m e n t a t i o n ) as w e l l as t h e m e t a c e r c a r i a and s i l k g l a n d s t a i n e d l i g h t g r e e n . T h e r e f o r e , t h e brown p i g m e n t a t i o n was c o n s i d e r e d t o be m e l a n i n . In a l l o f t h e m e l a n i z e d m e t a c e r c a r i a e s e c t i o n e d , the lumen of t h e s i l k g l a n d n e x t t o t h e p a r a s i t e had been t o t a l l y b l o c k e d by hemocytes. T h i s f i n d i n g and t h e f a c t t h a t m e l a n i z e d m e t a c e r c a r i a e were c o n s i s t e n t l y found o n l y a t t h e end o f t h e summers, s u g g e s t s t h a t the m e l a n i z a t i o n o f t h e p a r a s i t e o c c u r r e d o n l y a f t e r t h e p a r a s i t e had been w i t h i n t h e c a d d i s f l y l a r v a f o r a l o n g p e r i o d o f t i m e , perhaps two o r t h r e e months. I t may be n o t e d t h a t even a f t e r t h i s p e r i o d o f t i m e , t h e hemocytes r e p l a c e d t h e s i l k g l a n d e p i t h e l i u m b u t d i d n o t form a m a s s i v e unorganized, c a p s u l e . I n s t e a d , t h e y formed a s t r u c t u r e v e r y s i m i l a r i n d i a m e t e r and shape t o t h e o r i g i n a l s i l k g l a n d e p i t h e l i u m . The m e t a c e r c a r i a e w i t h i n t h e m e l a n i z e d s i l k g l a n d s u s u a l l y d i d n o t appear i n t a c t (P i n F i g u r e 2 5 ) . However, i t i s d i f f i c u l t t o d e t e r m i n e whether o r n o t t h i s i s an a r t i f a c t o f s e c t i o n i n g owing t o t h e tough m e l a n i n . J . Hemocytes I n v o l v e d i n t h e E n c a p s u l a t i o n Response I n b o t h t h e 35 day c a p s u l e formed around t h e Epon i m p l a n t s and t h e e s t a b l i s h e d m e t a c e r c a r i a l c a p s u l e , ' t h e c y t o p l a s m and n u c l e i o f t h e hemocytes f o r m i n g the dense i n n e r l a y e r o f t h e c a p s u l e had become so f l a t t e n e d and e x t e n d e d t h a t t h e e x t e n t o f t h e c y t o p l a s m and n u c l e i were d i f f i c u l t t o d e t e r m i n e and c o n s e q u e n t l y p a r t i c u l a r t y p e s o f hemocytes 81 were n o t r e c o g n i z a b l e . However, t h e hemocytes f o r m i n g t h e o u t e r l a y e r o f t h e o l d e r m e t a c e r c a r i a l c a p s u l e s , t h e o u t e r p a r t o f t h e 35 day i m p l a n t c a p s u l e , as w e l l as t h o s e t a k i n g p a r t i n t h e response t o the seven day i m p l a n t , and t h e new m e t a c e r c a r i a e were not as f l a t t e n e d and s e c t i o n s o f whole hemocytes were o f t e n v i s i b l e . To f a c i l i t a t e t h e comparison between t h e f r e e l y c i r c u l a t i n g hemocytes and c e l l t y p e s t a k i n g p a r t i n t h e e n c a p s u l a t i o n r e s p o n s e , a sample o f t h e l o n g e s t c e l l s seen i n each o f t h e r e a c t i o n s t o t h e seven day i m p l a n t s , 35 day i m p l a n t s , young m e t a c e r c a r i a e , and e s t a b l i s h e d m e t a c e r c a r i a e was t a k e n and s k e t c h e d t o s c a l e w i t h t h e a i d o f a d r a w i n g tube ( F i g u r e 2 6 ) . These were t h e n compared w i t h t h e t y p e s o f hemocytes seen i n hemolymph smears ( F i g u r e 1 1 ) . I t s h o u l d be s t r e s s e d t h a t w h i l e t h e s k e t c h e s o f f r e e c i r c u l a t i n g hemocytes a r e o f whole c e l l s , t h e s k e t c h e s o f t h e c a p s u l e c e l l s a r e o f c r o s s s e c t i o n s . Because o f t h e c o m p l i c a t i o n s t h a t may be i n v o l v e d w i t h c u t t i n g c r o s s s e c t i o n s o f n o n - s y m m e t r i c a l c e l l s a t v a r i o u s a n g l e s , I chose t o sample o n l y t h e l o n g e s t c e l l s seen i n t h e above c a p s u l e s ; i n i n t e r p r e t i n g s e c t i o n s showing s m a l l c e l l s , I had no way o f d i s t i n g u i s h i n g between s e c t i o n s c u t a c r o s s t h e a x i s o f l o n g t h i n c e l l s ( t h e r e f o r e , a p p e a r i n g as s m a l l c e l l s ) and s e c t i o n s c u t l o n g i t u d i n a l l y t h r o u g h t r u l y s m a l l c e l l s . However, two o f t h e m e l a n i z e d m e t a c e r c a r i a e were c u t s a g i t a l l y r a t h e r t h a n t r a n s v e r s e l y and hence show many o f t h e hemocytes i n v o l v e d i n t h e c a p s u l e s . 82 FIGURE 26 Drawings o f sample hemocytes from hemolymph smears and i n v o l v e d i n o u t e r l a y e r o f c a p s u l e s . Drawings done w i t h a i d o f a drawing t u b e , (a) F r e e c i r c u l a t i n g hemocytes from hemolymph smears; numbers c o r r e s p o n d t o t h o s e i n F i g u r e 11. (b) Hemocytes from c p a s u l e around seven day Epon i m p l a n t s , (c) Hemocytes from c a p s u l e around 35 day Epon i m p l a n t s , (d) Hemocytes from c a p s u l e around new m e t a c e r c a r i a e . (e) Hemocytes from c a p s u l e s around e s t a b l i s h e d m e t a c e r c a r i a e . 83 84 U n f o r t u n a t e l y t h e l i m i t s o f t h e c e l l s seen i n t h e s a g i t a l s e c t i o n s were not v i s i b l e . The s e c t i o n s o f hemocytes t a k i n g p a r t i n t h e c a p s u l e around t h e seven day. i m p l a n t s a r e shown'in F i g u r e 26b. These c e l l s appear t o be l o n g e r t h a n t h o s e t a k i n g p a r t i n the c a p s u l e around t h e new m e t a c e r c a r i a e ( F i g u r e 2 6 a ) . These c e l l s a r e as l o n g as c e l l #14 and c e l l #18, however, no c e l l s seen were as l o n g as c e l l #5. As t h e p r e p a r a t i o n s a r e o f c r o s s s e c t i o n s o f c e l l s , c h a r a c t e r s such as c y t o p l a s m a r e a , n u c l e u s a r e a , and c y t o p l a s m and n u c l e u s d e n s i t y c a n n o t be used , and s u b s e q u e n t l y a more d e t a i l e d c omparison w i t h t h e f r e e - c i r c u l a t i n g c e l l s c a n not be made. A sample o f t h e l a r g e hemocytes t a k i n g p a r t i n t h e response t o t h e new m e t a c e r c a r i a e ( F i g u r e 26d) show c e l l s t h a t . a r e s m a l l e r t h a n any o f t h e t h r e e l o n g e s t f r e e -c i r c u l a t i n g c e l l t y p e s , and c o r r e s p o n d more c l o s e l y t o the s i z e o f c e l l #8 and #3 from t h e f r e e - c i r c u l a t i n g hemocyte smears. A g a i n as t h e s e c e l l s a r e c r o s s s e c t i o n s no f u r t h e r c o r r e l a t i o n between t h e s e and t h e f r e e - c i r c u l a t i n g c e l l t y p e s can be made. F i g u r e 26c shows t h e l a r g e c e l l s seen t a k i n g p a r t i n t h e r e a c t i o n t o t h e 35 day i m p l a n t . These c e l l s a r e l o n g enough t o c o r r e s p o n d t o f r e e - c i r c u l a t i n g c e l l s #14, 18, and 5. S e v e r a l o f t h e c e l l s appear t o have d i s t i n c t l y e c c e n t r i c n u c l e i and t h e r e f o r e would c o r r e s p o n d most c l o s e l y t o #18. The c e l l s i n F i g u r e 26e a r e t h o s e seen t a k i n g p a r t i n 85 t h e response t o w e l l e s t a b l i s h e d m e t a c e r c a r i a e . Three o f t h e s e c e l l s a r e l o n g e r t h a n t h e l o n g e s t f r e e - c i r c u l a t i n g c e l l t y p e seen(#5). I t w i l l be n o t e d t h a t t h e edges o f t h e s e c e l l s a r e e x t r e m e l y t h i n ( c . f . t h o s e around t h e new m e t a c e r c a r i a e ) , perhaps i n d i c a t i n g e l o n g a t i o n o f t h e c e l l s as e n c a p s u l a t i o n p r o g r e s s e s . From t h i s s t u d y however, I am u n a b l e t o d e t e r m i n e whether t h e s e c e l l s r e p r e s e n t new c e l l s o r e l o n g a t e d v e r s i o n s o f t h e f r e e - c i r c u l a t i n g c e l l t y p e s seen. Only t h e n u c l e i o f hemocytes t a k i n g p a r t i n t h e m e l a n i z e d r e a c t i o n s were v i s i b l e ; t h e c y t o p l a s m i c b o u n d a r i e s o f t h e c e l l s were n o t v i s i b l e . These n u c l e i were of e q u a l d e n s i t y and had a s i z e range from 4 t o 9u. V7ithout t h e c y t o p l a s m i c b o u n d a r i e s o f t h e c e l l s , c o r r e l a t i o n s between t h e s e and t h e f r e e - c i r c u l a t i n g hemocyte t y p e s c o u l d n o t be made. In summary t h e n , i t appears t h a t a number o f d i f f e r e n t hemocytes may respond t o t h e m e t a c e r c a r i a e l o c a t e d w i t h i n t h e s i l k g l a n d s . I n p a r t i c u l a r i t i s suggested t h a t c e l l s s i m i l a r t o f r e e - c i r c u l a t i n g c e l l s , # 14,-18, 5, and s e v e r a l c e l l s l a r g e r t h a n #5 may p a r t i c i p a t e i n t h e r e a c t i o n . Those t h a t do respond may f l a t t e n and e x t e n d t h e i r c y t o p l a s m as t h e e n c a p s u l a t i o n p r o c e e d s . T h i s would account f o r t h e e x t r e m e l y f l a t t e n e d c e l l s i n t h e i n n e r l a y e r o f o l d e r c a p s u l e s . 86 IV. DISCUSSION A. P a r a s i t e s o f T r i c h o p t e r a and t h e I n c i d e n c e o f t h e P a r a s i t e  Bunodera m e d i o v i t e l l a t a i n i t s T r i c h o p t e r a n Host ' Of t h e f i v e major groups o f metazoan p a r a s i t e s , I n s e c t a , Digenea, Cestoda,Nematoda and A c a n t h o c e p h a l a , S a l t (196 3) s u g g e s t s t h a t digeneans a r e t h e o n l y group t h a t p a r a s i t i z e t h e T r i c h o p t e r a . To my knowledge t h i s s t a t ement remains t r u e t o d a t e . However, not a l l d i geneans are p a r a s i t e s o f c a d d i s f l i e s . I n f a c t , o f t h e t h i r t e e n f a m i l i e s o f Digenea o t h e r t h a n th e a l l o c r e a d i i d s , known t o p a r a s i t i z e a q u a t i c i n s e c t s , ( H a l l , p e r s o n a l communication) o n l y t h r e e have been r e p o r t e d from T r i c h o p t e r a . These i n c l u d e members o f t h e L e c i t h o d e n d r i i d a e , G o r g o d e r i d a e , and P l a g i o r c h i i d a e . The s p e c i e s w i t h i n each f a m i l y r e c o r d e d from T r i c h o p t e r a a r e g i v e n i n T a b l e 4. There was a l a r g e d i f f e r e n c e i n t h e i n c i d e n c e o f p a r a s i t i s m by B. m e d i o v i t e l l a t a between t h e two s p e c i e s o f T r i c h o p t e r a . S i n c e (1) c e r c a r i a e were seen t o p e n e t r a t e b o t h s p e c i e s o f c a d d i s f l y , and (2) t h e r e appeared t o be no d i f f e r e n c e between the c y s t s o f B. m e d i o v i t e l l a t a w i t h i n t h e s i l k g l a n d s o f L. r o a f i and t h o s e w i t h i n P. a l a s c e n s i s , i t appears t h a t once the c e r c a r i a e a r e i n t h e v i c i n i t y o f e i t h e r s p e c i e s o f c a d d i s f l y l a r v a t h e y w i l l p e n e t r a t e and e n c y s t . However, the f a c t t h a t fewer m e t a c e r c a r i a e were found n a t u r a l l y i n f e c t i n g l a r v a e o f L. r o a f i t h a n l a r v a e o f P. a l a s c e n s i s s u g g e s t s t h a t i n g e n e r a l , l a r v a e o f L. r o a f i were n o t i n t h e v i c i n i t y o f T a b l e 4. F a m i l i e s o f Digenea, e x c l u d i n g the A l l o c r e a d i i d a e , whose members p a r a s i t i z e T r i c h o p t e r a . G= Gorgoder'idae, L= L e c i t h o d e n d r i i d a e , P= P l a g i o r c h i i d a e , NM= I n f o r m a t i o n not mentioned i n r e f e r e n c e . S p e c i e s o f Digenean L a r v a l Stage T r i c h o p t e r a n Host S i t e i n Host Re f e r e n c e (L) A c a n t h a t r i u m oregonense Macy m e t a c e r c a r i a e unencysted m e t a c e r c a r i a e unencysted L i m n e p h i l u s sp. ( l a r v a e ) Dicosmoecus sp. ( l a r v a e ) f a t body abdominal hemocoel, g i l l s K n i g h t and P r a t t (1955) Burns (1961) (L) A l l a s o g o n o p o r u s v e s p e r t i l l i o n i s Macy m e t a c e r c a r i a e e n c y s t e d L i m n e p h i l u s sp. ( l a r v a e ) f a t body K n i g h t and P r a t t (1955) (L) L e c i t h o d e n d r i u m c h i l o s t o m i i (Mehl.) m e t a c e r c a r i a e 1° unencysted m e t a c e r c a r i a e 2° e n c y s t e d Phryganea g r a n d i s L. ( l a r v a e ) Phyrganea g r a n d i s (pupae and a d u l t s ) f r e e w i t h i n body c a v i t y , f a t body t h o r a c i c hemocoele Brown (1933) Brown (1933) (L) P l e u r o g e n e s medians (Olss.) m e t a c e r c a r i a e e n c y s t e d L i m e p h i l u s sp. ( l a r v a e ) S e r i c o s t o m a sp. ( l a r v a e ) c o n n e c t i v e t i s s u e o f g i l l , body c a v i t y nearby Neuhaus (1940) (G) P h y l l o d i s t o m u m s t a f f o r d i Pearse m e t a c e r c a r i a e unencysted T r i c h o p t e r a ( l a r v a e ) t h o r a c i c S c h e l l (1967) (P) Distomum endolobum m e t a c e r c a r i a e e n c y s t e d L i m n e p h i l u s l u n a t u s C u r t i s , L. g r i s e u s ( L . ) , L. rhombicus ( L . ) , A n a b o l i a n e r v o s a Leach NM L i n s t o w (1897) T a b l e 4. (continued) (P) Distomum isoporum m e t a c e r c a r i a e e n c y s t e d C h a e t o p t e r y x v i l l o s a F., A n a b o l i a n e r v o s a NM L i n s t o w (1897) (P) Distomum m y s t a c i d i s m e t a c e r c a r i a e e n c y s t e d M y s t a c i d e s n i g r a (L.) L i m n e p h i l u s f l a v i c o r n i s (Fab.) NM L i n s t o w (1897) (P) O p i s t h o g l y p h e l o c e l l u s m e t a c e r c a r i a e e n c y s t e d T r i c h o p t e r a ( l a r v a e ) NM Macy and Moore (1958) Kossack (P) P l a g i o r c h i s goodmani m e t a c e r c a r i a e e n c y s t e d L i m n e p h i l u s i n d i v i s u s NM N a j a r i a n (1952) N a j a r i a n Walker ( l a r v a e ) CO CO t h e c e r c a r i a e a t t h e t ime o f t h e i r r e l e a s e , whereas, t h e l a r v a e o f P. a l a s c e n s i s were. U n f o r t u n a t e l y I d i d not have time t o c a r r y out a d e t a i l e d s t u d y of t h e n a t u r a l h a b i t s o f each s p e c i e s o f c a d d i s f l y . However, I can s p e c u l a t e as t o t h r e e c i r c u m s t a n c e s w h i c h may account f o r L. r o a f i h a v i n g l e s s c o n t a c t w i t h B. m e d i o v i t e l l a t a . F i r s t l y , i t i s p o s s i b l e t h a t t h e two s p e c i e s o f c a d d i s f l y n o r m a l l y i n h a b i t a r e a s a t d i f f e r e n t depths i n t h e s t r e a m . There i s i n d i r e c t e v i d e n c e t o s u g g e s t t h a t t h i s i s t r u e . When b u c k e t s c o n t a i n i n g c a d d i s f l y l a r v a e c o l l e c t e d from t h e f i e l d were examined, l a r v a e o f L. r o a f i were c o n s i s t e n t l y found a t t a c h e d t o t h e w a l l s o f t h e b u c k e t s j u s t below t h e s u r f a c e o f t h e w a t e r . L a r v a e o f P. a l a s c e n s i s r a r e l y a t t a c h e d t o t h e w a l l s o f t h e b u c k e t s ; t h e y were u s u a l l y l o c a t e d on t h e f l o o r o f t h e b u c k e t s a l o n g w i t h t h e c l a m s . S e c o n d l y , i t i s p o s s i b l e t h a t t h e l i f e c y c l e s o f L. r o a f i and P. a l a s c e n s i s d i f f e r such t h a t d u r i n g t h e t i m e when c e r c a r i a e a r e r e l e a s e d from c l a m s , l a r v a e o f L. r o a f i a r e t o o s m a l l t o be i n f e c t e d , o r a r e l e s s abundant t h a n t h o s e o f P. a l a s c e n s i s . S e v e r a l t i m e s o v e r the 24 month c o l l e c t i o n p e r i o d no l a r v a e o f L. r o a f i were found. I n c o n t r a s t l a r v a e o f P. a l a s c e n s i s were always f o u n d , a l t h o u g h a t t i m e s i n s m a l l numbers. L a s t l y , i t i s p o s s i b l e t h a t owing t o t h e l a r g e r s i z e o f t h e c a s e , t h e l a r v a e o f P. a l a s c e n s i s have a strongc-v e n t i l l a t i o n f o r c e and t h u s a r e a b l e t o p u l l more c e r c a r i a e i n t o t h e i r v i c i n i t y . However, i f t h i s were t h e ca s e one would e x p e c t t h a t a n i m a l s w i t h l a r g e c a s e s would be i n f e c t e d w i t h more t h a n one m e t a c e r c a r i a e more o f t e n t h a n t h o s e w i t h s m a l l c a s e s . The a n a l y s i s o f v a r i a n c e d emonstrated however, t h a t t h e s i z e o f t h e case o f t h e i n d i v i d u a l d i d not appear t o a f f e c t t h e number o f c y s t s per i n d i v i d u a l . The i n c i d e n c e o f p a r a s i t i s m appeared t o be s e a s o n a l . I n b o t h l a r v a e , t h e r e was a low i n c i d e n c e o f p a r a s i t i s m a t t h e b e g i n n i n g o f s p r i n g , b u t t h i s t h e n i n c r e a s e d t o a h i g h i n c i d e n c e i n J u l y o f b o t h s a m p l i n g y e a r s . These s e a s o n a l d i f f e r e n c e s may r e f l e c t t h e anadramous n a t u r e o f t h e s t i c k l e b a c k d e f i n i t i v e h o s t . A c c o r d i n g t o Kennedy ( p e r s o n a l communication) s t i c k l e b a c k s move up t h e stream from i t s o u t l e t a t t h e ocean a t v a r i o u s t i m e s d u r i n g t h e y e a r . I t i s p o s s i b l e t h a t t h e i n f e c t e d s t i c k l e b a c k s move up th e s t r e a m i n May. A t t h i s p o i n t eggs from B. m e d i o v i t e l l a t a a r e r e l e a s e d w i t h t h e f e c e s o f t h e f i s h and t h u s t h e c y c l e o f t h e p a r a s i t e b e g i n s so t h a t by J u l y c e r c a r i a e have been r e l e a s e d from t h e clams and have i n f e c t e d t h e c a d d i s f l i e s . I n s u p p o r t o f t h i s , t h e a n a l y s i s o f v a r i a n c e i n d i c a t e d t h a t t h e date o f c o l l e c t i o n a f f e c t e d t h e number o f c y s t s p e r i n d i v i d u a l . F u r t h e r i n v e s t i g a t i o n , however, i n t o t h e t i m e s o f m i g r a t i o n o f th e s t i c k l e b a c k s i n T i n Can Creek i s n e c e s s a r y . 91 B. Encystment o f A l l o c r e a d i i d s B e f o r e d i s c u s s i n g t h e d e t a i l s o f e n c a p s u l a t i o n r e a c t i o n s t o a l l o c r e a d i i d s I s h a l l b r i e f l y d i s c u s s t h e h o s t s and s i t e s o f encystment o f t h e members o f t h e A l l o c r e a d i i d a e . I n a d d i t i o n t o Bunodera m e d i o v i t e l l a t a , t h e second i n t e r m e d i a t e h o s t and s i t e o f encystment have been d e t e r m i n e d f o r a number o f o t h e r members o f t h e A l l o c r e a d i i d a e . Not a l l a l l o c r e a d i i d s e n c y s t i n a q u a t i c i n s e c t s . M e t a c e r c a r i a e o f t h i s f a m i l y o f digeneans have a l s o been r e p o r t e d from b i v a l v e s and c r u s t a c e a n s (Table 5). I n c r u s t a c e a n s m e t a c e r c a r i a e have g e n e r a l l y been r e p o r t e d from t h e hemocoele o r c a r d i a c r e g i o n o f t h e h o s t . I n sh r i m p , m e t a c e r c a r i a e have sometimes been found i n t h e l e g s and eye s t a l k s . I n b i v a l v e s , t h e mantle i s the o n l y s i t e o f m e t a c e r c a r i a l encystment r e c o r d e d . However, as t h e c e l l u l a r d e f e n s e system o f b o t h b i v a l v e s and c r u s t a c e a n s d i f f e r s from t h e system o f i n s e c t s ( M i c h e l s o n , 1977) i t i s more s u i t a b l e t o compare t h e defence r e a c t i o n o f l a r v a e o f t h e t r i c h o p t e r a n P. a l a s c e n s i s t o t h e r e a c t i o n o f o t h e r i n s e c t s h o s t i n g a l l o c r e a d i i d s . A l l o c r e a d i i d s have p r e v i o u s l y been r e p o r t e d from f o u r o r d e r s of i n s e c t s namely t h e C o l e o p t e r a , D i p t e r a , Ephemeroptera and T r i c h o p t e r a . These r e p o r t s a r e summarized i n T a b l e 6. E n c y s t e d m e t a c e r c a r i a e have been r e p o r t e d from t h e subcutaneous c o n n e c t i v e t i s s u e s and s e r o s a l membranes o f c h i r o n o m i d l a r v a e ( B u n o d e r e l l a  m e t t e r i S c h e l l ; Anderson e t a l . 1965), t h e f a t body and T a b l e 5. A l l o c r e a d i i d s whose second i n t e r m e d i a t e h o s t s a r e i n v e r t e b r a t e s o t h e r t h a n i n s e c t s . NM= I n f o r m a t i o n not mentioned i n r e f e r e n c e . S p e c i e s o f A l l o c r e a d i i d L a r v a l stage Hosts L o c a t i o n i n Hosts R e f e r e n c e A l l o c r e a d i u m i c t a l u r i (Pearse) m e t a c e r c a r i a e e n c y s t e d u n i o n i d b i v a l v e s t h i c k m antle edge S e i t n e r (1951) A l l o c r e a d i u m isoporum (Looss) m e t a c e r c a r i a e e n c y s t e d f i n g e r n a i l clams (Sphaerium sp.) NM Looss (1894) A l l o c r e a d i u m lobatum ( W a l l i n ) m e t a c e r c a r i a e p r o g e n e t i c amphipods- Gammarus pseudolimnaeus B o u s f i e l d , C r a n g o n i x g r a c i l i s Smith NM De G u i s t i (1962) Bunodera l u c i o p e r c a e ( M u l l e r ) m e t a c e r c a r i e e n c y s t e d Daphnia p u l e x (de Geer) body c a v i t y -i n t e s t i n a l r e g i o n W i s n i e w s k i (1958) Bunodera s a c c u l a t a V i h C l e a v e e t M u e l l e r m e t a c e r c a r i a e e n c y s t e d Daphnia s i m i l i s C l a u s hemocoel a d j a c e n t t o g u t Cannon (1971) Crepidostomum cornutum (Osborn) m e t a c e r c a r i a e encysted c r a y f i s h - Cambarus b a r t o n i Rhodes • c a r d i a c r e g i o n Cheng (1957) O r i e n t o c r e a d i u m s i l u r i ( D u b i n i n a e t B y k n o w s k i i ) m e t a c e r c a r i a e e n c y s t e d ? shrimp- Limnomysis b e n e d e n i , Mesomysis kowalewskyi t h o r a c i c c a v i t y abdominal r e g i o n , l e g s , eye s t a l k s B y c h o w s k i i e t D u b i n i n i a (1954) to T a b l e 6. A l l o c r e a d i i d s whose second i n t e r m e d i a t e h o s t s a r e i n s e c t s . C = C o l e o p t e r a , D= D i p t e r a , E= Ephemeroptera, T= T r i c h o p t e r a , NM= I n f o r m a t i o n n o t mentioned i n r e f e r e n c e . S p e c i e s o f A l l o c r e a d i i d L a r v a l Stage Hosts S i t e i n Host Re f e r e n c e A l l o c r e a d i u m a l l o n e o t e n i c u m a d u l t s (T) L i m n e p h i l u s sp. abdominal hemocoele Wootten (1957) (Wootten) ( l a r v a e ) A l l o c r e a d i u m neotenicum :. a d u l t s (C) D y t i s c u s hemocoele P e t e r s (1957) (P e t e r s ) a c i l i u s Leach Bunodera m e d i o v i t e l l a t a m e t a c e r c a r i a e e n c y s t e d (T) Lepidostoma r o a f i s i l k g l a n d s s i l k g l a n d s Kennedy ( i n l i t . ) , C a i r a ( p r e s e n t study) Z i m b a l i k and Roytman (Milne) ( l a r v a e ) , (T) P s y c h o g l y p h a a l a s c e n s i s (Banks) B u n o d e r e l l a m e t t e r i m e t a c e r c a r i a e e n c y s t e d (T) R h y a c o p h i l a hemocoele, d e r m i s , subcutaneous c o n n e c t i v e t i s s u e , s e r o s a Anderson, e t a l . (1965) S c h e l l ( l a r v a e ) (D) Chironomidae ( l a r v a e ) Crepidostomum c o o p e r i m e t a c e r c a r i a e e n c y s t e d (E) Hexagenia r e c u r v a t a NM NM Choquette (1954) Hopkins (Morgan) ( n a i a d s ) , (E) P o l y m i t a r c y s sp. (naiads) Crepidostomum f a r i o n i s m e t a c e r c a r i a e e n c y s t e d ? (E) Ephemera d a n i c a f a t body, abdominal muscle Brown (1927) , C r a w f o r d (1943) ( M u l l e r ) M u l l , ( naiads) M e g a l o q o n i a i c t a l u r i m e t a c e r c a r i a e e n c y s t e d (E) M a y f l y g i l l s S u r b e r (1928) (Surber) muscle t i s s u e s o f n a i a d s o f t h e m a y f l y Ephemera d a n i c a M u l l . (Crepidostomum f a r i o n i s ( M u l l e r ) ; C r a w f o r d , 1943) , g i l l s o f m a y f l y n a i a d s (Megalogonia i c t a l u r i ( S u r b e r ) ; S u r b e r , 1928) and the hemocoele o f a d u l t d i v i n g b e e t l e s , D y t i s c u s a c i l i u s Leach ( A l l o c r e a d i u m neotenicum ( P e t e r s ) ; P e t e r s , 1957). A l l s i t e r e c o r d i n g s o f a l l o c r e a d i i d s o t h e r t h a n B. m e d i o v i t e l l a t a from c a d d i s f l i e s , have been from t h e abdominal o r t h o r a c i c hemocoele. Thus, t h e f i n d i n g o f m e t a c e r c a r i a e o f Bunodera  m e d i o v i t e l l a t a e n c y s t i n g s p e c i f i c a l l y w i t h i n t h e s i l k g l a n d s o f T r i c h o p t e r a l a r v a e , r e p r e s e n t s a new s i t e o f encystment f o r a l l o c r e a d i i d s . The f a c t t h a t e n c y s t e d m e t a c e r c a r i a e were d i s c o v e r e d i n t h e s i l k g l a n d s o f i n d i v i d u a l s r e p r e s e n t i n g two d i f f e r e n t f a m i l i e s o f T r i c h o p t e r a (L. r o a f i - L e p i d o s t o m a t i d a e , P. a l a s c e n s i s -L i m n e p h i l i d a e ) , s u g g e s t s t h a t t h i s s i t e i s t h e normal a r e a o f encystment f o r t h e m e t a c e r c a r i a e o f t h i s s p e c i e s . C. E n c a p s u l a t i o n R e a c t i o n s E x c l u d i n g the a d u l t s o f A l l o c r e a d i u m n eotenicum , which have n o t been examined i n d e t a i l , a l l a l l o c r e a d i i d s r e p o r t e d seem c a p a b l e o f e l i c i t i n g some response from t h e i r a q u a t i c i n s e c t h o s t s . The g e n e r a l p a t t e r n o f r e s p o n s e o f t h e h o s t seems t o be t h e f o r m a t i o n o f a c e l l u l a r o r ' s e c r e t e d ' l a y e r around t h e p a r a s i t e f o l l o w e d by the d e p o s i t i o n o f a b r o w n - y e l l o w pigment. U n f o r t u n a t e l y , t h e d e s c r i p t i o n s o f t h e h o s t r e s p o n s e s are g e n e r a l l y 95 l a c k i n g i n d e t a i l . Brown (1927), i n examining m e t a c e r c a r i a e o f C. f a r i o n i s from m a y f l i e s , mentions an 'outer c y s t ' p r o b a b l y o f m a y f l y o r i g i n . He does not mention t h e n a t u r e o f t h i s 'outer c y s t ' , however, h i s i l l u s t r a t i o n shows a f i b r o u s l a y e r somewhat r e m i n i s c e n t o f a hemocyte c a p s u l e around t h e e n c y s t e d m e t a c e r c a r i a e d e s c r i b e d h e r e i n t h e . T r i c h o p t e r a l a r v a e . C r a w f o r d (194 3) mentions t h a t as t h e h o s t ' c y s t s ' mature around t h e m e t a c e r c a r i a e o f C. f a r i o n i s , t h e y become surrounded by a b r o w n - y e l l o w pigment o f m a y f l y o r i g i n . T h i s d e s c r i p t i o n i s v e r y much l i k e a m e l a n i z a t i o n r e a c t i o n such as d e s c r i b e d h e r e i n around t h e o l d e r c y s t s o f B . m e d i o v i t e l l a t a i n t h e s i l k g l a n d o f P. a l a s c e n s i s . A g a i n C r a w f o r d (1943) does not mention t h e s t r u c t u r e o f t h e h o s t c a p s u l e , b u t i n h i s drawing t h e m e t a c e r c a r i a o f C. f a r i o n i s i s s urrounded by s e v e r a l l a y e r s o f s p i n d l e shaped c e l l s . Wootten (1957) r e p o r t e d f i n d i n g d e g e n e r a t e d a d u l t s o f A l l o c r e a d i u m a l l o n e o t e n i c u m (Wootten) w h i c h appeared as b l a c k e n e d amorphous masses, but s t i l l r e t a i n e d t h e shape o f t h e worm, a g a i n s u g g e s t i n g t h e p r o d u c t i o n o f m e l a n i n by t h e c a d d i s f l i e s around t h e p a r a s i t e s . The r e a c t i o n s o f T r i c h o p t e r a t o t h e i r i n t e r n a l metazoan p a r a s i t e s have been d i s c u s s e d by v e r y few a u t h o r s , and u s u a l l y o n l y s u p e r f i c i a l l y . L i n s t o w (1897) r e p o r t e d a h o s t r e a c t i o n t o t h e m e t a c e r c a r i a e o f t h r e e s p e c i e s o f d i s t o m e s w i t h i n a number o f T r i c h o p t e r a l a r v a e . Thompson (1915) mentioned a more o r l e s s opaque c a p s u l e composed o f t h e 'amoebocytes' of t h e h o s t around p a r a s i t e s l y i n g 96 f r e e w i t h i n t h e hemocoele o f T r i c h o p t e r a and N e u r o p t e r a l a r v a e . Brown (1933) r e p o r t e d an a d v e n t i t i o u s o u t e r s p h e r i c a l c y s t " s e c r e t e d 1 by t h e T r i c h o p t e r a l a r v a e around m e t a c e r c a r i a e o f L e c i t h o d e n d r i u m c h i l o s t o m u m (Mehl.) i n t h e t h o r a c i c hemocoele o f Phryganea g r a n d i s L. Burns (1961) d e s c r i b e d an u n u s u a l r e a c t i o n i n Dioesmoecus l a r v a e t o t h e p e n e t r a t i n g c e r c a r i a e o f A c a n t h a t r i u m oregonense Macy. He r e p o r t e d t h a t t h e hypodermis a d j a c e n t t o t h e p e n e t r a t i n g c e r c a r i a e t h i c k e n e d and t u r n e d a r e d d i s h brown; i n some c a s e s t h e r e a c t i o n was so r a p i d t h a t c e r c a r i a e f a i l e d t o p e n e t r a t e . Thus from t h e l i t e r a t u r e t o d a t e i t i s a p parent t h a t T r i c h o p t e r a l a r v a e and pupae have t h e a b i l i t y t o produce some s o r t o f c a p s u l e around t h e i r d i g e n e a n p a r a s i t e s . I n a d d i t i o n , a f t e r some p e r i o d o f t ime most digenean p a r a s i t e s seem t o e l i c i t a m e l a n i z a t i o n r e s ponse from th e t r i c h o p t e r a n h o s t . However, d e t a i l s o f t h e r e a c t i o n s o f T r i c h o p t e r a t o i n t e r n a l f o r e i g n o b j e c t s a r e s k e t c h y , and as d e t a i l s o f e n c a p s u l a t i o n i n o t h e r i n s e c t s a r e more e x t e n s i v e , t h e c e l l u l a r e n c a p s u l a t i o n r e a c t i o n s o f P. a l a s c e n s i s w i l l be d i s c u s s e d i n terms o f t h e s e r e a c t i o n s i n o t h e r i n s e c t s . I have d i v i d e d t h e d i s c u s s i o n o f t h e e n c a p s u l a t i o n r e s p o n s e s o f P. a l a s c e n s i s i n t o t h r e e s e c t i o n s i n c l u d i n g ( i ) t i m e of r e a c t i o n , ( i i ) l a y e r s o f t h e c a p s u l e s , and ( i i i ) hemocytes t a k i n g p a r t i n t h e r e a c t i o n s . (i) Time of Reaction As the infected Trichoptera larvae, i n which the encapsulation reactions to metacercariae were examined, were taken from the f i e l d , the ages of the capsules were not known. Consequently reaction times could not be examined. However, reactions to Epon implants in Trichoptera were examined both seven and 35 days a f t e r implantation and can be discussed here. Recently the hemocyte response of a number of insects to non-living implants such as cellophane, a r a l d i t e , and tissue implants such as Schistocerca gregaria Forsk. nerve cord, have been examined u l t r a s t r u c t u r a l l y , eg. i n the thysanuran Thermobia domestica (Packard) (Francois, 1975), the lepidopterans Ephestia kuehneilla Z e l l . (Grimstone et a_l. , 1967) , and Bombyx sp. (Sato et aJL. , 1976), and i n the phasmid Clitumnus extradentatus (Schmit and R a t c l i f f e , 1978). A l l of these authors report that the capsule was complete (no more hemocytes were seen to adhere to the surface of the capsule) by the t h i r d to seventh day a f t e r implantation. In contrast, my work on the capsule i n P. alascensis showed that the capsule had begun to form by the seventh day and the majority of the capsular material was l a i d down sometime betwen the eighth and t h i r t y ^ - f i f t h day a f t e r implantation. This comparably slow reaction time i n P. alascensis may r e f l e c t several phenomena. F i r s t , i t i s possible that the larvae of P. alascensis were affected by the C0 2 anesthetic. Unfortunately Zachary 98 e t a l . (1976), B r e h e l e i n e t a l . (1975) , and Schmit and R a t c l i f f e (1977, 1978) do n o t mention t h e i r method o f a n e s t h e t i z a t i o n , i f i n d e e d t h e y d i d a n e s t h e t i z e t h e a n i m a l s . F r a n c o i s (19 75) r e p o r t e d a r a p i d hemocyte r e s p o n s e , b u t a n e s t h e t i z e d t h e t h y s a n u r a n f o r 10 min a t 10°C. S a l t (1960) however, r e p o r t s t h a t w i t h c a t e r p i l l a r s o f t h e tomato moth D i a t a r a x i a , a l t h o u g h t h e y l i e i n e r t f o r two t o t h r e e hours a f t e r a n e s t h e t i z a t i o n w i t h CC^ / t h e y c o m p l e t e l y and t h i c k l y c o v e r eggs o f t h e p a r a s i t e N e m e r i t u s o n l y f o u r h o u r s a f t e r t h e l a t t e r were i n j e c t e d i n t o the hemocoele. The s i t u a t i o n however, may d i f f e r i n T r i c h o p t e r a . Second, i t i s p o s s i b l e t h a t owing t o t h e c o l d maintenance t e m p e r a t u r e (10°C) f o r a q u a t i c i n s e c t l a r v a e such as I P . a l a s c e n s i s , t h e m e t a b o l i s m o f t h e i n s e c t i s much l o w e r t h a n t h a t o f organisms m a i n t a i n e d a t h i g h e r t e m p e r a t u r e s , and s u b s e q u e n t l y t h e r e a c t i o n t ime o f t h e hemocytes i s extended. Grimstone e t a l . (196 7) , F r a n c o i s (1975) , B r e h e l e i m e t a l . (1975) , Sato e t a_l. (1976), Zachary e t a l . (1976) and Schmit and R a t c l i f f e (1977, 1978) a l l m a i n t a i n e d t h e i r a n i m a l s a t 25-38°C. However, i t i s p o s s i b l e t h a t c a p s u l e f o r m a t i o n i n t h e o r d e r T r i c h o p t e r a i s a c t u a l l y a much s l o w e r p r o c e s s t h a n i n the o t h e r o r d e r s o f i n s e c t s examined t o d a t e . A l t h o u g h t h e members o f the o r d e r most c l o s e l y r e l a t e d t o t h e T r i c h o p t e r a , namely t h e L e p i d o p t e r a (Hennig, 1969), complete t h e response w i t h i n t h r e e d a y s , a l l i n d i v i d u a l s were m a i n t a i n e d a t 25°C. To make the r e s u l t s comparable i t would be n e c e s s a r y t o examine the e n c a p s u l a t i o n r e s ponse o f a d u l t T r i c h o p t e r a which u n l i k e t h e l a r v a e may be m a i n t a i n e d a t 25°C. A l t e r n a t i v e l y , i t would be i n t e r e s t i n g t o s t u d y t h e e n c a p s u l a t i o n r e s ponse o f l e p i d o p t e r a n l a r v a e m a i n t a i n e d a t 10°C. I n a d d i t i o n , i t might be i n s t r u c t i v e t o examine t h e e n c a p s u l a t i o n r e s ponse o f t h e i n d i v i d u a l s o f an o r d e r t h a t p o s s e s s e s a q u a t i c l a r v a e and i s f a i r l y c l o s e l y r e l a t e d t o the T r i c h o p t e r a , namely t h e N e u r o p t e r a ( M e g a l o p t e r a ) . ( i i ) L a y e r s o f t h e C a p s u l e s Owing t o t h e d i f f e r e n c e i n n a t u r e between n o n - l i v i n g i m p l a n t s such as Epon and n e r v e c o r d , and l i v i n g p a r a s i t e s , t h e r e a c t i o n s o f i n s e c t s t o n o n - l i v i n g and l i v i n g f o r e i g n o b j e c t s must be d i s c u s s e d s e p a r a t e l y . I n a s e p a r a t e s e c t i o n I w i l l compare the r e a c t i o n s o f t h e two. (a) N o n - L i v i n g I m p l a n t s Schmit and R a t c l i f f e (1978) d i v i d e d t h e r e a c t i o n o f C l i t u m n u s e x t r a d e n t a t u s t o a r a l d i t e fragments i n t o two phases (1) a r e c o g n i t i o n p r o c e s s i n which s p e c i f i c c e l l t y p e s l y s e on t h e s u r f a c e o f t h e i m p l a n t p r o d u c i n g a l o c a l i z e d c l o t f o r m a t i o n , and (2) the a c c u m u l a t i o n of c e l l s around t h e i m p l a n t . I n G a l l e r i a m e l l o n e l l a L. Schmit and R a t c l i f f e (1977) r e p o r t e d t h a t a f t e r 20 m i n u tes p l a s m a t o c y t e s adhere s o l e l y a t t h e s i t e s where g r a n u l e c e l l l y s i s has o c c u r r e d p r o d u c i n g clumps of c e l l s on t h e s u r f a c e o f t h e i m p l a n t . Irt P. a l a s c e n s i s the seven day 100 i m p l a n t showed a s p o t t y hemocyte response d e m o n s t r a t i n g clumps o f c e l l s s i m i l a r t o t h o s e r e p o r t e d by Schmit and R a t c l i f f e (1977). I n some a r e a s o f the i m p l a n t l o n e n u c l e i were v i s i b l e . I n o t h e r a r e a s o f t h e i m p l a n t , however, f u l l y i n t a c t c e l l s were seen l y i n g d i r e c t l y a g a i n s t t h e s u r f a c e o f the i m p l a n t . I t i s p o s s i b l e t h a t some c e l l t y p e had l y s e d a t t h e a r e a s o f t h e hemocyte clumps, however, w i t h o u t u l t r a s t r u c t u r a l s t u d i e s i t i s d i f f i c u l t t o d e t e r m i n e whether o r n o t t h e c e l l s d i r e c t l y a d j a c e n t t o t h e i m p l a n t were u n d e r g o i n g l y s i s o r n o t . Mature c a p s u l e s around i m p l a n t s have been r e p o r t e d t o be composed o f two l a y e r s ( B r e h e l e i m e t al.,1975) t h r e e l a y e r s (Grimstone e t a l . , 1967; Schmit and R a t c l i f f e , 1977, 1978) and f o u r l a y e r s ( F r a n c o i s , 1975). I n most ca s e s a u t h o r s agree t h a t t h e r e i s an i n n e r l a y e r o f o r g a n i z e d f l a t t e n e d c e l l s a b u t t i n g an o u t e r l a y e r o f u n o r g a n i z e d , n o t so f l a t t e n e d c e l l s , sometimes c o n t a i n i n g c e l l s r e s e m b l i n g f r e e - c i r c u l a t i n g hemocytes. Schmit and R a t c l i f f e (1977, 1978), Grimstone e t a l . (1967) and F r a n c o i s (1975) r e p o r t e d a l a y e r between t h e f l a t t e n e d l a y e r o f c e l l s and t h e i m p l a n t . Schmit and R a t c l i f f e (1977) r e p o r t e d t h i s l a y e r t o be composed o f l y s e d g r a n u l a r c e l l s i n G. m e l l o n e l l a and l y s i n g c y s t o c y t e s and some g r a n u l a r c e l l s i n t h e phasmid C. e x t r a d e n t a t u s . F r a n c o i s (1975) d e s c r i b e d i t as a l a y e r o f c e l l s i n t h e p r o c e s s o f n e c r o s i s i n Thermobia  d d m e s t i c a . Grimstone e t a_l. (196 7) r e p o r t e d t h e i n n e r l a y e r t o be composed o f u n f l a t t e n e d c e l l s w i t h a l a r g e 101 number o f dense i n c l u s i o n s i n E. k u e h n i e l l a . In a d d i t i o n , F r a n c o i s (19 75) d e s c r i b e d one l a y e r even more p r o x i m a l t o t h e i m p l a n t t h a n t h e n e c r o s i n g l a y e r as a t h i n l i m i t i n g l a y e r , p o s s i b l y m e l a n i n . The c a p s u l e o f P. a l a s c e n s i s was t y p i c a l o f t h e two l a y e r e d c a p s u l e r e p o r t e d above. Two d i s t i n c t l a y e r s were seen even i n l i g h t m i c r o s c o p e t h i c k s e c t i o n s . These c o n s t i t u t e d an i n n e r l a y e r o f f l a t t e n e d compact c e l l s , and an o u t e r l a y e r o f l e s s o r g a n i z e d l e s s f l a t t e n e d c e l l s . A t b o t h t h e seven and 35 day s t a g e s , no hemocytes c o r r e s p o n d i n g t o round f r e e - c i r c u l a t i n g hemocytes were t a k i n g p a r t i n t h e o u t e r l a y e r o f t h e c a p s u l e and because o f t h e d i s r u p t e d f a t body t i s s u e l o c a t e d on t h e s u r f a c e o f t h e c a p s u l e i t i s d i f f i c u l t t o d e t e r m i n e i f t h e r e i s e v i d e n c e o f l y s e d hemocyte m a t e r i a l beneath th e l a y e r o f f l a t t e n e d hemocytes. (b) L i v i n g P a r a s i t e s W i t t i g (1962) and S a l t (1970) have suggested t h a t t h r e e t y p e s o f e n c a p s u l a t i o n r e a c t i o n s i n v o l v i n g hemocytes e x i s t i n r e s p o n s e t o p a r a s i t e s i n i n s e c t s . F i r s t l y , t h e r e a r e t h o s e i n which d i s c o n t i n u o u s groups o f hemocytes s p a r s e l y c o v e r p a r a s i t e s i n c l u s t e r s o r p l u g s around t h e i r o r a l and/or a n a l r e g i o n s . Such r e a c t i o n s have been d e s c r i b e d by P o i n a r and Leutenegger (1971), Vey and Gotz (1975) and u s u a l l y f o l l o w t h e p r o d u c t i o n o f m e l a n i n ; hemocytes may o n l y be p r e s e n t f o r a s h o r t p e r i o d o f t i m e . S a l t (1970) c a l l e d t h i s r e a c t i o n 'sheath c a p s u l e f o r m a t i o n ' . 102 The second type of encapsulation response i s one i n which the parasite i s surrounded by i r r e g u l a r l y shaped clumps of loosely packed hemocytes. The.surface of t h i s capsule i s often sticky and i r r e g u l a r . This type of reaction was found by Salt (1956) around eggs of the ichneumonid Venturia canescens (Grav.) (= Nemeritis canesens)in the st i c k insect Carausius morosus Br. The l a s t type of capsule i s one which Salt (1970) termed 'ordinary c e l l u l a r encapsulation' as i t appears to be the most frequent type of capsule. These capsules are characterized by smooth surfaces and are composed of densely packed hemocytes. According to Salt (1970) these capsules are composed of two d i f f e r e n t layers; (1) an inner layer of hemocytes, the c e l l walls of which are. i n v i s i b l e , giving the layer the appearance of a continuous mass of cytoplasm with nuclei v i s i b l e at i n t e r v a l s , and (2) an outer layer of uncoalesced c e l l s , some round i n shape and some flattened. Salt (1956) found t h i s type of capsule around eggs and larvae of the ichneumonid V. canescens i n c a t e r p i l l a r s of the genus Diatraxia. The reaction produced by P. alascensis to the metacercariae of B. mediovitellata corresponds most cl o s e l y to the 'ordinary encapsulation reaction' type . of Salt (1970). The capsule i n P. alascensis has a f a i r l y smooth appearance and at least two layers are v i s i b l e ; an inner layer of densely packed c e l l s distinguishable only by the presence of chromatin granules i n the n u c l e i , 103 and an o u t e r l a y e r o f l o o s e l y packed c e l l s . Two l a y e r s c o r r e s p o n d i n g t o t h e ' o r d i n a r y c e l l u l a r e n c a p s u l a t i o n ' r e a c t i o n have a l s o been r e p o r t e d by s e v e r a l a u t h o r s (Nappi and Streams, 196 9; Nappi and S t o f f a n o l o , 1971; G i b s o n and B e r b e r e t , 1974), however, t h e d e t a i l s g i v e n of each l a y e r a r e s p a r s e . F o r t u n a t e l y , P o i n a r e t a l . (1968) and M isko (1972) have examined t h e u l t r a s t r u c t u r e o f ' o r d i n a r y c e l l u l a r e n c a p s u l a t i o n ' i n b e e t l e s and c o c k r o a c h e s r e s p e c t i v e l y , and c o n s e q u e n t l y our knowledge o f t h e n a t u r e o f each l a y e r i n t h e s e i n s e c t s i s more c o m p l e t e . P o i n a r e t a_l (1968) d e s c r i b e d t h e u l t r a s t r u c t u r e o f t h e 72 hour c a p s u l e s formed by t h e b e e t l e s D i a b r o t i c a  b l a t e a t a Lec. and D. u n d e c i n o p u n c t a t a (Mann) around t h e t h e nematode F i l i p j e v i v e r m i s l e i p s a n d r a as h a v i n g f o u r major r e g i o n s . These i n c l u d e d : (1) an i n n e r r e g i o n t h a t was n o n c e l l u l a r and p a r t i a l l y m e l a n i z e d , (2) a s i n g l e i r r e g u l a r l a y e r o f s p h e r i c a l n e c r o t i c c e l l s i n w h i c h n u c l e i and fragments o f c y t o p l a s m were v i s i b l e , (3) t h r e e t o f o u r l a y e r s o f d e n s e l y packed c e l l s which were e x t r e m e l y f l a t t e n e d , and whose c y t o p l a s m i c b o u n d a r i e s were d i f f i c u l t t o d i s t i n g u i s h , and (4) an o u t e r zone of l o o s e l y a t t a c h e d c e l l s r e s e m b l i n g f r e e hemocytes. The d e s c r i p t i o n o f t h e t h i r d l a y e r o f t h e c a p s u l e d e s c r i b e d by P o i n a r e t a l . (1968) seems t o most c l o s e l y resemble S a l t ' s (1970) d e s c r i p t i o n o f t h e i n n e r l a y e r o f such c a p s u l e s . However, i n a d d i t i o n t h e a u t h o r s have 104 d e s c r i b e d two l a y e r s more p r o x i m a l t o t h e p a r a s i t e t h a n t h e c e l l u l a r l a y e r . I t i s p o s s i b l e t h a t d e t a i l s o f t h e s e l a y e r s a r e o n l y v i s i b l e i n e l e c t r o n m i c r o g r a p h s , and t h a t t h e s e i n n e r l a y e r s were p r e s e n t , b u t were n o t seen by S a l t (1970) as no u l t r a s t r u c t u r a l i n v e s t i g a t i o n was u n d e r t a k e n . The f o u r t h l a y e r d e s c r i b e d by P o i n a r e t a l . (1968) i s somewhat s i m i l a r t o t h e d e s c r i p t i o n o f t h e o u t e r l a y e r by S a l t (1970), a l t h o u g h no f l a t t e n e d hemocytes w i t h c y t o p l a s m i c b o u n d a r i e s were v i s i b l e as r e p o r t e d by P o i n a r e t a l . (1968). M i s k o (1972), a c c o r d i n g t o Nappi (1974), s t u d i e d t h e u l t r a s t r u c t u r e o f t h e c a p s u l e formed by t h e c o c k r o a c h P e r i p l a n e t a americana L. around th e nematode C a e n o r h a b d i t e s b r i g g s a e . He found t h a t t h e hemocytes a t t h e s u r f a c e o f t h e nematode were more e l e c t r o n dense, and c o n t a i n e d more membrane bound i n c l u s i o n s t h a n t h o s e hemocytes i n t h e o u t e r r e g i o n s ; as e n c a p s u l a t i o n p r o g r e s s e d t h e s e membrane bound v e s i c l e s became pigmented and f u s e d t o form masses o f pigment which were s u b s e q u e n t l y d e p o s i t e d on t h e p a r a s i t e . The d e s c r i p t i o n o f t h i s l a y e r by M i s k o (1972) seems c o n s i s t e n t w i t h t h e d e s c r i p t i o n o f t h e f i r s t p a r t i a l l y m e l a n i z e d l a y e r o f t h e c a p s u l e s by P o i n a r e t a l . (1968). I n a d d i t i o n M isko (19 72) s u g g e s t e d t h a t d u r i n g t h e e a r l i e r s t a g e s o f c a p s u l e f o r m a t i o n t h e e x t r a c e l l u l a r spaces a t t h e s u r f a c e o f t h e p a r a s i t e c o n t a i n e d amorphous m a t e r i a l i n w h i c h were found f r e e n u c l e i and c y t o p l a s m i c fragments o f d i s r u p t e d hemocytes. T h i s l a y e r appears t o be i d e n t i c a l 105 t o t h e l a y e r o f n e c r o t i c c e l l s ( l a y e r 2) r e p o r t e d by P o i n a r e t a l . (1968). I n b o t h c a s e s n u c l e i and c y t o p l a s m i c fragments were v i s i b l e . From t h e m i c r o g r a p h s o f Misko (1972) ( i n N a p p i , 19 7 4 ) , t h e n e x t l a y e r of t h e c a p s u l e appears t o be composed o f e x t r e m e l y f l a t t e n e d hemocytes whose n u c l e i and c y t o p l a s m i c b o u n d a r i e s a r e v i s i b l e . I n the m i c r o g r a p h s no hemocytes r e s e m b l i n g f r e e - c i r c u l a t i n g hemocytes a r e v i s i b l e . However, i t i s p o s s i b l e t h a t they were mer e l y not p r e s e n t i n t h i s a r e a o f t h e m i c r o g r a p h , as t h e a r e a c o v e r e d i s s m a l l . T h i s l a y e r c o r r e s p o n d s more c l o s e l y t o t h e o u t e r l a y e r d e s c r i b e d by S a l t (1970) t h a n does t h a t o f t h e o u t e r l a y e r d e s c r i b e d by P o i n a r e t a l . (196 8 ) . I n P. a l a s c e n s i s t h e i n n e r l a y e r o f t h e n o n - m elanized c a p s u l e s t a i n e d a d a r k b l u e w i t h p o l y c h r o m a t i c methylene b l u e and c o n t a i n e d n u c l e i , and l a r g e v a c u o l a r s t r u c t u r e s which s t a i n e d w i t h a p i n k c o l o u r . T h i s l a y e r i s s i m i l a r t o t h a t d e s c r i b e d i n S a l t (1970). However, w i t h o u t u l t r a s t r u c t u r a l work i t i s d i f f i c u l t t o d e t e r m i n e whether o r not the c e l l s i n t h i s a r e a were n e c r o t i c , o r i f c e l l u l a r d e b r i s was p r e s e n t as d e s c r i b e d i n t h e c a p s u l e s o f P o i n a r e t a l . (1968) and Misko (1972). The o u t e r l a y e r o f t h e c a p s u l e was composed of b o t h f l a t t e n e d hemocytes and c e l l s r e s e m b l i n g f r e e - c i r c u l a t i n g hemocytes. T h e r e f o r e , t h e s t r u c t u r e i s c o n s i s t e n t w i t h t h a t d e s c r i b e d by S a l t (1970), and i s i n t e r m e d i a t e between t h o s e d e s c r i b e d by P o i n a r e t a l . (1968) and M i s k o (1972). 106 I n the m e l a n i z e d c a p s u l e s s t u d i e d i n P. a l a s c e n s i s i t i s o n l y p o s s i b l e t o say t h a t t h e m e l a n i n was d e p o s i t e d on t h e i n n e r l a y e r o f t h e c a p s u l e , d i r e c t l y i n c o n t a c t w i t h t h e p a r a s i t e . A g a i n , w i t h o u t u l t r a s t r u c t u r a l work i t i s i m p o s s i b l e t o det e r m i n e whether o r n o t e l e c t r o n dense v e s i c l e s as d e s c r i b e d by Misko (1972) were p r e s e n t i n t h e s e c e l l s . j (c) Comparison o f t h e R e a c t i o n s t o L i v i n g and N o n - L i v i n g F o r e i g n O b j e c t s The c e l l u l a r e n c a p s u l a t i o n r e a c t i o n o f l a r v a e o f P. - a l a s c e n s i s t o Epon i m p l a n t s and t o m e t a c e r c a r i a e o f B. m e d i o v i t e l l a t a were s i m i l a r i n t h a t two d i s t i n c t l a y e r s t o b o t h c a p s u l e s were v i s i b l e . These l a y e r s i n c l u d e d an i n n e r l a y e r o f f l a t t e n e d hemocytes w h i c h were d e n s e l y p a cked, and an o u t e r l a y e r o f hemocytes wh i c h were n o t as f l a t t e n e d and n o t as d e n s e l y packed. Both c a p s u l e s c o r r e s p o n d e d t o t h e " o r d i n a r y c e l l u l a r e n c a p s u l a t i o n ' r e a c t i o n o f S a l t (1970). The r e a c t i o n s d i f f e r e d i n t h a t an homogeneous su b s t a n c e which s t a i n e d p i n k w i t h p o l y c h r o m a t i c methylene b l u e was v i s i b l e towards t h e i n s i d e o f the c a p s u l e around m e t a c e r c a r i a e , b u t was n o t v i s i b l e around Epon i m p l a n t s . W h i l e c e l l s i n t h i s i n n e r l a y e r around t h e p a r a s i t e appeared f l a t t e n e d , t h e i r c e l l b o u n d a r i e s were n o t as c l e a r as t h o s e i n t h e i n n e r l a y e r o f t h e 35 day c a p s u l e around t h e Epon i m p l a n t . I n a d d i t i o n , some o f t h e c a p s u l e s around m e t a c e r c a r i a e 107 were seen t o m e l a n i z e ; no m e l a n i n was seen around Epon i m p l a n t s . I t i s p o s s i b l e , however, t h a t m e l a n i n i s n o t d e p o s i t e d u n t i l t he r e a c t i o n i s s e v e r a l months o l d . I n t h i s c a s e i t i s p o s s i b l e t h a t t h e i m p l a n t s had n o t been i n t h e l a r v a e l o n g enough f o r m e l a n i n t o be d e p o s i t e d . ( i i i ) Hemocytes I n v o l v e d i n t h e R e a c t i o n A l t h o u g h I have proposed t h e p r o d u c t i o n o f a Summary F a t e Map o f t h e hemocytes as a good method o f d e t e c t i n g t h e a c t u a l o r d e r among hemocytes, no o t h e r a u t h o r s have d e a l t w i t h hemocytes i n t h i s manner. Hence, any c o m p a r i s o n o f t h e i r work t o mine i n terms o f a Summary F a t e Map would be c o n f u s i n g . T h e r e f o r e i n t h i s s e c t i o n on hemocytes I have a t t e m p t e d t o d i s c u s s my work i n terms o f th e ambiguous hemocyte t e r m i n o l o g y and t y p e s p r e s e n t l y u sed i n t h e l i t e r a t u r e . The d e f i n i t i o n s o f each hemocyte t y p e as p r e s e n t e d by Jones (1977) have been used as r e f e r e n c e . I s h a l l t h e n d i s c u s s t h e problems w i t h t h e p r e s e n t hemocyte c l a s s i f i c a t i o n schemes, and attempt t o demonstrate how t h e p r o d u c t i o n o f a Summary F a t e Map would reduce some o f t h e i n c o n s i s t e n c i e s o f c l a s s i f i c a t i o n . L a s t l y , I s h a l l a t t e m p t t o p l a c e t h e c e l l t y p e s seen t a k i n g p a r t i n each e n c a p s u l a t i o n r e a c t i o n , on t h e Summary F a t e Map. (a) N o n - l i v i n g I m p l a n t s The e n c a p s u l a t i o n r e sponse o f an i n s e c t t o a non-l i v i n g i m p l a n t seems t o i n v o l v e a number o f d i f f e r e n t hemocyte t y p e s . I n a d d i t i o n , t h e t y p e s o f hemocytes t a k i n g 108 p a r t i n t h e r e a c t i o n s seem t o v a r y between i n s e c t s . I n e x a m i n i n g t h e r e a c t i o n s o f t h e phasmid C l i t u m n u s  e x t r a d e n t a t u s t o i m p l a n t e d a r a l d i t e f r a g m e n t s , Schmit and R a t c l i f f e (1978) d e s c r i b e d c y s t o c y t e s , g r a n u l a r c e l l s , p l a s m a t o c y t e s , and s p h e r u l e c e l l s t a k i n g p a r t i n t h e c a p s u l e f o r m a t i o n a f t e r one h our. A f t e r f o u r hours o n l y g r a n u l a r c e l l s and p l a s m a t o c y t e s were found i n the c a p s u l e and t h e s e were d i f f i c u l t t o d i s t i n g u i s h . I n c o n t r a s t , Schmit and R a t c l i f f e (1977) su g g e s t e d t h a t a f t e r t h e f o r m a t i o n o f t h e i n n e r l a y e r o f l y s e d g r a n u l a r c e l l s o n l y p l a s m a t o c y t e s t a k e p a r t i n t h e c a p s u l e f o r m a t i o n around i m p l a n t s i n G a l l e r i a m e l l o n e l l a . Grimstone e t a_l. (196 7) d i d n o t name th e c e l l s t a k i n g p a r t i n t h e r e a c t i o n o f E p h e s t i a k u e h n i e l l a t o a r a l d i t e i m p l a n t s ; however, th e m i c r o g r a p h s show c e l l s c o r r e s p o n d i n g t o t h e g r a n u l a r c e l l s o f Schmit and R a t c l i f f e (1977). F r a n c o i s (1975) mentioned t h r e e . t y p e s o f hemocytes t a k i n g p a r t i n t h e c a p s u l e i n a t h y s a n u r a n (Thermobia  d o m e s t i c a ) . He s u g g e s t e d t h a t a f t e r f o u r hours c o a g u l o c y t e s , p r e dominate. A f t e r e i g h t hours th e hemocytes t a k i n g p a r t i n t h e c a p s u l e f o r m a t i o n a r e d i v i d e d as f o l l o w s : 6 0% c o a g u l o c y t e s , 30% p l a s m a t o c y t e s , and 10% g r a n u l o c y t e s . B r e h e l i e n e t a l . (1975) r e p o r t e d t h a t t h e m a j o r i t y o f t h e c e l l s f o r m i n g l a y e r s o f t h e c a p s u l e a r e g r a n u l a r hemocytes, though p l a s m a t o c y t e s a r e o c c a s i o n a l l y embedded i n the t i s s u e , and c o a g u l o c y t e s a r e o c c a s i o n a l l y o b s e r v e d i n t h e o u t e r c a p s u l e . A l t h o u g h most a u t h o r s agree t h a t g r a n u l o c y t e s , 109 plasmatocytes, and the various intermediates between these two c e l l types take part i n formation of the bulk of the capsule, the difference between these hemocyte types i s not comparable between authors. For example, in a paper on the ultrastructure of the hemocytes of Calliphora  erythrocephala (Meig),Zachary and Hoffman (1973) demonstrated a c e l l type which they term plasmatocyte I I I ; I f i n d t h i s c e l l type indistinguishable from the granular c e l l of Schmit and R a t c l i f f e (1978). Even within t h e i r own work Schmit and R a t c l i f f e (1977) did not define the terms 'granulocyte' and 'plasmatocyte' well enough for other authors to distinguish between the two. As u l t r a s t r u c t u r a l c r i t e r i a such as number of packets of microtubules, number of mitochondria and presence and number of dense inclusions were used by Schmit and R a t c l i f f e (1978) and Francois (1975) to distinguish the various hemocyte types, i t i s d i f f i c u l t for me to correlate my work with that of these authors. Nevertheless, i t appears that the usual c e l l type taking part i n the formation of a capsule around i n e r t implants i n P. alascensis i s a large c e l l with a f a i r l y large nucleus, and as i t appears that granules cannot be detected with methylene blue s t a i n , i t can be said that the c e l l s taking part are either plasmatocytes, granulocytes or intermediates between the two. Even in smears of f r e e - c i r c u l a t i n g hemocytes no c e l l s corresponding to the cystocytes or spherule c e l l s of Schmit and R a t c l i f f e (1978) were seen. 110 (b) Hemocytes I n v o l v e d i n t h e R e a c t i o n t o P a r a s i t e s W i t t i g (1962) s u g g e s t e d t h a t p l a s m a t o c y t e s were the p r i m a r y hemocyte t y p e i n v o l v e d i n t h e e n c a p s u l a t i o n o f p a r a s i t e s . T h i s s u g g e s t i o n has been s u p p o r t e d by t h e work o f P o i n a r e t al. (1968) and Na p p i and Streams (1969) who r e p o r t e d t h a t p l a s m a t o c y t e s e n l a r g e d and d i f f e r e n t i a t e d i n t o e x t r e m e l y f l a t d i s c - s h a p e d c e l l s w h i c h t h e y termed 1 l a m e l l o c y t e s ' . G i b s o n and B e r b e r e t (1974) d i d n o t name th e hemocytes i n v o l v e d i n t h e r e a c t i o n o f t h e a l f a l f a w e e v i l Hypera p o s t i c a ( G y l l e n h a l ) t o eggs o f t h e p a r a s i t o i d B a t h y p l e c t e s c u r c u l i o n i s (Thompson) and i t i s d i f f i c u l t t o d i s t i n g u i s h hemocyte t y p e s from t h e i r p h o t o g r a p h s . Lo e t a_l. (1975) p r e s e n t e d S.E.M. photographs o f t h e hemocyte r e a c t i o n t o an o p e c o e l i d d i g e n e a n e n c y s t i n g i n m a y f l y n a i a d s . A l t h o u g h t h e y d i d not name t h e hemocyte t y p e s t a k i n g p a r t i n t h e e n c a p s u l a i o n r e a c t i o n , t h e i r p h otographs show f l a t t e n e d hemocytes, some w i t h f i l a m e n t o u s pseudopodia e x t e n d i n g t o n e i g h b o u r i n g c e l l s . M i s k o (1972) ( i n N a p p i , 1974) p r e s e n t e d m i c r o g r a p h s o f c a p s u l e s i n t h e c o c k r o a c h P. americana around t h e nematode C a e n o r h a b d i t e s b r i g g s a e , b o t h one hour and one week o l d . The hemocytes f o r m i n g c a p s u l e s around t h e one hour o l d i n f e c t i o n were f u l l o f e l e c t r o n dense i n c l u s i o n s and resemble t h e g r a n u l o c y t e s found around a n o n - l i v i n g i m p l a n t by Schmit and R a t c l i f f e (1978). I n t h e one week c a p s u l e , t h e e l e c t r o n dense i n c l u s i o n s appear t o have t o t a l l y v a n i s h e d from t h e e l o n g a t e d c e l l s g i v i n g them t h e appearance o f f l a t t e n e d p l a s m a t o c y t e s . The l a r g e hemocytes p a r t i c i p a t i n g i n t h e c a p s u l e around t h e m e t a c e r c a r i a e o f P. a l a s c e n s i s s u g g e s t s t h a t t h e y c o r r e s p o n d e d t o t h e p l a s m a t o c y t e s o f Jones (1977). Owing t o t h e d i f f i c u l t i e s o f f i x i n g t h e m e t a c e r c a r i a l c y s t s , I was un a b l e t o d i s t i n g u i s h hemocyte t y p e s from t h e e l e c t r o n m i c r o g r a p h s o f t h e c a p s u l e around m e t a c e r c a r i a e o f B. m e d i o v i t e l l a t a . However, t h e e l o n g a t e d n a t u r e o f the c e l l s seen t a k i n g p a r t i n t h e r e a c t i o n t o t h e e s t a b l i s h e d I m e t a c e r c a r i a e i n l i g h t m i c r o s c o p e s e c t i o n s ( l o n g e r t h a n t y p e #5 i n F i g u r e 13) i s s u g g e s t i v e o f a p l a s m a t o c y t e - l i k e c e l l . That t h e c e l l s r e s p o n d i n g t o t h e young m e t a c e r c a r i a e were n o t as extended as t h e c e l l s r e s p o n d i n g t o t h e e s t a b l i s h e d m e t a c e r c a r i a e may r e f l e c t two f a c t s : (1) the hemocytes ( p l a s m a t o c y t e s ? ) which respond t o t h e p a r a s i t e , f l a t t e n as t h e e n c a p s u l a t i o n p r o c e e d s , o r (2) t h e d i f f e r e n c e i n f i x a t i o n between t h e two c a p s u l e s a f f e c t s t h e d e t a i l s o f the hemocytes p r e s e r v e d . The young m e t a c e r a r i a l c a p s u l e s were f i x e d w i t h B o u i n ' s f i x a t i v e whereas t h e e s t a b l i s h e d m e t a c e r c a r i a e were f i x e d w i t h g l u t a r a l d e h y d e ; t h e g l u t a r a l d e h y d e i s a much b e t t e r f i x a t i v e as i t p r e s e r v e s t h e d e t a i l s and e x t e n s i o n s o f c e l l s t o a g r e a t e r e x t e n t t h a n does B o u i n ' s . (c) Summary F a t e Map as a S o l u t i o n V a r i o u s a u t h o r s have d i s t i n g u i s h e d from one (Moran, 1971) t o f o u r t e e n (Yeager, 1945) t y p e s o f i s e c t hemocytes from v a r i o u s o r d e r s o f i n s e c t s . T h i s d i v e r s i t y o f o p i n i o n stems from t h e f a c t t h a t r e s e a r c h e r s i n t h e p a s t , have been u n a b l e t o dete r m i n e i f hemocyte t y p e s a r e m o r p h o l o g i c a l r e p r e s e n t a t i o n s o f phases i n t h e l i f e o f a b l o o d c e l l , each phase w i t h a d i f f e r e n t f u n c t i o n ( s i n g l e c e l l t h e o r y ) , o r s e v e r a l d i s t i n c t t y p e s o f c e l l s w hich a r e immutable and s e r v e d i f f e r e n t r o l e s i n i n s e c t p h y s i o l o g y ( m u l t i p l e c e l l t h e o r y ) . A c c o r d i n g t o A r n o l d (1979), t h r e e main s o u r c e s o f t h i s c o n t r o v e r s y e x i s t : (1) t h e inadequacy o f our d e f i n i t i o n o f hemocyte t y p e - t h e r e a r e no c y t o l o g i c a l c h a r a c t e r s t h a t d i s t i n g u i s h each w i t h c e r t a i n t y i n a l l i n s e c t s - a t l e a s t n o t t o t h e s a t i s f a c t i o n o f d i f f e r e n t a u t h o r s , (2) t h e d i f f e r e n c e s i n t h e hemocyte t y p e s a r e not homologous i n d i f f e r e n t g r o u p s , and (3) t h e tendency o f hemocytes t o r e a c t d i f f e r e n t l y t o changes i n t h e i r e n v ironment, and t h u s t o d i f f e r e n t t e c h n i q u e s used t o p r e p a r e them. What i s l a c k i n g i n t h e hemocyte work, t h e n , i s an o b j e c t i v e method o f r e c o g n i z i n g any c o n s i s t e n t g e n e r a l p a t t e r n o f r e l a t i o n s h i p among t h e v a r i o u s hemocytes and s u b s e q u e n t l y p r o d u c i n g a g e n e r a l r e f e r e n c e system. I n t h i s s t u d y I have attem p t e d t o produce such a r e f e r e n c e system o f hemocyte c l a s s i f i c a t i o n u s i n g q u a n t i t a t i v e parsimony a n a l y s i s ( F a r r i s , 1970) o f m o r p h o l o g i c a l d a t a f o r a number o f hemocytes from P. a l a s c e n s i s . T h i s method o f a n a l y s i s was o r i g i n a l l y d e v e l o p e d as an a i d t o r e c o n s t r u c t i n g p h y l o g e n i e s ( c l a d i s t i c s ) , b u t has been g e n e r a l i z e d as t h e method f o r p r o d u c i n g t h e most e f f i c i e n t , 113 l e a s t ambiguous summations o f any d a t a s e t ( F a r r i s , 1979). I n o r d e r t o ensure t h a t t h e hemocytes were s t r i c t l y comparable I examined o n l y hemocytes h e a t - f i x e d and t h e n g i e m s a - s t a i n e d . The c l a d i s t i c a n a l y s i s l e d t o t h e p r o d u c t i o n o f a c o n s i s t e n t p a t t e r n o f r e l a t i o n s h i p among t h e hemocytes examined. T h i s p a t t e r n was h e i r a r c h i c a l . A t t h e base o f th e p a t t e r n was a s m a l l c e l l w i t h a s m a l l n u c l e u s / c y t o p l a s m r a t i o , and a dense n u c l e u s , t h i s c e l l c o r r e s p o n d s t o t h e prohemocyte o r suggested p r e c u r s o r y c e l l o f Jones (19 77) and A r n o l d (19 7 9 ) . Because t h e p a t t e r n produced i s a h e i r a r c h i c a l one, i t f o l l o w s t h a t t h e f u r t h e r o u t on t h e b r a n c h i n g d i a g r a m you go, t h e more t r a i t s t h e hemocytes a t t h i s p o i n t s h a r e . Each t r a i t r e p r e s e n t s a p r o g r e s s i v e t r a n s f o r m a t i o n o f a c h a r a c t e r from t h e prohemocyte s t a t e o f t h a t c h a r a c t e r . Many o f t h e v a r i o u s t r a n s f o r m a t i o n s a r e r e p r e s e n t e d i n t h e o b s e r v e d c e l l s . T h e r e f o r e , because each o f t h e o b s e r v e d c e l l s r e p r e s e n t s some s u b s e t o f t h o s e t r a n s f o r m a t i o n s , t h e n th e more t r a n s f o r m a t i o n s two c e l l s s h a re i n terms o f t h e cla d o g r a m , t h e more l i k e l y i t i s t h a t t h e y a r e d e r i v e d from a common s e t o f p r e c u r s o r c e l l s . I n o t h e r words t h e cla d o g r a m may be viewed as a Summary F a t e Map i n d i c a t i n g b o t h t h e o n t o g e n e t i c p o t e n t i a l a v a i l a b l e t o a p a r t i c u l a r c e l l and t h e o n t o g e n e t i c c o n s t r a i n t s p l a c e d on a c e l l by p r o g r e s s i v e s p e c i a l i z a t i o n . F o r example i n F i g u r e 27 c e l l #2 c o u l d p o t e n t i a l l y become #16, 22, «(ii), 15, 11, 17, o r any o f t h e i r i n t e r m e d i a t e s . I t c o u l d n o t however, become c e l l #3, f o r example. The i d e a t h a t hemocytes pass 114 FIGURE 27 Summary F a t e Map ( F i g u r e 1 3 ) . L i n e a g e s of hemocyte t y p e s i m p l i c a t e d i n p a r t i c i p a t i o n i n e n c a p s u l a t i o n o f n o n - l i v i n g and l i v i n g o b j e c t s i n T r i c h o p t e r a a r e c i r c l e d . 1 1 5 116 from one d e v e l o p m e n t a l s t a g e t o a n o t h e r i s s u p p o r t e d by t h e work o f many a u t h o r s ( C r o s s l e y , 1964; Hoffman, 1966; Hoffman e t a l . , 1968; Moran, 1971; P r i c e and R a t c l i f f e , 1974). A u t h o r s up t o now have been t r y i n g t o put each c e l l i n t o a 'type' c l a s s when i n f a c t each o b s e r v e d c e l l s i m p l y r e p r e s e n t s t h e p r o g r e s s i o n o f t h e ontogeny o f an i n d i v i d u a l c e l l t o a p a r t i c u l a r p o i n t . The e n t i r e ontogeny f o r c e l l #24 i s 19-* • ( i ) -*• • ( i i i ) -*• 12-* 1-* 24 . But f o r c e l l #20 t h e e n t i r e ontogeny i s 19-* • ( i ) -* • ( i i i ) -* 8-+ 20 . I f we want t o u n d e r s t a n d t h e common d e v e l o p m e n t a l elements we would use t h a t d e s i g n a t i o n w h i c h would show us t h a t 19-* 7-*» ( i ) H> • ( i i i ) i s common t o th e ontogeny o f b o t h #24 and 20. However i n terms o f u n d e r s t a n d i n g t h e p r o g r e s s i v e d i f f e r e n t i a t i o n o f #24 and 20 i t i s more i n f o r m a t i v e t o c o n s i d e r t h e r e s t r i c t e d l i n e a g e s 12-*- 1-* 24 and 8-+ 20 r e s p e c t i v e l y . Thus t h e Summary F a t e Map i s u s e f u l f o r e x a m i n i n g b o t h s i m i l a r i t i e s and d i f f e r e n c e s However, t h e r e a r e some problems i n c l a s s i f y i n g hemocytes, f o r example, o b s e r v i n g a c e l l w h i c h c o r r e s p o n d s t o «(iii) i n F i g u r e 27 does n o t t e l l us i f t h a t c e l l would have d e v e l o p e d i n t o a c e l l l i k e #12 o r 8. T h e r e f o r e i f we r e c o g n i z e #24 and 20 as r e p r e s e n t i n g d i f f e r e n t s p e c i a l i z e d c e l l l i n e a g e s and t h e r e f o r e r e q u i r i n g d i f f e r e n t i a l c l a s s i f i c a t i o n , we would have t o p l a c e c e l l s c o n f o r m i n g t o # »(iii) i n t h e c a t e g o r y c o n t a i n i n g #24 and i n t h e c a t e g o r y c o n t a i n i n g #20. I n any c l a s s i f i c a t i o n , t h e r e f o r e , t h e o n l y c e l l s w hich can be unambiguously c l a s s i f i e d a r e the n o n - s u b d i v i d i n g l i n e a g e s . I n o t h e r words i n F i g u r e 27, c e l l s #19 , 6 , 12-+ 1-* 24 , 23 , 18 , 5-*-14 , 3, 4 , 20, 16-* 22, 15-*11, 17, 13, 9 and 21 c o u l d be c l a s s i f i e d . The o t h e r c e l l s i n c l u d i n g 2, 7, 8, 10, ©(i), • ( i i ) , • ( i i i ) , ©(iv) • ( v ) , and ©(vi), would have t o be i n c l u d e d i n a t l e a s t two d i f f e r e n t c a t e g o r i e s i n t h e c l a s s i f i c a t i o n . I n t h e case o f t h e s e c e l l s t h e n , the d e t e r m i n a t i o n o f t h e p o s i t i o n o f t h e s e c e l l s i n t h e Summary F a t e Map, as t o t i p o t e n t c e l l s , r a t h e r t h a n c l a s s i f y i n g them as c e l l ' t y p e s ' would l e a d t o much l e s s c o n f u s i o n t h a n a t t e m p t i n g t o c l a s s i f y them as c e l l ' t y p e s ' . A l t h o u g h n o n - s u b d i v i d i n g l i n e a g e s may be c l a s s i f i e d t h e y s h o u l d n o t be c l a s s i f i e d i n terms o f each c e l l i n t h e l i n e a g e . R a t h e r t h e s e c e l l s s h o u l d be c l a s s i f i e d i n terms of t h e l i n e a g e i t s e l f , as each c e l l i n t h i s l i n e a g e r e p r e s e n t s p a r t o f a t r a n s f o r m a t i o n s e r i e s , i t i s l e s s c o n f u s i n g and more e x a c t . Only i f t h e l i n e a g e i s composed o f a s i n g l e c e l l w i t h one c h a r a c t e r s t a t e change can t h i s c e l l be g i v e n a t y p e name. For example i n F i g u r e 27 c e l l s # 12, 1 and 24 would be c o n s i d e r e d as one ty p e o f hemocyte l i n e a g e . I f t h e p r e s e n t c l a s s i f i c a t i o n scheme o f hemocyte t y p e s such as t h a t d e s c r i b e d by Jones (1977) i s a n a t u r a l scheme, my s u g g e s t i o n o f a method o f c l a s s i f i c a t i o n w i l l not e l i m i n a t e t h e terms used i n t h i s scheme, b u t w i l l m e r e l y e x t e n d them t o a l i n e a g e o f hemocyte t y p e s so t h a t we w i l l speak i n terms o f t h e ' g r a n u l o c y t e l i n e a g e ' 118 and t h e 'p l a s m a t o c y t e l i n e a g e ' . I f however, t h e p r e s e n t method o f c l a s s i f i c a t i o n i s not a n a t u r a l scheme, f o r example i f t h e c e l l s p r e s e n t l y termed p l a s m a t o c y t e s do n o t r e p r e s e n t a s i n g l e l i n e a g e o f c e l l s , t h e n such terms w i l l have t o be a l t e r e d o r dropped a l t o g e t h e r from t h e c l a s s i f i c a t i o n scheme. I n o r d e r t o det e r m i n e t h e r o b u s t n e s s o f t h e hemocyte terms now i n common usage, c e l l s c l a s s i f i e d u s i n g t h e s e terms s h o u l d be compared t o t h e c e l l s i n t h e Summary F a t e Map.. U n f o r t u n a t e l y , n o t much work has been done on t h e hemocytes o f T r i c h o p t e r a and t h e r e f o r e i t i s d i f f i c u l t f o r me t o compare t h e hemocyte l i n e a g e s o f t h e Summary F a t e Map t o a p u b l i s h e d c l a s s i f i c a t i o n scheme o f T r i c h o p t e r a hemocytes. P r i c e and R a t c l i f f e (19 74) examined t h e hemocytes o f t h e l i m n e p h i l i d A n a b o l i a n e r v o s a and r e p o r t e d t h a t t h e l a r v a e p o s s e s s e d prohemocytes, p l a s m a t o c y t e s , g r a n u l o c y t e s , c y s t o c y t e s and p o s s i b l e o e n o c y t o i d s . The a u t h o r s , however, d i d n o t p u b l i s h photographs o f any o f t h e s e hemocyte 'ty p e s ' and t h u s no com p a r i s o n o f t h e s e hemocytes w i t h t h e Summary F a t e Map can be made. Throughout t h i s s t u d y t h e n , I have d e a l t w i t h i n d i v i d u a l hemocyte t y p e s r a t h e r t h a n hemocyte l i n e a g e s and a t t e m p t e d t o c o r r e l a t e them w i t h t h e c l a s s i f i c a t i o n scheme o f Jones (1977), s i n c e d e a l i n g w i t h l i n e a g e s would make t h e s t u d y non-comparable t o t h o s e o f o t h e r s . A t t h i s p o i n t , i t i s however p o s s i b l e t o s p e c u l a t e as t o whic h l i n e a g e s o f hemocytes t h e c e l l s seen t a k i n g p a r t i n t h e e n c a p s u l a t i o n r e s p o n s e s may b e l o n g . From the l e n g t h s o f t h e c e l l s seen t a k i n g p a r t i n t h e r e a c t i o n t o t h e seven and 35 day i m p l a n t s , as w e l l as t o t h e e s t a b l i s h e d m e t a c e r c a r i a e , i t appears t h a t t h e s e c e l l s c o r r e s p o n d most c l o s e l y t o c e l l s #18, 5 and 14. T h i s s u g g e s t s t h a t t h e c e l l s p a r t i c i p a t i n g i n t h e e n c a p s u l a t i o n r e a c t i o n may have d e v e l o p e d from two l i n e a g e s o f c e l l s . These c e l l l i n e s a r e c i r c l e d i n F i g u r e 27. That c e l l s even more e l o n g a t e d t h a n t y p e s #18, 5 and 14 were found i n t h e c a p s u l e s s u g g e s t s t h a t t h e s e e l o n g a t e d hemocytes a r e subsequent s t a g e s i n t h e development a l o n g t h e s e two l i n e a g e s o f c e l l s . D. Hemocyte A c t i v a t i o n A t p r e s e n t t h e mechanism o f hemocyte a c t i v a t i o n remains u n c l e a r . S a l t (1970) m a i n t a i n e d t h a t ' t h e main q u e s t i o n t o be examined i s whether t h e hemocytes a r e a t t r a c t e d t o a f o r e i g n o b j e c t from a d i s t a n c e o r whether the hemocytes make c o n t a c t w i t h t h e s u r f a c e o f an o b j e c t d u r i n g t h e i r c i r c u l a t i o n and a r e s u b s e q u e n t l y s t i m u l a t e d t o adhere. S a l t (1970) f a v o u r e d t h e former e x p l a n a t i o n . He s u g g e s t s t h a t i f hemocytes were seen t o f l a t t e n b e f o r e t h e i r a c t u a l c o n t a c t w i t h t h e c a p s u l e i t would show a s t i m u l u s had r e a c h e d them b e f o r e t h e y f o r t u i t o u s l y c o n t a c t e d t h e p a r a s i t e , b u t he s u g g e s t s t h a t t h i s has n o t been seen. However, p r e c o c i o u s f l a t t e n i n g o f hemocytes has been o b s e r v e d i n d i p t e r a n s by Nappi and 120 Streams (1969) and N a p p i and S t o f f a n o l o (1971). S e v e r a l t h e o r i e s as t o t h e n a t u r e o f a p o s s i b l e s t i m u l u s a t t r a c t i n g hemocytes t o t h e p a r a s i t e have been s u g g e s t e d . Nappi (1974) q u e s t i o n e d how any o b j e c t i n t r o d u c e d i n t o t h e body o f an i n s e c t c o u l d a v o i d c o n t a m i n a t i o n from s u b s t a n c e s r e l e a s e d from t i s s u e s i n j u r e d d u r i n g t h e e n t r y o f t h e body, and s u b s e q u e n t l y a t t r a c t hemocytes. I n f a c t , Cherbas (1973) i d e n t i f i e d an i n j u r y f a c t o r w h i c h she c a l l e d 'hemokinin' from e p i d e r m a l c e l l s and t h e hemolymph of s a t u r n i i d pupae. She s u g g e s t e d t h a t h e m o k i n i n may a l s o be a s s o c i a t e d w i t h o t h e r c e l l s such as hemocytes. A second t h e o r y m a i n t a i n e d by N a p p i (1974), i s t h a t a change i n t h e normal t i t r e o f h o s t hormones i s b r o u g h t about by p a r a s i t i z a t i o n . Such changes a l t e r t h e p e r m e a b i l i t y o f c e l l membranes t o c e r t a i n m e t a b o l i t e s and b r i n g about p r e c o c i o u s d i f f e r e n t i a t i o n and m i g r a t i o n o f hemocytes. I n t h e p r e s e n t s t u d y I have examined a s i t u a t i o n i n w h i c h a p a r a s i t e has moved t o an a r e a , t h e lumen o f t h e s i l k g l a n d s , where hemocytes a r e n o t n o r m a l l y p r e s e n t . F u r t h e r , t h e c e l l u l a r d e f e n s e system o f t h e c a d d i s f l y was t e s t e d by i m p l a n t i n g p i e c e s o f Epon i n t o t h e hemocoele, an a r e a where hemcoytes a r e p l e n t i f u l . Had hemocytes n o t responded t o t h e p a r a s i t e s i n t h e lumen o f t h e s i l k g l a n d s , b u t o n l y t o i m p l a n t s l o c a t e d w i t h i n t h e hemocoele, i t would have been e v i d e n c e i n s u p p o r t o f the 'random bumping' h y p o t h e s i s o f S a l t (1970). However, hemocytes d i d respond t o t h e p a r a s i t e w i t h i n t h e lumen o f the s i l k g l a n d . A l t h o u g h t h i s s u g g e s t s t h a t t h e hemocytes a r e b e i n g drawn i n t o an a r e a where t h e y would not n o r m a l l y go, i t t e l l s us n o t h i n g about which s t i m u l u s t h e hemocytes a r e r e s p o n d i n g t o . I n t h i s c a s e i t c o u l d be: (1) s e c r e t i o n s (hemokinin?) from t h e damaged e p i t h e l i a l c e l l s o f t h e s i l k g l a n d , (2) s u b s t a n c e s r e l e a s e d from o t h e r c e l l s t h a t were damaged by t h e p a r a s i t e as i t moved from t h e c u t i c l e i n t o t h e s i l k g l a n d , (3) s u b s t a n c e s r e l e a s e d from t h e m e t a c e r c a r i a e , o r (4) a change i n t h e t i t r e o f h o s t hormones r e s u l t i n g from p a r a s i t i s m . E. Wound H e a l i n g The f a c t t h a t t h e e p i t h e l i a l l a y e r o f t h e s i l k g l a n d o f P. a l a s c e n s i s around m e t a c e r c a r i a e o f B. m e d i o v i t e l l a t a was t o t a l l y r e p l a c e d by hemocytes has so f a r been d i s c u s s e d i n terms o f a d e f e n s i v e c e l l u l a r e n c a p s u l a t i o n r e a c t i o n d i r e c t e d a t t h e p a r a s i t e . However, s i n c e i t i s p o s s i b l e t h a t t h e hemocytes were r e s p o n d i n g t o t h e e p i t h e l i a l c e l l s o f t h e s i l k g l a n d which had been damaged by t h e encystment o f t h e m e t a c e r c a r i a e w i t h i n t h e lumen o f t h e g l a n d , r a t h e r t h a n t h e p a r a s i t e i t s e l f , i t i s now a p p r o p r i a t e t o d i s c u s s t h e r o l e o f hemocytes i n the wound h e a l i n g r e a c t i o n s o f i n s e c t s . I n t h e p a s t , wound h e a l i n g has been examined i n t h e a s s a s s i n bug Rhodnius p r o l i x u s S t a l . ( W i g g l e s w o r t h , 1937; L o c k e , 1966; L a i - F o o k , 1968, 1970), t h e gut o f t h e c o c k r o a c h P e r i p l a n e t a americana (Day, 1952), t h e gut o f t h e mosquito 122 Aedes a e q y p t i (L.) and t h e l e a f h o p p e r Orosium a r g e n t a t u s (Ev.) (Day and B e n n e t t s , 1953), and c a t e r p i l l a r s o f t h e wax moth G a l l e r i a m e l l o n e l l a (Rowley and R a t c l i f f e , 1978). In s e v e r a l c a s e s hemocytes were n o t i n v o l v e d i n t h e wound h e a l i n g r e a c t i o n . W i g g l e s w o r t h (1937) and Day and B e n n e t t s (1953) found t h a t hemocytes would n o t r e s p o n d t o c u t i c u l a r wounds u n l e s s the wound was more t h a n s u p e r f i c i a l , and t h e basement membrane was damaged. However, e p i t h e l i a l c e l l s responded n o r m a l l y . I n a d d i t i o n , Day and B e n n e t t s (1953) d e m o n s t r a t e d t h a t h e a l i n g o c c u r r e d i n t h e gut o f A. a e g y p t i w i t h o u t t h e p r e s e n c e o f hemocytes. The a u t h o r s a t t r i b u t e d t h i s t o t h e r a p i d r e g e n e r a t i v e powers o f t h e gut e p i t h e l i u m . N e v e r t h e l e s s , i n most i n s e c t s so f a r examined, hemocytes have been i n v o l v e d t o some e x t e n t i n t h e r e p a i r o f i n j u r y . I n most c a s e s hemocytes were r e p o r t e d t o form a p l u g o v e r t h e wound t h u s p r e v e n t i n g t h e l o s s o f m a t e r i a l from t h e gut (Day, 1952; Day and B e n n e t t s , 195 3) o r hemocoele (Wigglesworth,1937; L a i - F o o k , 1968, 1970; Rowley and R a t c l i f f e , 1978) . The p l u g so formed appeared t o be a temporary s t r u c t u r e , p r e s e n t o n l y u n t i l t h e e p i t h e l i a l l a y e r was a b l e t o m i g r a t e o v e r t h e hemcoytes and r e - e s t a b l i s h i t s e l f ( W i g g l e s w o r t h , 1937; Day, 1952; Day and B e n n e t t s , 1953; Locke, 1966; L a i - F o o k , 1968, 1970). W i g g l e s w o r t h (1937) s u g g e s t e d t h a t a f t e r t h e e p i t h e l i a l c e l l s had c r o s s e d o v e r and s e a l e d t h e wound, t h e hemocytes f o r m i n g the p l u g would d i s s o l v e and d i s a p p e a r . Day (1952) demonstrated t h a t when t h e e p i t h e l i u m had c o m p l e t e l y r e - e s t a b l i s h e d i t s 123 c o n t i n u i t y , t h e 'wound t i s s u e ' formed o f hemocytes was reduc e d . He suggested t h a t t h i s was because hemocytes had l e f t t h e wound and begun t o c i r c u l a t e a g a i n . I n t h e i r s t u d y Rowley and R a t c l i f f e (1978) s u g g e s t e d t h a t f o u r hemocyte t y p e s were i n v o l v e d i n the wound h e a l i n g r e a c t i o n o f G a l l e r i a m e l l o n e l l a t h e s e i n c l u d e d : p l a s m a t o c y t e s , g r a n u l a r c e l l s , s p h e r u l e c e l l s and o e n o c y t o i d s . They s u g g e s t e d t h a t p l a s m a t o c y t e s and g r a n u l a r c e l l s were t h e most abundant o f t h e c e l l t y p e s . The o t h e r a u t h o r s d i d n o t mention t h e t y p e s o f hemocytes i n v o l v e d i n t h e wound h e a l i n g r e a c t i o n s . I n my o p i n i o n t h e n , t h r e e c h a r a c t e r i s t i c s o f t h e hemocyte r e a c t i o n i n P. a l a s c e n s i s suggest t h a t i t i s an e n c a p s u l a t i o n r a t h e r t h a n a wound h e a l i n g r e a c t i o n : (1) i n a l l c a s e s o f wound h e a l i n g so f a r examined t h e hemocytes form a temporary p l u g u n t i l t h e e p i t h e l i a l c e l l s t a k e o v e r ; i n no s l i d e s o f t h e r e a c t i o n o f P. a l a s c e n s i s were s i g n s o f r e g e n e r a t i o n o f t h e s i l k g l a n d e p i t h e l i u m v i s i b l e . I t appeared t h a t r a t h e r t h a n f o r m i n g a temporary wound p l u g u n t i l t h e e p i t h e l i u m was a b l e t o r e g e n e r a t e i t s e l f , t h e hemocytes took on t h e shape o f t h e s i l k g l a n d e p i t h e l i u m and remained as a permanent s t r u c t u r e around t h e p a r a s i t e . Even i n t h e o l d e r m e t a c e r c a r i a l i n f e c t i o n s w h i c h had been m e l a n i z e d , hemocytes were w e l l o r g a n i z e d and no s i g n o f e p i t h e l i a l r e g e n e r a t i o n was v i s i b l e ; (2) a l t h o u g h R a t c l i f f e and Rowley (1978) de m o n s t r a t e d t h e f o r m a t i o n o f a c a p s u l e - l i k e s t r u c t u r e around t h e damaged a r e a i n G. meHone11a t h e s t r u c t u r e was u n o r g a n i z e d and n o t l i k e t h e o r g a n i z e d two l a y e r e d c a p s u l e seen i n P. a l a s c e n s i s ; (3) i n a normal wound h e a l i n g r e a c t i o n hemocytes move t o an a r e a where t i s s u e has been damaged and t e m p o r a r i l y t a k e t h e p l a c e o f t h e s e damaged t i s s u e s . I n t h e s i l k g l a n d s o f P. a l a s c e n s i s hemocytes were seen, n o t o n l y t o r e p l a c e t h e e p i t h e l i a l l a y e r o f t h e s i l k g l a n d , b u t a l s o t o e n t e r i n t o t h e lumen o f t h e g l a n d and f l a t t e n a g a i n s t a r e a s o f t h e p a r a s i t e s which had never been i n c o n t a c t w i t h damaged t i s s u e . V. BIBLIOGRAPHY Anderson, G.A., S.C. S c h e l l and I.D. P r a t t . 1965. The l i f e c y c l e o f B u n o d e r e l l a m e t t e r i ( A l l o c r e a d i i d a e : B u n o d e r i n a e ) , a trematode p a r a s i t e o f Ascaphus t r u e i . J . P a r a s i t . 51: 579-582. A n d r e a d i s , T.G. and D.W. H a l l . 1976. N e o a p l e c t a n a c a r p o c a p s a e n c a p s u l a t i o n i n Aedes a e g y p t i and changes i n h o s t hemolymph p r o t e i n s . Exp. P a r a s i t . 39: 252-261. A r n o l d , J.W. 1979. 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APPENDIX Protocol for Spurrs Sections FIXATION; 2.5% glutaraldehyde, .05 M sodium cacodylate, 1% sucrose, .01 M calcium chloride, 1 drop d i l u t e d photoflow overnight i n vacuum WASH: 5% sucrose, .05 M sodium cacodylate, 1 drop d i l u t e d photoflow overnight i n vacuum POST FIX: 1% osmium tetroxide, .05 M sodium cacodylate, 2% sucrose.. 6 hrs i n vacuum EN-BLOCK STAINING: D i s t i l l e d water 2x 10 min 2.5% uranyl acetate i n d i s t i l l e d water 2 hrs i n dark DEHYDRATION: 35% ethanol 15 min' 50% ethanol .. 15 min 70% ethanol.... 15 min 80% ethanol 15 min 90% ethanol 15 min 100% ethanol.... 2x 1 hr 100% ethanol: propylene oxide 3:1 overnight i n vacuum etOH:PO- 2:1.... overnight i n vacuum etOH:PO- 1:1 overnight i n vacuum etOH:PO- 1:2 overnight i n vacuum etOH:PO- 1:3 overnight i n vacuum 100% PO 2x 30 min i n vacuum INFILTRATION: PO:resin- 3:1 overnight i n vacuum PO:resin- 2:1 overnight i n vacuum PO:resin- 1:1 overnight i n vacuum VI. APPENDIX c o n t i n u e d 133 1 P O : r e s i n - 1:2 o v e r n i g h t i n vacuum P O : r e s i n - 1:3 o v e r n i g h t i n vacuum 100% r e s i n o v e r n i g h t i n vacuum EMBEDDING: 100% r e s i n i n beam c a p s u l e . . . . 8 h r s , 60°C i n 151bs ^ ' vacuum 

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